Copyright

by

William Maurice Doyon

2005

The Dissertation Committee for William Maurice Doyon certifies that this is the

approved version of the following dissertation:

The Effect of Ethanol Consumption on Dopamine and Ethanol

Concentrations in the Nucleus Accumbens during the

Development of Reinforcement and the Involvement

of the κ-Opioid Receptor in the Modulation of Dopamine

Activity during Ethanol Self-administration Committee: Rueben A. Gonzales, Supervisor Richard A. Morrisett Richard E. Wilcox Christine L. Duvauchelle Hitoshi Morikawa

The Effect of Ethanol Consumption on Dopamine and Ethanol

Concentrations in the Nucleus Accumbens during the

Development of Reinforcement and the Involvement

of the κ-Opioid Receptor in the Modulation of Dopamine

Activity during Ethanol Self-administration

by

William Maurice Doyon, B.A., B.S.

Dissertation

Presented to the Faculty of the Graduate School of

the University of Texas at Austin

in Partial Fulfillment

of the Requirements

for the Degree of

Doctor of Philosophy

The University of Texas at Austin

December, 2005

DEDICATION I dedicate this work to my family, particularly my parents, William M. Doyon Sr. and Maria E. Doyon, both of whom have always supported my endeavors with love and devotion.

ACKNOWLEDGEMENTS

I am especially appreciative of Dr. Rueben A. Gonzales for his professionalism and excellent mentorship. For their outstanding advice and guidance, I would also like to thank my dissertation committee – Drs. Richard A. Morrisett, Christine L. Duvauchelle, Richard E. Wilcox, and Hitoshi Morikawa. Additionally, I am grateful to the academic community within the College of Pharmacy at the University of Texas for their exceptional support, including Mickie S. Sheppard, Deborah K. Brand, and Anita L. Mote. I also thank the current and former members of the Gonzales laboratory with whom I worked for their technical assistance and friendly encouragement.

v

The Effect of Ethanol Consumption on Dopamine and Ethanol

Concentrations in the Nucleus Accumbens during the

Development of Reinforcement and the Involvement

of the κ-Opioid Receptor in the Modulation of Dopamine

Activity during Ethanol Self-administration

Publication No. ______________

William Maurice Doyon, Ph.D. The University of Texas at Austin, 2005

Supervisor: Rueben A. Gonzales

A key process associated with the development of reinforcement is the release of

dopamine in the nucleus accumbens from mesolimbic cells of the ventral

tegmental area. There is evidence that accumbal dopamine activity increases

during operant ethanol self-administration, but it is unknown whether this effect is

related to appetitive or consummatory aspects of behavior. Neuroadaptations in

the mesolimbic system during the development of ethanol reinforcement are also

unclear. Studies were undertaken to address these issues, all of which

measured dopamine and ethanol concentrations in the nucleus accumbens of

rats using a procedure in which a fixed number of operant responses was

followed by 20 min of ethanol availability. An initial exposure to 5 or 10% ethanol

vi

(with sucrose) resulted in reduced intake levels compared to controls and no

alterations in dopamine concentrations during consumption. In contrast, during

limited self-administration (about 7 days) of 10% ethanol (with sucrose) or long-

term self-administration (over 40 days) of 10% ethanol, a brief rise in dopamine

levels occurred within 5 min of access that corresponded to consumption. During

the period in which the dopamine response occurred, brain ethanol

concentrations were low but increased progressively thereafter, indicating a rapid

dissociation between the two time courses. Together these results suggest that

the dopamine response was due to factors other than the pharmacological

properties of ethanol, such as the stimulus cues of the solution during acquisition.

These data also suggest that a dopamine response to ethanol occurs after the

development of motivated ethanol drinking. Furthermore, accumbal dopamine

activity during ethanol self-administration may be under the regulation of the

endogenous κ-opioid system, acting to suppress dopamine activity and ethanol

intake. We tested this hypothesis by blocking the κ-opioid receptor with nor-

binaltorphimine during ethanol self-administration. Nor-binaltorphimine treatment

resulted in a brief increase in dopamine concentration 20 min after ethanol

drinking commenced. The latent rise in dopamine levels correlated positively

with accumbal ethanol concentration, suggesting that kappa blockade uncovered

a pharmacological stimulation of dopamine activity by ethanol.

vii

TABLE OF CONTENTS

1.0 General introduction………………………………………………...........……1

1.1 Operant reinforcement……………………………………………….................1

1.2 Dopaminergic substrates of operant reinforcement......................................4

1.2.1 Pharmacological manipulations of dopamine during ICSS........................7

1.2.2 Additional substrates for ICSS reinforcement………………………............8

1.2.3 Dopamine transmission during ICSS……………………………….............12

1.2.4. ICSS in relation to general reinforcement …………………………...........14

1.3 Anatomy and cellular properties of dopamine systems………………..........15

1.4 Behavioral significance of dopamine………………………………..........…...18

1.4.1 Dopamine responses to incentive stimuli…………………………..............19

1.4.2 Dopamine responses to aversive stimuli……………………………............20

1.4.3 Dopamine responses to novel stimuli……………………………….............22

1.5 Dopamine, glutamate, and learning…………………………………...............23

1.6 Summary………………………………………………………………...........…..24

1.7 Dopamine and ethanol reinforcement………………………………................25

1.7.1 Blockade of dopamine during ethanol reinforcement……………...............29

1.8 Opioid modulation of mesolimbic dopamine………………………….............30

1.8.1 Opioid modulation of ethanol reinforcement……………………..........…....35

1.9 Specific aims…………………………………………………...………...............36

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2.0 Dopamine Activity in the Nucleus Accumbens during Consummatory

Phases of Oral Ethanol Self-administration...................................................41

Introduction…………………………………………………………...........….42

Materials and Methods…………………………………………...................45

Results………………………………………………………………...............57

Discussion…………………………………………………………...........…..67

3.0 Accumbal dopamine concentration during operant self-administration

of a sucrose or a novel sucrose with ethanol solution.................................79

Introduction………………………………………………………...........……80

Materials and Methods …………………………………………...........…...82

Results……………………………………………………………..................92

Discussion…………………………………………………………............…103

4.0 Effect of Operant Self-Administration of 10% Ethanol plus 10% Sucrose

on Dopamine and Ethanol Concentrations in the Nucleus

Accumbens.......................................................................................................112

Introduction……………………………………………………………...........113

Materials and Methods …………………………………………..........…....116

Results………………………………………………………...…..........…....126

Discussion...............................................................................................142

5.0 The Effect of κ-Opioid Blockade on Accumbal Dopamine

Concentrations during Operant Ethanol Self-Administration.....................152

Introduction.............................................................................................153

ix

Materials and Methods............................................................................155

Results....................................................................................................166

Discussion...............................................................................................179

6.0 General discussion....................................................................................187

6.1 Future directions..........................................................................................195

Bibliography.......................................................................................................198

Vita.....................................................................................................................245

x

1.0 General introduction

The objectives of this dissertation were (1) to determine the effect of ethanol

consumption on extracellular dopamine concentrations in the nucleus

accumbens during different phases of ethanol reinforcement using procedures in

which we segregated operant responding from ethanol ingestion and (2) to

determine whether the κ-opioid receptor is involved in the modulation of

accumbal dopamine concentrations during ethanol self-administration. The

subsequent sections of this introduction will review the concept of operant

reinforcement and the putative neuronal mechanisms that underlie it, which will

form the theoretical basis for studying operant self-administration of ethanol.

1.1 Operant reinforcement

Whether alcohol is taken for pleasure, the alleviation of negative mental states,

or perhaps other reasons, behaviors are modified and repeated to acquire the

drug. In some cases alcohol consumption can progress into a compulsive

dysfunction, in which the individual loses the ability to regulate intake and is said

to be dependent. The development of alcohol drinking and dependence is due to

complex interactions between environmental and genetic factors that are not

completely understood. The fact that behavioral changes, such as increased

alcohol intake and preference, are observable suggests that there is a learning

component involved. Presumably, this process begins through exposure to

alcohol whereby the subject develops a sense of the positive pharmacological

1

effects of the drug. This incentive may eventually become associated with

specific behaviors and environmental contexts that the subject will want to

repeat. In an attempt to model motivated alcohol drinking in humans, the use of

operant self-administration paradigms has gained widespread acceptance.

Consequently, it is important to recognize the theoretical and historical basis for

their utilization.

E.L. Thorndike pioneered some of the first studies of operant reinforcement,

which he termed associative processes. Thorndike concluded when a behavioral

response elicits a satisfying outcome – such as a cat pulling a string to escape

from an enclosed space – the connection between the response and the

outcome is strengthened (Thorndike, 1911). The Law of Effect, as it became

known, suggested that the behavioral response to the outcome was merely an

unconscious reaction, which had become “stamped in” by the experience and

could be likened to a simple stimulus-response relationship. Thorndike’s later

work in this area led to an interesting observation. Subjects not only recalled

previous verbal responses better when the experimenter rewarded their

responses with a reply of “right” (the Law of Effect), they also showed significant

recall of verbal responses that occurred just before or after the reinforced

response (Thorndike, 1933). These results suggested that certain stimuli could

enhance the retention of temporally contiguous stimulus-response relationships,

which may be important for understanding the behavioral and neurochemical

2

responses that drug-associated stimuli are capable of eliciting.

B.F. Skinner refined the concepts of operant reinforcement to make the approach

more objective and quantitative. Skinner (1938) extended the work of Thorndike

by eliminating the subjective aspect of the outcome and simply defining a

reinforcer as any stimulus that increases the frequency of a specific behavior

(i.e., the operant response). He developed an important conceptual distinction

between a behavioral response to a stimulus (a Pavlovian response) and a

response manifested or “emitted” by the animal for a stimulus (Skinner, 1938).

Skinner posited that as random behaviors developed into goal-directed

responses certain stimuli come to predict the occurrence of the reinforcer

whereas others do not, in which case they are ignored. Therefore, predictive or

discriminative stimuli exert control over the initiation of the operant response

through their correlation with the reinforcer. In this way, the discriminative

stimulus, the operant response, and the reinforcer form a three-component

model of operant reinforcement, which can be thought of in terms of a stimulus-

response-stimulus relationship. There are two general categories of operant

reinforcement: positive and negative, both of which can strengthen behavior. In

positive reinforcement, the reinforcing stimulus elicits approach behavior and

increases the frequency of the operant response because of its addition (e.g.,

pressing a lever to obtain a sugar pellet). In negative reinforcement, the

reinforcing stimulus is negative or aversive and increases the frequency of the

3

operant response as a result of its removal (e.g., pressing a lever to halt the

presence of a loud noise). Negative reinforcement will not be discussed in detail

because it does not pertain to the subsequent work. Lastly, Skinner recognized

that the timing or schedule of the reinforcement could significantly affect operant

behavior. In a continuous reinforcement schedule, every response emitted by

the animal is reinforced, whereas in a fixed-ratio schedule or a variable-interval

schedule the behavior is reinforced after the completion of a certain number of

responses or after a certain amount of time has passed. The latter are types of

intermittent reinforcement that often produce persistent behavior that is more

difficult to extinguish, such as gambling.

1.2 Dopaminergic substrates of operant reinforcement

Tremendous interest in the neuronal mechanisms mediating operant

reinforcement and motivation developed after the seminal experiments of Olds

and Milner (1954), which demonstrated that rats could be trained to deliver

electrical stimulation to distinct regions of their brains. For example, electrical

stimulation of the septal area of the forebrain induced the animals to press a

lever between 285-742 times per hour, whereas stimulation of a nearby site

within the caudate nucleus produced very few responses. After years of

mapping the anatomical sites that support intra-cranial self-stimulation (ICSS), it

can be concluded that several brain regions, connected by distinct pathways, can

elicit reinforcement (Phillips, 1984; Wise and Rompre, 1989). The regions that

4

have received the most attention, and which appear to be most sensitive to

ICSS, are those that lie in close proximity to the medial forebrain bundle. The

medial forebrain bundle is a massive, heterogeneous fiber tract composed of

about 50 different cell groups that course between the brain stem and multiple

forebrain areas in an ascending and descending direction (Nieuwenhuys et al.,

1982). Remarkably, early experiments demonstrated that the reinforcement

produced by self-stimulation of the medial forebrain bundle was of such strength

that animals did not exhibit satiation (Olds, 1958) and persisted in the operant

response despite hunger and weight loss (Routtenberg and Lindy, 1965).

The major ascending dopaminergic (A9-A10) cell groups originate in the

ventromedial mesencephalon or midbrain tegmentum and project in a caudal to

rostral direction, fasciculating at the medial forebrain bundle (Beckstead et al.,

1979; Nieuwenhuys et al., 1982). The dopaminergic fibers of the A9 nuclei, or

substantia nigra pars compacta, course through the lateral region of the bundle

and innervate the majority of the caudate-putamen. This pathway is generally

referred to as the nigrostriatal dopamine system. The dopaminergic fibers of the

A10 nuclei, or ventral tegmental area, also course through the lateral region of

the bundle and innervate the nucleus accumbens, olfactory tubercle, and bed

nucleus of the stria terminalis most densely, which is collectively known as the

mesolimbic dopamine system. Several other regions are also innervated by the

A10 cells, including the posterior hypothalamus, the arcuate nucleus, the

5

olfactory bulb, the amygdala, the lateral septum, the hippocampus, the ventral-

medial caudate putamen. Mesencephalic dopamine cells also project to cortical

sites, such as the prefrontal cortex, but the function of these cells may be

regulated by different mechanisms than those associated with the mesolimbic

system (Jackson and Moghaddam, 2004). Other smaller and less studied

dopaminergic pathways also exist, but they will not be discussed here.

Several early mapping experiments showed that ICSS is attenuated by

catecholamine antagonists (Milner, 1991), which together with the anatomical

evidence led to the hypothesis that activation of catecholamine neurons within

the medial forebrain bundle contributes to ICSS (German and Bowden, 1974).

Subsequent experiments with highly specific movable electrodes supported this

concept by demonstrating that electrode placement directly within the ventral

tegmentum and substantia nigra induced ICSS (Corbett and Wise, 1980). It was

shown that the current (µA) needed to maintain responding (current threshold)

correlated negatively with the proximity of the electrode to dopaminergic nuclei,

suggesting that the sensitivity to the stimulation was due to dopamine activation.

In contrast, direct stimulation of the locus coeruleus (the A6 noradrenergic

nucleus) failed to induce ICSS (Corbett and Wise, 1979), making it apparent that

the ascending noradrenergic system was not as critical for the induction of ICSS

as the ascending dopaminergic pathways. Note that these studies only indicate

the specific regions involved in ICSS, they do not provide cellular and

6

neurochemical specificity for the phenomenon. Although historically difficult to

interpret, focal lesion studies generally support the idea of a more prominent role

of dopamine in ICSS. Severe lesions of the dorsal noradrenergic pathway were

shown to not significantly affect ICSS (Clavier et al., 1976; Corbett et al., 1977),

whereas lesions of the mesencephalic dopaminergic system can severely disrupt

ICSS (Phillips et al., 1976; Fibiger et al., 1987).

1.2.1 Pharmacological manipulations of dopamine during ICSS

Further support for the involvement of dopamine systems in ICSS came from

experiments in which dopaminergic transmission was altered by pharmacological

agents during self-stimulation. For example, systemic blockade of dopamine

receptors consistently reduces ICSS behavior (Olds et al., 1956; Zarevics and

Setler, 1979; Gallistel and Davis, 1983; Stellar et al., 1983; Nakajima and

McKenzie, 1986; Gilbert et al., 1995). Local blockade of dopamine receptors

within the terminal regions of the mesolimbic pathway also inhibits ICSS

(Mogenson et al., 1979; Stellar et al., 1983; Duvauchelle et al., 1998). However,

a recurrent confound associated with blockade of dopamine transmission is the

fact that dopamine antagonists can also produce impairments in normal motor

function (Fibiger et al., 1976; Stellar et al., 1983; Tombaugh et al., 1979), in

addition to any effects on reinforcement processes.

7

Although this issue is difficult to parse, many studies have attempted to

distinguish these effects and several have provided evidence that blockade of

dopamine transmission may lessen the reinforcing efficacy of ICSS. For

example, changes in the frequency (pulses/sec) of the electrical stimulation can

alter responding for ICSS, and a minimum frequency is needed to elicit even a

low level of responding. The use of frequency variation, as opposed to current

variation, is thought to be a more quantitative approach to assessing the efficacy

of ICSS, since varying frequency does not appear to alter the number stimulated

neurons (Gallistel and Freyd, 1987). At certain doses dopamine antagonists can

increase the frequency required to maintain responding (a rightward shift in the

rate-frequency function), while not necessarily affecting the performance involved

in attaining the self-stimulation (Stellar et al., 1983; Gallistel and Karras, 1984;

Nakajima and McKenzie, 1986; Gallistel and Freyd, 1987), suggesting that

dopamine blockade can have an effect on ICSS reinforcement that is not due to

motor impairment. Studies of the effects of enhanced dopamine transmission on

ICSS are in accordance with this idea. Administration of amphetamine

systemically (Gallistel and Karras, 1984; Gallistel and Freyd, 1987) or locally

within the nucleus accumbens (Colle and Wise, 1988) can reduce the frequency

needed to maintain ICSS (a leftward shift in the rate-frequency function). These

results suggest that the pharmacological effects of amphetamine can substitute

for self-stimulation and that increased catecholamine activity, possibly dopamine,

is involved.

8

1.2.2 Additional substrates for ICSS reinforcement

Despite this evidence, closer examination of the relationship between

dopaminergic densities and specific stimulation points suggests an incongruity

and that dopamine neurons are not the only cells through which ICSS of the

medial forebrain bundle is mediated. For example, descending ICSS-activated

fibers from the lateral hypothalamic nuclei project to the ventral tegmental area

(Shizgal et al., 1980) and electrical stimulation of this site consistently supports

ICSS (Wise and Rompre, 1989). Gratton and Wise (1983) mapped the ICSS-

sensitive sites within the medial forebrain bundle between the lateral

hypothalamus and the ventral tegmentum and found that the most sensitive sites

(as measured by current threshold) are towards the center of the medial

forebrain bundle. Considering that A9 and A10 dopamine fibers are dispersed

diffusely within the lateral region of the bundle (Beckstead et al., 1979) and

constitute only about 15% of the cell types (Yeomans, 1989), ICSS sites should

presumably have a different distribution. Additionally, stimulation of the terminal

regions innervated by ascending dopamine neurons, specifically the nucleus

accumbens and olfactory tubercle, also elicits very robust ICSS (German and

Bowden, 1974). However, the current thresholds are not always proportional to

the density of dopaminergic terminals (Prado-Alcala and Wise, 1984; Prado-

Alcala et al., 1984). This is exemplified by the finding that sites within the

9

olfactory tubercle with the highest dopaminergic innervation did not support

ICSS, whereas other sites within the structure with less innervation did so.

Studies examining the in vivo electrophysiological properties of ICSS-activated

cells provide evidence that ICSS does not necessarily activate dopamine

neurons directly; rather ICSS appears to excite a subset of descending

myelinated fibers within the medial forebrain bundle that in turn may activate

dopamine neurons in the mesencephalon. The evidence comes from

observations that most ICSS-sensitive neurons have different excitability

properties and opposite anatomical directions of conduction compared to

dopamine neurons. Paired-pulse experiments show that the refractory period

(the time required for a neuron to repolarize following an action potential) for

ICSS-sensitive neurons of the medial forebrain bundle (0.4-1.2 ms) is shorter

than that of dopamine neurons (1.2-2.5 ms; Yeomans, 1979). The shorter

refractory periods are consistent with those estimated for myelinated axons.

Gratton and Wise (1985) characterized two distinct lateral hypothalamic

populations of ICSS-sensitive cells with anatomical linkage to the ventral

tegmentum and showed that one population had short refractory periods (0.4-0.6

ms) and was sensitive to cholinergic blockade. Furthermore, Bielajew and

Shizagal (1982) and others have demonstrated that the estimated conduction

velocity of the action potentials for ICSS-activated neurons is greater than that of

dopamine neurons (Maeda and Mogenson, 1980). Conduction velocities can be

10

estimated by stimulating ICSS-sensitive axonal fibers from different ends with the

paired-pulse technique and then determining the conduction time based on a

behavioral measure (e.g., increased lever pressing). The paired pulses can only

enhance behavior when the interval between the pulses is long enough;

otherwise the two signals interfere with each other due to a depolarization block.

The conduction velocity is then derived by knowing the conduction time, the

distance between the two electrodes, and the refractory period of the axons.

Importantly, this procedure also allows one to determine the direction of the

nerve impulses that contribute to ICSS along a given pathway. Bielajew and

Shizgal (1986) showed that inactivation of the ventral tegmental area with one

electrode (using the depolarization block) decreased the efficacy of ICSS from

the lateral hypothalamus (a site rostral to the ventral tegmentum). This effect

was not observed when the lateral hypothalamus was inactivated and the ventral

tegmentum was stimulated. Presumably if information flow traveled in a caudal

to rostral direction along the medial forebrain bundle, then the stimulation

induced by ICSS should not have been altered by inactivation of the ventral

tegmentum. This finding demonstrates that lateral hypothalamic ICSS can occur

through a ventral tegmental-dependent mechanism. Consistent with this

conclusion, lesions of the lateral hypothalamus can increase the number of

responses and the current needed to maintain self-stimulation of the medial

forebrain bundle (Murray and Shizgal, 1996). Together, these results strongly

suggest that the reinforcement elicited by ICSS is mediated, at least in part, by

11

myelinated axons descending through the medial forebrain bundle, which in turn

stimulate mesencephalic dopamine neurons. These data do not suggest that

unmyelinated neurons do not directly contribute to ICSS, because there is

sufficient evidence that unmyelinated fibers, including dopaminergic axons, can

be excited by ICSS (Yeomans, 1989). However, in general these cells require

higher current levels to be activated and thus appear less sensitive to

reinforcement.

1.2.3 Dopamine transmission during ICSS

Regardless of the mechanism, direct or indirect activation of ascending

dopamine neurons should elicit increased neurotransmitter efflux at the terminal

regions, such as the nucleus accumbens or caudate/putamen, assuming that

these cells are involved in ICSS. Studies quantifying the extracellular

concentration of dopamine during ICSS demonstrate that dopamine activity can

indeed increase in response to the reinforcement. However, early work using

microdialysis and electrochemical detection was contradictory and difficult to

interpret. Some studies showed that the dopamine concentrations in the nucleus

accumbens increased during ICSS (Phillips et al., 1989; Phillips et al., 1992;

Fiorino et al., 1993), whereas others showed no significant changes (Nakahara et

al., 1989; Miliaressis et al., 1991). One issue with the studies that demonstrated

a positive response was that the amplitude of the ICSS current was varied during

the testing periods. The number of neurons that are activated by ICSS is

12

presumed to be directly proportional to the amount of current applied (Gallistel

and Freyd, 1987). Although these experiments suggest that alterations in

stimulus strength or neuronal ensemble activity can enhance dopamine efflux,

they do not necessarily allow us to relate self-stimulation behavior to a specific

neuronal event because the stimulation parameters were not held constant

during reinforcement. Miliaressis et al. (1991) showed that ICSS stimulation

parameters, which elicited similar behavior, could have significantly different

effects on accumbal dopamine release. This study determined a strength-

duration function for ICSS-relevant behavior by varying current amplitude and the

duration of each pulse within an ICSS train. A combination of low current and

long pulse duration was found to be as equally reinforcing as high current and

short pulse duration, which should have resulted in similar effects on dopamine

output if this transmitter is involved in ICSS. The combination of high current and

short pulse duration resulted in enhanced dopamine concentration during ICSS

using a 1-hr microdialysis sampling interval. However, low current and long

pulse duration failed to increase dopamine levels during self-stimulation. In

addition to showing the importance of examining the stimulus parameters for

ICSS, this study also provided early evidence that ICSS reinforcement could

occur in the absence of changes in extracellular dopamine activity, as measured

by microdialysis.

Recent work by Garris et al. (1999) further clarifies this issue by demonstrating

13

that dopamine release in the nucleus accumbens is only observed during ICSS

under specific conditions. Using real-time voltammetric detection, this group

analyzed rapid changes in extracellular dopamine efflux in the accumbens during

self-stimulation of the ventral tegmentum and during experimenter delivered

stimulation of the same region. First, dopamine release was not observed in rats

that failed to be trained for ICSS, suggesting that ICSS reinforcement and

learning may be dopamine dependent. In trained rats, dopamine concentrations

increased during 6 out of 48 ICSS trials, most of which apparently occurred

during the first few trials of the test session. By contrast, in ICSS-naïve rats,

unpredicted intra-cranial stimulation delivered by the experimenter consistently

enhanced dopamine concentrations (about 13 times greater than that seen

during voluntary stimulation). Very similar findings were reported by Kilpatrick et

al. (2000) using the same procedure in the dorsal striatum. Together, these

findings strongly suggest that dopamine release is not required for maintenance

of ICSS reinforcement per se, but may be necessary for acquisition of the

operant response.

1.2.4. ICSS in relation to general reinforcement

The concepts that arose from the earlier ICSS studies provided an important

framework from which the theories of natural (e.g., food) and psychoactive drug

reinforcement developed (Rompre and Wise, 1989). A former theory of the role

of dopamine in reinforcement processes proposed that mesencephalic dopamine

14

systems mediate the hedonic sensation elicited by reinforcing substances (Wise,

1982). It was hypothesized that a reduction in dopamine neurotransmission

caused by dopamine antagonists suppressed operant behavior because of their

ability to blunt the positive feelings associated with obtaining the reinforcer, which

was independent of the effects of these drugs on motor performance. The

results of the recent ICSS studies by Wightman and colleagues are clearly not

compatible with much of this theory since responding for the reinforcing stimulus

continued in the absence of dopamine activity. However, they are in agreement

with a growing body of data indicating that dopamine systems are involved in the

prediction of salient environmental stimuli and in associative learning and

memory (Spanagel and Weiss, 1999; Kelley, 2004), all of which are inextricable

components of goal-directed behavior and reinforcement.

1.3 Anatomy and cellular properties of dopamine systems

As constituents of the basal ganglia, the dorsal and ventral striatum to which the

mesencephalic dopamine cells project are in an optimal position for the

integration of sensory and motor information that is necessary for such complex

processes to occur (Mogenson et al., 1980). For succinctness, subsequent

discussion will focus primarily on studies concerning the mesolimbic system, with

an emphasis on the ventral tegmental area and nucleus accumbens. The

nigrostriatal system is certainly a key component of the circuitry involved in goal-

directed behavior (Packard and Knowlton, 2002); but it is simply beyond the

15

scope of the introduction. The nucleus accumbens is complex and

heterogeneous in organization, containing two major subregions – the core and

shell – and possibly a third rostral extension. These distinctions are based on

cytoarchitecture and neurochemical markers (Zaborszky et al., 1985; Jongen-

Relo et al., 1994; Zahm, 1999), but it is accepted that even these subregions are

not homogeneous and further compartmentalization is possible. The principle

cells of the nucleus accumbens are the medium spiny neurons, which comprise

approximately 90% of the cell types in the region (O’Donnell and Grace, 1993)

and release GABA as a primary neurotransmitter. These cells are also reactive

for neurotensin, enkephalin, dynorphin, and substance P (Meredith, 1999). The

medium spiny neurons are thought to receive convergent synaptic input from

ventral tegmental dopaminergic neurons and glutamatergic neurons from the

hippocampus, amygdala, and various cortical regions (Brog et al., 1993). The

dopamine signal is transduced by two categories of G protein-coupled receptors:

D1-like (D1 and D5) and D2-like (D2-4). Activation of D1-like receptors is generally

excitatory and leads to stimulation of adenylate cyclase, whereas activation of

D2-like receptors suppresses adenylate cyclase activity. Glutamate release

excites the post-synaptic cell through the activation of ionotropic AMPA/kainate

and NMDA receptors and though metabotropic glutamate receptors. The major

output pathways for the nucleus accumbens are the ventral pallidum and the

ventral tegmental area/substantia nigra (Zhou et al., 2003). The anatomical

arrangement between dopamine and glutamate systems within the mesolimbic

16

system is thought to be critical for the expression of the neuroadaptational

processes associated with operant learning and drug reinforcement (Kelley,

2004).

In addition to autoreceptor regulation and a potent uptake system at the

terminals, the extracellular concentration of dopamine within the nucleus

accumbens is maintained by the electrophysiological activity of the dopamine

cells in the ventral tegmental area. In behaving rats, these cells fluctuate

between periods of single-spike action potentials and periods of rapid burst

activity. The single-spike mode is characterized by consistent but low frequency

firing rates (approximately 4 Hz), whereas the burst or phasic mode is

characterized by higher frequency firing rates (approximately 15-20 Hz; Freeman

and Bunney, 1987; Hyland et al., 2002). Notably, the percentage of cells that

exhibit burst firing is very high (90%) in behaving animals (Freeman and Bunney,

1987) compared to recordings taken in anesthetized animals, which in some

cases can be reduced to 0% (Schultz and Romo, 1987). In freely moving

animals, the occurrence of burst firing has been reported to coincide with sensory

stimulation and attentive behavioral responses. Irrespective of the firing pattern,

dopamine is released by exocytosis from terminals within the nucleus

accumbens on a continuous basis, and dopamine molecules appear to diffuse

readily from the synaptic cleft following release (Garris et al., 1994).

Extrasynaptic transporters (Nirenberg et al., 1997) for dopamine are the primary

17

mechanism by which the effects of dopamine are terminated. The extracellular

concentration of dopamine under basal conditions is estimated to be in the low

nanomolar range with microdialysis (Parsons and Justice, 1992; Tang et al.,

2003). High affinity D2 autoreceptors located extrasynaptically (Richfield et al.,

1989) may be activated by these low basal levels of dopamine to regulate

release (Grace, 2000). However, during phasic burst firing the amount of

dopamine released may exceed the capacity of the uptake system and dopamine

concentration in the extracellular space can increase substantially for a brief

period (Chergui et al., 1994). Using electrochemical detection, Gonon (1988)

calculated that this concentration may be approximately 6 times greater than

baseline levels. Theoretically, excess dopamine molecules could then diffuse

and activate receptors at distances remote from their site of release. The

transient changes in extracellular dopamine levels that occur during bursting are

thought to represent an important mechanism by which dopamine cells modulate

cellular activity within the accumbens (Grace, 2000). Pertinent to the present

work, the relative contribution of burst activity to tonic extracellular dopamine

concentration measured with microdialysis is unclear. However, it is assumed

that tonic levels increase transiently following burst activity (Wightman and

Robinson, 2002).

1.4 Behavioral significance of dopamine

Examination of the range of conditions and stimuli that activate mesolimbic

18

dopamine neurons suggests a very broad functional role for these cells in

behavior. The subsequent section is not comprehensive but is meant to illustrate

the involvement of dopamine in positive reinforcement and associative learning

processes. The focus will be primarily on dopaminergic responses to natural and

aversive stimuli, since this is most relevant to our understanding of how this

system functions under physiological conditions that an animal may manifest in

its natural environment.

1.4.1 Dopamine responses to incentive stimuli

Single unit recordings from mesencephalic dopamine neurons indicate that the

firing rate of many of these cells increases transiently based on the ability of the

animal to predict the occurrence of an incentive. For example, primates that are

thirsty exhibit transient bursts of dopamine activity following the acquisition of a

liquid incentive stimulus (Schultz et al., 1997). However, after the animals learn

that a light cue always precedes the availability of the liquid stimulus, the

neuronal activity ceases to occur in response to the liquid and occurs instead

after the presentation of the light, which has become a conditioned stimulus that

now predicts the timing of the reinforcer. If the reinforcer is omitted, the firing

activity of the dopamine neurons is suppressed. In a related experiment, it was

shown that the ability of the liquid reinforcer to stimulate dopamine neurons is

also related to the reliability of the reinforcement (Hollerman and Schultz, 1998).

As the animals learned when to expect acquisition of the liquid, dopamine activity

19

decreased progressively as a function of the number of testing days. The work

of Fiorillo et al. (2003) supports this by demonstrating that the magnitude of the

dopamine activity observed is dependent on the probability of obtaining the

reinforcer.

However, dopaminergic responses to incentive stimuli are strongly influenced by

the motivational state of an animal. Although sucrose is highly reinforcing,

microdialysis studies show that dopamine concentrations only increase during

consumption in animals that are food or water deprived (Hajnal and Norgren,

2001; Hajnal et al., 2004; Genn et al., 2004). In contrast, voltammetric

measurements of dopamine release in unrestricted rats show that dopamine

activity is absent during periods of sucrose consumption following operant

responding (Roitman et al., 2004). However, conditioned cues that predicted

sucrose availability were shown to stimulate dopamine levels, which together is

consistent with the previous data obtained by single unit recordings. In fact,

genetically engineered mice that cannot synthesize dopamine still show sucrose

preference and “liking” (Cannon and Palmiter, 2003), whereas hyper-

dopaminergic mice show greater performance capacity (“wanting”) to attain

sweet substances (Pecina et al., 2003), further suggesting that dopamine is

involved in functions other than hedonic processes of reinforcement. One

hypothesis is that the motivational state of an animal can augment the incentive

value or salience of a reinforcing stimulus (Kelley and Berridge, 2002). For

20

example, a hungry animal should attribute greater incentive value to food than a

satiated animal. Indeed, differential activation of the mesolimbic dopamine

system has been shown under these conditions (Wilson et al., 1995).

1.4.2 Dopamine responses to aversive stimuli

These transient changes in mesencephalic dopamine neuron excitability have

been argued to code for errors in the prediction of positive reinforcement, rather

than of negative or other salient events (Schultz, 1998; Ungless, 2004). These

views are based mainly on electrophysiological evidence, in awake or

anesthetized animals, showing that a large percentage of dopamine neurons are

either inhibited/unaffected (Schultz and Romo, 1987; Mantz et al, 1989; Guarraci

and Kapp, 1999; Ungless et al., 2004) or activated weakly (Mirenowicz and

Schultz, 1996) by aversive conditional stimuli (e.g., air puffs to the hand, oral

hypertonic saline, or tail pinch). In contrast, microdialysis studies indicate that

stressful stimuli, such as shock (Sorg and Kalivas, 1991; Abercrombie et al.,

1989), handling (Enrico et al., 1998), or sensory cues that predict aversive stimuli

(Young et al., 1998), can increase extracellular dopamine concentration. Recent

work by Young (2004) illustrates this point. Using 1-min microdialysis sampling it

was shown that dopamine concentration increased after each presentation of a

mild foot shock. Importantly, when the foot shocks were paired with a

conditioned cue, the cue by itself elicited a rise in dopamine activity. These

results are even more convincing considering that the procedure took into

21

account the lag time inherent in microdialysis, indicating that this was not a

delayed effect from the previous sample.

There are two criticisms associated with the electrophysiological data. Although

it has been claimed that the neurophysiological responses of dopamine neurons

are not strongly affected by the anesthesia used (Ungless, 2004), the results are

simply not relevant to the behavior of an animal during reinforcement. Horovitz

(2000) has also pointed out that the negative data may be due to the mild nature

of the aversive stimuli utilized in some of these experiments. Clearly, a puff of air

to the hand is not as arousing or salient as a foot shock, for example.

Conversely, the positive results from the microdialysis experiments could be

related to a slow onset time for the dopaminergic response caused by opponent

processes (Daw et al., 2002), which the single unit recordings would fail to detect

because of time resolution (Ungless, 2004). As an alternative explanation, the

removal of an aversive event (or negative reinforcement) may also be mediated

by dopaminergic processes similar to those involved in positive reinforcement,

and thus increased dopamine may signal stimulus removal (Ikemoto and

Panksepp, 1999). However, the results of Young (2004) are not consistent with

this hypothesis, since it was shown that a dopamine activity increased in

response to a conditioned cue prior to onset and removal of the foot shock.

Although these issues are not completely settled, these studies generally indicate

that dopamine neurons are activated by reinforcing or aversive stimuli and to

22

cues that predict the occurrence of these events.

1.4.3 Dopamine responses to novel stimuli

The degree of novelty that a stimulus imparts is another condition that facilitates

the activation of mesolimbic dopamine neurons. Ingestion of highly palatable

food, for example, can enhance extracellular dopamine concentration in the

nucleus accumbens in deprived (Anh and Phillips, 1999) or non-deprived animals

(Bassareo and Di Chiara, 1997). However, a second exposure to the same

substance soon after results in a blunted dopamine response during

consumption (Bassareo and Di Chiara, 1997), unless the subject is presented

with a different palatable substance during the second exposure, in which case

dopamine levels rise again (Anh and Phillips, 1999). Furthermore,

somatosensory stimuli, which have no apparent reinforcing or biological value,

are also capable of activating dopamine neurons. Single cell recordings taken in

behaving cats show that unconditioned auditory and visual cues can elicit burst

firing activity in ventral tegmental dopaminergic cells (Horvitz et al., 1997).

Similar to the observations of Freeman and Bunney (1987), this study reported

that burst activity under basal conditions coincided with attentiveness in the

animal.

1.5 Dopamine, glutamate, and learning

Increasing evidence suggests that many of the neuroadaptational processes that

23

underlie natural and drug reinforcement are similar to those involved in some

types of learning and memory (Kelley, 2004). The predominant medium spiny

neurons of the nucleus accumbens are thought to receive convergent synaptic

input from ventral tegmental dopaminergic neurons and glutamatergic neurons

from the hippocampus, amygdala, and various cortical regions (Brog et al.,

1993). At the cellular level, glutamatergic NMDA receptor-mediated currents are

potentiated by coincident activation of dopamine D1 receptors (Cepeda et al.,

1993; Harvey and Lacey, 1997). The expression of immediate early genes by

amphetamine or dopamine is dependent on NMDA receptor-mediated entry of

calcium in striatal preparations, suggesting that coordinated activation can have

lasting consequences. Dopamine-dependent plasticity has been reported in the

nucleus accumbens (Floresco et al., 2001) and the striatum in combination with

excitatory cortical input (Wickens et al., 1996; Kerr and Wickens, 2001; Reynolds

et al., 2001). Importantly, in terms of behavior, the co-activation of glutamate

receptors (NMDA, AMPA/kainate) and dopamine D1 receptors within the

accumbens and the prefrontal cortex is a necessary condition for incentive-

related operant learning to occur (Smith-Roe and Kelley, 2000; Baldwin et al.,

2002; Hernandez et al., 2005). Additionally, Pecina et al. (2003) found that task

performance is enhanced in hyper-dopaminergic mice. Together, these findings

support the notion that interactions between dopamine and glutamate systems

are an important process that may be involved simple forms of learning.

24

1.6 Summary

Viewed in a broad sense, this collection of data suggests that the mesolimbic

dopamine system contributes to the detection of salient environmental events

and the associative learning processes involved in goal-directed behavior. This

system does not appear to be critical for the maintenance of positive

reinforcement under conditions in which acquisition is highly probable. However,

dopamine transmission does seem necessary for the development of positive

reinforcement. During the transition from random responses to goal-directed

behavior, a simple type of learning is required in which the subject must form an

association between a wanted stimulus and a behavior that leads to the

acquisition of the stimulus. In this way, increased dopamine activity may act as a

teaching signal during the development of reinforcement, continually updating the

current state of events to help modulate behavior accordingly. If acquisition and

salience of the reinforcer is predictable, then new learning is not required.

However, when deviations from the expected outcome occur (e.g., changes in

the timing or strength of the reinforcer), then it is necessary to update prior

reference points as an adaptive response to a changing environment.

1.7 Dopamine and ethanol reinforcement

Operant models of ethanol reinforcement consistently show that animals are

willing to perform behaviors that result in acquisition of ethanol (Winger and

Woods, 1973; Samson, 1986; Samson et al., 1988; Suzuki et al., 1988; Weiss et

25

al., 1993; Williams and Woods, 1998; Czachowski and Samson, 1999). The

precise mechanisms by which ethanol elicits reinforcement are unclear due to its

ubiquitous action in the brain, but mesolimbic dopamine is certainly implicated in

the process. Based on current models of dopamine function, ethanol should

stimulate this system at some point during the development of reinforcement or

during certain phases associated with a reinforcement session. A systematic

examination of dopaminergic activity during different stages of ethanol-reinforced

behavior has not been performed (for example, from an initial exposure period

until well-maintained behavior), which would helpful for understanding the

plasticity involved in motivated ethanol drinking. However, several microdialysis

studies in rodents have quantified changes in dopamine concentration during

certain phases of ethanol self-administration. The first of this kind was

conducted by Weiss et al. (1993), which demonstrated that accumbal dopamine

concentration increased (1) during the period preceding ethanol access after the

animals were transferred from their home cages into the operant chamber and

(2) during periods following ethanol responding and ingestion. Subsequent

studies have replicated these findings (Weiss et al., 1996; Gonzales and Weiss,

1998; Katner and Weiss, 1999; Melendez et al., 2002), including one that

measured the mesolimbic dopamine response within the ventral pallidum

(Melendez et al., 2004). The finding that dopamine concentrations rise when the

animals are placed into the operant environment is consistent with the known

effects of salient stimulus cues on dopamine activity. However, because of

26

evidence indicating that novelty and tactile stimulation associated with physical

handling can also increase mesolimbic dopamine levels (Enrico et al., 1998;

Adams and Moghaddam, 2000), it is unclear whether the enhancement reported

in certain studies represents an effect of handling the animals or, as the authors

suggest, an incentive motivational response to cues that predict ethanol

availability.

Furthermore, there are also issues concerning the finding that ethanol stimulates

dopamine activity during the consummatory phase of ethanol self-administration.

Significant dopamine responses during and after ingestion and lack thereof in the

control groups originally led to the interpretation that ethanol may increase

dopamine concentration through its pharmacological action on the mesolimbic

system (Weiss et al., 1993). Previous procedures utilized a schedule of

continuous reinforcement in which 1-3 responses emitted by the animal were

reinforced with a small amount of ethanol over a 30-60 min session. Therefore,

during this period there were two separate behavioral responses occurring within

the span of a single microdialysis sample (5-10 min): the appetitive response of

lever pressing for ethanol and the consummatory response of ingesting the

ethanol. This is an important distinction because changes in dopamine activity

could be involved in either of these behaviors, independent of the

pharmacological effects of ethanol. Beyond the experimental design, another

issue that may alter the interpretation of these data regards the statistical

27

procedures used to asses the changes in dialysate dopamine levels.

Examination of the data from Weiss and colleagues indicates that the dialysate

dopamine concentrations during the self-administration period were compared to

the home-cage baseline concentrations, which were significantly lower than

those that occurred after the rats were moved into the operant environment.

From a conservative standpoint, the increased dopamine response after the

transfer period is the more appropriate baseline to use in this situation.

Depending on the study, these dopamine levels were either indistinguishable

(Gonzales and Weiss, 1998) or only marginally higher (Weiss et al., 1993) than

those that occurred during self-administration in Wistar animals. Based on these

concerns, it is uncertain whether the dopamine response observed during the

drinking period is in fact elevated compared to the adjusted baseline and, if so,

whether the appetitive or consummatory phases of behavior are involved in the

process.

Of the previous operant studies of ethanol reinforcement, those that provide the

strongest evidence for activation of the mesolimbic dopamine system during

ethanol self-administration are those that utilized alcohol-preferring P rats as

subjects (Weiss et al., 1993; Melendez et al., 2002; Melendez et al., 2004). The

P line display a marked dopamine response to ethanol stimulus cues that is not

related to handling and show robust elevations in dopamine during self-

administration (60-100% above basal). The P rats, like other alcohol-preferring

28

lines, are bred for their alcohol preference and are known to display abnormal

neurochemical and behavioral traits. These lines exhibit lower tissue levels of

dopamine (Murphy et al., 1987; Gongwer et al., 1989; Katner and Weiss, 2001)

and greater dopaminergic responsiveness to oral ethanol intake (Weiss et al.,

1993; Katner and Weiss, 2001) compared with non-preferring lines. Table 1.0

illustrates differences between ethanol intake and peak dopamine response to

systemic ethanol administration in selected rat lines (adapted from Katner and

Weiss, 2001). While alcohol-preferring rats are a valuable model for studying the

possible neuronal mechanisms underlying high-alcohol drinking behavior, they

may not be optimal for understanding low or moderate ethanol self-

administration. Nonetheless, further characterization of ethanol-preferring and

heterogeneous rats using operant procedures that distinguish appetitive

responding from ethanol consumption is needed to clarify the time course of

ethanol’s effects on dopamine during self-administration and to determine the

extent to which continuous reinforcement affects this response.

1.7.1 Blockade of dopamine during ethanol reinforcement

Apart from directly measuring dopamine efflux, another effective method for

determining the involvement of mesolimbic dopamine in ethanol reinforcement is

to manipulate dopamine transmission during ethanol self-administration.

Numerous studies have tested the effects of systemic application of dopamine

agonists (Pfeffer and Samson, 1988; Rassnick et al., 1993; Cohen et al., 1999)

29

TABLE 1.0. Summary of daily ethanol preference and peak accumbal dopamine response to i.p. ethanol (1.5 g/kg) in ethanol preferring and non-preferring rat lines, adapted from Katner and Weiss, 2001.

Rat line *Preference ratio

% DA increase (above basal)

Alko alcohol 0.51±0.04 51±19 Alko nonalcohol 0.04±0.00 25±7

High alcohol-preferring 0.51±0.06 38±12 Low alcohol-preferring 0.10±0.03 23±8

Wistar 0.17±0.03 34±15

*Preference calculated as a ratio of daily ethanol intake to ethanol plus water intake

30

and antagonists (Pfeffer and Samson, 1988; Files et al., 1998; Czachowski et al.,

2002) on ethanol-reinforced behavior. While these works are important, the

nonspecific action of these drugs due to route of administration makes their

results difficult to interpret fully. An alternative approach is to examine the

literature on the effects of local administration of dopaminergic agents within the

terminal fields of the mesolimbic pathway on ethanol self-administration. In

studies in which continuous reinforcement schedules were implemented,

microinfusion of a dopamine D1 and D2 antagonist into the nucleus accumbens

reduced the total number of responses emitted for ethanol (Rassnick et al., 1992;

Samson et al., 1993; Hodge et al., 1997). A role for the D2 receptor in the

mediation of ethanol reinforcement is supported by evidence that mice lacking

the D2 receptor show reduced responding for ethanol (Risinger et al., 2000).

Conversely, activation of accumbal D2 receptors can increase responding at

certain doses, whereas D1 activation does not significantly alter ethanol-seeking

behavior (Hodge et al., 1997). Czachowski et al. (2001) distinguished the effects

of operant responding and ethanol consumption by training rats to emit a fixed

number of responses for 20 minutes of access to 10% ethanol. This work

demonstrated that D2 blockade in the accumbens delayed the initiation of

responding and only affected intake levels at high doses, suggesting a selective

effect on appetitive responses as opposed to consummatory behavior. As a

caveat, it is important to note that differences in drug efficacy and selectivity for

dopamine receptors (vs. non-dopamine receptors) could potentially influence the

31

outcome of these types of experiments. Although more work of this kind is

obviously needed, these data are together consistent with the predicted

involvement of mesolimbic dopamine system in acquisitional processes and

suggest that reinforcing effects of ethanol are mediated through dopamine

receptors, particularly D2-like receptors.

1.8 Opioid modulation of mesolimbic dopamine

The endogenous opioid system is hypothesized to have a key role in modulating

mesolimbic dopamine function and ethanol reinforcement processes (Hertz,

1997; Cowen and Lawrence, 1999). Opioid neurotransmission involves at least

four major types of endogenous peptides: β-endorphin, endomorphin,

enkephalin, and dynorphin (Terenius and Wahlstrom, 1975; Hughes et al., 1975;

Li and Chung, 1976; Goldstein et al., 1979; Zadina et al., 1997). The opioid

peptide signal is transduced by three major populations of receptors (µ, δ, κ),

each of which has known subtypes. β-endorphin and endomorphin have high

affinities for the µ-opioid receptor (Zadina et al., 1997). β-endorphin and

enkephalin have similar affinities for the δ-opioid receptor (Lord et al., 1977).

Dynorphin binds with high affinity to the κ-opioid receptor (Chavkin et al., 1982).

These receptors couple to inhibitory G proteins, and their activation has a

suppressive effect on adenylate cyclase, calcium currents, and overall cellular

activity. Opioid receptors are expressed throughout the mesolimbic circuitry

(Mansour et al., 1987). For example, the µ- and κ-opioid receptors are found

32

within the ventral tegmental area and are in particularly high density within the

nucleus accumbens. Examination of their cellular distribution within the

accumbens indicates that the µ-opioid receptor is located mainly on post-synaptic

structures, but a fair number (about 27%) are also presynaptic (Svingos et al.,

1996). Within the ventral tegmental area, mu receptors are found primarily on

non-dopaminergic cells (Garzon and Pickel, 2001). The cellular localization of

the κ-opioid receptor is distinct in that it is predominantly on presynaptic

accumbal terminals (many of which appear to be dopamine-containing) and to a

much smaller degree on postsynaptic sites (Svingos et al., 1999; Meshul and

McGinty, 2000; Svingos et al., 2001). Electrophysiological evidence suggests

that the kappa receptor is found on dopamine cell bodies and on presynaptic

glutamatergic neurons within the ventral tegmental area (Margolis et al., 2003;

Margolis et al., 2005). However, additional work using ultrastructural staining

techniques is needed to determine the precise cellular distribution of kappa

receptors within the mesencephalon.

As its neuroanatomical arrangement suggests, the endogenous opioid system

can modulate mesolimbic dopamine activity significantly. The major current view

is that the mu and delta receptors mediate cellular responses that are opposite to

those conducted by kappa receptors, and this dynamic interaction is

accompanied by changes in accumbal dopamine and motivational responses.

For example, a variety of experiments show that activation of µ-opioid receptors,

33

either locally within the ventral tegmental area or systemically, stimulates

mesoaccumbens dopamine activity (Gysling and Wang, 1983; Matthews and

German, 1984; Di Chiara and Imperato, 1988; Leone et al., 1991; Spanagel et

al., 1992; Devine et al., 1993; Yoshida et al., 1993). Administration of selective

mu agonists directly into the nucleus accumbens has produced mixed results,

with studies showing increases in dopamine levels (Yoshida et al., 1999) or no

effect at all (Spanagel et al., 1992). This discrepancy may be related to the

activation of other opioid receptors due to the high dose utilized in the former

study or to the use of anesthesia in the latter study. Behavioral data support the

idea that mu effects on mesolimbic activity are mediated through a ventral

tegmental mechanism. Rats prefer to spend more time in a room where they

received an infusion of a mu agonist into the ventral tegmentum (suggesting

positive reinforcement), but this behavior is absent in accumbens-treated animals

(Bals-Kubik et al., 1993; Nader and van der Kooy, 1997). The process by which

dopamine activity is enhanced may involve indirect excitation of tegmental

dopamine cells through mu receptor-mediated inhibition of GABA interneurons

(Johnson and North, 1992; Margolis et al., 2003).

Conversely, κ-opioid receptor activation consistently reduces basal dopamine

activity (Di Chiara and Imperato, 1988; Heijna et al., 1990; Spanagel et al., 1992;

Devine et al., 1993; Xi et al., 1998; Margolis et al., 2003). Microdialysis studies

have suggested that this effect is mediated by kappa receptors in the nucleus

34

accumbens since dopamine levels are decreased by accumbal administration of

kappa agonist, whereas administration of these agents into the ventral tegmental

area is without effect (Spanagel et al., 1992; Devine et al., 1993). However,

Margolis et al. (2003; 2005) demonstrated that a subpopulation of ventral

tegmental dopamine cells and apparently certain excitatory inputs are directly

inhibited by kappa activation. Furthermore, behavioral data indicate that the

nucleus accumbens and the ventral tegmental area are involved in this response,

since rats avoid rooms where they received an infusion of a kappa agonist into

either of these sites, suggesting negative reinforcement at both regions (Bals-

Kubik et al., 1993). The results of the previous microdialysis experiments may

have been influenced by the fact that they were performed under anesthesia,

which can alter the firing pattern of dopamine neurons (Hyland et al., 2002).

Therefore, the relative contribution of the ventral tegmental area and the nucleus

accumbens in mediating the dopamine suppression induced by kappa activation

is unclear.

A few studies have also determined the effects of κ-opioid blockade on accumbal

dopamine levels (Spanagel et al., 1992; Maisonneuve et al., 1994), but the

results have thus far been difficult to interpret. It was reported that dopamine

concentrations increased transiently in response to accumbal or systemic

administration of the kappa antagonist, nor-binaltorphimine after acute treatment.

Nor-binaltorphimine is a potent and long-lasting kappa antagonist (Portoghese et

35

al., 1987; Horan et al., 1992), but it is known to have nonselective action when

administered acutely (Endoh et al., 1992). Therefore, the transient dopamine

response observed may have been partially due to activation of other receptor

types by the antagonist, which a longer pretreatment time would resolve. A

recent study by Chefer et al. (2005) suggests that dopamine release is enhanced

after long-term kappa blockade with nor-binaltorphimine, but that basal dopamine

concentration remains unchanged due to an increase in dopamine uptake from

the extrasynaptic space. Thus, the κ-opioid system may interact with the

dopamine transporter to regulate basal dopamine levels. In conclusion, the

effects of opioid receptor manipulation on mesolimbic dopamine activity have

been studied extensively. Currently, however, experiments of this type have not

been conducted during ethanol reinforcement, which would provide important

information about the possible interactions between the mesolimbic opioid and

dopamine systems.

1.8.1 Opioid modulation of ethanol reinforcement

It is hypothesized that the release of endogenous opioids contributes to ethanol

reinforcement. Several experiments have demonstrated that tissue levels of the

major opioid peptides are elevated within brain regions associated with

reinforcement following ethanol administration (Schulz et al., 1980; Seizinger et

al., 1983; De Waele et al., 1994; Lindholm et al., 2000). Microdialysis work has

indicated that β-endorphin and dynorphin concentrations increase shortly after an

36

i.p. ethanol injection (Olive et al., 2001; Marinelli et al., 2004; Marinelli et al.,

2005). It is well documented that nonselective opioid antagonists, such as

naltrexone, can reduce alcohol intake and relapse in humans (Volpicelli et al.,

1992). In animal models, ethanol-reinforced responding is curtailed by µ- and δ-

opioid receptor antagonists (June et al., 1999; Hyytia and Kiianmaa, 2001).

Further, mice lacking the µ-opioid receptor also show reduced operant ethanol

responding (Roberts et al., 2000). The specific role of the κ-opioid system in

ethanol self-administration is less clear. Ethanol-preferring rodents show lower

basal dynorphin levels in the accumbens compared to ethanol-avoiding rats

(Nylander et al., 1994). However, kappa receptor blockade with nor-

binaltorphimine has not been shown to alter ethanol reinforcement (Williams and

Woods, 1998; Holter et al., 2000), which may be related to the low doses used

(3-5 mg/kg). By contrast, activation of κ-opioid receptors effectively attenuates

ethanol self-administration (Lindholm et al., 2001; Cosgrove and Carroll, 2002).

Unfortunately, kappa agonists also produce an array of adverse side effects,

including sedation, conditioned place aversion, reduced drug and food self-

administration, reduced sexual behavior, and dysphoria in humans (Pfeiffer et al.,

1986; Leyton and Stewart, 1992; Bals-Kubik et al., 1993; Mello and Negus, 1998;

Walsh et al., 2001). Due to these general effects on behavior, it has proven

difficult to determine the specific role of kappa receptor activation in ethanol

seeking. Future studies using operant reinforcement paradigms that distinguish

appetitive and consummatory behavior and that utilize higher doses of nor-

37

binaltorphimine may be helpful for determining the involvement of the κ-opioid

system during motivated ethanol drinking.

1.9 Specific aims

The following section represents the specific aims of this dissertation:

1. Determine the effect of 10% ethanol consumption on dopamine and ethanol

concentrations in the nucleus accumbens in male Long-Evans rats using

microdialysis and a training procedure in which the animals have developed

established patterns of ethanol reinforcement. To evaluate the effect of oral

ethanol consumption on dopamine activity apart from responding, the behavior of

the rats will be segregated into discrete epochs. During the appetitive or operant

period the animals will complete a single fixed-ratio response requirement

(approximately 20 lever presses), followed by 20 min of unrestricted access to a

10% ethanol solution. We will quantify dopamine concentrations throughout

these periods and consumption patterns and intra-accumbal ethanol levels will

be monitored after ethanol availability for comparison. The experiments will

include two control groups: one trained to respond for tap water rather than

ethanol and another that is not trained for operant self-administration and simply

placed into the operant environment.

2. Determine the effect of 10% sucrose (plus 5 or 10% ethanol) consumption on

38

dopamine and ethanol concentrations in the nucleus accumbens in male Long-

Evans rats using microdialysis and a training procedure in which the animals

have not had prior exposure to an ethanol solution. The goal is to establish

whether a first-time exposure to ethanol prior to the development of

reinforcement can enhance dopamine levels. A single fixed-ratio response

requirement (approximately 20 lever presses) will precede 20 min of access to

the drinking solution. During the dialysis experiment one group of animals will

self-administer 10% sucrose as trained and a second group will self-administer a

novel 10% sucrose (plus 5 or 10% ethanol) solution. Consumption patterns and

intra-accumbal ethanol levels will also be monitored for comparison with

dopamine concentrations after ethanol availability.

3. Determine the effect of 10% ethanol (plus 10% sucrose) consumption on

dopamine and ethanol concentrations in the nucleus accumbens in male Long-

Evans rats using microdialysis and a training procedure in which the animals are

at an early stage in the development of reinforcement. One goal is to test the

hypothesis that increased ethanol intake (by the addition of sucrose) will

potentiate the dopamine response observed during self-administration. The rats

will self-administer 2-10% ethanol (plus 10% sucrose) over 6 days. A single

fixed-ratio response requirement (2-4 lever presses) will precede 20 min of

access to a drinking solution. Consumption patterns and intra-accumbal ethanol

levels will also be quantified for comparison with dopamine concentrations after

39

ethanol availability. The experiments will include two control groups: one trained

to respond for 10% sucrose without ethanol and another that is not trained for

operant self-administration and simply placed into the operant environment.

4. Test the hypothesis that κ-opioid receptor blockade will potentiate dopamine

concentrations in the nucleus accumbens during operant ethanol self-

administration. The ethanol training procedure and reinforcement schedule used

in specific aim 3 will be repeated here. We will inject the rats with the kappa

antagonist, nor-binaltorphimine or saline subcutaneously 15-20 hrs prior to the

experiment. Consumption patterns and intra-accumbal ethanol levels will also be

quantified for comparison with dopamine concentrations after ethanol availability.

The effective dose of nor-binaltorphimine will be determined prior to these

experiments by assessing its ability to block the pharmacological effects of

U50488 (a κ-opioid receptor agonist).

40

2.0 Dopamine Activity in the Nucleus Accumbens during Consummatory

Phases of Oral Ethanol Self-administration

[published in Alcoholism: Clinical and Experimental Research (2003) 27 (10):

1573-1582; by William M. Doyon, Jennifer L. York, Laurea M. Diaz, Herman H.

Samson, Cristine L. Czachowski, and Rueben A. Gonzales reproduced with

permission from Lippincott Williams & Wilkins]

ABSTRACT

The present study was designed to clarify the role of dopamine in the nucleus

accumbens during operant ethanol self-administration by separating bar pressing

(ethanol seeking) from ethanol consumption. Furthermore, we sought to define

the relationship between ethanol in the brain and the accumbal dopamine

response after oral self-administration of ethanol. Two separate groups of male

Long-Evans rats were trained to bar press using 10% ethanol or water and a

procedure that distinguished between bar pressing and consumption of ethanol

(or water). Rats were trained to elicit an escalating number of bar presses

across daily sessions before gaining access to the drinking solution for 20 min.

Microdialysis was performed before (during a waiting period), during and after

bar pressing and drinking. A handling control group was included, but did not

receive training. Dopamine and ethanol were analyzed from the dialysis

samples. There was a significant increase in accumbal dopamine during

41

placement of the rats into the operant chamber in both trained rats and handling

controls. The lever-pressing period did not produce an increase in dialysate

dopamine. Accumbal dopamine was increased in the first 5 min of ethanol, but

not water, consumption, followed by a return to baseline. Ethanol appeared in

the dialysates in the first 5 min sample following ethanol availability, and peak

concentrations were reached at 10 min. Most of the ethanol and water

consumption occurred in the first 5 minutes after access to the solution. The

probe placements were distributed in the core (32%), shell (32%), and core plus

shell (36%) regions of the nucleus accumbens. The enhancement of dopamine

activity during transfer into the operant chamber does not depend on anticipation

or operant training using ethanol or water reinforcement. Furthermore, the

difference between the time course of the accumbal dopamine response and

ethanol in dialysates suggests that the dopamine response is not solely due to

pharmacological effects of ethanol. Instead, the dopamine response may be

associated with the stimulus properties of ethanol presentation, which would be

strongest during consumption.

INTRODUCTION

There is substantial evidence indicating that dopamine in the nucleus accumbens

is involved in the process of ethanol reinforcement (Spanagel and Weiss, 1999;

Wise and Rompre, 1989). For example, the microinjection of dopamine agonists

into the nucleus accumbens has been shown to increase ethanol intake, whereas 42

dopamine antagonists decrease ethanol intake (Hodge et al., 1992; Samson et

al., 1993). Heterogeneous Wistar rats and genetically selected alcohol-preferring

P rats directly self-administer ethanol into the ventral tegmentum (Gatto et al.,

1994; Rodd-Henricks et al., 2000). Furthermore, ethanol elevates dopamine in

the nucleus accumbens of P rats and Wistar rats trained to self-administer

ethanol orally (Gonzales and Weiss, 1998; Melendez et al., 2002; Weiss et al.,

1993).

Studies using operant procedures to examine ethanol self-administration

(Gonzales and Weiss, 1998; Melendez et al., 2002; Weiss et al., 1993) show that

accumbal dopamine increases during two separate phases of the experiment: (1)

in the period preceding ethanol access during which the rat is waiting in the

operant chamber, and (2) during ethanol self-administration. It has been

suggested that the sensory cues in the environment that predict the reinforcer

cause the dopamine activity observed in the first phase, whereas the direct

pharmacological properties of ethanol cause the dopamine activity observed in

the second phase (Weiss et al., 1993). However, these previous studies utilized

continuous reinforcement schedules in which a single bar press resulted in short-

term access to ethanol on a repeated basis throughout the self-administration

period. This design does not allow for the separation of appetitive (seeking) and

consummatory phases of behavior because during the self-administration period,

both an appetitive behavior (lever pressing) and a consummatory behavior

43

(drinking) are occurring during the period of a single microdialysis sample.

The present study sought to clarify the potential roles of accumbal dopamine

during appetitive and consummatory phases of ethanol self-administration by

monitoring dopamine efflux in the nucleus accumbens using microdialysis. In

addition to reinforcement processes, midbrain dopamine systems are also

involved in voluntary movement associated with appetitive responding (Nishino et

al., 1987; Salamone, 1992). Therefore we employed (1) a waiting period in the

operant chamber with no additional cues present, (2) an appetitive phase of

responding with a single, discrete response requirement that increased

incrementally across daily sessions, and (3) a drinking period free of any

additional response requirement (Czachowski and Samson, 1999) to circumvent

possible motor confounds related to bar pressing and drug self-administration. In

this way, we temporally and neurochemically distinguished the motor activity

involved in appetitive behavior from the motor activity involved in consummatory

behavior (i.e., the act of bar pressing was separated from the act of ethanol

drinking).

In addition, another major goal of this study was to define the relationship

between the ethanol that reached the brain during self-administration and the

corresponding dopamine response. Because microdialysis samples will contain

both dopamine and ethanol that diffused into the probe during the sampling

44

period, we followed the time course of the drug to determine whether the

dopamine response is directly related to the concentration of ethanol reaching

the nucleus accumbens.

MATERIALS AND METHODS

Subjects

Our experiments used male Long-Evans rats (Charles River Laboratories,

Wilmington, MA) that weighed between 423-720 g at the time of testing. Long-

Evans were chosen based on previous behavioral studies indicating that this line

consumes moderate levels of ethanol (Czachowski et al., 2001). Each rat lived

individually in a humidity and temperature-controlled (22°C) environment under a

12-hr light/dark cycle (on at 7:00 A.M.; off at 7:00 P.M.). Each rat had food and

water available ad libitum in the home cage except during the procedures

indicated below. All procedures complied with guidelines specified by the

National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Behavioral apparatus

Standard operant chambers (Med Associates Inc., St. Albans, VT) modified for

microdialysis perfusion were used for self-administration training and

microdialysis testing. One wall of each chamber featured a retractable lever on

the left side (2 cm above grid floor), which upon activation triggered the entry of a

retractable drinking spout on the right side (5 cm above grid floor). The floor was 45

a grid of metal bars, and this was connected to the metal spout of the drinking

bottle with a lickometer circuit (Med Associates). A cubicle, with the front doors

left open during training and testing, housed each operant chamber. PC

software provided by Med Associates controlled operant chamber components

and acquisition of lickometer data. Activation of an interior chamber light and a

sound-attenuating fan accompanied the start of each operant session.

Self-administration training

Rats were handled and weighed at least 5 days prior to training. Operant

sessions occurred once a day for 5 days/week. Subjects were divided into 2

groups (ethanol or water drinkers) and trained to bar press for access to 10-15%

(w/v) sucrose. The ethanol group received 10% ethanol (v/v) as the sole source

of fluid in the home cage for 2-3 days prior to the operant training. Acquisition of

the operant behavior was facilitated by water deprivation (10-22 hr) prior to the

session. A reliable bar pressing response for sucrose occurred in approximately

3-6 days. Subsequently, rats had water available ad lib. We mildly water

deprived one rat in the ethanol group and one rat in the water group the night

before a few sessions during the training procedure. However, none of the rats

were water deprived within 7 weeks of or during the microdialysis session.

A major goal of this experiment was to compare dopamine responses during self-

administration of ethanol versus water. We initially used a training procedure in

46

which the response requirement was gradually increased over several days until

a response requirement of 20 was reached. Most of the initial rats in the study

successfully attained this criterion, but it soon became apparent that this

behavior was not consistent in all rats when the rat was exposed to this schedule

continuously. We were able to carry out microdialysis with a few of the rats in

this study using this procedure in which rats had a daily response requirement of

20 (n=3 in the ethanol group, n=2 in the water group). However, because of the

inconsistent behavior in most of the rats, we changed the procedure to one in

which we determined whether the rat would consistently reach a response

requirement of 20 by using a reinforcement schedule in which the response

requirement increased incrementally across daily sessions, as described below.

The ethanol group (n=11) was trained to self-administer 10% ethanol using the

sucrose fading procedure (Samson, 1986). Only rats that consumed ≥ 0.3 g/kg

of ethanol during microdialysis testing were included in the ethanol group.

Consumption was monitored during training and during the microdialysis session

by measuring the volume of liquid in the drinking bottle before and after the

session, taking care to account for spillage. Body weights were measured at

least twice a week. The water group had water substituted for sucrose

immediately after bar pressing behavior was established, and never received

ethanol (n=11). We gradually incorporated (2 min/2 days) a 15-20 min waiting

period prior to the extension of the lever into the operant chamber during

subsequent training sessions. Completion of the response requirement resulted

47

in retraction of the lever from the chamber and 20 min of free access to the

drinking solution (ethanol or water). The bottle then retracted from the operant

chamber and the rat remained inside for an additional 20 min, followed by

removal of the rat to the home cage. The response requirement gradually

increased to 4 during this period of training.

After approximately 4 weeks, we switched both groups of rats to a reinforcement

schedule used by Czachowski and Samson (1999), in which the response

requirement increased incrementally across daily sessions (4, 8, 12, 16, 20, 24,

28, 32, 40; a few rats were trained beyond 40). During this period, we delayed

the completion of the response requirement by programming “time-outs” into the

lever-pressing period. A time-out refers to a brief period of time (30-45 sec)

between bar presses during which the bar retracts from the chamber, so that a

given response requirement would take at least 3.5 min to complete. This design

ensured the acquisition of a sufficient dialysate sample volume during testing.

The rats continued on this reinforcement schedule until a cessation in operant

responding occurred or a maximum response requirement of 28-40 was reached,

after which the response requirement was returned to 4. The across session

escalating response requirement schedule was repeated two additional times.

Microdialysis was then performed during a fourth across-session escalating

response requirement series, and corresponded to the day on which the

response requirement was 20 (for those rats that reliably performed a response

48

requirement of 20 or above) or the highest response requirement reached in

each of the three previous across-session escalating response requirement

series (for those rats that did not reliably perform a response requirement of 20).

A third group of rats (n=7) served as a handling control. These animals were

placed into the operant chamber for the same periods of time and corresponding

number of days as the other groups, except that they did not receive training for

self-administration; i.e., they were never exposed to a lever or a drinking bottle in

the chamber. Each rat in the handling group was paired surgically and

experimentally with a rat in either the ethanol or the water group.

Surgery

After completion of the second across-session escalating response requirement

series, we surgically prepared the rats for microdialysis by inserting a stainless

steel guide cannula (21 gauge; Plastics One, Roanoke, VA) above the left

nucleus accumbens. The surgery occurred while the rats were under halothane

or isoflurane anesthesia (1.5-2.5% in 95%/5% O2/CO2, 1-2 l/min), using standard

stereotaxic equipment. The following coordinates were used (in mm relative to

bregma): +1.6 AP, +1.0 lateral, -4.0 ventral to the skull surface (Paxinos and

Watson, 1998). The guide cannula was cemented to the skull by embedding

three stainless steel screws into the skull and covering the entire unit around the

base of the cannula with dental cement (Plastics One, Roanoke, VA). We also

49

placed a single steel bolt vertically into the hardening cement as an anchor for

the microdialysis tether. An obdurator was placed inside the guide cannula to

prevent blockage prior to the microdialysis session. The rats resumed self-

administration training after one week of recovery.

Microdialysis

The microdialysis probes were constructed according to the methods described

by Pettit and Justice (1991). Briefly, fused silica tubing (i.d. = 40 µm; Polymicro

Technologies, Phoenix, AZ) formed the inlets and outlets of the probes, and

hollow cellulose fiber (i.d. = 200 µm; molecular weight cutoff = 13,000; Spectrum,

Rancho Dominguez, CA) formed the dialysis membrane. The active dialysis

membrane spanned 2.2 mm (the distance between the end of the inlet and the

epoxy that sealed the membrane).

Habituation to the microdialysis tethering apparatus occurred within the week

preceding testing. The habituation procedure consisted of tethering the rats

overnight in the operant testing room, with continued tethering throughout the

subsequent day of self-administration training. Rats were tethered either by

gently restraining the conscious animal or by sedating the animal with halothane

for a few minutes. On the day preceding the dialysis session we perfused (flow

rate = 2 µl/min) the microdialysis probes with artificial cerebral spinal fluid (149

mM NaCl, 2.8 mM KCl, 1.2 mM CaCl2, 1.2 mM MgCl2, and 0.25 mM ascorbic

50

acid, 5.4 mM D-glucose) and slowly inserted them into the brain through the

guide cannula while the rat was briefly anesthetized (15-20 min) with 2%

halothane in air. This procedure occurred at least 14 hr before the start of the

experiment. We used a syringe pump (CMA102; CMA, Solna, Sweden) to pump

the perfusate through a fused-silica transfer line into a single channel swivel

(Instech Laboratories, Plymouth, PA), which hung from a counterbalanced lever

arm (Instech Laboratories). The swivel then connected to the inlet of the probe.

A spring tether secured the animal to the swivel. After the rat recovered from the

probe implantation procedure (usually within 15 min), the perfusion flow rate was

decreased to 0.2 µl/min overnight. The flow rate was returned to 2.0 µl/min 2

hours before the baseline-sampling period began. We manually changed each

sample vial, which were immediately frozen on dry ice (except for samples used

for ethanol analysis, see below) and then stored at -80˚C until analyzed.

Experiments with a few rats were modified from this procedure by implanting the

probe more than once. This was done if the probe lines were damaged during

the overnight recovery period. In these cases (3 out of 29 rats in the study), the

probe was carefully removed from the rat under halothane sedation, and the rat

was allowed to resume self-administration training for at least another week. In

all of these cases, we confirmed that the basal dopamine concentrations were

calcium-dependent.

51

Experimental design

Dialysis samples were taken every 5 min except as indicated below. Baseline

consisted of 30 min in the home cage (6 samples). The period during which the

rat was transferred into the operant chamber consisted of one 5 min sample.

The waiting period preceding the introduction of the bar consisted of 15-20 min of

sampling (3-4 samples). The bar pressing period varied, usually lasting from 3.5-

5 min (but in a few animals more than 5 min). A 20 min drinking period with free

access began after completion of the response requirement (4 samples). A post-

drinking period in the chamber without access to the solution followed (20 min, 4

samples). At the end of this period, the rat was moved back into the home cage,

and sampling continued for another hour at 10 min intervals to obtain dialysates

for ethanol analysis. After obtaining all samples, the perfusion solution was

switched to one without calcium for 45-60 min, and a sample was taken to

monitor the calcium dependency of the dialysate dopamine.

Histology

After the experiment the rats were overdosed with chloral hydrate (600 mg/rat),

and saline was perfused through the heart, followed by 10% (v/v) formalin. The

brains were removed and immersed in 10% formalin/30% (w/v) sucrose for at

least 3 days. Brains were cut into coronal sections (48 µm thick) with a cryostat

(Bright Instrument Co., Cambs, England), and the sections stained with cresyl

violet. The slides were examined to confirm the placement of the active dialysis

52

membrane.

Dopamine analysis

Several chromatography systems were used to separate and quantify dopamine

during the course of these experiments due to equipment failure and sensitivity

requirements. All systems were based on reversed phase chromatography using

an ion pairing agent and electrochemical detection. Initial analyses (samples

from 9 subjects) were done with a coulometric detector (Model 5011, E1 set to -

100 mV, E2 set to +200 mV; ESA, Bedford, MA) using a Shimadzu LC10AD

pump (obtained through ESA), Model 465 autosampler (ESA), and a Hypersil

BDS C18 column (3 x 100 mm, 3 µm particle size; Thermo Hypersil-Keystone,

Bellefonte, PA). The mobile phase (2 mM disodium ethylenediaminetetraacetic

acid, 71 mM sodium dihydrogen phosphate, 0.41%, (w/v) octanesulfonic acid, pH

5.6, with 8-9% (v/v) acetonitrile or 13-15% methanol added) was pumped at a

rate of 0.5 ml/min, and the column temperature was set to 40-45°C. Seven µl of

the dialysate was incubated with 21 µl ascorbate oxidase (102.3 units/mg;

Sigma, St. Louis, MO) for 2 min, and 20 µl was injected into the system.

Detection limit for this system was 0.5 nM (signal to noise = 3), and this did not

prove to be sensitive enough for all of the dialysis samples. Therefore, we also

used amperometric detection in subsequent analyses. For example, a Model

5041 analytical cell (potential set to 350 mV, ESA) was used in combination with

a 2.1 x 100 mm column (Hypersil BDS C18), and this provided detection limits of

53

0.3 nM (samples from 17 subjects were analyzed with this configuration).

Another configuration that was used included an ISCO 260D pump (Lincoln, NE),

FAMOS autosampler (LC Packings, San Francisco, CA), VT-03 cell (2 mm

working electrode diameter, potential set to 0.45 V against Ag/AgCl reference;

Antec, Leyden, Netherlands) connected to an Intro controller (GBC Separations,

Hubbardson, MA) and a 1 x 150 mm column (C18, 5 µm particle size, LC

Packings). In this case 7 µl of the dialysate was directly injected using the

FAMOS autosampler (samples from 2 subjects were analyzed with this

configuration). For these amperometric systems, the mobile phase composition

was altered appropriately using either higher concentrations of octanesulfonic

acid (0.41-1.15%, w/v) or decanesulfonic acid (0.15%, w/v) in combination with

methanol (10-15%) to sufficiently resolve dopamine, and the flow rate was also

adjusted appropriately for the smaller bore columns (0.2-0.3 ml/min for the 2.1

mm id column, 0.08-0.1 ml/min for the 1 mm id column). A Shimadzu C-R3A

integrator (Houston, TX), HP 3396A integrator (Hewlett-Packard, Dallas, TX), or

a computer data acquisition system (EZ Chrome Elite; Scientific Software,

Pleasanton, CA) recorded the dopamine peaks, and quantitation was carried out

by comparing dopamine peak heights from dialysate samples to external

standards. Variability in the external standards is minimized in our laboratory by

confirming that HPLC technicians are capable of replicating standards within a

relative standard deviation of 0.01-0.05.

54

Ethanol analysis

Ethanol was analyzed in all dialysis samples taken after consumption of ethanol

began. Before freezing the dialysis sample, 2 µl were transferred into a glass vial

and sealed with a teflon-backed septum for ethanol analysis later that day. A gas

chromatograph (Varian CP 3800; Varian, Walnut Creek, CA) equipped with a

flame ionization detector (220°C) measured the dialysate ethanol. The sealed

vial was heated to 65°C using an Autotherm (Strumenti Scientifici, Padova, Italy)

for at least 20 min before injection. A Varian 8200 headspace autosampler

equipped with a solid phase microextraction fiber assembly (75 µm carboxen-

PDMS; Supelco, Bellefonte, PA) injected the samples (3 min absorption and a 1

min desorption) into the heated injection port (175 or 220°C). Helium was the

carrier gas (2.2 psi on constant pressure mode or 8.5 ml/min on constant flow

mode) and separation occurred under isothermal conditions (65°C) using either a

Supelcowax-10 capillary column (15 m x 0.53 mm x 1.0 µm film thickness;

Supelco) or an HP Innowax capillary column (30 m x 0.53 mm x 1.0 µm film

thickness). The stationary phase, injector temperature, and gas flow rate

parameters were changed to improve the conditions of our method. The limit of

detection was 0.03 mM (signal to noise = 3), and quantification of ethanol in

dialysates was carried out by comparison of peak areas obtained from the Star

chromatographic analysis system (Varian) to external standards.

Statistical analysis

55

Analysis of variance (ANOVA) with repeated measures was used for analysis of

dialysate dopamine levels. Dopamine concentrations (nM) were log transformed

to maintain homogeneity of variance. The average of the 6 baseline samples

defined the basal dopamine values. Technical problems associated with either

sample collection or HPLC analysis resulted in the loss of some samples (14 out

of 549). We estimated these values by averaging adjacent time points, and then

adjusting the degrees of freedom in the ANOVA to account for this. Separate

ANOVA tests were conducted to test for group by time interactions for three

separate phases of the experiment (basal and transfer + waiting period; basal

and lever press period; basal and drinking + post-drinking periods). We

performed post-hoc contrasts comparing individual time points to baseline after

finding significant effects of time or a significant group by time interaction.

Bonferroni corrections were used in the case of post-hoc contrasts for this and

subsequent analyses. A secondary analysis was carried out on the last waiting

period point, the lever press point, and the 4 samples taken during the drinking

session for the ethanol and water groups only. This was done to adjust for the

effect of a drift in the baseline during the waiting period. Post hoc tests were

done using simple interactions for this secondary analysis. ANOVA was carried

out using the Manova routine in SPSS for Windows, and post-hoc contrasts were

carried out using the GLM procedure. Significance in this and subsequent

analyses was determined when p < 0.05.

56

Dialysate ethanol concentrations were analyzed using repeated measures

ANOVA followed by post-hoc contrasts to determine whether the concentrations

of ethanol obtained in first 5 min sample were significantly different from

subsequent samples. For this analysis, only the first 5 samples were used. This

was done because one rat out of the eleven in the group had dialysate ethanol

lower than our detection limit after that sample. Therefore, we had a complete

set of data for times up to 25 min after drinking began.

Analysis of drinking parameters was carried out using multivariate and univariate

ANOVA (GLM procedure in SPSS). Several of the parameters were transformed

to maintain homogeneity of variance (lever pressing time was transformed to its

reciprocal, latency was log transformed, and a power transformation was used

for the licking rate for the first half of the initial bout). One of the rats in the water

group was excluded from this analysis because we were unable to obtain a value

for the duration of bar pressing. Therefore, the behavioral data shown in table 1

reflects this change in sample size.

RESULTS

Histological analysis and calcium-dependence of basal dopamine

The histological analysis showed that most of the probes had at least 50% of the

active dialysis membrane located in the medial part of the nucleus accumbens,

and a majority intersected both the core and shell subregions (Figure 2.0). Five

57

of the probes were located in the very medial part of the nucleus accumbens,

including one that appeared to be located through the islands of Calleja, which is

the medial border of the shell of the nucleus accumbens. Overall the probe

locations in the core (32%), shell (32%), or core and shell (36%) were not very

different between the three groups (ethanol group: 3 core, 5 shell, 2 core and

shell; water group: 4 core, 2 shell, 5 core and shell; handling group: 2 core, 2

shell, and 3 core and shell). Because of this mixed distribution of the probe

locations, we did not analyze the results with respect to functional differences

between core and shell subregions. Confirmation of the probe placement could

not be verified in one rat due to the loss of the section containing the probe track.

However, this subject was included in the study because the dialysate samples

had clear dopamine signals and HPLC chromatograms contained unidentified

peaks that displayed regional selectivity for the accumbens. Overall, the basal

dialysate samples showed a calcium dependency of 71 ± 3% for 28 out of the 29

subjects (calcium-dependency data was not collected for one subject due to

probe problems), which is consistent with previous data (Westerink and De Vries,

1988). Basal samples for one subject had a low level of calcium-dependency

(32%), but the remaining rats showed greater than 50% calcium-dependency.

Dopamine during handling, waiting and appetitive phases of self-

administration

Rats drinking ethanol and water displayed similar increases in dopamine above

58

FIGURE 2.0. Coronal sections showing microdialysis probe placement within the nucleus accumbens. Lines indicate the active dialysis regions. Numbers below the figure represent the position of the slice relative to Bregma. The figure was adapted from Paxinos and Watson (1998).

59

basal (30 ± 9% and 21 ± 8%, respectively) during the period in which they were

transferred from the home cage into the operant chamber (Figure 2.1A and B).

However, the handling control group, which did not receive operant training, also

showed an elevation in dopamine during this period (25 ± 5% above basal;

Figure 2.1C). All three groups showed a similar and general decline in dopamine

during the waiting period following the transfer. ANOVA revealed a significant

effect of time (F(4,102) = 10.6, p < 0.05) but no group by time interaction

(F(8,102) = 0.6, p > 0.05). Post hoc contrasts indicated that dopamine increased

significantly above basal during the transfer period and during each of the three

samples that constitute the waiting period (F(1,28) = 14.3, p < 0.05 for all four

contrasts). Some rats in both ethanol and water groups were trained to wait 20

min before access to the active lever (n=4 for each group), but we did not

observe any difference between the dopamine concentrations in dialysates

obtained from the 15 min period compared with the 20 min period (p > 0.05 by

paired t test, t = 0.23, df = 7). Therefore, the full ANOVA described above was

done using only the points up to 15 min of waiting in these 8 subjects.

Both groups of trained rats responded similarly to obtain reinforcement (18 ± 1

bar presses for the ethanol group, 15 ± 2 bar presses for the water group). In

both the ethanol and water groups, dopamine concentrations during the bar

press period were only slightly higher than their basal values (11 ± 5% and 12 ±

7% for the ethanol and water groups, respectively; Figure 2.1A and B). For the

60

FIGURE 2.1. Effect of operant ethanol (panel A) and water (panel B) self-administration on accumbal dopamine in dialysate. Baseline indicates that the rat is in its home cage. Transfer (closed arrow) refers to the period in which the rat is transferred from the home cage into the operant chamber. Wait refers to the time in which the rat is in the operant chamber without access to the lever or drinking solutions. Lever press (open arrow) refers to the period during which the rat is lever pressing prior to access to the drinking solution. Drink refers to the 20 min free-access drinking period. Post-drink refers dopamine while the rat is in the operant chamber in the absence of the drinking solution. Panel C shows the effect of the handling procedure on accumbal dopamine in dialysate. Each point represents the mean ± sem (n=11 for the ethanol and water groups, n=7 for the handling group). a denotes significance compared to basal for the transfer and waiting periods in all three groups by ANOVA and post hoc simple contrasts (p < 0.05). b denotes significance compared to basal in the ethanol group during the drinking and post-drinking periods after a significant group x time interaction and post hoc simple contrasts (p < 0.05).

61

analysis of dopamine during the bar press period we specifically compared the

data of the ethanol plus water groups (n=22) with that of the handling group

(n=7). The time point chosen for the handling group corresponded to the period

in which bar pressing occurred for the trained groups (Figure 2.1C). This test

was a group by time analysis comparing a single sampling point across groups

to basal, and was done to determine whether the act of bar pressing altered

dialysate dopamine concentrations. Percent of basal dopamine during bar

pressing for the ethanol plus water group was 111 ± 4%, and at an equivalent

point for the handling group was 103 ± 3%. ANOVA did not show a significant

within-subjects effect of time (F(1,25) = 3.35, p > 0.05) or a group by time

interaction (F(1,25) = 0.77, p > 0.05). There also was a main effect of group

(F(1,25) = 6.36, p < 0.05), and this was likely due to differences in the basal

dopamine response between the ethanol/water group (1.8 ± 0.3 nM) and the

handling group (0.9 ± 0.2 nM). Based on the group by time interaction and a

single lever-pressing period (> 3.5 min) in which the lever was only accessible

periodically, bar pressing to obtain access to liquid reinforcement did not

significantly increase dopamine in the core and shell subregions of the

accumbens.

Dopamine during consumption of ethanol or water

Basal levels of dialysate dopamine obtained while the rat was in its home cage

were not significantly different among the three groups in the study (1.5 ± 0.2 nM

62

for the ethanol group, 2.1 ± 0.5 nM for the water group, 0.92 ± 0.16 nM for the

handling group; F(2,26) = 3.17, p > 0.05). Consumption of ethanol and water

during the 20 min session was similar in the two groups (3.3 ± 0.2 ml and 3.3 ±

0.5 ml for the ethanol and water groups, respectively). The ethanol group

ingested 0.45 ± 0.04 g/kg. Accumbal dopamine in the ethanol group increased to

21 ± 4% above basal in the first 5 min of the drinking period, with levels returning

to basal during the subsequent sample periods (Figure 2.1A). In contrast, the

water group showed an increase in dopamine of 6 ± 7% above basal in the first 5

min of the drinking period, with levels remaining similar during the subsequent

sample periods (Figure 2.1B). Statistically the ethanol group differed from the

water group during the periods constituting drinking and post drinking (group by

time interaction; F(8,150) = 2.17, p < 0.05). Post hoc analysis by simple

contrasts showed that a significant increase in dopamine compared with basal

occurred in the first sample obtained during the drinking period only in the

ethanol group (F(1,10) = 41.7, p < 0.05).

In addition, a secondary analysis was carried out to test whether the transient

increase in dopamine suggested by the above analysis could be detected when

using a new baseline defined by the last waiting period sample. In this analysis

the last waiting period, the lever press point, and the 4 samples taken during the

drinking period were used for both the ethanol and water groups only. Again, we

confirmed that the dopamine response in the ethanol group differed from the

63

FIGURE 2.2. Dialysate ethanol from the nucleus accumbens during drinking and post-drinking periods. Data are from the same rats shown in Figure 2.1, and ethanol was analyzed in the same samples from which the dopamine analysis was done. Each point is the mean ± sem (n=11). Asterisks denote significance compared with the first sample (p < 0.05).

64

water group before and during the drinking period (group x time interaction;

F(5,96) = 2.32, p < 0.05). Post hoc analysis using simple interactions showed

that the last waiting period point was not different from the lever press period

across the two groups (group x time interaction; F(1,96) = 1.57; p > 0.05). This

confirms the analysis shown above for the appetitive behavior. Furthermore, the

first drinking point was significantly higher than the waiting period point in the

ethanol group compared with the comparable two points in the water group

(group x time interaction; F(1,96) = 10.8, p < 0.05). Altogether, the statistical

analyses showed that the first point during ethanol drinking was the only one that

was significantly higher than baseline (either obtained in the home cage or using

the last waiting period point).

Brain ethanol during self-administration

We measured dialysate ethanol in each sample taken during and after ethanol

consumption (Figure 2.2). A low concentration of ethanol (0.59 ± 0.17 mM)

appeared in the dialysate in the first 5 min of the drinking period, and increased

significantly (F(1,10) = 19.4, p < 0.05) in the next sample (1.17 ± 0.26 mM).

Ethanol concentrations peaked 10-15 min after ethanol access began and then

declined over time to levels below our detection limit. Examination of the

individual ethanol time courses showed that peak ethanol levels in the brain and

ethanol clearance from the brain displayed a large degree of variability among

the animals (data not shown), which was not related to intake (r2 = 0.24 for the

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TABLE 2.0. Lickometer parameters for ethanol or water self-administration during microdialysis

Parameter Ethanol (n=11) Watera (n=10)

lever pressing time (min) 4.5 ± 0.5 9.4 ± 3.3 latency to begin drinking (min) 0.10 ± 0.05 0.16 ± 0.09

number of bouts 1.64 ± 0.29 1.60 ± 0.32 total licks 637 ± 41 637 ± 70

initial bout response rate (licks/min) 310 ± 25 247 ± 23 response rate for ½ of first bout (licks/min) 401 ± 7 304 ± 18b

Values shown as mean ± sem a – the water group was significantly different from the ethanol group by multivariate ANOVA (p < 0.05) b –significantly different from the ethanol group by univariate ANOVA (p < 0.05)

66

correlation between ethanol (area under the curve) and consumption, p > 0.05).

Rats consuming similar volumes of ethanol did not necessarily have similar time

courses, in terms of peak ethanol concentrations in the dialysate and ethanol

clearance from the dialysate.

Licking behavior during consumption of ethanol and water

We also determined several parameters that characterize licking behavior during

consumption of ethanol and water during the dialysis procedure (Table 2.0). The

ethanol group differed significantly from the water group according to the

parameters listed in Table 2.0 (F(7,13) = 131.8, p < 0.05 by multivariate ANOVA).

The group difference was primarily due to a faster lick rate during the first half of

the first bout of consumption in the ethanol group compared with the water group.

This parameter was the only one found to be significantly different between the

two groups by univariate ANOVA (F(1,19) = 38.2, p < 0.05). Lickometer records

indicated that 95 ± 2% of the total volume of ethanol consumed (as measured by

spout licks) during access to the reinforcer occurred in the first 5 min of the

drinking period. Similarly, in the water group 89 ± 6% of consumption occurred

within the first 5 min of the drinking period.

DISCUSSION

The nucleus accumbens represents an interface between motor and limbic

neural networks and has been hypothesized to be an important functional

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connection between motivational processes and behavioral action (Mogenson et

al., 1980). The accumbens is anatomically heterogenous, consisting of two

primary subregions: the core and shell, and functional differences in dopamine

transmission have been found to exist between the subregions (Bassereo and Di

Chiara, 1999; Jones et al., 1996). The present study did not attempt to

distinguish between the core and the shell, in terms of regional probe placement

or extracellular dopamine response. The dialysis sampling was performed in the

core (32%), shell (32%), and core plus shell (36%) regions of the accumbens.

This study is the first to relate dopamine response with ethanol concentration by

measuring both dopamine and ethanol from the same dialysis sample obtained

from the nucleus accumbens of rats that are self-administering alcohol. A major

finding of our study is that there is an increase in accumbal dopamine during

ethanol self-administration that is not directly related to the concentration of

ethanol reaching the nucleus accumbens. Comparison of the dopamine

response with the ethanol concentrations from accumbal dialysates during the

drinking session showed that there was a clear dissociation between the two.

This was particularly evident when looking at the first 10 min of the drinking

session. During this period the dopamine response had already reached its peak

and was declining back to basal levels, while the ethanol concentrations were

low in the first 5 min and reached their peak at 10 min. This observation holds

true when examining the mean responses across a group of rats or when looking

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at the correlation between dialysate ethanol concentration and dopamine

response within individual rats.

One interpretation regarding the dissociation between ethanol concentration and

dopamine response is that the dopamine response is not solely determined by

the pharmacological effects of the ethanol reaching the brain, but may be more

related to the stimulus properties of the presentation of ethanol, its consumption,

or an interaction between the two. If the dopamine response was due to

pharmacological actions of ethanol, an oral dose of ethanol would be expected to

cause an elevation in dopamine that is a function of the concentration of ethanol

in the brain. However this did not occur. Analysis of the licking behavior during

the drinking session supports the idea that the stimulus properties of ethanol

were involved since 95% of total ethanol consumption occurred within the first 5

min of ethanol access. It was during this period that the stimulus properties

associated with the consumption of ethanol (its taste and smell and the onset of

its pharmacology) were strongest. In contrast, the water group did not exhibit an

increase in dopamine during this period, suggesting that exposure to a fluid

stimulus with a relatively neutral value in the operant environment does not

necessarily produce an accumbal response. In the present study we did not

include another group that received a naturally strong stimulus value such as

sucrose alone or saccharin. Therefore, it is possible that the response we

observed for the ethanol group may also occur in groups that receive solutions

69

with stronger reinforcing properties than water. Analysis of the lickometer

records indicated that there was an overall difference between the ethanol and

water groups in their licking behavior during the session taken as a whole. This

was primarily due to a greater lick rate during the first half of the initial bout in the

ethanol group compared with the water group. This may reflect the stronger

stimulus properties of the ethanol solution compared with water, and this

difference during the first 5 min of the drinking period may also have contributed

to the difference in the dopamine response between these groups. In addition,

the motor effects related to licking the spout and swallowing the solution may

have contributed to the transient dopamine response observed during ethanol

self-administration. However we do not think this is a major factor because an

increase in accumbal dopamine was not observed in the group that was trained

to drink water.

There are several issues that could impact the interpretation that the accumbal

dopamine response was not solely due to the pharmacological action of ethanol.

One is that the nucleus accumbens is not likely the primary target involved in the

mechanism by which low doses of ethanol produce stimulation of mesolimbic

dopamine (Yim et al., 1998). Previous studies from numerous groups suggest

that the mechanism of action for ethanol’s stimulatory effects on dopamine in the

accumbens is via an enhancement of the firing rate of dopaminergic cell bodies

in the ventral tegmental area (Brodie et al., 1999; Gessa et al., 1985; Yim et al.,

70

1998; Yim and Gonzales, 2000). Therefore it is possible that the time course of

ethanol concentrations reaching the ventral tegmental area is different from that

reaching the nucleus accumbens, and that direct measurement of the ethanol

time course from the ventral tegmental area would more closely match the

dopamine response. Although this possibility cannot be ruled out, it is unlikely

due to the known pharmacokinetic properties of ethanol. Ethanol is a small

molecule that is rapidly distributed to tissues that are highly perfused such as the

brain (Nurmi et al., 1994; Nurmi et al., 1999). Although differences in the normal

perfusion of basal ganglia and midbrain areas have been reported (Otsuka et al.,

1991), it does not seem plausible that these differences would be responsible for

a reversal of the pharmacokinetic profile of these two regions within the first 10

min of ethanol absorption and distribution after oral self-administration.

Another caveat is that the dissociation between the observed ethanol and

dopamine time courses may be partially due to an extremely rapid acute

tolerance to ethanol. It is possible that the ethanol reaching the brain during the

rising phase of the ethanol time course may have more of a stimulatory effect on

accumbal dopamine compared with that at peak ethanol concentrations or later

during the falling phase. This mismatch between the effects of ethanol during the

rising and falling phases of the ethanol time course is well known in both human

studies (Hiltunen, 1997a; Hiltunen, 1997b) and in animal studies (Erwin and

Dietrich, 1996; Lewis and June, 1990; Ponomarev and Crabbe, 2002; Waller et

71

al., 1983). However, in previous animal studies this phenomenon is usually

determined using conditions dramatically different from the present study. For

example, ethanol doses are usually > 1.7 g/kg, motor impairment is commonly

used as a behavioral measure, and it usually takes at least 30 min to

demonstrate the phenomenon (Erwin and Dietrich, 1996; Le and Kalant, 1992;

Tampier and Mardones, 1999). Previous work from our lab showed that the

accumbal dopamine response dissociates from ethanol concentrations after 45-

60 min (Yim et al., 2000). If our present observations are partially due to rapid

acute tolerance, this would be the first demonstration of this occurring within the

first 10 min of ethanol self-administration.

A novel aspect of the present work is that we have defined for the first time the

dialysate concentrations of ethanol that are associated with an enhancement of

accumbal dopamine activity during ethanol self-administration. The dialysate

concentrations reported here have not been corrected for in vivo recovery, and

therefore, represent a fraction of the concentrations that are actually in the tissue.

Our previous work shows that quantification of extracellular concentrations of

ethanol is possible with microdialysis (Robinson et al., 2000), but we did not

attempt this characterization in the present study. However, the microdialysis

probes we used in the present study were nearly identical in design to those

used in the previous quantitative microdialysis study, which makes it possible to

draw some tentative conclusions about our current results. Our previous study

72

indicated that the in vivo extraction fraction for ethanol under conditions similar to

those used in the present study is 0.13. Therefore we estimate the concentration

of ethanol in the extracellular space of the nucleus accumbens to be 4.5 mM in

the first 5 min sample after consumption of ethanol began. Nurmi et al. (1994)

reported similar concentrations of ethanol within 10 min of self-administration of

ethanol in AA and Wistar rats. This concentration is lower than that usually used

in studies to determine the mechanism of action of ethanol in vitro (Brodie et al.,

1990; Brodie et al., 1999; Verbanck et al., 1990). This suggests that a full

understanding of the mechanism of action of ethanol on dopamine systems,

relevant to the reinforcing properties of ethanol, requires the use of

concentrations in the 5-10 mM range.

Finally, we also have to consider the possibility that the intake of ethanol and the

subsequent tissue concentrations of ethanol attained after absorption and

distribution in the brain may not be high enough to produce a pharmacological

response on accumbal dopamine activity. As discussed in the previous

paragraph, the expected brain concentrations of ethanol are quite low in our

experiments. Further experiments are required to determine whether higher

intakes of ethanol using the present methods will produce an increase in

accumbal dopamine concentrations that are related to the brain ethanol

concentrations. It is also possible that ethanol may produce a pharmacological

effect on accumbal dopamine early in the development of the reinforcement

73

process, but that this becomes blunted after long-term training. In any event, our

data show that in rats that have clearly exhibited the development of ethanol

reinforcement with long-term training, the dopamine response is not sustained

beyond 5 min after the commencement of drinking.

Although our data were not fully consistent with the idea that the increase in

accumbal dopamine is primarily due to the pharmacological effects of ethanol

reaching the brain (with the caveats described above), recent literature may

provide an explanation for our observation of a transient dopamine response.

For example, midbrain dopamine activity has been shown to increase during the

presentation of an unpredicted reinforcer (Schultz et al., 1997). However, after

the establishment of a conditioned stimulus by repeated pairing of the

unconditioned stimulus with the reinforcer, midbrain dopamine neurons no longer

respond to the presentation of the reinforcer. Instead, dopamine activity

increases during the presentation of the conditioned stimulus. Direct

measurement of extracellular dopamine in response to intracranial self-

stimulation of the medial forebrain bundle also shows that the dopamine

response is much larger during experimentally administered stimulation

compared with that observed subsequently during established self-stimulation

(Garris et al., 1999). These studies show that after the animal has learned the

association between an operant response and access to a reinforcer, midbrain

dopamine neurons no longer respond to the reinforcer alone. Accordingly, it is

74

possible that in the present study accumbal dopamine was responding to the

stimulus properties of the presentation of ethanol rather than the pharmacological

effects of ethanol that would presumably take place several minutes after its

consumption. Further work will be necessary to substantiate this possibility.

There is evidence that dopamine in the accumbens is involved in the appetitive

phases of motivation (Nishino et al., 1987; Salamone, 1992). To determine

whether accumbal dopamine is involved in the appetitive, seeking response that

precedes ethanol reinforcement, the bar pressing phase of the present

experiment was specifically separated from the drinking phase. By doing this, we

sought to separate the potentially confounding effect of appetitive plus

consummatory motor activity on accumbal dopamine that may occur in a

continuous reinforcement situation. Under a continuous reinforcement schedule,

a response requirement (bar pressing) leads to the consumption of a small

amount of the reinforcer, and additional response-reinforcement cycles may

follow (Gonzales and Weiss, 1998; Melendez et al., 2002; Weiss et al., 1993).

Thus, in these previous studies, the motor activity involved in bar pressing could

have contributed to the dopamine response observed during the drinking periods.

The present study demonstrates that motivated bar pressing does not produce

an enhancement of dopamine levels in the nucleus accumbens. In animals

trained to bar press for access to a water or ethanol solution, the dialysate

concentration of dopamine was similar in the sample taken during bar pressing

compared with a sample taken at an identical time in a group of rats that were

75

placed into the operant chamber but did not receive operant training. This was

further substantiated by comparing the lever press sample in the ethanol and

water groups to the last sample taken during the waiting period and finding no

differences between the two. Ito et al. (2000) obtained similar results with rats

trained to bar press using cocaine reinforcement. However, acceptance of this

conclusion should be done cautiously since it is based on single lever-pressing

period (> 3.5 min) in which the lever bar was only accessible periodically.

Transient changes in dopamine could be occurring episodically during the brief

lever-pressing period, but at a level undetectable with microdialysis. Further

experiments are necessary to confirm our finding.

In both the ethanol and water drinking groups, dopamine significantly increased

during the period in which the rats were transferred into the operant chamber

from the home cage, and during the 15 min waiting period that preceded lever

pressing. However, the handling control group, which was not trained for self-

administration, also showed a significant elevation in dopamine during the period

in which they were transferred into the chamber and during the subsequent 15

min period in the chamber. Together these results indicate that the observed

dopamine response is caused by the physical handling of the rats as they are

placed into the operant chamber, a change of environment, or a combination of

the two. This conclusion does not agree with previous studies in which the

transfer of rats from one setting to another was not accompanied by an increase

76

in accumbal dopamine (Damsma et al., 1992; Humby et al., 1996; Weiss et al.,

1993). The reason for this discrepancy is not clear, but there are several

procedural variables that could contribute to the difference between the present

results and those of previous studies. For example, in the study by Damsma et

al. (1992) there were small (11% above baseline), but non-significant increases

in accumbal dopamine during transfer into an experimental setting after several

training sessions. However, this study used sexual experience during the

training procedure, and no operant responses were required. Similarly, the

procedure used by Humby et al. (1996) was a simple transfer from one

environment to another, and no operant training was involved. In the previous

study by Weiss et al. (1993) a control group was trained to lever press for water

reinforcement during water deprivation, but subsequent training involved

exposure to the operant environment in the absence of deprivation, and operant

responding ceased. Under these conditions, no increase in accumbal dopamine

was observed in response to the transfer to the operant chamber. However,

there are differences between controls used in the study of Weiss et al. (1993)

and the present study including rat strain (Wistar vs. Long-Evans), training

procedure (operant training vs. no operant training), and conditions during the

exposure to the operant chamber (closed environment with no external stimuli vs.

open doors of the outer cubicle that allowed some external stimuli to be present).

It is not clear which, if any, of the above variables could contribute to the

discrepancy between control groups. Nonetheless, our results provide evidence

77

that the transient accumbal dopamine response prior to access to a reinforcer

may not reflect anticipation of the reinforcer.

In summary, the present study shows that there is a significant and transient

increase in accumbal dopamine during the first 5 min of ethanol self-

administration following operant responding that does not occur during water

self-administration. This dopamine response is not directly related to the

concentration of ethanol in the dialysates, but we cannot rule out the possibility

that higher intakes of ethanol may lead to a direct relationship between dopamine

release and brain ethanol concentrations. Additionally, bar pressing, an

appetitive behavior, did not significantly alter accumbal dopamine. Moreover, we

found that the physical handling of the rat causes an elevation in dopamine that

is not specifically related to the subsequent availability of ethanol or water

reinforcement. Although we cannot rule out an extremely rapid tolerance to

ethanol-stimulated dopamine release after oral self-administration, our results

suggest that the enhancement of accumbal dopamine is caused by the stimulus

properties related to the presentation and consumption of ethanol, rather than a

direct consequence of the presence of pharmacologically relevant concentrations

of ethanol in the brain.

78

3.0 Accumbal dopamine concentration during operant self-administration

of a sucrose or a novel sucrose with ethanol solution

[published in Alcohol (2004) 34: 261–271; by William M. Doyon, Vorani

Ramachandra, Herman H. Samson, Cristine L. Czachowski, Rueben A.

Gonzales]

ABSTRACT

The goal of the present study was to determine the effect of operant self-

administration of (1) 10% sucrose and (2) a first-time solution of 10% sucrose

with 5 or 10% ethanol, on dopamine concentration in the nucleus accumbens.

We used an operant procedure that distinguished lever pressing (an appetitive

behavior) from drinking to better assess the effect of fluid consumption on

accumbal dopamine activity. Male Long-Evans rats were trained to bar press

using 10% sucrose reinforcement, and they were required to emit an escalating

number of bar presses across daily sessions. Completion of the response

requirement resulted in 20 min of access to the solution. Microdialysis samples

were collected before, during, and after bar pressing and drinking, and ethanol

and dopamine content was determined. Dialysate dopamine was slightly, but

significantly increased in both groups during lever pressing, but dopamine

increased after consumption began, in the sucrose, but not the sucrose with

ethanol group, followed by a return to baseline. Ethanol consumption was low

79

(0.27 ± 0.02 g/kg) and corresponded to low dialysate ethanol concentrations,

which appeared within 5 min of drinking. These results demonstrate that operant

self-administration of sucrose increases accumbal dopamine during

consummatory phases of behavior that are not apparent when a novel, perhaps

aversive, solution (sucrose with ethanol) is presented. This difference may be

due to the sensory-related stimulus properties of each solution. Additionally, oral

self-administration of 0.27 ± 0.02 g/kg ethanol over 20 min is not sufficient for

stimulation of dopamine activity in the nucleus accumbens.

INTRODUCTION

Consumption of ethanol stimulates dopamine activity in the nucleus accumbens

using operant paradigms in which reinforcement has been well-established

(Doyon et al., 2003; Gonzales & Weiss, 1998; Melendez et al., 2002; Weiss et

al., 1993). In contrast, the effect of ethanol consumption on dopamine activity

during incipient stages of reinforcement has not been adequately addressed.

Recent work in our laboratory used a procedure that defined the ethanol-induced

dopamine response that is associated with consumption of ethanol but is

independent of appetitive behaviors such as lever pressing (Doyon et al., 2003).

Interestingly, we observed a small, but significant, increase in accumbal

dopamine within five min of the drinking period before returning to baseline, but

not during the subsequent 30 min period in which accumbal ethanol levels

continued to escalate with no apparent effect on dopamine activity. Due to these

80

findings, we speculated that the dopamine response became blunted over time

with the long-term ethanol reinforcement schedule that was used (2-4 months of

training). This phenomenon has been shown to occur with repeated presentation

of food reinforcement (Bassareo & Di Chiara, 1997; Schultz et al., 1997). During

an earlier period in the development of reinforcement, it is plausible that the

dopaminergic response to ethanol consumption could be more robust.

Therefore, the present study was undertaken to determine behavioral and

neurochemical responses to ethanol during an initial exposure in ethanol-naïve

rats.

Natural and drug-related rewards share the capacity to stimulate mesolimbic

dopamine activity and likely modulate common neural substrates during the

development of reinforcement (Kelley & Berridge, 2002; Spanagel & Weiss,

1999). The ability of natural reinforcers to activate dopamine systems, however,

seems to be more dependent upon the conditions under which they are

administered. For example, deprivation generally increases dopamine activity in

response to food reward (Ahn & Phillips, 1999; Mirenowicz & Schultz, 1994;

Wilson et al., 1995). In non-deprived animals, highly palatable substances can

also increase dopamine levels when presentation is novel, occurring for the first

time; but this response is attenuated with repeated exposures (Ahn & Phillips,

1999; Bassareo & Di Chiara, 1997). Sucrose-induced dopamine efflux in water-

deprived animals has also been demonstrated (Hajnal & Norgren, 2001; Hajnal

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et al., 2004). The effect of sucrose reward on accumbal dopamine concentration,

however, has not been tested previously with microdialysis in an operant

condition, nor has it been examined in non-deprived animals. This information

would be important for understanding the general role of dopamine in

reinforcement. Therefore, a second aim of the present study was to determine

the effect of operant self-administration of 10% sucrose on accumbal dopamine

activity, in non-deprived rats.

To accomplish these aims, we trained male Long-Evans rats to lever press for

limited access (20 min) to 10% sucrose. Temporal and neurochemical

segregation of consumption was achieved by using an operant procedure that

separated appetitive (lever pressing) from consummatory behavior (Doyon et al.,

2003). The microdialysis experiment consisted of self-administration of 10%

sucrose or a first-time solution of 10% sucrose with 5 or 10% ethanol. For the

sucrose plus ethanol group, we followed the time course of intra-accumbal

dopamine and ethanol concentrations during the consummatory period to

determine the relationship, if any, between intracerebral ethanol concentration

and the accompanying dopamine response. Consumption patterns of sucrose

and sucrose plus ethanol during drinking were also quantified for comparison

with ethanol and dopamine concentrations over the same period.

MATERIALS AND METHODS

82

Subjects

The present study used 18 male Long-Evans rats (Charles River Laboratories,

Wilmington, MA) that weighed between 297-538 g at the time of testing. Rats

were handled and weighed for at least 5 days upon arrival prior to surgery or

training. Each rat lived individually in a humidity and temperature-controlled

(22°C) environment under a 12-hr light/dark cycle (on at 7:00 A.M.; off at 7:00

P.M.). Each rat had food and water available ad libitum in the home cage except

during the procedures indicated below. All procedures complied with guidelines

specified by the National Institutes of Health Guide for the Care and Use of

Laboratory Animals.

Behavioral apparatus

Standard operant chambers (Med Associates Inc., St. Albans, VT) modified for

microdialysis perfusion were used for self-administration training and

microdialysis testing. One wall of each chamber contained a retractable lever on

the left side (2 cm above grid floor), which upon activation triggered the entry of a

retractable drinking spout on the right side (5 cm above grid floor). A grid of

metal bars formed the floor, which was connected to the metal spout of the

drinking bottle with a lickometer circuit (Med Associates). A cubicle with the front

doors left open during training and testing housed each operant chamber. PC

software provided by Med Associates controlled operant chamber components

and acquisition of lickometer data. Activation of an interior chamber light and a

83

sound-attenuating fan accompanied the start of each operant session.

Surgery

Prior to operant training and testing, we surgically prepared the rats for

microdialysis by inserting a stainless steel guide cannula (21 gauge; Plastics

One, Roanoke, VA) above the left nucleus accumbens. The surgery occurred

while the rats were under halothane or isoflurane anesthesia (1.5-2.5% in

95%/5% O2/CO

2, 1-2 l/min), using standard stereotaxic equipment. The following

coordinates were used (in mm relative to bregma): +1.7 AP, +1.0 lateral, -4.0

ventral to the skull surface (Paxinos and Watson, 1998). The guide cannula was

cemented to the skull by embedding three stainless steel screws into the skull

and covering the entire unit around the base of the cannula with dental cement

(Plastics One, Roanoke, VA). We also placed a single steel bolt vertically into

the hardening cement as an anchor for the microdialysis tether. An obturator

was placed inside the guide cannula to prevent blockage prior to the

microdialysis session. The rats began operant self-administration training after

one week of recovery.

Self-administration training

Operant sessions occurred once a day for 5 days/week. Subjects were divided

into 2 groups (10% sucrose and 10% sucrose with 5 or 10% ethanol) and trained

to bar press for access to 15% (w/v) sucrose. Animals were water deprived (10-

84

22 hr) prior to each session (30 min) to facilitate acquisition of operant

responding. A reliable bar pressing response for sucrose occurred in

approximately 2-6 days. Rats were not water restricted during subsequent

training sessions.

After reliable bar-pressing behavior was established, both groups continued to

receive 10% sucrose reinforcement for approximately eight additional days.

During this period, we gradually habituated the rats to (1) a 15 min wait time (2

min added every 2 days) that preceded access to the lever and drinking solution;

(2) an escalating response requirement (RR) across daily training sessions (i.e.,

RR = 2, 4, 8, 12, 16, 20, 24; Czachowski and Samson, 1999); and (3) a response

requirement in which the completion was delayed using “time-outs.” A time-out

refers to a brief period (e.g., 45 sec) within the lever-press epoch during which

the lever retracts from the chamber, so that a response requirement of 24, for

example, would require at least 3.5 min to perform (Doyon et al., 2003). Upon

completion of the response requirement, the drinking solution was available for

consumption for 20 min, followed by a 20-min post-drinking period in the absence

of the lever and drinking solution. The rats were scheduled for dialysis the day

that the response requirement was set at 24. Two rats were not able to complete

this RR. In these cases, the RR was lowered to 8 and 20, respectively. During

the microdialysis test, one group of rats received 10% sucrose reinforcement,

and two other groups received 10% sucrose with 5 or 10% ethanol for the first

85

time. The sucrose group (n=8) was never exposed to ethanol, and the sucrose +

ethanol groups (n=10, combined) were not exposed to ethanol except on the day

of the experiment. Consumption was monitored during training and during the

microdialysis session by a lickometer and by measuring the volume of liquid in

the drinking bottle before and after the session, taking care to account for

spillage. Body weights were measured each day.

Microdialysis

The microdialysis probes were constructed according to the methods described

by Pettit and Justice (1991). Briefly, fused-silica tubing (i.d. = 40 µm; Polymicro

Technologies, Phoenix, AZ) formed the inlets and outlets of the probes, and

hollow cellulose fiber (i.d. = 200 µm; molecular weight cutoff = 13,000; Spectrum,

Rancho Dominguez, CA) formed the dialysis membrane. The active dialysis

membrane spanned 2.2 mm (the distance between the end of the inlet and the

epoxy that sealed the membrane).

Habituation to the microdialysis tethering apparatus occurred within the week

preceding testing. The habituation procedure consisted of tethering the rats

overnight in the operant testing room, with continued tethering throughout the

subsequent day of operant training. Rats were tethered either by gently

restraining the conscious animal or by sedating the animal with halothane for a

few minutes. On the day preceding the dialysis session, we perfused (flow rate =

86

2 µl/min) the microdialysis probes with artificial cerebral spinal fluid (149 mM

NaCl, 2.8 mM KCl, 1.2 mM CaCl2, 1.2 mM MgCl

2, and 0.25 mM ascorbic acid,

5.4 mM D-glucose) and slowly inserted them into the brain through the guide

cannula while the rat was briefly anesthetized (15-20 min) with 2% halothane in

air. This procedure occurred at least 14 hr before the start of the experiment.

We used a syringe pump (CMA102; CMA, Solna, Sweden) to pump the perfusate

through a fused-silica transfer line into a single channel swivel (Instech

Laboratories, Plymouth, PA), which hung from a counterbalanced lever arm

(Instech Laboratories). The swivel then connected to the inlet of the probe. A

spring tether secured the animal to the swivel. After the rat recovered from the

probe implantation procedure (usually within 15 min), the perfusion flow rate was

decreased to 0.2 µl/min overnight. The flow rate was returned to 2.0 µl/min 2

hours before the baseline-sampling period began. We manually changed each

sample vial, which were immediately frozen on dry ice (except for samples used

for ethanol analysis, see below) and then stored at -80°C until analyzed.

Experimental design

Dialysis samples were taken every 5 min except as indicated below. Baseline

consisted of 30 min in the home cage (6 samples; data not shown). The period

in which the rat was transferred into the operant chamber consisted of one 5 min

sample that preceded activation of the operant program. Upon activation of the

program, a waiting period preceded the introduction of the lever and consisted of

87

15 min of sampling (3 samples). The bar pressing period varied, lasting between

3.8-6 min (but in a few animals more than 6 min). Completion of the response

requirement was followed by a 20 min drinking period with unrestricted access (4

samples) and then a post-drinking period within the chamber in the absence of

the solution (20 min, 4 samples). At the end of this time, the rat was moved back

into the home cage. For the ethanol group, sampling continued for another hour

at 10 min intervals to obtain dialysates for ethanol analysis. After obtaining all

samples, the perfusion solution was switched to one lacking calcium for 45-60

min. A sample was then taken to monitor the calcium dependence of the

dopamine in dialysates.

Histology

After the experiment, the rats were overdosed with chloral hydrate (600 mg/rat)

and saline was perfused through the heart, followed by 10% (v/v) formalin. The

brains were removed and immersed in 10% formalin/30% (w/v) sucrose for at

least 3 days. Brains were cut into coronal sections (48 µm thick) with a cryostat

(Bright Instrument Co., Cambs, England) and the sections stained with cresyl

violet. The slides were examined to confirm the placement of the active dialysis

membrane (2.2 mm).

Dopamine analysis

Two chromatography systems were used to separate and quantify dopamine

88

during these experiments. Both HPLC systems were amperometric and based

on reversed phase chromatography using an ion-pairing agent with

electrochemical detection. The first system included a LC-10AD pump

(Shimadzu, Kyoto, Japan), a 5041 analytical cell (potential set to 350 mV; ESA,

Chelmsford, MA) and a 465 autosampler (ESA) was used in combination with a

2.1 x 100 mm column (Hypersil BDS C18, 3-µm particle size). Samples from 7

subjects were analyzed with this configuration. The second system included an

ISCO 260D pump (Lincoln, NE), FAMOS autosampler (LC Packings, San

Francisco, CA), and a VT-03 cell (2 mm working electrode diameter, potential set

to 450 mV against an Ag/AgCl reference; Antec, Leyden, Netherlands) in

connection with an Intro controller (GBC Separations, Hubbardson, MA) and a 2

x 50 mm column (C18, 3-µm particle size, Polaris). Samples from 10 subjects

were analyzed with this configuration. One additional subject was analyzed with

a 1 x 150 mm column (C18, 5-µm particle size, LC Packings). For these

systems, the mobile phase composition was altered appropriately, using

octanesulfonic acid (0.41-0.77%, w/v) in combination with methanol (12-15%) to

resolve dopamine sufficiently. The flow rates were set at 0.2-0.35 ml/min for the

2.1 mm id column, 0.3 ml/min for the 2 x 50 mm id column, and 0.12 ml/min for

the 1 mm id column). A Shimadzu C-R3A integrator (Houston, TX), HP 3396A

integrator (Hewlett-Packard, Dallas, TX), or a computer data acquisition system

(EZ Chrome Elite; Scientific Software, Pleasanton, CA) recorded the dopamine

peaks. Quantification was carried out by comparing dopamine peak heights

89

from dialysate samples to external standards.

Ethanol analysis

Ethanol was analyzed in all dialysis samples taken after consumption of ethanol

+ sucrose began. Before freezing the dialysis sample, 2 µl were transferred into

a glass vial and sealed with a septum for analysis of ethanol later that day. A

gas chromatograph (Varian CP 3800; Varian, Walnut Creek, CA) equipped with a

flame ionization detector (220°C) measured the ethanol in dialysates. Specific

details concerning the treatment of dialysate ethanol samples and the

components of the gas chromatograph are described by Doyon et al. (2003).

The limit of detection was 0.03 mM (signal to noise = 3). Quantification of

ethanol in dialysates was carried out by comparison of peak areas obtained from

the Star chromatographic analysis system (Varian) to external standards.

Statistical analysis

Analysis of the sucrose plus ethanol group was performed on the pooled data for

the 10% sucrose plus 5% ethanol group and the 10% sucrose plus 10% ethanol

group, due to comparable ethanol intakes (g/kg). Analysis of variance (ANOVA)

with repeated measures was used for the analysis of dialysate dopamine levels.

Dopamine concentrations (nM) were log transformed to maintain homogeneity of

variance. The six home cage samples served as the baseline response to which

the transfer, wait, and lever samples were compared. The average of the

90

transfer-period sample, the three wait samples, and the lever-press sample

defined the baseline dopamine response to which the drink and post-drink

samples were compared. Technical problems associated with either sample

collection or HPLC analysis resulted in the loss of some samples (3 of 247). To

account for this we estimated these values by averaging adjacent time points and

then adjusting the degrees of freedom in the ANOVA. Separate ANOVA tests

were conducted to test for group by time interactions during the two main phases

of the experiment (the drink and post-drink periods). We performed post-hoc

contrasts comparing individual time points to basal after determining a significant

group by time interaction. Bonferroni corrections were used in the case of post-

hoc contrasts. ANOVA was performed using the Manova routine in SPSS for

Windows, and post-hoc contrasts were carried out using the GLM procedure.

Significance for this and other analyses was determined when p < 0.05.

Analysis of consumption parameters was done using multivariate ANOVA (GLM

procedure in SPSS). Several of the parameters were log transformed to

maintain homogeneity of variance (latency to drink, number of bouts, total licks,

and initial bout licks). One rat in the sucrose group and three rats in the sucrose

+ ethanol group were excluded from this analysis because we were unable to

obtain a value for the duration of bar pressing due to a technical problem. The

behavioral data shown in Table 1 reflects this change in sample size.

91

RESULTS

Histological analysis and calcium-dependence of dopamine

All probes had at least 50% of the active dialysis membrane located within the

nucleus accumbens. The majority of the probes traversed both the core and

shell subregions towards the medial part of the accumbens (Figure 3.0). Overall,

44% of the probes were positioned within the core, 6% in the shell, and 50% in

the core plus shell. Within each group the placements were not very different

(sucrose group: 4 core, 1 shell, 3 core and shell; sucrose + ethanol group: 4 core,

0 shell, 6 core and shell). Differences in dopamine function between the core

and shell subregions were not examined due to the mixed distribution of the

probes. Overall, the dialysate samples showed very robust calcium dependence

(75 ± 2% for the 18 subjects). Only the rats in which calcium dependency

exceeded 50% were included in the study.

Accumbal dopamine and operant activity prior to consumption

ANOVA indicated that the groups were not significantly different during the

baseline, transfer, wait, and lever-press periods (group by time interaction;

[F(5,80) = 1.45, p > 0.05]). There was, however, an effect of time across the

groups [F(5,80) = 23.21, p < 0.05]. Post hoc contrasts showed a significant

increase in dopamine during the period in which the rats were transferred into the

operant chamber from the home cage ([F(1,17) = 50.04, p < 0.05]; data not

shown) and during each of the samples that followed within the wait period. Due

92

1.20 mm 1.60 mm1.70 mm2.20 mm

FIGURE 3.0. Coronal sections showing microdialysis probe placement within the nucleus accumbens. Lines indicate the active dialysis regions. Numbers below the figure represent the position of the slice relative to Bregma. The figure was adapted from Paxinos and Watson (1998).

93

to this non-specific increase in dopamine we did not utilize the home cage

samples as a baseline for the subsequent analyses. Instead, it is more

appropriate to compare potential effects of the lever press and drinking periods

with the dopamine concentrations obtained just before the experimental phases,

rather than that obtained during the home cage baseline when the rat was

quiescent and resting. The groups did not differ during the pre-consumption

periods within the operant chamber (group by time: F(4,64) = 1.92, p > 0.05;

group: F(1,16) = 0.28, p > 0.05; 5 samples), nor were there significant changes

over time across the two groups (time: F(4,64) = 1.66, p > 0.05). Therefore, in

our analyses of the subsequent periods the dopamine values across the transfer

and wait epochs were averaged (4 samples), and this served as the baseline

response to which the lever press, drink and post-drink periods were compared.

Mean dialysate dopamine concentrations during this baseline period were 1.6 ±

0.2 nM for the sucrose group and 1.8 ± 0.2 nM for the sucrose plus ethanol

group.

Both groups displayed similar operant response patterns to obtain reinforcement

during the dialysis session. The sucrose group completed a response

requirement of 24, except for one rat in which the RR was decreased to 8 due to

inconsistent responding. Similarly, the sucrose plus ethanol group completed a

response requirement of 24, except for one rat in which the RR was lowered to

20. The time required to complete the response requirement was also similar

94

among the groups (sucrose: 5.7 ± 1.0 min; sucrose plus ethanol: 5.7 ± 0.7 min;

Table 3.0). Dopamine concentrations obtained during the lever press period were

slightly, but significantly increased (5.6% ± .02) compared with the average

baseline obtained during the transfer and wait periods when both groups are

combined (main effect of time: F(1,16) = 7.44, p < 0.05). However, there was no

significant interaction between group and time, indicating that the effect of the

lever pressing behavior on the dopamine concentration did not differ between the

two groups [F(1,16) = 3.28, p > 0.05]. It should be restated that before the drink

and post-drink periods of the experiments, both groups were trained to self-

administer 10% sucrose and, therefore, are not expected to be statistically

different from each other at this stage.

Accumbal dopamine and drinking behavior during consumption

Upon completion of the response requirement, dopamine concentration

increased (15 ± 3% above baseline) within 5 min of access to 10% sucrose

(Figure 3.1A). The elevation in dopamine was transient, and levels returned to

baseline during the subsequent seven samples. In contrast, dopamine

concentrations remained at baseline levels during and after consumption of 10%

sucrose with 5 or 10% ethanol (Figure 3.1B). Separate ANOVAs across each

point during the drink and post-drink periods showed a significant group by time

interaction during the first dopamine sample of the drink period (F(1,16) = 18.3, p

< 0.05) compared with baseline. Post hoc contrasts indicated a significant

95

80

90

100

110

120

0 10 20 30 40 50 60 70

DIAL

YSAT

E DO

PAM

INE

(% o

f bas

al)

TIME (minutes)

drink post-drinkwaitlever

SUCROSE*transfer

80

90

100

110

120

0 10 20 30 40 50 60 70

drink post-drinkwait

lever

SUC/ETOH

TIME (minutes)

DIAL

YSAT

E DO

PAM

INE

(% o

f bas

al)

transfer

A

B

FIGURE 3.1. Effect of operant sucrose (panel A) and sucrose plus ethanol (panel B) self-administration on accumbal dopamine in dialysate. Transfer (closed arrow) refers to the period in which the rat was transferred from the home cage into the operant chamber. Wait refers to the time in which the rat was in the operant chamber without access to the lever or drinking solution. Lever press (open arrow) refers to the period in which the rat was lever pressing prior to access to the drinking solution. Drink refers to the 20 min free-access drinking period in the absence of the lever. Post-drink refers dopamine while the rat is in the operant chamber in the absence of the drinking solution and the lever. Each point represents the mean ± sem (n=8 for the sucrose group, n=10 for the sucrose plus ethanol group). Note the transient dopamine increase within 5 min of the drink period for the sucrose, but not the sucrose plus ethanol group. Asterisk denotes significance compared with the transfer, wait, and lever periods in both groups by ANOVA and post hoc simple contrasts (p < 0.05).

96

0

2

4

6

8

10

12

14

16 Session prior to dialysisDialysis

SUC ETOH

INTA

KE (m

l)

SUC SUC

*

FIGURE 3.2. Mean fluid intake during dialysis (black bars) compared with the training session prior to dialysis (white bars) for the sucrose (left pair; n=8) and sucrose plus ethanol groups (right pair; n=10). SUC indicates that 10% sucrose was consumed, and ETOH indicates that 10% sucrose plus ethanol was consumed. Asterisk denotes significance (p < 0.05) compared with the preceding training session by ANOVA and post hoc simple contrasts.

97

increase in dopamine concentration during this period compared with baseline for

the sucrose group (F(1,7) = 22.27, p < 0.05; Figure 3.1A), but not for the sucrose

plus ethanol group (F(1,9) = 0.28, p > 0.05; Figure 3.1B).

Fluid intake was significantly different between the groups across the last two

days of self-administration (group x time: F(1,16) = 22.82, p < 0.05; Figure 3.2).

The sucrose plus ethanol group ingested significantly less fluid during the dialysis

session compared with the preceding training day (F(1,9) = 63.18, p < 0.05),

whereas intake during these times did not differ in the sucrose group (F(1,7) =

2.67, p > 0.05). The sucrose group ingested 9.9 ± 2.0 ml of 10% sucrose during

the dialysis session, whereas the sucrose with ethanol group ingested 2.3 ± 0.4

ml (0.27 ± 0.02 g/kg).

Licking behavior during consumption of sucrose and sucrose with ethanol

The licking behavior of the sucrose group differed significantly from the sucrose

plus ethanol group in three of the seven parameters listed in Table 3.0 (F(8,5) =

2337, p < 0.05 by multivariate ANOVA). The sucrose group exhibited higher

values for (1) the number of total licks of solution, (2) the number of licks within

the first drinking bout, and (3) the rate at which the solution was licked during the

first drinking bout compared with the sucrose + ethanol group. No significant

differences between the groups were observed for number of bouts, latency to

begin drinking, or initial bout duration. The sucrose group showed a trend for a

98

longer duration of the initial drinking bout compared with the sucrose + ethanol

group (Table 3.0), although this difference between groups did not attain

statistical significance (F(1,12) = 3.63, p = 0.081). The lickometer records

showed that 73 ± 10% of the total volume of 10% sucrose consumed (as

measured by spout licks) during the 20-min drink period occurred within the first

5 min, and 98 ± 2% within the first 10 min (n=7; Figure 3.3A). Most consumption

of 10% sucrose with 5 or 10% ethanol (73 ± 14%) also occurred within 5 min of

the drink period (n=10; Figure 3.3B). To determine whether licking behavior

might influence the dopamine response obtained during the first drinking sample,

we correlated the two parameters for the sucrose-drinking rats. The correlation

was not significant (r2 = 0.026, p > 0.05).

Accumbal ethanol during consumption

We quantified the ethanol concentration in each accumbal dialysate sample

collected during and after sucrose plus ethanol consumption. Overall, the

dialysate ethanol concentrations were very low (Figure 3.4), reflecting the small

amount of ethanol that the rats consumed (0.27 ± 0.02 g/kg). The mean ethanol

concentrations were very similar throughout the drink period. The highest mean

concentration of dialysate ethanol occurred 10 min after sucrose plus ethanol

access (0.16 ± 0.06 mM). Individual ethanol time courses varied greatly among

the animals with respect to peak ethanol concentration in the accumbens and

ethanol clearance from the accumbens, and data representative of the entire set

99

0

200

400

600

800

1000

1200

1400

1600

5 10 15 20TIME (minutes)

SUC/

ETO

H LI

CKS

B

0

200

400

600

800

1000

1200

1400

1600

5 10 15 20

SUCR

OSE

LIC

KS

TIME (minutes)

A FIGURE 3.3. Total number of spout licks during the 20-min drink period for the sucrose (n=7) panel A) and sucrose plus ethanol group (n=10; panel B). 73 ± 10% of 10% sucrose consumption (as measured by spout licks) occurred within the first 5 min, and 98 ± 2% within the first 10 min. 73 ± 14% of 10% sucrose plus 5 or 10% ethanol consumption occurred within 5 min.

100

TABLE 3.0. Lickometer parameters during sucrose or sucrose plus ethanol self-administration during the microdialysis session.

Parameterc Sucrose

a

n=7 Sucrose + ethanol

n=7 lever pressing time (min) 5.7 ± 0.7 5.7 ± 1.0

latency to begin drinking (min) 0.09 ± 0.05 0.05 ± 0.02 number of bouts 1.1 ± 0.2 1.6 ± 0.3

total licks 2407 ± 545 557 ± 194b

initial bout licks 2346 ± 522 331 ± 83b

initial bout duration (min) 8.1 ± 1.6 3.9 ± 1.8 lick rate for initial bout (licks/min) 294 ± 28 108 ± 18

b

Values shown as mean ± sem a – the sucrose group was significantly different from the sucrose plus ethanol group by multivariate ANOVA (p < 0.05) b –significantly different from the sucrose group by univariate ANOVA (p < 0.05) c –parameters are defined as follows: lever pressing time = time needed to complete response requirement (RR), latency = time between completion of RR and first spout lick, bout = period of at least 25 licks with no more than 2 min between licks, total licks = number of licks per session (20 min), initial bout licks = number of licks in first bout, initial bout duration = time needed to complete initial bout, initial bout lick rate = initial bout licks divided by initial bout duration

101

0

0.1

0.2

0.3

0.4

0.5

0 5 10 15 20 25 30 35 40

JR33JR31EJ9EJ5

Rat g/kg0.290.210.240.24

NAc

DIA

LYSA

TE

ETH

ANO

L (m

M)

TIME (minutes)

FIGURE 3.4. Representative dialysate ethanol concentrations from the nucleus accumbens of individual rats during drinking and post-drinking periods. Data are from four of the ten rats shown in Figure 3.1B. Ethanol was analyzed in the same samples from which the dopamine concentrations derived. Peak ethanol concentrations and ethanol clearance from the accumbens displayed individual variability.

102

are shown in Figure 3.4 (excluding 3 rats which had ethanol concentrations

which were below our detection limit). Rats consuming similar amounts of

ethanol (e.g., EJ9, EJ5) did not necessarily display similar ethanol time courses.

There was no correlation (r2 = .0002, p > 0.05) between consumption (g/kg) and

dialysate ethanol (AUC). In addition, we investigated the possible relationship

between ethanol concentrations in the dialysate (AUC) and the peak dopamine

response (as percent of basal) in the ethanol drinking rats. However, there was

no correlation between these parameters (r2 = 0.40, p > 0.05).

DISCUSSION

The major findings of the present study are that sucrose consumption produced a

transient increase in nucleus accumbens extracellular dopamine concentration,

whereas consumption of a small volume of ethanol (in sucrose) in a naïve rat had

no effect. In addition, appetitive responding (lever pressing) did not produce a

significant enhancement of accumbal dopamine. Although consumption of a

sucrose solution has previously been shown to increase accumbal dopamine

activity with microdialysis measurements (Hajnal & Norgren, 2001; Hajnal et al.,

2004), our study is the first to demonstrate this effect in rats that are not water

deprived.

The lack of effect of consumption of ethanol in sucrose on accumbal dopamine

could be due to several factors. The mean intake of 5-10% ethanol (plus 10%

103

sucrose) was rather low (2.3 ± 0.4 ml; 0.27 ± 0.02 g/kg) compared with the rats

that drank 10% sucrose only (9.9 ± 2.0 ml). Consequently, peak dialysate

ethanol levels were low (0.16 ± 0.06 mM). This dose of ethanol may not possess

the pharmacological activity or the stimulus strength necessary to stimulate

accumbal dopamine activity. Results from most previous microdialysis studies of

effects of systemic injections of ethanol in naïve rats have shown that accumbal

dopamine concentration is increased at doses ≥ 1.0 g/kg (Blomqvist et al., 1993;

Heidbreder & De Witte, 1993; Kiianmaa et al., 1995; Yim et al., 2000; Yoshimoto

et al., 1992), although a few studies have reported stimulatory effects at 0.25

g/kg (Blanchard et al., 1993; Imperato & Di Chiara, 1986; Tanda & Di Chiara,

1998). Furthermore, under limited-access models, previous studies of oral

ethanol reinforcement report increases in accumbal dopamine concentration in

response to intakes of 0.45 – 1.4 g/kg (Doyon et al., 2003; Melendez et al., 2002;

Weiss et al., 1993), values that were not achieved in the present study.

Therefore, we cannot exclude the possibility that alterations in dopamine activity

may occur with consumption of ethanol higher than that achieved in the present

study.

Another factor that may contribute to the lack of stimulation of accumbal

dopamine during ethanol consumption is that the reinforcing properties of ethanol

were not yet established in the ethanol naïve rats. Indeed, our behavioral results

support the suggestion that ethanol was aversive, because the fluid intake

104

decreased by 77% when ethanol was present in the drinking fluid for the first time

compared with the previous session in which 10% sucrose was available. We

previously showed that ethanol consumption produces a transient stimulation of

accumbal dopamine activity in rats in which ethanol reinforcement is well

established after sucrose has been completely faded out of the solution (Doyon

et al., 2003). Together, the present results with ethanol-naïve rats and the

previous findings with ethanol-experienced rats support the idea that accumbal

dopamine activity may contribute, at least in part, to neural signals that encode

the incentive salience of ethanol. This interpretation is consistent with findings

from a recent study that indicate that the magnitude of dopamine efflux from the

accumbens is related to the incentive salience of sucrose reward (Genn et al.,

2004).

A significant finding of this study is the demonstration that operant self-

administration of 10% sucrose can induce elevations in accumbal dopamine

concentrations in non-deprived rats. Studies using non-operant paradigms of

consumption generally indicate that novelty and motivational state (e.g.,

deprivation) are important factors that influence food-induced dopamine efflux

(Ahn & Phillips, 1999; Bassareo & Di Chiara, 1997; Hajnal et al., 2004; Wilson et

al., 1995). The present study is the first to detect a dopamine response

associated with sucrose drinking in an operant condition and in non-deprived

animals using microdialysis. The increase in dopamine concentration during

105

sucrose consumption was transient, and was observed only in the first dialysis

sample of the drinking period (5 min) before returning to baseline. Consumption

of sucrose occurred predominantly within the first 5-10 min of access to the

solution (73 ± 10% of total consumption, from lickometer data), indicating that

feeding (licking) was coincident with the neurochemical response. The

magnitude of the transient increase in dialysate dopamine we observed during

sucrose consumption (15% above baseline) is lower than that observed in

experiments in which rats bar press to obtain food reinforcement (greater than

50% increase above baseline; McCullough et al., 1993; Salamone et al., 1994;

Cousins et al., 1999; McCullough et al., 1992; Sokolowski et al., 1998). This

could be due to the use of food deprivation in the previous studies, whereas we

did not use deprived rats. It is also possible that the short lag period caused by

the delivery of the dialysate from the probe tip to the collection vial may

contribute to the smaller increase we observed. This lag period would cause

mixing of the dopamine concentration (about 20% of the 5 min sample) from the

end of the bar press period with changes produced during the beginning of the

drinking period, which would effectively dilute the signal.

Based on the consummatory data alone, these results seem to indicate that (1)

the stimulus properties of the sucrose solution (taste, smell), (2) the motor activity

involved in drinking the solution, or (3) a combination of the two, were involved in

the elevated dopamine response. The fact that the ethanol (plus sucrose) group

106

consumed a comparatively smaller amount of fluid during the dialysis session,

corresponding with a clear absence of dopamine activity, supports the idea that

motor performance during drinking may have been a contributing factor here. In

contrast, the lack of a significant correlation between number of licks and the

accumbal dopamine response indicates that licking behavior per se did not

significantly influence the neurochemical response. Moreover, our recent data

from Long-Evans rats self-administering water do not support the notion that the

motoric processes during licking enhance accumbal dopamine. Using an

operant procedure similar to the one used in the present study, water

consumption did not induce elevations in dopamine during drinking (Doyon et al.,

2003). Additionally, periodic (low intake) feeding can elevate accumbal

dopamine activity, but bout-like feeding that is unrestricted has no effect

(McCullough & Salamone, 1992), further supporting the idea that increased

dopamine is a response to something other than intake alone. Moreover, the

increase in accumbal dopamine produced during operant self-administration of

food pellets in food deprived rats was not correlated with the amount of food

consumed (Sokolowski et al., 1998). Collectively, these findings seem to indicate

that the motor activity that occurred during drinking behavior does not adequately

account for our results. Therefore, we suggest that the sensory properties of

sucrose, which were strongest within the first 5 min of consumption, contributed

to the transient dopamine activity.

107

An alternative explanation for our sucrose-induced dopamine increase is that the

operant procedure used in this study contained an element of unpredictability as

to exactly when receipt of the drinking solution would occur. Dopamine neurons

fire in response to liquid reward presented at unpredictable times, but not reliably

when its delivery is predictable (Fiorillo et al., 2003; Mirenowicz & Schultz, 1994).

We used an escalating response requirement (RR) across daily training sessions

so that during the dialysis session a sample could be collected during lever

pressing, but there was no habituation to the escalating response requirement.

Therefore, each successive trial during the training period consisted of an RR

whose completion time was longer than that of the preceding day, and the day of

the dialysis test corresponded to the highest RR to which the animal was

exposed. In this way, the animal had learned that the sucrose reward would be

available, but not the time that it would be available. Fiorillo et al. (2003) recently

showed that uncertainty in reward prediction is associated with a sustained

increase in dopamine neuron activity. It is possible that this type of sustained

dopamine activity contributed to the transient increase in dialysate dopamine we

observed.

It should be noted that our finding of a transient increase in accumbal dopamine

activity that coincides with sucrose consumption does not agree with previous

reports in which no increase in accumbal dopamine was found during sucrose

consumption (Bassareo et al., 2002; Bassareo et al., 2003; Datla et al., 2002).

108

However, these previous studies did not use an operant procedure, and

therefore, direct comparison between them and the present study is not possible.

It is interesting that the procedure used by Datla et al. (2002) involved

associative conditioning, and when a new stimulus was presented along with

cues that predicted sucrose availability, accumbal dopamine was elevated during

sucrose consumption. This finding is consistent with the idea that the accumbal

dopamine response we observed could be due, in part, to the conditioning that

was taking place during the operant procedure.

Dopamine systems can also be activated during certain aspects of appetitive

behavior (e.g., lever pressing; Nishino et al., 1987; Roop et al., 2002). Findings

obtained from previous microdialysis studies show that the amount of food-

reinforced lever pressing is correlated with the magnitude of dopamine release in

the nucleus accumbens in food-deprived rats (McCullough et al., 1993;

Salamone et al., 1994; Sokolowski et al., 1998; Cousins et al., 1999). However,

these studies used reinforcement schedules in which lever pressing occurs along

with food consumption during the collection of the microdialysis sample, and

direct comparison of these results with the present study is not possible.

Segregation of operant responding allowed us to examine dopamine activity

during active lever pressing and, importantly, during fluid consumption alone.

When we focus on the dopamine concentration obtained during the lever press

period we found a small increase (approximately 6%) above that obtained during

109

the preceding period in which the rat was waiting in the operant chamber. The

present results do not agree with our previous findings in which rats were

extensively trained (2 months or more) to bar press for ethanol or water

reinforcement (Doyon et al., 2003). One possible reason for this discrepancy

may be that the longer training procedure had blunted the dopamine response

during the lever press period.

In general our findings agree with previous reports of enhanced mesolimbic

dopamine activity during lever pressing behavior (Nishino et al., 1987;

Richardson & Gratton, 1996; Roitman et al., 2004), although the magnitude of

our response is lower. The greater time resolution of the measurements of firing

of dopamine neurons (Nishino et al., 1987) or electrochemical signals

(Richardson & Gratton, 1996; Roitman et al., 2004) compared with our

microdialysis measurements likely contributes to the difference in magnitude.

Thus, a single lever-press period (> 3.5 min), in which responding can only occur

periodically, may not be sufficient to produce robust neurochemical changes in

the mesoaccumbal dopamine system, at least when measured using

microdialysis. Specifically, in our study, rats only performed a low number of

lever presses (24 or less), and this level may be too low to produce a large

increase in accumbal dialysate dopamine concentrations. In contrast, Roitman et

al. (2004) recently showed that transient (within a few seconds) dopamine

signals were detected just before lever-pressing for sucrose reward using fast-

110

scan cyclic voltammetry. Similarly, Richardson and Gratton (1996) showed that

dopamine signals change within a minute of lever pressing. Therefore, a similar

effect could be occurring in our experiments, but the larger transient response is

diluted with the lower concentrations of dopamine during the normal tonic state of

the dopamine system. Additional microdialysis studies using shorter sampling

times (subminute) are required to resolve this issue.

In summary, the present study demonstrates that operant self-administration of

sucrose in non-deprived rats can cause significant, transient increases in

accumbal dopamine within 5 min of consumption. This dopamine response may

be related to the stimulus properties of sucrose, which were strongest during the

first 5-10 min of drinking. We do not rule out the possibility, however, that the

operant procedure may have promoted the response by maintaining

unpredictability of sucrose presentation. Finally, we show that first-time

consumption of 5 or 10% ethanol (0.27 ± 0.02 g/kg) with sucrose elicits low levels

of intake and no alterations in accumbal dopamine. These results provide a

reference point to which other operant self-administration studies can be

compared and support the idea that, at some point in the development of ethanol

reinforcement, there is a shift in the incentive value of ethanol corresponding to

an increase in consumption and dopamine activity within the accumbens.

111

4.0 Effect of Operant Self-Administration of 10% Ethanol plus 10% Sucrose

on Dopamine and Ethanol Concentrations in the Nucleus Accumbens

[published in Journal of Neurochemistry (2005) 93: 1469-1481; by William M.

Doyon, Sheneil K. Anders, Vorani S. Ramachandra, Cristine L. Czachowski, and

Rueben A. Gonzales]

ABSTRACT

Although operant ethanol self-administration can increase accumbal dopamine

activity, the relationship between dopamine and ethanol levels during

consumption remains unclear. We trained Long-Evans rats to self-administer

escalating concentrations of ethanol (with 10% sucrose) over 7 days, during

which 2-4 lever presses resulted in 20 min of access to the solution with no

further response requirements. Accumbal microdialysis was performed in rats

self-administering 10% ethanol (plus 10% sucrose) or 10% sucrose alone. Most

ethanol (1.6 ± 0.2 g/kg) and sucrose intake occurred during the first 10 min of

access. Sucrose ingestion did not induce significant changes in dopamine

concentrations. Dopamine levels increased within the first 5 min of ethanol

availability followed by a return to baseline, whereas brain ethanol levels reached

peak concentration more than 40 min later. We found significant correlations

between intake and dopamine concentration during the initial 10 min of

112

consumption. Furthermore, ethanol-conditioned rats consuming 10% sucrose

showed no effect of ethanol expectation on dopamine activity. The transient rise

in dopamine during ethanol ingestion suggests that the dopamine response was

not solely due to the pharmacological properties of ethanol. The dopamine

response may be related to the stimulus properties of ethanol presentation,

which were strongest during consumption.

INTRODUCTION

The role of accumbal dopamine in ethanol reinforcement remains a complex

issue. Blockade of dopamine transmission interferes with responding for ethanol

reinforcement (Rassnick et al., 1992; Samson et al., 1993; Hodge et al., 1997;

Czachowski et al., 2001), whereas ethanol consumption is not as sensitive to this

manipulation (Samson et al., 1993; Czachowski et al., 2001). Several studies

have shown, however, that extracellular dopamine concentrations increase

during operant ethanol self-administration (Weiss et al., 1993; Gonzales and

Weiss, 1998; Melendez et al., 2002; Doyon et al., 2003). Furthermore, rats self-

administer ethanol directly into the ventral tegmental area (Gatto et al., 1994;

Rodd-Henricks et al., 2000), the region from which the neurons of the

mesoaccumbens system originate, suggesting a direct link between ethanol

reinforcement and dopamine activation.

113

We recently examined the consummatory component of ethanol reinforcement in

more detail by (1) using an operant procedure that distinguished lever

responding from ethanol consumption and (2) measuring intra-accumbal ethanol

and dopamine concentrations concurrently during limited-access drinking (Doyon

et al., 2003). A small, transient increase in accumbal dopamine concentration

was observed within five minutes of ethanol access, after which point accumbal

ethanol levels continued to rise with no apparent stimulatory effect on dopamine

activity. We hypothesized that this rapid dissociation between the dopamine and

ethanol time courses could be related to the stimulus properties of ethanol (taste,

smell) rather than its pharmacological action. However, two possible alternative

explanations for this transient dopaminergic response are that the mean ethanol

intake among the rats was not high enough to induce further neurochemical

activity or that the response desensitized over time due to the long-term ethanol

exposure that the animals underwent (over 40 days). The latter phenomenon

can occur when a reward becomes predictable (Bassareo and Di Chiara, 1997;

Schultz et al., 1997).

Therefore, a more definitive examination of these issues is required to clarify the

relationship between intra-accumbens ethanol and dopamine in response to

limited-access consumption of ethanol. The present experiments were designed

to determine the effect of ethanol intake on dopamine and ethanol concentrations

within the accumbens during an early period in the development of reinforced

114

responding. To accomplish these goals, we trained male Long-Evans rats to

press a lever for limited access to ethanol (plus sucrose) over seven days, using

an operant procedure that segregated lever pressing from drinking behavior.

Microdialysis was performed during self-administration of 10% ethanol (plus 10%

sucrose). We followed the time course of intra-accumbal dopamine and ethanol

concentrations during the limited-access drinking period to determine the

relationship, if any, between intra-accumbal ethanol levels and the accompanying

dopamine response. Consumption patterns for both treatment groups were also

quantified for comparison with the ethanol and dopamine concentrations over

time.

MATERIALS AND METHODS

Subjects

The present study used 41 male Long-Evans rats (Charles River Laboratories,

Wilmington, MA) that weighed between 327-496 g at the time of testing. Rats

were handled and weighed for at least 5 days upon arrival prior to surgery or

training. Each rat lived individually in a humidity and temperature-controlled

(22°C) environment under a 12-hr light/dark cycle (on at 7:00 A.M.; off at 7:00

P.M.). Each rat had food and water available ad libitum in the home cage except

during the procedures indicated below. All procedures complied with guidelines

specified by the Institutional Animal Care and Use Committee of the University of

Texas at Austin and the National Institutes of Health Guide for the Care and Use 115

of Laboratory Animals.

Behavioral apparatus

Standard operant chambers (Med Associates Inc., St. Albans, VT) modified for

microdialysis perfusion were used for self-administration training and

microdialysis testing. One wall of each chamber contained a retractable lever on

the left side (2 cm above grid floor), which upon activation triggered the entry of a

retractable drinking spout on the right side (5 cm above grid floor). The floor was

a grid of metal bars in connection with the spout of the drinking bottle, which

formed a lickometer circuit (Med Associates Inc.). A cubicle with the front doors

left open during training and testing housed each operant chamber. PC software

provided by Med Associates controlled operant chamber components and

acquisition of lickometer data. Activation of an interior chamber light and a

sound-attenuating fan accompanied the start of each operant session.

Surgery

Prior to operant training and testing, we surgically prepared the rats for

microdialysis by inserting a stainless steel guide cannula (21 gauge; Plastics One

Inc., Roanoke, VA) above the left nucleus accumbens. The surgery occurred

while the rats were under isoflurane anesthesia (1.5-2.5% in 95%/5% O2/CO2, 1-

2 L/min), using standard stereotaxic equipment. The following coordinates were

used (in mm relative to bregma): +1.7 AP, +1.0 lateral, -4.0 ventral to the skull

116

surface (Paxinos and Watson, 1998). The guide cannula was cemented to the

skull by embedding three stainless steel screws into the skull and covering the

entire unit, around the base of the cannula, with dental cement (Plastics One

Inc.). We also placed a single steel bolt vertically into the hardening cement as

an anchor for the microdialysis tether. An obturator was placed inside the guide

cannula to prevent blockage prior to the microdialysis session. After one week of

recovery, the rats began the training procedure.

Self-administration training

Operant sessions occurred once a day for 5 days/week. Subjects were initially

divided into 2 groups (10% ethanol plus 10% sucrose or 10% sucrose) and all

were trained to lever press for access to 15% sucrose (w/v). Animals were water

deprived (10-22 hours) prior to each session (30 min) to facilitate acquisition of

the operant response. A reliable bar pressing response for sucrose occurred in

approximately 2-6 days. Rats were not water restricted at any time during the

subsequent training periods.

After reliable lever-pressing behavior was established, subjects in the ethanol

plus sucrose group were trained for self-administration of 10% ethanol with 10%

sucrose using a modified version of the sucrose fading procedure (Samson,

1986), in which we increased the concentration of ethanol (v/v) in the drinking

solution across sessions (2-10% over six days), but we did not subsequently

117

remove the sucrose (Table 4.0). Following lever training, the subjects in the

sucrose group (n=10) were switched to 10% sucrose (w/v) as reinforcement over

the same number of days as the ethanol group. During this period, we gradually

habituated the rats to (1) a 15 min “wait time”, which preceded access to the

lever and drinking solution, and (2) a response requirement (RR) that increased

from 2 to 4 across sessions. Upon completion of the RR, the drinking solution

became accessible for 20 min, followed by a 20-min post-drinking period in the

absence of the lever and drinking solution. For the dialysis experiment, the

response requirement was set at 4 and the reinforcer was either 10% ethanol

(plus 10% sucrose) or 10% sucrose. The sucrose group was never exposed to

ethanol. Consumption was monitored during training and during the

microdialysis session by a lickometer and by measuring the volume of liquid in

the drinking bottle before and after the session, taking care to account for

spillage. Body weights were measured each day.

A third group of rats (n=7) was included to control for the non-specific effects of

handling on dopamine activity. These animals were placed into the operant

chamber for the same periods of time and corresponding number of days as the

other groups, except that they did not receive training for self-administration.

These rats were never exposed to a lever or a drinking bottle in the chamber.

Each rat in the handling group was paired surgically and experimentally with a rat

in the ethanol plus sucrose or the sucrose group.

118

TABLE 4.0. Summary of operant self-administration training protocol for ethanol plus sucrose self-administration

Day Drink solution^ Pre-drink wait (min) Response requirement

1 10S 2 2 2 10S 2E 4 2 3 10S 2E 6 2 4 10S 5E 8 2 5 10S 5E 10 4 6 10S 10E 12 4 7* 10S 10E 15 4

The sucrose control group followed the same schedule except that ethanol was not faded into the drink solution. ^ S equals sucrose and E equals ethanol. Numeral for drink solution represents percentage (w/v for sucrose; v/v for ethanol). * - dialysis session.

119

We subsequently included an additional experimental group (n=8) that was

trained in exactly the same manner as the ethanol group, except that rather than

receiving 10% ethanol (plus 10% sucrose) during the dialysis session, these rats

self-administered a solution of 10% sucrose, which did not contain ethanol.

Therefore, the only procedural difference between this group (i.e., unexpected

sucrose) and the ethanol group occurred on the experiment day, during which

time each group consumed a distinct reinforcer.

Microdialysis

The microdialysis probes were constructed according to the methods described

by Pettit and Justice (1991). Briefly, fused-silica tubing (inner diameter = 40 µm;

Polymicro Technologies, Phoenix, AZ) formed the inlets and outlets of the

probes, and hollow cellulose fiber (inner diameter = 200 µm; molecular weight

cutoff = 13,000; Spectrum Laboratories Inc., Rancho Dominguez, CA) formed the

dialysis membrane. The active dialysis membrane spanned 2.2 mm (the

distance between the end of the inlet and the epoxy that sealed the membrane).

Habituation to the microdialysis tethering apparatus occurred within the week

preceding testing. This procedure consisted of tethering the rats overnight in the

operant testing room, with continued tethering throughout the subsequent day of

operant training. Rats were tethered by gently restraining the conscious animal

or by sedation with halothane for a few minutes. On the day preceding the

dialysis session, we perfused (flow rate = 2 µl/min) the microdialysis probes with

120

artificial cerebral spinal fluid (149 mM NaCl, 2.8 mM KCl, 1.2 mM CaCl2, 1.2 mM

MgCl2, and 0.25 mM ascorbic acid, 5.4 mM D-glucose) and slowly inserted them

into the brain through the guide cannula while the rat was briefly anesthetized

(15-20 min) with 2% halothane in air. This procedure occurred at least 14 hours

before the start of the experiment. We used a syringe pump (CMA102; CMA,

Solna, Sweden) to pump the perfusate through a fused-silica transfer line into a

single channel swivel (Instech Solomon, Plymouth Meeting, PA), which hung

from a counterbalanced lever arm (Instech Solomon). The swivel formed a

connection with the inlet of the probe and a spring tether secured the animal to

the swivel. After the rat recovered from the probe implantation procedure

(usually within 15 min), the perfusion flow rate was decreased to 0.2 µl/min

overnight. The flow rate was returned to 2.0 µl/min 2 hours prior to the baseline-

sampling period. We manually changed each sample vial, which was

immediately frozen on dry ice (excluding the fraction of dialysate that was

removed for ethanol analysis, see Ethanol analysis) and then stored at -80˚C

until analyzed.

Experimental design

Dialysis samples were taken every 5 min except as indicated below. Six

samples were collected during a baseline period in the home cage (30 min; data

not shown). One sample was collected during the period in which the rat was

transferred into the operant chamber prior to activation of the operant program (5

121

min; data not shown). Upon activation of the program, three samples were

collected prior to introduction of the drinking spout: two 5 min samples during a

waiting period and a third waiting sample (approximately 5.7 min) that included at

its end a brief lever-pressing period (0.7 ± 0.2 min; excluding one rat that

required 15.3 min). Completion of the response requirement was followed by a

20 min drinking period with unrestricted access (four samples) and then a post-

drinking period within the chamber in the absence of the solution (20 min, four

samples). At the end of this time, the rat was moved back into the home cage.

For the ethanol group, we then collected an additional 6 samples (at 10-min

intervals) to monitor ethanol concentrations in the dialysates, but these sample

were not analyzed for their dopamine content. After obtaining all samples, the

perfusion solution was switched to one lacking calcium for 45-60 min. A sample

(10 min) was then taken to determine the calcium dependency of the dopamine

in dialysates.

Histology

After the experiment, the rats were overdosed with chloral hydrate (600 mg/rat)

and saline was perfused through the heart, followed by 10% (v/v) formalin. The

brains were removed and immersed in 10% formalin/ 30% sucrose (w/v) for at

least 3 days. Brains were cut into coronal sections (48 µm thick) with a cryostat

(Bright Instrument Co., Cambridgeshire, England), and the sections stained with

cresyl violet. The slides were examined to confirm the placement of the active

122

dialysis membrane (2.2 mm). We determined subregional placement within the

core, shell, or core and shell if at least 30% of the dialysis membrane bisected

any of these areas.

Dopamine analysis

Two chromatography systems were used to separate and quantify dopamine

during these experiments. Both HPLC systems were amperometric and based

on reversed phase chromatography using an ion-pairing agent with

electrochemical detection. The majority of samples were analyzed with the first

system, which included one of three pumps [ISCO 260D (Lincoln, NE), LC-10AD

(Shimadzu Scientific Instruments Inc., Columbia, MD, and LC-10ADVP

(Shimadzu Scientific Instruments Inc.)], a FAMOS autosampler (LC Packings,

Sunnyvale, CA), a VT-03 cell (2 mm working electrode diameter, potential: 450

mV against a Ag/AgCl reference; Antec Leyden BV, Zoeterwoude, Netherlands)

in connection with an Intro controller (GBC Separations Inc., Hubbardston, MA)

and a Polaris 2 x 50 mm column (C18, 3-µm particle size; Varian, Palo Alto, CA).

Samples from 39 subjects were analyzed with this configuration. The second

system consisted of a LC-10ADVP pump (Shimadzu, Kyoto, Japan), a 5041

analytical cell (potential: 350 mV; ESA Inc., Chelmsford, MA), and a 465

autosampler (ESA Inc.) used in connection with a BDS Hypersil 2.1 x 100 mm

column (C18, 3-µm particle size; Thermo Hypersil-Keystone, Bellefonte, PA).

Samples from 2 subjects were analyzed with this configuration. For these

123

systems, the mobile phase composition was altered appropriately, using

octanesulfonic acid (0.72-0.77%, w/v) or octanesulfonic acid (0.5%, w/v) plus

decanesulfonic acid (0.05%, w/v) in combination with methanol (12-15%, v/v) to

resolve dopamine sufficiently. The flow rates were set at 0.3 ml/min for both

systems. For the first system, 7 µl of the dialysate was injected using the

microliter pickup mode and the transfer fluid was ascorbate oxidase (EC

1.10.3.3; 102.3 units/mg; Sigma, St. Louis, MO). For the second system, 7 µl of

the dialysate was incubated with 21 µl of ascorbate oxidase (102.3 units/mg;

Sigma) for 1.5 min, and 20 µl was injected. A Shimadzu C-R3A integrator

(Houston, TX) or a computer data acquisition system (EZ Chrome Elite; Scientific

Software Inc., Pleasanton, CA) recorded the dopamine peaks. Quantification

was carried out by comparing dopamine peak heights from dialysate samples to

external standards.

Ethanol analysis

Ethanol was analyzed in all dialysis samples collected after the lever-press

period for subjects in the ethanol plus sucrose group. Before freezing the

dialysis sample, 2 µl of fluid were transferred into a glass vial and sealed with a

septum for analysis of ethanol later that day. A gas chromatograph (Varian CP

3800; Varian, Walnut Creek, CA) with flame ionization detection (220°C)

measured the ethanol in the dialysates. Specific details concerning the treatment

of dialysate ethanol samples and the components of the gas chromatograph are

124

described by Doyon et al. (2003). The limit of detection was 0.03 mM ethanol

(signal to noise = 3). Quantification of ethanol in dialysates was done by

comparing peak areas obtained with a Star chromatographic analysis system

(Varian) to external standards.

Statistical analysis

Dialysate dopamine levels (nM) were analyzed using analysis of variance

(ANOVA) with repeated measures. The six home cage samples served as the

baseline response to which the transfer and wait samples were compared. The

average of the transfer and wait periods (4 samples), including the last wait

sample that encompassed the lever-press period, defined the baseline response

to which the drink and post-drink samples were compared. The lever press

period was included as a basal sample because of its short duration. Any

potential dopamine activity resulting from this period would be collected in the

next sample due to the brief time lag inherent in microdialysis. Technical

problems associated with sample collection or HPLC analysis resulted in the loss

of some samples (8 of 429). To account for this we estimated these values by

averaging adjacent time points and then adjusting the degrees of freedom in the

ANOVA. Separate ANOVA tests were conducted to test for group by time

interactions during the main phases of the experiment (i.e., basal plus

transfer/wait periods; transfer/wait periods plus drink and post-drink periods).

Post-hoc contrasts comparing individual time points to baseline within groups

125

were performed after determining a significant group by time interaction during

these periods. Bonferroni corrections were used in the case of post-hoc

contrasts. ANOVA was performed using the Manova routine in SPSS for

Windows, and post-hoc contrasts were carried out using the GLM procedure.

Significance for these analyses was determined when p < 0.05.

Analysis of the consumption parameters was carried out using multivariate

ANOVA (GLM procedure) and F-values derived from Wilks’ Lambda. Two

parameters were log transformed to maintain homogeneity of variance (latency to

drink, lever-pressing time). Due to a technical issue, one rat in the ethanol plus

sucrose groups and two rats in the sucrose group were excluded from this

analysis because we were unable to obtain a value for the duration of bar

pressing. The behavioral data shown in Table 2 reflects this change in sample

size.

RESULTS

Histological analysis and calcium-dependence of dopamine concentrations

At least 50% of the active dialysis membrane for each probe was within the

nucleus accumbens. Examination of the probe positions within subregions of the

accumbens showed that, overall, 42% were within the core, 33% in the shell, and

24% bisected both the core and shell (Figure 4.0). The placements were random

in distribution between each experimental group (sucrose plus ethanol groups: 5

126

1.00 mm1.20 mm 1.60 mm 1.70 mm 2.20 mm

FIGURE 4.0. Coronal sections showing microdialysis probe placement within the nucleus accumbens. Lines indicate the active dialysis regions. Numbers below the figure represent the position of the slice relative to Bregma. The figure was adapted from Paxinos and Watson (1998).

127

core, 6 shell, 5 core and shell; sucrose group: 6 core, 2 shell, 2 core and shell;

handling group: 2 core, 3 shell, 2 core and shell). The core and shell subregions

were not examined with regard to differences in dopaminergic activity due to the

dispersion of the probes. The dialysate dopamine samples showed very robust

calcium dependence (78 ± 3% for the 33 subjects). The calcium dependency of

dialysate dopamine exceeded 50% in all subjects except for one, which showed

41%. In addition, approximately 50% of all subjects incurred a certain amount of

ventricular damage caused by the insertion of the microdialysis probe. However,

these subjects were randomly distributed within each experimental group.

Ethanol intake patterns during the training procedure

Due to clear differences in ethanol preference during training and dialysis (Figure

4.1), we divided the rats in the ethanol plus sucrose group into two subgroups: a

high ethanol group (with intakes ≥ 0.8 g/kg; n = 10) and a low ethanol group (with

intakes ≤ 0.5 g/kg; n = 6), based on intake during the dialysis session. The

ethanol intake levels between the high and low ethanol groups differed

significantly across the training period [group: F(1,14) = 6.38, p < 0.05; group x

time: F(5,70) = 8.59, p < 0.05]. The separation into subgroups was also justified

by a histogram analysis of the ethanol intakes during dialysis, which showed that

the population as a whole did not follow a normal distribution, with the low

ethanol-drinking rats skewed to the left of the distribution.

128

0

0.5

1

1.5

2

0 1 2 3 4 5 6 7

ETH

AN

OL

INTA

KE

(g/k

g)

TRAINING DAY

high ethanol grouplow ethanol group

**

FIGURE 4.1. Ethanol intake levels (g/kg) for the high and low ethanol groups during the operant training procedure. Intakes differed significantly across the training period between the groups, most noticeably on days 5-6 when the ethanol concentration in the drinking solutions increased to 10%. Asterisks denote significance compared with the low ethanol group by ANOVA.

129

Dopamine concentrations and operant activity prior to consumption

Mean dialysate dopamine concentrations during the home-cage baseline period

were 1.5 ± 0.1 nM for the high ethanol plus sucrose group, 1.7 ± 0.6 nM for the

low ethanol plus sucrose group, 1.9 ± 0.4 nM for the sucrose group, and 1.4 ±

0.1 nM for the handling group. Home-cage baseline dopamine concentrations

were not significantly different among the groups [group: F(3,29) = 0.04, p > 0.05;

group by time interaction: F(15,145) = 1.05, p > 0.05]. Examination of the home

cage baseline, transfer, and wait periods also showed that none of the groups

differed significantly from one another across this time frame [group: F(3,25) =

0.03, p > 0.05; group by time interaction: F(12,112) = 0.77, p > 0.05]. An effect of

time, however, was observed across all groups [F(4,112) = 12.68, p < 0.05],

including the handling group, which was exposed to the operant chamber but did

not self-administer a solution. Post hoc contrasts indicated that dopamine

increased significantly during the period in which the rats were transferred from

the homecage [F(1,32) = 54.68, p < 0.05; data not shown] into the operant

chamber and during each of the samples that followed (i.e., the wait period). In

the subsequent analysis of the drink and post-drink periods we used the

dopamine samples from the transfer and wait periods as a baseline rather than

the home-cage samples. All groups showed stable and similar dopamine

responses during the transfer and wait periods [time: F(3,83) = 1.85, p > 0.05;

group by time: F(9,83) = 0.79, p > 0.05; Figure 4.2A-D]. Mean dialysate

dopamine concentrations during these periods were 1.9 ± 0.2 nM for the high

130

TABLE 4.1. Lickometer parameters for rats self-administering ≥ 0.8 g/kg ethanol (High ethanol), ≤ 0.5 g/kg ethanol (Low ethanol), and sucrose during dialysis

Parameter High ethanol n=9

Sucrosen=8

Low ethanolan=6

lever-pressing time (min) 0.8 ± 0.4 0.6 ± 0.3 3.3 ± 2.7 latency to begin drinking (min) 0.07 ± 0.02 0.27 ± 0.19 0.47 ± 0.23c

number of bouts 1.7 ± 0.2 1.9 ± 0.2 1.3 ± 0.2 initial bout duration (min) 6.6 ± 1.2 8.3 ± 0.8 2.0 ± 0.6b

total licks 1855 ± 275 2359 ± 299 349 ± 120b

licks during initial bout 1692 ± 293 2202 ± 297 298 ± 126b

initial bout response rate (licks/min) 266 ± 20 256 ± 27 184 ± 55

response rate for ½ of initial bout (licks/min) 343 ± 15 328 ± 22 220 ± 63

Bout refers to a period of at least 25 licks, with no more than 2 min between licks. Values shown as mean ± sem. a – significantly different from the High ethanol and Sucrose group by multivariate ANOVA (p < 0.05); b – significantly different from the High ethanol and Sucrose group by univariate ANOVA (p < 0.05); c – significantly different from the High ethanol group by univariate ANOVA (p < 0.05).

131

ethanol plus sucrose group, 2.0 ± 0.5 nM for the low ethanol plus sucrose group,

2.2 ± 0.4 nM for the sucrose group, and 1.7 ± 0.2 nM for the handling group.

ANOVA indicated that the high ethanol, low ethanol, and sucrose groups did not

differ significantly in terms of the duration of the operant response during the

dialysis experiment. The time required to complete the response requirement

was 0.8 ± 0.4 min for the high ethanol group, 0.6 ± 0.3 min for the sucrose group,

and 3.3 ± 2.7 min for the low ethanol group [F(2,23) = 1.42, p > 0.05; Table 4.2].

The variability seen in the low ethanol group was due to a single rat that stalled

during the lever-pressing period (15.3 min) but eventually finished the response

requirement.

Drinking behavior and dopamine concentrations during consumption

The low ethanol group (n=6), consumed 0.32 ± 0.06 g/kg, ranging from 0.2-0.5

g/kg. In contrast, the high ethanol group (n=10) drank 1.6 ± 0.2 g/kg with intakes

ranging from 0.8-2.8 g/kg. The sucrose and high ethanol groups ingested similar

amounts of fluid within the 20 min drink period (sucrose group: 11.4 ± 1.6 ml;

high ethanol group: 8.3 ± 1.1 ml). The low ethanol group ingested 1.7 ± 0.3 ml,

which was significantly lower than that consumed by the sucrose and high

ethanol groups [F(1,25) = 27.77, p < 0.05].

Upon completion of the response requirement, mean dopamine levels increased

132

70

80

90

100

110

120

130

0 10 20 30 40 50 6

SUCROSE

TIME (minutes)

B

0

drink post-drinkwait

leverpress

70

80

90

100

110

120

130

0 10 20 30 40 50 60DIA

LYSA

TE D

OPA

MIN

E (%

of b

asal

)

TIME (minutes)

HIGH ETOH

leverpress

drink post-drinkwait

A *

70

80

90

100

110

120

130

0 10 20 30 40 50 60

LOW ETOH

DIA

LYSA

TE D

OPA

MIN

E (%

of b

asal

)

TIME (minutes)

C

drink post-drinkwait

leverpress

70

80

90

100

110

120

130

0 10 20 30 40 50 6

HANDLING D

TIME (minutes)

wait in operant chamber

0

FIGURE 4.2. Effect of operant self-administration of 10% ethanol plus 10% sucrose and 10% sucrose (panel B) on extracellular accumbal dopamine levels. The rats in the ethanol plus sucrose group were subdivided into high (≥ 0.8 g/kg; panel A) and low (≤ 0.5 g/kg; panel C) drinking groups based on intake. A significant increase in mean dopamine levels occurred briefly during the first 5 min of the drink period for the high ethanol group, but not at any point for the sucrose or low ethanol group. Wait refers to the time in which the rat was in the operant chamber prior to access of the drinking solution. Lever press (open arrow) is the time at which lever pressing occurred. Drink refers to the 20-min free-access drinking period in the absence of lever pressing. Post-drink refers to dopamine while the rat was in the operant chamber in the absence of the drinking solution. Each point represents the mean ± sem (n=10 for the high ethanol group, n=10 for the sucrose group; n=6 for the low ethanol group). Panel D is the effect of the operant environment on extracellular accumbal dopamine in rats not trained for operant self-administration (n=7). Asterisk denotes significance compared with the wait period by post hoc simple contrasts (p < 0.05). 133

(20 ± 6% above baseline) within 5 min of access to 10% ethanol plus 10%

sucrose for the high ethanol group (Figure 4.2A). This elevated state was b

and values returned to baseline during the subsequent seven samples. Eight of

ten rats in the high ethanol group showed peak dopamine responses within the

first 10 min (two samples) of the 20 min drink period. In contrast, dopamine

concentrations remained at baseline levels during this period for the sucrose

low ethanol groups (Figure 4.2B-C). ANOVA showed a significant overall group

by time interaction [F(8,90) = 2.70, p < 0.05] during the drink period between the

high ethanol, low ethanol, and sucrose groups. Further analysis revealed that

the groups differed during the first dopamine sample of the drink period. At this

point a group by time interaction existed between the high ethanol group and (1)

the sucrose group [F(1,18) = 5.04, p < 0.05] and (2) the low ethanol group

[F(1,14) = 7.90, p < 0.05], but not between the sucrose and low ethanol gro

[F(1,14) = 1.01, p > 0.05]. Within-group post hoc contrasts indicated a significant

increase in dopamine levels during the first drink sample compared with baseline

for the high ethanol group [F(1,9) = 12.13, p < 0.05; Figure 4.2A]. In contrast, the

sucrose and the low ethanol groups did not show a significant dopamine

response at any point during the drink period. Furthermore, because of th

apparent trend towards a decrease in dopamine levels following low ethanol

consumption, we also analyzed the dopamine response between the low etha

and handling groups. However, there was no significant difference between

these groups [group x time: F(8,88) = 1.70, p > 0.05].

rief,

and

ups

e

nol

134

Licking behavior during consumption of ethanol and sucrose

igh ethanol,

ose

cks

iod.

ups were

ccumbal ethanol concentrations during consumption

ethanol

Multivariate analysis revealed an overall group effect between the h

low ethanol, and sucrose groups during consumption with respect to several

parameters [F(16,26) = 2.15, p < 0.05; Table 4.2]. The high ethanol and sucr

groups were not statistically different [F(4,12) = 0.64, p > 0.05], whereas the low

ethanol group differed from both the high ethanol [F(4,10) = 7.96, p < 0.05] and

sucrose groups [F(4,9) = 10.57, p < 0.05]. Univariate ANOVA showed that high

ethanol and sucrose groups differed from the low ethanol group in three

parameters (Table 4.2): (1) duration of the first bout, (2) total number of li

during the first drinking bout, and (3) total number of licks during the drink per

This analysis also showed that the latency to begin drinking after completion of

the response requirement was only different between the high ethanol and low

ethanol groups (Table 4.2). Consumption (licking) in all groups began almost

immediately after completion of the operant response (i.e., latency to begin

drinking), with 89 ± 4% of spout licks occurring during the first bout.

Consumption during the first bout for the sucrose and low ethanol gro

comparable (93 ± 2% and 83 ± 12%, respectively). Figure 4.3 (inset) shows the

average number of licks within each 5 min epoch of the drink period for the high

ethanol group.

A

In addition to dopamine in dialysates, we also quantified the

135

0

0.4

0.8

1.2

1.6

2

2.4

2.8

3.2

80

90

100

110

120

0 10 20 30 40

ethanol dopamine D

IALY

SA

TE E

THA

NO

L (m

M)

DIA

LYS

ATE

DO

PA

MIN

E

(% o

f bas

al)

TIME (minutes)

HIGH ETOH LICKS

1600

FIGURE 4.3. Mean dialysate dopamine and ethanol levels from the nucleus accumbens during drinking and post-drinking periods for the high ethanol group.

e

Dopamine data are from the same rats shown in Figure 4.2A. Ethanol was analyzed in the same samples from which the dopamine analysis was done. Inset shows periods of ethanol ingestion during the drink period for the high ethanol group. Although there was no direct relationship between the dialysatdopamine and ethanol time courses during any phase of the experiment, thepeak dopamine response coincided with the period in which most ethanol intake occurred. Each point is the mean ± sem (n=10).

136

concentration in each sample collected after completion of the response

requirement for the ethanol plus sucrose groups. Ethanol appeared in dialysates

within 5 min of ethanol availability in all rats. For the high ethanol group, mean

dialysate ethanol concentrations increased progressively (Figure 4.3), reaching

peak concentration (2.8 ± 0.5 mM) approximately 40 min after drinking began

before declining. The low ethanol group showed low mean dialysate ethanol

levels (data not shown), which reflected the amount of ethanol this group

consumed (1.7 ± 0.3 ml). The peak ethanol concentration for the low ethanol

group was 0.4 ± 0.3 mM. Examination of the time course data indicates that

ethanol levels remained very close to this value throughout the drink and post-

drink periods. Overall, individual ethanol time courses varied substantially

between the animals, including parameters such as peak ethanol concentration

and clearance from the dialysates. Pooling the data from the high and low

ethanol groups, regression analysis indicated that a significant, positive

correlation existed between (1) intake (g/kg) and the area under curve [F(1,13) =

27.23, p < 0.05; Figure 4.4A] and (2) intake and peak dialysate ethanol

concentration [F(1,13) = 33.77, p < 0.05].

Dose-effect relationships between ethanol intake and dopamine response

Regression analysis showed significant, positive correlations between intake and

ethanol-induced dopamine activity during the drink period for the high and low

ethanol groups combined. For example, ethanol intake (g/kg) correlated

137

-10

0

10

20

30

40

50

60

70

0 0.5 1 1.5 2 2.5 3

DIA

LYS

ATE

ETH

AN

OL

(AU

C)

ETHANOL INTAKE (g/kg)

A

60

80

100

120

140

160

180

0 500 1500 2500 3500

drink DApost-drink DA

DO

PAM

INE

RE

SP

ON

SE

(%

of b

asal

)

INITIAL BOUT LICKS

C60

80

100

120

140

160

180

0 0.5 1 1.5 2 2.5 3

drink DApost-drink DA

DO

PAM

INE

RE

SP

ON

SE

(%

of b

asal

)

ETHANOL INTAKE (g/kg)

B

FIGURE 4.4. Dose-effect relationships between (1) ethanol intake (g/kg) and ethanol area under curve (panel A), (2) ethanol intake (g/kg) and peak dopamine response (DA) during the initial 10 minutes of the drink period and first 5 min of the post-drink period (panel B), and (3) consummatory behavior (licks during first drinking bout) and peak dopamine response (DA) during the first 10 min of the drink period and first 5 min of the post-drink period (panel C). Regression curves represent data pooled from the high and low ethanol-drinking groups (n=16). Vertical dashed lines distinguish the low ethanol from the high ethanol group (panel A and B). Bolded data points on panel C identify the high ethanol group, whereas the unbolded points are of the low ethanol group. Ethanol intake (g/kg) correlated positively with accumbal ethanol levels (area under curve; r = 0.83, p < 0.05) and peak dopamine response during the initial 10 min of the drink period (r = 0.69, p < 0.05), but not with the dopamine response during the first 5 min of the post-drink period (r = 0.33, p > 0.05). Similarly, initial bout licks correlated positively with peak dopamine levels during the initial 10 min of the drink period (r = 0.55, p < 0.05), but not during the post-drink period (r = 0.31, p > 0.05).

138

positively with dopamine levels during the first 5 min of the drink period [F(1,15) =

8.42, p < 0.05] and with the peak response within the first 10 min of the drink

period [F(1,15) = 11.36, p < 0.05; Figure 4.4B], when most animals showed their

maximal dopamine response. However, intake did not correlate with dopamine

levels during the post-drink period [F(1,15) = 1.73, p > 0.05; first 5 min sample],

in which the drinking spout was absent and consumption could not occur. Since

the majority of ethanol consumption occurred during the first drinking bout (i.e.,

85 ± 5% of all licks occurred within 6.6 ± 1.2 min), we also analyzed initial bout

licks with the dopamine response at various time points. Figure 4.4C shows that

initial bout licks correlated positively with dopamine levels during the first 5 min

[F(1,15) = 5.06, p < 0.05] and the peak response during the first 10 min [F(1,15)

= 5.97, p < 0.05] of the drink period, but not during the post-drink period [F(1,15)

= 1.48, p > 0.05; first 5 min sample]. In contrast, for the sucrose-drinking rats we

found no significant correlations between licking and dopamine levels (e.g., initial

bout licks versus peak dopamine response during the first 10 min of the drink

period [F(1,8) = 0.07, p > 0.05]). Lastly, there was no significant relationship

between peak ethanol and peak dopamine levels in dialysates [F(1,13) = 2.01, p

> 0.05].

Dopamine concentrations during unexpected sucrose self-administration

We next examined the potential effect of expectation of ethanol reinforcement on

the transient dopamine response observed in the high ethanol group. For this

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experiment, we trained a group of rats in exactly the same manner as the high

and low ethanol groups. However, on the experiment day, these animals self-

administered a solution of 10% sucrose, which did not contain ethanol. As with

the other treatment groups, at least 50% of the active dialysis membrane for

each probe was located within the nucleus accumbens, with the probe positions

distributed randomly within the core and shell subregions. The dopamine

samples showed excellent calcium dependence (81 ± 4%). Daily ethanol intake

levels across the training procedure mirrored those of the high ethanol group.

For example, these rats ingested 1.4 ± 0.3 g/kg ethanol on the day prior to

dialysis. The unexpected sucrose group also displayed comparable mean

dialysate dopamine concentrations during the baseline periods (home-cage: 1.9

± 0.4 nM; operant wait: 2.2 ± 0.4 nM) with respect to the other treatment groups.

Dopamine concentrations remained at baseline levels during consumption

(Figure 4.5). The high ethanol group differed significantly from the unexpected

sucrose group across the first drink sample [group x time: F(1,16) = 4.98, p <

0.05; n=8]. The unexpected sucrose group showed very similar lickometer-

parameter values compared with the sucrose and high ethanol groups (i.e., total

licks: 2209 ± 203; initial bout licks: 2182 ± 208; initial bout duration: 9.1 ± 1.0 min;

latency to begin drinking: 0.11 ± 0.07 min). Figure 4.5 (inset) shows the mean

number of licks within each 5 min epoch of the drink period. The lever press time

for this group (0.50 ± 0.11 min) was also comparable to those of the other

groups.

140

70

80

90

100

110

120

130

0 10 20 30 40 50 60

UNEXPECTED SUCROSEleverpress

drink post-drinkwait

DIA

LYSA

TE D

OPA

MIN

E (%

of b

asal

)

TIME (minutes)

800 UNEXP. SUC. LICKS

FIGURE 4.5. Effect of operant self-administration of 10% sucrose on extracellular accumbal dopamine levels in rats previously reinforced with 2-10% ethanol (plus 10% sucrose). In contrast to the high ethanol group, no significant changes in dopamine occurred during the consumption period. Each point represents the mean ± sem (n=8). The dialysis sampling periods shown here are identical to those in Figure 4.2. Inset shows the periods of sucrose ingestion during each 5 min epoch within the drink period.

141

DISCUSSION

This is the second study to examine the relationship between intra-accumbal

dopamine and ethanol concentrations using an operant procedure that

specifically distinguished ethanol consumption from lever-pressing behavior

(Doyon et al., 2003). This study extends previous findings (Weiss et al., 1993;

Melendez et al., 2002; Doyon et al., 2003) by demonstrating that accumbal

dopamine levels can clearly undergo transient elevations in response to ethanol

(plus sucrose) intake. The magnitude of the dopamine response was dependent

upon the amount of ethanol consumed, but not on the concentration of ethanol

reaching the accumbens. Examination of the time courses of individual rats

showed that the rise in dopamine levels occurred predominantly within 5-10 min

of ethanol access before declining to baseline. At this time, accumbal ethanol

concentrations were still in the rising phase of their time course, reaching peak

levels over 40 min later. Although quantitative microdialysis was not performed,

we estimated the mean tissue concentration of ethanol to be 13.8 ± 1.7 mM 10

min into the drink period and 21.7 ± 3.5 mM at its peak. These estimations were

based on dialysate concentrations and an in vivo extraction fraction for ethanol of

0.13 (Robinson et al., 2000) and are comparable to previous reports of brain

ethanol levels following oral self-administration (Nurmi et al., 1999).

Our previous results also demonstrated a transient dopamine response to

limited-access ethanol consumption (Doyon et al., 2003), which was smaller in

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magnitude but with a remarkably similar time course to the one observed here.

We originally suggested that the discrepancy between the ethanol and dopamine

time courses could be due to (1) intakes (0.45 ± 0.04 g/kg) that were not

sufficiently high enough to produce a sustained ethanol-induced dopamine

response or (2) desensitization with a long-term reinforcement schedule, thereby

causing a blunted ethanol-induced dopamine response over time. The present

study indicates that ethanol intakes of 1.6 ± 0.2 g/kg (over three times higher

than the previous report) were not sufficient for stimulation of mean dopamine

activity beyond the first 5 min of consumption, suggesting that low levels of

ethanol intake do not adequately explain our previous data. Moreover, the brief

increase in extracellular dopamine concentration during drinking was similar to

that found by Doyon et al. (2003), suggesting that the dopaminergic response

does not desensitize with training.

The transience of the ethanol-induced dopamine response observed here is

inconsistent with prior studies of operant ethanol self-administration (Weiss et al.,

1993; Gonzales and Weiss, 1998; Melendez et al., 2002), in which dopamine

levels remained elevated above basal throughout a limited-access drinking

period. This discrepancy cannot be attributed to differences in consumption,

since mean intakes (g/kg) were comparable to, if not greater than, those reported

previously. It is possible that intrinsic neurochemical differences between the rat

strains used in these studies could account for some of these data. The present

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study employed male Long-Evans rats, whereas others have used alcohol-

preferring male (Weiss et al., 1993) and female P rats (Melendez et al., 2002)

and male Wistar rats (Weiss et al., 1993; Gonzales and Weiss, 1998). Alcohol-

preferring rat lines display lower accumbal dopamine levels (Murphy et al., 1987;

Gongwer et al., 1989; Katner and Weiss, 2001) and exhibit greater dopaminergic

responsiveness to oral ethanol (Weiss et al., 1993; Katner and Weiss, 2001)

compared with non-preferring lines. Although critical neurochemical differences

could exist between Wistar and Long-Evans rats, it does not seem likely

considering that both lines are genetically heterogeneous and are closely related

in background. A notable aspect of this study that distinguishes it from others is

the operational distinction between consumption and appetitive responding,

which resulted in a clearly defined dopamine response during the initial phases of

ethanol self-administration, providing a possible explanation for the

inconsistencies between these studies. However, the motivational and

neurochemical consequences of dispensing reinforcement in “lump sum,”

compared with small amounts that are contingent upon further behavior (Weiss

et al., 1993; Gonzales and Weiss, 1998; Melendez et al., 2002), are not

understood. Further work is necessary to determine whether differences exist

between these types of response-outcome procedures and which approach best

models human alcohol drinking.

The present study strongly supports the suggestion by Doyon et al. (2003) that

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the ethanol-induced dopamine response produced during operant self-

administration may not be solely pharmacological in nature, as others have put

forward (Weiss et al., 1993; Gonzales and Weiss, 1998), but may instead be

related to the stimulus properties of ethanol presentation. The occurrence of

prominent elevations in accumbal ethanol concentrations well after the dopamine

response had subsided indicates that any pharmacological effect of ethanol was

transient at best. Regression analysis showed significant correlations between

intake and dopamine response within the first 10 min of the drink period but not

15 min later during the post-drink period. The fact that dialysate ethanol levels

were not related to the peak dopamine response further suggests that the

dopaminergic activity was not entirely due to the pharmacological actions of

ethanol. Importantly, the absence of a dopamine response in ethanol-

conditioned rats self-administering 10% sucrose indicates that the observed

dopamine response is dependent on the presence of ethanol, and is not merely

an artifact related to operant responding or the expectation of ethanol

reinforcement, for example. These results are consistent with a previous report

by Katner et al. (1996), which showed no effect of ethanol expectation in

heterogeneous Wistar rats. On the contrary, our hypothesis concerning a cue-

induced dopamine increase is not fully supported by recent data from rats

performing a second-order schedule of reinforcement for cocaine, in which a

conditioned stimulus preceding cocaine presentation failed to evoke an increase

in accumbal dopamine activity (Ito et al., 2000). However, several

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methodological differences could contribute to this apparent discrepancy. Ito et

al. (2000) utilized a delay (up to 20 min) between the conditioned stimulus and

the onset of reinforcement, whereas in the present study the stimulus cues of

ethanol (i.e., taste and odor) coincided with the acquisition of the reinforcer.

Therefore, the temporal contiguity between a conditioned stimulus and the onset

of reinforcement may be an important factor for predicting cue-related dopamine

responses measured with microdialysis. Alternatively, the stimulus strength of

the visual stimulus used in the Ito et al. (2000) study may not be as strong as the

sensory stimuli in the present study for eliciting an accumbal dopamine response.

The results of the current study indicate that some mechanism must be

functioning to stimulate extracellular dopamine activity transiently during ethanol

consumption. We propose that this mechanism could involve (1) an increase in

the firing rate of VTA dopamine cells due to sensory-mediated excitatory drive or

(2) a very rapid acute functional tolerance to ethanol within the mesoaccumbens

system. According to Grace (2000), the extracellular dopamine response to

ethanol could be mediated by phasic increases in the firing rate of VTA dopamine

cells. Burst-mediated release of dopamine is significantly higher in conscious

animals compared with anesthetized ones (Freeman and Bunney, 1987) and a

variety of salient environmental stimuli evoke burst activity (Overton and Clark,

1997; Horvitz, 2000), indicating that these events are linked to sensory

stimulation. Excitatory glutamatergic activity within the VTA, possibly conveyed

146

by sensory input, is one source of this phasic dopaminergic activtiy (Murase et

al., 1993; Zheng and Johnson, 2002; Floresco et al., 2003). In the present study,

ethanol drinking occurred predominantly within the first 10 min of access,

corresponding to the period in which all but two rats showed peak dopamine

responses. During this period, the stimulus properties of the ethanol solution

(i.e., taste, smell) were maximal. Rats consuming sucrose for a comparable

amount of time did not show such an enhancement of dopamine, suggesting that

this effect was specific to ethanol and not common to all reinforcing stimuli.

Therefore, the mismatch between the dopamine and ethanol time courses

observed here could be due to transient sensory-mediated stimulation of the

dopamine system that occurred with ethanol ingestion. This hypothesis is

supported by the positive correlation between ethanol licks and the dopamine

response during the initial drinking bout and by the absence of a dopamine

response in sucrose-drinking rats previously conditioned for ethanol self-

administration. The operational segregation of the consummatory phase of

operant ethanol self-administration may have revealed or enhanced this effect by

providing the ethanol stimulus in a bolus-like manner.

Alternatively, the brief dopamine response may be partially due to an extremely

rapid tolerance to the acute effects of ethanol within the mesoaccumbens

system. Therefore, ethanol concentrations reaching the brain during the

ascending phase of the ethanol time course could exert greater regulation over

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extracellular dopamine accumulation than could peak ethanol concentrations or

falling phase concentrations. Functional tolerance has been widely

demonstrated in both human (Hiltunen, 1997a,b) and animal studies (Waller et

al., 1983; Lewis and June, 1990; Le and Kalant, 1992; Erwin and Deitrich, 1996;

Ponomarev and Crabbe, 2002). Doses of ethanol that are self-administered by

rats can stimulate locomotor activity during the ascending phase of blood-ethanol

concentrations, an effect that is absent during the descending phase (Lewis and

June, 1990). There is a lack of information, however, regarding neurochemical

tolerance to acute administration of ethanol. Previous work demonstrates that

the dopamine response to systemically administered ethanol does not

desensitize or undergo tolerance in animals given the drug chronically (Rossetti

et al., 1993). Yim et al. (2000) showed that intra-accumbal ethanol

concentrations dissociate from the dopamine response about 45 min after i.p.

injection. The pharmacokinetics of acute ethanol administration, however, are

clearly different from those produced by the oral route. Therefore, a direct

comparison of these routes of administration with the dissociation between their

respective dopamine and ethanol time courses is not fully valid. Taken together,

if acute functional tolerance contributed to the present results, this would be an

extremely rapid instance of the phenomenon within the mesoaccumbens system.

All experimental groups displayed significant elevations in extracellular dopamine

during the period in which they were transferred into the operant chamber from

the home cage and during the 15-min wait period that preceded drinking. This

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pre-drinking increase in dopamine across the groups, however, does not appear

to be related to expectation of ethanol or sucrose reinforcement per se, since the

handling group that was not trained for operant reinforcement showed a similar

response during the same periods. Our previous data demonstrating a non-

specific effect of handling on dopamine activity corroborate these results (Doyon

et al., 2003). We concluded that this phenomenon was due to the physical

handling of the rats as they are placed into the operant chamber, a change of

environment, or a combination of these factors. These conclusions are

supported by studies showing that tactile stimulation can evoke increases in

extracellular dopamine in a variety of terminal areas (Inglis and Moghaddam,

1999; Adams and Moghaddam, 2000). Our observations, however, are not in

agreement with certain studies in which a rise in dopamine levels did not occur

during the transfer of rats from one environment to another (Weiss et al., 1993;

Damsma et al., 1992; Humby et al., 1996). The reason for these inconsistencies

is unclear, but could be due to procedural differences between these studies.

For perspective, we should also note that a role for dopamine in ethanol

reinforcement is not as widely accepted as dopamine’s role in psychostimulant

reinforcement, for example. Although there is a large and diverse body of work

that supports a dopamine hypothesis of ethanol reinforcement (Gonzales et al.,

2004; Weiss and Porrino, 2002), there is also a substantial amount of negative

data. Microinjection studies consistently show that disruption of dopamine

149

transmission within the accumbens reduces responding for ethanol (Rassnick et

al., 1992; Samson et al., 1993; Hodge et al., 1997; Czachowski et al., 2001). In

terms of ethanol consumption, however, most studies show that blockade of

dopamine receptors has little or no effect on ethanol intake (Samson et al., 1993;

Silvestre et al., 1996; Czachowski et al., 2001). Furthermore, ablation of

accumbal neurons does not disrupt ethanol consumption (Ikemoto et al., 1997) or

operant responding for ethanol (Rassnick et al., 1993) in rats previously

conditioned to ethanol. In addition, microdialysis studies do not show that

ethanol strongly stimulates extracellular dopamine to the degree that

psychostimulants do (Mocsary and Bradberry, 1996; Nurmi et al., 1996;

Bradberry, 2002; Doyon et al., 2003). These studies, along with the results of the

present one, indicate that the functional significance of accumbal dopamine

activity in ethanol reinforcement remains complex and further work in this area is

certainly needed.

In summary, our study clearly demonstrates the occurrence of a rapid

dissociation between accumbal dopamine and ethanol time courses during

consummatory periods of ethanol self-administration. Although the dopamine

response observed during ethanol drinking correlated with the amount of ethanol

consumed (g/kg or licks of ethanol), a pharmacological relationship between

ethanol and dopamine is not fully supported here due to the transient nature of

the effect. These results may be due to the stimulus-mediated properties of

150

ethanol, which may evoke phasic increases in dopamine activity during

consummatory phases of self-administration. We do not discount the possibility,

however, that other factors contributed, such as a very rapid tolerance to the

acute effects of ethanol within the mesoaccumbens system.

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5.0 The Effect of κ-Opioid Blockade on Accumbal Dopamine

Concentrations during Operant Ethanol Self-Administration

ABSTRACT

Our study sought to determine the effect of κ-opioid receptor blockade on ethanol

consumption and accumbal dopamine concentrations during operant ethanol

self-administration. Long-Evans rats were trained to self-administer 10% ethanol

(with 10% sucrose) over 7 days, during which 2-4 responses resulted in 20 min

of ethanol access with no further response requirements. Rats were treated with

the long-lasting κ-opioid receptor antagonist nor-binaltorphimine (NorBNI; 0 or 20

mg/kg) 15-20 hrs prior to testing, and accumbal microdialysis was performed in

rats self-administering 10% ethanol (plus 10% sucrose). NorBNI did not alter

operant responding or ethanol intake. The control group displayed a transient

elevation in dopamine concentration within 5 min of ethanol access. NorBNI-

treated rats did not exhibit this response, but showed a latent increase in

dopamine concentration at the end of the access period. The rise in dopamine

levels correlated positively with accumbal ethanol concentration for the NorBNI

group but not in controls. The transient dopamine activity during ethanol

acquisition in controls is consistent with a sensory-mediated dopamine response,

but the reason for its absence in the NorBNI group is unclear. These data

suggest that κ-opioid receptor blockade by NorBNI temporarily uncovered a

pharmacological stimulation of dopamine release by ethanol.

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INTRODUCTION

Converging evidence suggests that interactions between mesolimbic dopamine

and opioid systems contribute to the development of ethanol reinforcement and

dependence (Herz, 1997; Cowen and Lawrence, 1999). Endogenous opioid

systems appear to act in opposition to modulate mesolimbic dopamine activity

and motivational processes. For example, activation of µ-opioid receptors in the

ventral tegmentum can stimulate basal dopamine release in the nucleus

accumbens (Leone et al., 1991; Spanagel et al., 1992; Devine et al., 1993) and

lead to conditioned place preference (Bals-Kubik, 1993; Nader and van der Kooy,

1997). In contrast, activation of κ-opioid receptors within the mesolimbic circuitry

can result in decreased dopamine neuron firing (Margolis et al., 2003) and an

inhibition of accumbal dopamine release (Heijna et al., 1990; Spanagel et al.,

1992; Xi et al., 1998). Additionally, the administration of κ-opioid selective

agonists can cause conditioned place aversion (Mucha and Herz, 1985; Bals-

Kubik, 1993) and a suppression of drug self-administration (Lindholm et al.,

2001; Mello and Negus, 1998; Xi et al., 1998).

Although the functional significance of mesolimbic dopamine activity during

ethanol reinforcement remains a subject of debate, there is evidence that

dopamine transmission is involved in certain aspects of ethanol self-

administration. Local blockade of dopamine transmission within the accumbens

curtails operant responding for ethanol (Rassnick et al., 1992; Samson et al.,

153

1993; Hodge et al., 1997) but not necessarily intake (Czachowski et al., 2001).

Disruption of dopamine signaling can alter consumption in ethanol naïve animals

but not those with prior drinking experience (Ikemoto et al., 1997), suggesting

that a functional dopamine system facilitates the learning of ethanol reward.

Furthermore, operant procedures, in which responding is segregated from

ethanol drinking, demonstrate that accumbal dopamine levels increase briefly

upon acquisition of the ethanol reinforcement but not thereafter (Doyon et al.,

2003; Doyon et al., 2005), which points to an incentive salience role for

dopamine. One unresolved question is to what extent do specific opioid receptor

subtypes regulate mesolimbic dopamine activity during operant ethanol self-

administration.

Endogenous opioids can also modulate ethanol-reinforced behavior (Gonzales

and Weiss, 1998; Roberts et al., 2000; Hyytia and Kiianmaa, 2001). However,

the involvement of the κ-opioid system is not clear. Blockade of kappa receptors

with low doses (3-5 mg/kg) of nor-binaltorphimine (NorBNI) does not alter ethanol

consumption during reinforcement (Williams and Woods, 1998; Holter et al.,

2000). Support for kappa-ethanol interactions comes from studies demonstrating

that tissue levels of dynorphin (the endogenous κ-opioid receptor ligand) in the

accumbens increase within 30 min of ethanol administration (Lindholm et al.,

2000; Marinelli et al., 2005) and κ-opioid receptor mRNA in the accumbens and

ventral tegmentum is down-regulated after repeated ethanol exposure (Rosin et

154

al., 1999). The latter finding is suggestive of a compensatory response to

enhanced dynorphin stimulation of κ receptors.

The aim of the present study was to determine the effect of systemic κ-opioid

receptor blockade on ethanol consumption and accumbal dopamine

concentrations using an operant self-administration procedure that specifically

distinguished lever responding from ethanol ingestion. Our working hypothesis

was that ethanol self-administration induces the release of endogenous

dynorphin peptides, which act on the κ-opioid receptor that functions to inhibit

ethanol consumption and dopamine activity. Microdialysis was performed during

self-administration of 10% ethanol (plus 10% sucrose) in rats pretreated with

saline or the long-lasting κ-opioid receptor antagonist, nor-binaltorphimine

(Endoh et al., 1992; 20 mg/kg). We quantified intra-accumbal dopamine and

ethanol concentrations following ethanol access to assess the relationship

between ethanol levels and the accompanying dopamine response. Ethanol

intake and patterns of ingestion for both treatment groups were also quantified

for comparison with the neurochemical data.

MATERIALS AND METHODS

Subjects

Our study utilized 30 male Long-Evans rats (Charles River Laboratories Inc.,

155

Wilmington, MA, USA) that weighed between 295-534 g at the time of testing.

Rats were handled and weighed for at least 4 days upon arrival prior to surgery

and training. Each rat lived individually in a humidity and temperature-controlled

(22°C) environment under a 12-hr light/dark cycle (on at 7:00 A.M.; off at 7:00

P.M.). Each rat had food and water available ad libitum in the home cage except

during the procedure indicated below. All procedures complied with guidelines

specified by the Institutional Animal Care and Use Committee of the University of

Texas at Austin and the National Institutes of Health Guide for the Care and Use

of Laboratory Animals.

Surgery

Prior to operant training or testing, we surgically prepared the rats for

microdialysis by inserting a stainless steel guide cannula (21 gauge; Plastics One

Inc., Roanoke, VA, USA) above the left nucleus accumbens. The surgery

occurred while the rats were under isoflurane anesthesia (1.5-2.5% in 95%/5%

O2/CO2, 1-2 L/min), using standard stereotaxic equipment. The following

coordinates were used (in mm relative to bregma): +1.7 anterior-posterior, +1.0

medial-lateral, -4.0 ventral to the skull surface (Paxinos and Watson, 1998). The

guide cannula was cemented to the skull by embedding three stainless steel

screws into the skull and covering the entire unit, around the base of the cannula,

with dental cement (Plastics One Inc). We also placed a single steel bolt

vertically into the hardening cement as an anchor for the microdialysis tether. An

156

obturator was placed inside the guide cannula to prevent blockage prior to the

microdialysis session. After at least 4 days of recovery, the rats began the

training procedure.

Microdialysis

The microdialysis probes were constructed according to the methods described

by Pettit and Justice (1991). Briefly, fused-silica tubing (inner diameter = 40 µm;

Polymicro Technologies, Phoenix, AZ, USA) formed the inlets and outlets of the

probes, and hollow cellulose fiber (inner diameter = 200 µm; molecular weight

cutoff = 18,000; Spectrum Laboratories Inc., Rancho Dominguez, CA, USA)

formed the dialysis membrane. The active dialysis membrane spanned 2.2 mm

(the distance between the end of the inlet and the epoxy that sealed the

membrane).

Habituation to the microdialysis tethering apparatus occurred within the week

preceding testing. This procedure consisted of tethering the rats overnight in the

operant testing room, with continued tethering throughout the subsequent day of

self-administration training. Rats were sedated with 2% isoflurane in air for a few

minutes to attach the tether. On the day preceding the dialysis session, we

perfused (flow rate = 2 µl/min) the microdialysis probes with artificial cerebral

spinal fluid (149 mM NaCl, 2.8 mM KCl, 1.2 mM CaCl2, 1.2 mM MgCl2, and 0.25

mM ascorbic acid, 5.4 mM D-glucose), and slowly inserted the probes into the

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brain through the guide cannula while the rat was briefly anesthetized (15-20

min) with 2% isoflurane in air. This procedure occurred at least 14 hours before

the start of the experiment. We used a syringe pump (CMA102; CMA, Solna,

Sweden) to pump the perfusate through a fused-silica transfer line and into a

single channel swivel (Instech Solomon, Plymouth Meeting, PA, USA), which

hung from a counterbalanced lever arm (Instech Solomon). The swivel formed a

connection with the inlet of the probe and a spring tether secured the animal to

the swivel. The perfusion flow rate was set at 2.0 µl/min during the probe

implantation, after which it was decreased to 0.2 µl/min overnight. The flow rate

was returned to 2.0 µl/min 2 hours prior to baseline sampling. We manually

changed each of the sample vials, which were immediately frozen on dry ice

(excluding the fraction of dialysate that was removed for ethanol analysis, see

Ethanol analysis) and then stored at -80˚C until analyzed.

NorBNI blockade of U50488H

Rats were injected with 20 mg/kg (Endoh et al., 1992; Sofuoglu et al., 1992) nor-

binaltorphimine dihydrochloride (National Institute on Drug Abuse) in 1.5 ml

saline (subcutaneously, s.c.) 15-20 hrs prior to the experiment. The microdialysis

probes were inserted into the nucleus accumbens, and the protocol for the

preparation of the microdialysis experiments was identical to that described in the

Microdialysis section, excluding the tethering procedure. On the subsequent

testing day, 3 baseline samples (10 min each) were taken using standard ACSF

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as the perfusate. After removal of the third baseline sample, we switched the

perfusate to one containing the κ-opioid receptor agonist (-)-trans-(1S,2S)-U-

50488 hydrodrochloride (Clark and Pasternak, 1988; 0.4 or 1.6 µM; Sigma-

Aldrich Inc.). Six 10-min samples were taken following the switch to U50488H.

The flow rate was set at 2.0 µl/min for the entire experiment. Control rats

received an injection of saline (1.5 ml, s.c.) 15-20 prior to the experiment. Each

group contained 3-4 subjects.

Behavioral apparatus

Standard operant chambers (Med Associates Inc., St. Albans, VT, USA) modified

for microdialysis perfusion were used for self-administration training and

microdialysis testing. One wall of each chamber contained a retractable lever on

the left side (2 cm above grid floor), which upon activation triggered the entry of a

retractable drinking spout on the right side (5 cm above grid floor). Each lever

press activated a chamber light above the lever. The floor consisted of a grid of

metal bars in connection with the spout of the drinking bottle, which upon contact

formed a circuit to record licks (Med Associates Inc.). A cubicle with the front

doors left open during training and testing housed each operant chamber. PC

software provided by Med Associates controlled operant chamber components

and acquisition of lickometer data. Activation of an interior chamber light and

a sound-attenuating fan accompanied the start of each operant session.

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Self-administration training

Operant sessions occurred once a day for 5 days per week. The subjects were

trained initially to lever press for access to 10% sucrose (w/v). Animals were

water deprived (10-22 hours) prior to each session (approximately 45 min) to

facilitate acquisition of the operant response. A reliable lever-pressing response

for sucrose occurred in approximately 2-5 days. Rats were not water restricted

at any time during the subsequent training periods.

After reliable operant behavior was established, subjects were trained for self-

administration of 10% ethanol with 10% sucrose using a modified version (Doyon

et al., 2005) of the sucrose fading procedure (Samson, 1986), in which we

increased the concentration of ethanol (v/v) in the drinking solution across daily

sessions (2-10% over six days), but did not subsequently remove the sucrose.

During this period, we gradually habituated the rats to (1) a 15 min “wait time”,

which preceded access to the lever and drinking solution, and (2) a response

requirement (RR) that increased from 2 to 4 across seven sessions (Table 5.0).

Upon completion of the RR, the lever retracted and the drinking solution became

available for 20 min, followed by a 20-min post-drinking period in the absence of

the drinking solution. For the dialysis experiment, the response requirement was

set at 4 and 10% ethanol (plus 10% sucrose) served as the reinforcer.

Consumption was monitored during training and during the microdialysis session

by a lickometer and by measuring the volume of liquid in the drinking bottle

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TABLE 5.0. Summary of operant training protocol for ethanol (plus sucrose) self-administration

Day Drink solution^ Pre-drink wait (min) Response requirement

1 10S 2 2 2 10S 2E 4 2 3 10S 2E 6 2 4 10S 5E 8 2 5 10S 5E 10 4 6 10S 10E 12 4 7* 10S 10E 15 4

The sucrose control group followed the same schedule except that ethanol was not faded into the drink solution. ^ S equals sucrose and E equals ethanol. Numeral for drink solution represents percentage (w/v for sucrose; v/v for ethanol). * - dialysis session.

161

before and after the session, taking care to account for spillage. Body weights

were measured each day.

Experimental design

Rats were injected with NorBNI (20 mg/kg in 1.5 ml saline, s.c.) or saline (1.5 ml,

s.c.) 15-20 hrs prior to the experiment. Dialysate samples were collected every 5

min except where indicated below. The microdialysis experiments consisted of

four general sampling epochs: (1) home cage baseline, (2) transfer/wait, (3)

drink, and (4) post-drink. Six samples were collected during a baseline period in

the home cage (30 min). One sample was collected during the period in which

we transferred the rat into the operant chamber prior to activation of the operant

program (5 min). Upon activation of the program, three samples were collected

prior to introduction of the drinking spout: two 5 min samples during a waiting

period and a third waiting sample (approximately 5.7 min), which included at its

end a brief lever-pressing period (0.7 ± 0.2 min). Completion of the response

requirement was followed by a 20 min drinking period with unrestricted access

(four samples) and then a post-drinking period within the chamber in the absence

of the solution (20 min, four samples). At the end of this time, the rat was moved

back into the home cage. We then collected an additional 6 samples (at 10-min

intervals) to monitor ethanol concentrations in the dialysates, but these sample

were not analyzed for their dopamine content. After obtaining all samples, the

perfusion solution was switched to one lacking calcium for 60-90 min. A 10-min

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sample was then taken to determine the calcium dependency of the dopamine in

dialysates, which is a measure of the functionality of the nerve terminals.

Histology

Within 5 days after each experiment, the rats were overdosed with pentobarbital

(300 mg/kg) and saline was perfused through the heart, followed by 10% formalin

(v/v). The brains were removed and immersed in 10% formalin/ 30% sucrose

(w/v) for at least 3 days. Brains were cut into coronal sections (48 µm thick) with

a cryostat (Bright Instrument Co., Cambridgeshire, England), and the sections

stained with cresyl violet. The slides were examined to confirm the placement of

the active dialysis membrane (2.2 mm). We determined correct placement within

the accumbens when at least 55% of the active dialysis membrane bisected the

core, shell, or core plus shell.

Dopamine analysis

Our HPLC systems were amperometric and based on reversed phase

chromatography using an ion-pairing agent with electrochemical detection.

These systems included one of two pumps [a LC-10AD (Shimadzu Scientific

Instruments Inc., Columbia, MD, USA), or a LC-10ADVP (Shimadzu Scientific

Instruments Inc.)], one of two autosamplers [FAMOS (LC Packings, Sunnyvale,

CA, USA) or ALEXYS (Antec Leyden BV, Zoeterwoude, Netherlands], a VT-03

cell (2 mm working electrode diameter, potential: 450 mV against a Ag/AgCl

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reference; Antec Leyden BV) in connection with an Intro controller (GBC

Separations Inc., Hubbardston, MA, USA) and a Polaris 2 x 50 mm column (C18,

3-µm particle size; Varian Inc., Palo Alto, CA, USA). For these systems, the

mobile phase consisted of octanesulfonic acid (2.0 mM), decanesulfonic acid (0.2

mM), methanol (15%, v/v), sodium dihydrogen phosphate dehydrate (71.0 mM),

and ethylenediaminetetraacetic acid (0.3 mM). The flow rates were set at 0.3

ml/min for both systems. For the NorBNI-U50488H experiments, 17 µl of the

dialysate was injected using the microliter pickup mode and the transfer fluid was

ascorbate oxidase (EC 1.10.3.3; 102.3 units/mg; Sigma-Aldrich Inc., St. Louis,

MO, USA). For the ethanol self-administration experiments, 7 µl of the dialysate

was injected. A computerized data acquisition system (EZ Chrome Elite;

Scientific Software Inc., Pleasanton, CA, USA) recorded the dopamine peaks.

Quantification was carried out by comparing dopamine peak areas from dialysate

samples to external standards.

Ethanol analysis

We analyzed ethanol concentrations in all dialysis samples collected after the

lever-press period for subjects in the ethanol self-administration experiments.

Before freezing the dialysis sample, 2 µl of fluid were transferred into a glass vial

and sealed with a septum for analysis of ethanol later that day. A gas

chromatograph (Varian CP 3800; Varian Inc.) with flame ionization detection

(220°C) measured the ethanol in the dialysates. Specific details concerning the

164

components of the gas chromatograph are described by Doyon et al. (2003).

Quantification of ethanol in dialysates was done by comparing peak areas

obtained with a Star chromatographic analysis system (Varian Inc.) to external

standards.

Statistical analysis

Dialysate dopamine and ethanol concentrations (nM) were analyzed using

analysis of variance (ANOVA) with repeated measures. For the

NorBNI/U50488H experiments, the average of the three basal samples served as

the baseline dopamine response to which the six U50488H samples were

compared. For the ethanol self-administration experiments, the six home cage

samples served as the baseline response to which the transfer and wait samples

were compared. The average of the wait period (3 samples), including the last

wait sample that encompassed the lever-press period, defined the baseline

response to which the drink and post-drink samples were compared. The lever-

press period was included as a basal sample because of its short duration. Any

potential dopamine activity resulting from this period would be collected in the

next sample due to the brief time lag inherent in microdialysis. ANOVA did not

indicate a difference between the lever period and the two previous wait samples

(p > 0.05). Technical problems associated with sample collection or HPLC

analysis resulted in the loss of some samples (5 of 405). To account for this we

estimated these values by averaging adjacent time points and then adjusting the

165

degrees of freedom in the ANOVA. Separate ANOVA tests were conducted to

test for group by time interactions during the main phases of the experiment (i.e.,

basal plus transfer/wait period; wait period plus drink and post-drink periods).

Post-hoc contrasts comparing individual time points to baseline within groups

were performed after determining a significant group by time interaction during

these periods. Bonferroni corrections were used in the case of post-hoc

contrasts. ANOVA was performed using the Manova routine in SPSS for

Windows, and post-hoc contrasts were performed using repeated measures in

the GLM procedure. Regression analyses were carried out in EXCEL.

Significance for all analyses was determined when p < 0.05. The area-under-

curve was calculated by summing the values for dopamine and ethanol

concentrations, respectively. Analysis of the consumption parameters was

carried out using multivariate ANOVA (GLM procedure) and F-values derived

from Wilks’ Lambda.

RESULTS

Histological analysis and calcium-dependence of dopamine concentrations

Each probe contained at least 55% of the active dialysis membrane within the

nucleus accumbens. Examination of the probe positions within subregions of the

accumbens showed that, overall, 48% were in the shell, 4% were in the core, and

48% bisected both the core and shell (Figure 5.0). The core and shell

subregions were not examined with regard to differences in dopaminergic

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1.00-1.20 mm 1.60-1.70 mm 2.20 mm 0.70 mm FIGURE 5.0. Coronal brain sections indicating microdialysis probe placement within the nucleus accumbens. Lines denote the active dialysis regions. Numerals below the sections denote the position of the slice in relation to Bregma. Figure was adapted from Paxinos and Watson (1998).

167

function due to the dispersion of the probes between the groups. The dialysate

dopamine samples showed excellent calcium dependence (89 ± 2% for the 30

subjects). The calcium dependency of dialysate dopamine exceeded 69% in all

subjects. We excluded one subject that showed a calcium dependency of less

than 50%.

NorBNI blockade of U50488H

To confirm that the dose of NorBNI resulted in κ-opioid blockade, we tested the

ability of the antagonist to block U50488H-induced suppression of accumbal

dopamine activity (Heijna et al., 1990; Xi et al., 1998). Pretreatment with NorBNI

(20 mg/kg, s.c.) 15-20 hrs prior to the experiment did not significantly alter basal

dopamine levels in the accumbens (1.6 ± 0.3 nM; n=7) compared to the saline

controls (1.3 ± 0.2 nM; n=8). ANOVA showed no group effect [F(3,11) = 0.78, p

> 0.05] or group by time effect [F(6,20) = 1.02, p > 0.05]. Within 30 min of

perfusion through the dialysis probe, U50488H (0.4 or 1.6 µM) decreased

dopamine concentrations by approximately 40% of their basal levels in both

control groups (Figure 5.1). In contrast, NorBNI administration blocked the effect

of 0.4 µM U50488H [time: F(6,35) = 0.22, p > 0.05; n=4; Figure 5.1], but not the

effect of 1.6 µM U50488H [time: F(6,30) = 2.95, p < 0.05; n=3].

Dopamine concentrations and operant behavior prior to ethanol access

Pretreatment with NorBNI (20 mg/kg, s.c.) 15-20 hrs prior to the experiment did

168

20

40

60

80

100

120

140

0 20 40 60 80 100

1.6 U50NOR + 1.6 U50

0.4 U50NOR + 0.4 U50

baseline

U50 (1.6 or 0.4 µM) DO

PA

MIN

E (%

of b

asal

)

TIME (min)

FIGURE 5.1. Effect of NorBNI pretreatment (20 mg/kg) on U50488H-induced suppression of basal dopamine concentrations in the nucleus accumbens. NorBNI pretreatment occurred 15-20 hrs prior to the experiment. Microdialysis perfusion of the accumbens with U50488H (1.6 and 0.4 µM) reduced basal dopamine levels in the control groups. NorBNI pretreatment failed to reverse the effects of 1.6 µM U50488H but effectively blocked 0.4 µM U50488H. All groups showed a significant effect of time (p < 0.05) except for the 0.4 µM U50488H group pretreated with NorBNI (NOR+0.4 U50). Baseline dopamine concentrations: 0.4 U50 = 1.3 ± 0.3 nM, 1.6 U50 = 1.2 ± 0.3 nM, NOR + 0.4 U50 = 1.8 ± 0.4 nM, NOR + 1.6 U50 = 1.4 ± 0.4 nM. Each point represents the mean ± sem [n=4 for each group except NOR+1.6 U50 (n=3)].

169

not affect basal dopamine concentrations in the home cage [NorBNI group (n=7):

1.8 ± 0.4 nM; control group (n=8): 2.3 ± 0.5 nM]. There was not a significant

group effect [F(1,13) = 0.24, p > 0.05] or a group by time effect [F(5,65) = 0.99, p

> 0.05] during this period. Although both groups showed increased dopamine

concentrations upon the transfer into the operant chamber [time: F(4,52) = 10.59,

p < 0.05], the relative magnitude of each dopamine response above the home-

cage baseline differed (NorBNI: 16 ± 4 %; control: 25 ± 3%). ANOVA revealed a

group by time effect for the transfer sample [F(1,13) = 8.53, p < 0.05]. Due to the

nonspecific increase in dopamine concentration following the home-cage

baseline period, we used the three dopamine samples from the wait period as a

baseline for the subsequent analysis of the drink and post-drink periods. Both

groups showed stable and similar mean dialysate dopamine concentrations

(NorBNI: 2.0 ± 0.4 nM; control: 2.4 ± 0.5 nM) during the wait period [group:

F(1,13) = 0.28, p > 0.05; group x time: F(2,26) =0.03, p > 0.05; Figure 5.2].

ANOVA indicated that the NorBNI-treated group (0.7 ± 0.3 min) did not differ

significantly from the control group (0.6 ± 0.2 min) in terms of the time required to

complete the operant response prior to ethanol consumption [F(1,13) = 0.13, p >

0.05; lever-pressing time; Table 5.2].

Drinking behavior and dopamine concentrations following ethanol access

Both groups ingested similar amounts of 10% ethanol (plus 10% sucrose) during

170

70

80

90

100

110

120

130

0 10 20 30 40 50 60

CONTROLNORBNI

leverpress

drink post-drinkwait

TIME (min)

DO

PA

MIN

E (%

of b

asal

)

**

FIGURE 5.2. Effect of norbinaltorphimine (20 mg/kg; NorBNI) or saline (control) pretreatment on extracellular accumbal dopamine concentrations during operant self-administration of 10% ethanol (plus 10% sucrose). Pretreatment occurred 15-20 hrs prior to the experiment. Dopamine concentration increased significantly within 5 min of ethanol access for the control group and returned to basal levels thereafter. The NorBNI group displayed no change in dopamine concentrations initially, followed by a transient elevation at the end of the drink period. Wait refers to the time in which the rat was in the operant chamber prior to access of the drinking solution. Lever press (open arrow) is the time at which operant responding occurred. Drink refers to the 20-min ethanol access period free of operant responding. Post-drink refers to the 20-min period in which the rat remained in the chamber in the absence of the ethanol solution. Each point represents the mean ± sem (n=8 for the saline control group; n=7 for the NorBNI group). Asterisk denotes significance compared with the wait period by post hoc simple contrasts (p < 0.05).

171

TABLE 5.1. Lickometer parameters for the NorBNI and control groups during operant ethanol self-administration.

Parameter NorBNI n=7

Controln=8

lever-pressing time (min) 0.7 ± 0.3 0.6 ± 0.2 latency to begin drinking (min) 0.12 ± 0.05 0.05 ± 0.01

number of bouts 1.9 ± 0.3 1.5 ± 0.2 initial bout duration (min) 4.8 ± 1.2 5.8 ± 0.9

total licks 1321 ± 253 1541 ± 288 licks during initial bout 1164 ± 257 1457 ± 299

Initial bout response rate (licks/min) 257 ± 28 244 ± 30 response rate for ½ of initial bout

(licks/min) 266 ± 38 326 ± 37

Bout refers to a period of at least 25 licks, with no more than 2 min between licks. Values shown as mean ± sem.

172

the dialysis experiment [NorBNI (n=7): 1.5 ± 0.3 g/kg; control (n=8): 1.8 ± 0.3

g/kg]. The mean fluid intake during the 20-min drink period was also similar

between the groups (control: 8.9 ± 1.5 ml; NorBNI: 7.2 ± 1.2 ml).

The NorBNI and control groups displayed distinct dopamine time courses

following ethanol (plus sucrose) access. The groups differed significantly across

the drink and post-drink periods [group x time: F(8,101) = 2.49, p < 0.05] and

across the drink period alone [group x time: F(4,50) = 6.13, p < 0.05]. Within the

control group, post hoc contrasts indicated that the first dopamine sample of the

drink period was significantly elevated (14 ± 5%) compared to baseline [F(1,101)

= 9.79, p < 0.05], with dopamine concentrations then returning to basal levels by

the end of the 20-min drink period (Figure 5.2). In contrast, the NorBNI-treated

group did not exhibit a dopamine response during the first drink sample but

showed a small, transient increase in dopamine concentration at the end of the

drink period (13 ± 3% above baseline). Within-group contrasts for this group

showed a significant increase in dopamine levels only during the fourth sample of

the drink period [F(1,101) = 7.58, p < 0.05; Figure 5.2].

Accumbal ethanol concentrations following ethanol access

Ethanol appeared in the dialysates within 5 min of ethanol access in all rats, and

the mean ethanol concentration in each sample increased thereafter (Figure 5.3).

The ethanol time courses did not significantly differ between the two treatment

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0.0

1.0

2.0

3.0

4.0

5.0

0 20 40 60 80 100

NORBNI CONTROL

drink post-ingestion

TIME (min)

DIA

LYS

AT

E E

TH

AN

OL

(mM

) FIGURE 5.3. Dialysate ethanol concentrations (mM) from the nucleus accumbens following ethanol access for the NorBNI and saline control groups. Ethanol was analyzed from the same samples in which dopamine concentrations were measured. Both treatment groups showed very comparable ethanol pharmacokinetics. Accumbal ethanol concentrations increased rapidly after the first 5-min of the ethanol access period (drink) and remained elevated thereafter (post-ingestion). Each point is the mean ± sem.

174

groups [group: F(1,13) = 0.02, p > 0.05; group x time: F(9,116) = 0.19, p > 0.05].

Overall, the individual ethanol time courses and peak ethanol concentration

varied among the animals. As a result, the mean dialysate ethanol levels

reached peak concentration (approximately 3 mM) 15 min after drinking

commenced in both groups. Regression tests showed that both groups

displayed significant and positive correlations between (1) intake (g/kg) and

dialysate ethanol area-under-curve (p < 0.05; Figure 5.4A) and (2) intake (g/kg)

and peak dialysate ethanol concentration (p < 0.05).

Licking behavior during ethanol consumption

NorBNI treatment (20 mg/kg, s.c.) did not significantly alter licking behavior.

Multivariate [group: F(9,5) = 0.53, p > 0.05] and univariate analyses (p > 0.05)

revealed no group differences between the NorBNI and control groups with

respect to several behavioral parameters (Table 5.2). Ethanol consumption in

both groups began almost immediately after completion of the operant response

(latency to begin drinking: 0.08 ± 0.02 min). The majority of consumption (licks)

occurred during the first bout (91 ± 3% for the two groups). Figure 5.5 illustrates

the average number of licks within each 5 min epoch of the drink period for each

treatment. The NorBNI group ingested 92 ± 5% of its total intake within 10 min of

the ethanol access period and the control group ingested 99 ± 1% of its total

intake within this period.

175

A

0

10

20

30

40

50

60

70

80

0.5 1 1.5 2 2.5 3

NORBNICONTROLD

IALY

SATE

ETH

ANO

L (A

UC

)

ETHANOL INTAKE (g/kg)

0

2

4

6

8

10

0 2 4 6 8 10 12

NORBNI

DIA

LYSA

TE D

OPA

MIN

E (A

UC

)

DIALYSATE ETHANOL (AUC)

B

FIGURE 5.4. Dose-effect relationships between (1) ethanol intake (g/kg) and dialysate ethanol levels (AUC; panel A), and (2) dialysate ethanol levels (AUC) and dialysate dopamine levels (AUC) during the last 10 min of the drink period (panel B). NorBNI and saline control groups showed positive correlations between ethanol intake and accumbal ethanol levels (p < 0.05). Dialysate dopamine concentration correlated positively with dialysate ethanol concentration during the last 10 min of the drink period for the NorBNI group (p < 0.05).

176

0

300

600

900

1200

1500

5 10 15 20

CONTROLNORBNI

ETH

AN

OL

LIC

KS

TIME (min)

FIGURE 5.5. Ethanol consumption (spout licks) during each 5-min epoch of the ethanol access period. The groups showed very similar patterns of ethanol ingestion throughout each period. The majority of ethanol consumption occurred within the first 5-10 min. Each plot is the mean ± sem.

177

Dose-effect relationships between brain ethanol levels and dopamine

response

The dialysate ethanol levels (area-under-curve; AUC) during the drink period

correlated positively with dialysate dopamine levels (AUC) during the drink period

[F(1,5) = 14.05, p < 0.05; r = 0.86] for the NorBNI group, but not for the control

group [F(1,6) = 0.25, p > 0.05; r = 0.20]. Because the NorBNI group showed a

significant dopamine response at the end of the 20-min drink period, when

ethanol concentrations had reached peak levels, we also examined the

relationship between dopamine and ethanol concentrations during the last two

samples of the drink period. Regression analysis revealed a significant and

positive correlation between dialysate ethanol levels (AUC) and dialysate

dopamine levels (AUC) during this period for the NorBNI group [F(1,5) = 10.31, r

= 0.82; p < 0.05; (Figure 5.5)]. The control group showed no significant

correlations during the drink period.

Relationships between operant responding and dopamine across studies

We performed additional post hoc analyses by pooling the control groups trained

to drink 10% ethanol (plus 10% sucrose) from the current study and Doyon et al.

(2005). The animals were run under identical conditions, with the exception that

one group was injected with saline prior to testing. Changes in dopamine

concentration during the lever-press sample compared with the previous wait

sample were assessed between the combined ethanol-drinking group (n=18) and

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the NorBNI group (n=7). There was no significant difference between the groups

during the lever period [F(1,23) = 0.04, p > 0.05], nor was there a change in

dopamine levels over time after collapsing the groups [F(1,23) = 0.51, p > 0.05].

Due to evidence suggesting phasic dopamine activity is dependent on the

probability and uncertainty involved in obtaining an incentive (Fiorillo et al.,

2003), we also performed a regression analysis on the duration of the operant

response (0.68 ± 0.24 min) and the percent of basal dopamine response (19 ±

4%) during the first 5 min of ethanol access for only the combined ethanol group

(n=17). Lever-pressing duration correlated positively with the magnitude of the

dopamine response during the first 5 min of ethanol availability [F(1,16) = 5.57, r

= 0.52; p < 0.05].

DISCUSSION

These present findings demonstrate that blockade of κ-opioid receptors with

NOR-BNI resulted in a latent increase in accumbal dopamine concentration

following ethanol access, which showed a positive correlation with brain ethanol

levels. Despite this alteration in dopamine activity, NOR-BNI did not significantly

alter operant responding or consumption of 10% ethanol (plus 10% sucrose).

The NOR-BNI treatment effectively blocked the reduction of accumbal dopamine

levels by the κ-opioid agonist, U50488H, indicating that the antagonist occupied

the kappa receptor.

179

The NorBNI- and saline-treated rats displayed similar appetitive responses (as

measured by lever-response time and latency to begin licking), indicating that

inactivation of kappa receptors did not alter motor activity associated with

operant behavior. NorBNI (20 mg/kg) did not affect ethanol intake or any of the

consumption parameters measured. These results extend previous work that

examined the role of κ-opioid receptors in operant ethanol reinforcement, which

utilized lower doses of NorBNI that also failed to alter ethanol consumption

(Williams and Woods, 1998; Holter et al., 2000). Overall, ethanol intake during

the access period (about 1.5-1.8 g/kg in 20 min) was considerably higher than

that reported in the two previous studies within a comparable access period (1-3

hr), indicating that low brain ethanol levels do not adequately account for the

negative findings. These results further suggest that the endogenous κ-opioid

system is not involved in operant responding or consumption associated with

operant ethanol self-administration. However, considering the subtle effects of κ-

opioid blockade on dopamine activity in the present study, it is possible that the

behavioral response to NorBNI is not manifested until long after its initial

administration. More studies are needed to determine whether extended ethanol

reinforcement following κ-opioid blockade can alter drinking behavior beyond a 1-

day pretreatment period. Changes in extracellular dopamine signaling and

continuous-access ethanol drinking after prolonged NorBNI treatment have been

reported recently (Chefer et al., 2005; Mitchell et al., 2005), which may support

this possibility.

180

The saline control group exhibited a small and temporary increase in dopamine

concentrations during ethanol (plus sucrose) consumption, followed by a rapid

decline to basal levels. The transient dopamine response occurred at a time

when brain ethanol levels were low (1.0 ± 0.3 mM) relative to their peak

concentrations (3.0 ± 0.8 mM). This finding supports past results (Doyon et al.,

2003; Doyon et al., 2005) and further demonstrates that ethanol reinforcement

can induce a temporary elevation in accumbal dopamine activity that is not

consistent with a pharmacological response to ethanol. Doyon et al. (2005)

hypothesized that the brief increase in dopamine concentration during ethanol

drinking was due to the stimulus properties of ethanol and that these sensory

cues were strongest during ingestion, which is also in accordance with the

present findings. It is unlikely that the transient dopamine activity was the result

of overflow from the operant response period or ethanol anticipation since

dopamine stimulation did not occur during operant self-administration of a novel

sucrose solution in rats conditioned to drink ethanol in Doyon et al. (2005). A

non-pharmacological response to ethanol is also supported by the positive

correlation between operant response time and the percent of basal dopamine

response during the initial period of ethanol availability that was revealed by the

across-study analyses. Fiorillo et al. (2003) reported that the magnitude of

mesencephalic dopamine activity is proportional to the probability or uncertainty

involved in obtaining an incentive stimulus. A longer lever-pressing time may

reflect a larger degree of uncertainty or surprise regarding the timing of ethanol

181

acquisition, which is encoded for by the transient dopamine response, further

suggesting that this activity is involved in some aspect of obtaining the ethanol

solution.

NorBNI treatment resulted in a latent increase in dopamine concentration during

ethanol self-administration compared to the control group. The enhanced

dopamine response was brief and occurred as brain ethanol concentrations

reached peak levels. During this period, accumbal dopamine levels correlated

positively with ethanol concentrations, which is consistent with a pharmacological

response. This effect was specific to the NorBNI group, as a similar relationship

was not revealed at any point during the ethanol access period for controls. Our

original hypothesis was that ethanol administration elicits the release of

endogenous dynorphin peptides, which then act on the κ-opioid receptor that

functions to inhibit dopamine release. These results suggest that NorBNI

blockade of the κ-opioid receptor may have briefly uncovered dopamine activity

that is normally suppressed by ethanol-induced dynorphin release (Marinelli et

al., 2005). A pharmacological dopamine response to ethanol revealed by kappa

blockade agrees with work by Zapata et al. (personal communication), which

showed that an equivalent dose of NorBNI can potentiate the dopamine

response to i.p. ethanol administration. Our results extend this work by

suggesting that the κ-opioid system may counter the stimulatory effects of

ethanol on dopamine concentrations following operant ethanol self-

182

administration.

The duration of the latent dopamine response during ethanol self-administration

was not as long as expected, when considering that NOR-BNI is a long-acting

kappa antagonist and that brain ethanol levels remained elevated far beyond the

period in which the response occurred. These results may reflect a rapid

adaptive response within the mesolimbic system. Previous studies clearly

demonstrate that intra-accumbal dopamine and ethanol concentrations

dissociate rapidly following i.p. or oral ethanol administration (Yim et al., 2000;

Doyon et al., 2005). These data suggest that other mechanisms in addition to

the κ-opioid system exert strong regulation over ethanol-induced dopamine

activity. Alternatively, there is evidence that stimulation of accumbal dynorphin

release by ethanol is transient and occurs only within 30 min of ethanol

administration (Marinelli et al., 2005), which is consistent with our time course of

effects and suggests that further dopaminergic activation would not have

occurred regardless of κ-opioid blockade and the presence of ethanol.

One aspect of the dopamine profile observed under NorBNI blockade that is

difficult to explain is the absence of the transient dopamine activity during the

initial 5 min of ethanol access exhibited by controls. The idea that NorBNI

disrupted what is suggested to be a non-pharmacological, and possibly sensory

183

mediated, dopamine response is supported by the blunted dopamine levels

during the transfer period into the operant chamber for the NorBNI group,

compared to controls. We concluded previously that this phenomenon was due

to transient physical stimulation or environmental change (Doyon et al., 2003;

Doyon et al., 2005), which suggests that NorBNI is reliably disrupting similar

physiological processes. It is possible that these effects are mediated by the

blockade of κ-opioid receptors located on non-dopamingeric neurons that are

normally under tonic dynorphin inhibition. In a simple example, a GABAergic cell

with mesolimbic connectivity would thus be disinhibited by κ-opioid blockade,

which in turn would have a suppressive effect on dopamine activity. These

interactions could be mediated by neuronal pathways that are specifically

activated by sensory input and functionally independent from those involved in

the latent, possibly pharmacological, dopamine response. An examination of the

local effects of NorBNI within the ventral tegmental area or nucleus accumbens

during ethanol self-administration may help clarify this issue.

Because the ethanol drinking solution used in these experiments also contained

10% sucrose there is also the possibility that these results are related to the

effects of NorBNI on sucrose-induced dopamine activity. Increased accumbal

dopamine concentration during sucrose ingestion is consistently observed in

deprived (Hajnal and Norgren, 2001; Hajnal et al., 2004; Genn et al., 2004), but

not satiated, animals (Roitman et al., 2004), indicating that motivational state is

184

an important contributing factor. Previous results from our laboratory in non-

deprived rats show that dopamine levels can increase transiently during sucrose

consumption using a single fixed-ratio 20 response requirement (Doyon et al.,

2004), but not under a fixed-ratio 4 schedule (Doyon et al., 2005). This

discrepancy may be due to differences in the timing and expectancy involved in

obtaining sucrose between the reinforcement schedules, which could potentially

result in differential expression of dopamine activity. The response requirement

and training schedule in the present study was identical to that in Doyon et al.

(2005). Therefore, in the ethanol control group, it is not likely that the dopamine

response during ethanol reinforcement was related to the presence of sucrose in

the solution. The effects of κ-opioid blockade on sucrose-induced dopamine

activity, however, are not known. Thus, additional work using sucrose

reinforcement is needed to expand our findings and to confirm that the NorBNI

effect on dopamine concentrations is indeed specific to ethanol self-

administration.

NorBNI did not significantly affect basal dopamine concentrations, which is

consistent with a recent study by Chefer et al. (2005) that utilized a similar

NorBNI pretreatment time (24 hrs). Previous work has shown that NorBNI can

increase basal dopamine levels in the accumbens after acute administration

(Spanagel et al., 1992; Maisonneuve et al., 1994), but the effects are short-lived

and not characteristic of a long-acting antagonist. Other studies have suggested

185

that NorBNI is a slow onset κ-opioid antagonist that may have non-selective

action under acute conditions (Endoh et al., 1992; Williams and Woods, 1998).

Chefer et al. (2005) has suggested that dopamine release is in fact enhanced

after long-term kappa blockade with NorBNI, but that basal dopamine

concentration remains unchanged due to an increase in dopamine uptake. Thus,

the κ-opioid receptor system may interact with the dopamine transporter to

regulate basal dopamine levels, a possibility supported by their anatomical

localization (Svingos et al., 2001). How this interaction may affect dopamine

dynamics during ethanol self-administration is unknown at present.

In summary, these findings demonstrate that blockade of κ-opioid receptors with

NOR-BNI resulted in a latent increase in accumbal dopamine concentration

following ethanol access, which correlated with brain ethanol levels and is

consistent with a pharmacological relationship. Blockade of the κ-opioid receptor

with NOR-BNI may have temporarily disinhibited accumbal dopamine activity that

is normally suppressed by ethanol-induced dynorphin release. NOR-BNI did not

significantly affect operant responding or consumption of 10% ethanol or 10%

sucrose. However, a more complete interpretation of this work requires further

understanding of the precise mechanisms involved in κ-opioid regulation of

mesoaccumbens dopamine activity.

186

6.0 General discussion

In summary, the major findings of these studies are (1) that dopamine

concentration in the nucleus accumbens increases transiently during

consumption of ethanol in Long-Evans rats that have developed ethanol-

reinforced behavior, (2) that the increase in dopamine concentration during

consumption is not directly related to brain ethanol levels, and (3) that blockade

of the κ-opioid receptor can enhance accumbal dopamine concentration following

ethanol consumption in a manner consistent with pharmacological stimulation.

The present studies utilized an operant procedure that specifically separated two

major components of reinforcement, operant responding and acquisition of the

incentive stimulus. This operational distinction is important because it permitted

us to relate changes in extracellular dopamine concentration to discrete

behaviors associated with ethanol self-administration on a level not previously

possible. Earlier work in the field suggested that ethanol self-administration can

produce a pharmacological stimulation of dopamine levels in alcohol-preferring P

rats and in heterogeneous Wistar rats (Weiss et al., 1993; Gonzales and Weiss,

1998; Melendez et al., 2002). However, interpretation of these results was

limited by the fact that the animals in these experiments were performing an

operant response and ingesting ethanol during the same period in which

dopamine activity was measured. Additional analysis of the data collected from

Wistar rats also suggests that the reported percent increase in dopamine levels

187

would have been smaller, and possibly not statistically significant, had the

investigators used a more conservative baseline dopamine response. Therefore,

it was unclear whether the dopamine response observed during ethanol self-

administration was truly elevated above basal and, if so, whether the appetitive

or consummatory phases of behavior were involved in the process.

Our studies extend these findings by demonstrating that extracellular dopamine

concentrations in the nucleus accumbens can indeed increase during

consumption of ethanol. However, the dopamine response is transient and is

only reliably observed during the first 5 min of the ethanol-access period.

Concurrent analysis of dialysate ethanol concentrations indicated that brain

ethanol levels were low during this initial period, relative to their peak values, and

increased progressively thereafter. This lack of correspondence between

dialysate dopamine and ethanol concentrations is inconsistent with a

pharmacological response. Alternatively, ethanol intake commenced almost

immediately after completion of the operant response and typically consisted of

one to two “bouts” that occurred contemporaneously with the dopamine

response. Therefore, it was suggested that the increase in extracellular

dopamine is related to the sensory stimuli associated with ethanol drinking, such

as the taste or smell of the solution, and is not a pharmacological response to the

presence of ethanol in the brain per se. A sensory-mediated response is

supported by the finding that the transient dopamine response was not observed

188

during self-administration of an unexpected 10% sucrose solution in rats

responding for 10% ethanol (plus 10% sucrose), which suggested that effect is

not due to dopamine activity involved in lever pressing or expectation of ethanol

reinforcement. The post hoc regression analyses reported in Chapter 4 for high

and low intake groups drinking 10% ethanol (plus 10% sucrose) are also

consistent with this possibility. Intake (g/kg or total licks) correlated positively

with the percent of basal dopamine response during the first 5 minutes of ethanol

availability, suggesting that the dopamine response was related to the amount of

ethanol consumed during that period.

However, if we examine all control animals that drank 10% ethanol (plus 10%

sucrose) across studies (n=18), and exclude the low intake group from the

analysis, a correlation between intake and dopamine response is not revealed.

Therefore, intake levels may not adequately account for the variability in the

magnitude of the transient dopamine response between rats, which ranged from

-6 to +54% relative to basal in the ethanol plus sucrose groups. Recent work by

Fiorillo et al. (2003) demonstrates that the firing rate of mesencephalic dopamine

cells is directly related to the probability or uncertainty involved in obtaining an

incentive stimulus. In our procedures, the time needed to complete the operant

response varied between animals, which may be considered one index of

uncertainty. A regression analysis of all control rats drinking ethanol plus

sucrose revealed a positive correlation between operant response time and the

189

80

100

120

140

160

180

0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

r = 0.52

OPERANT RESPONSE TIME (min)

DIA

LYS

ATE

DO

PA

MIN

E

(%

of b

asal

)

FIGURE 6.0. Regression analysis showed a positive correlation between lever-pressing duration and dialysate dopamine response (percent of basal) during the first 5-min of the ethanol drinking period (p < 0.05). Data is from rats trained to respond for 10% ethanol (plus 10% sucrose) [n=17].

190

percent of basal dopamine activity during the first 5 minutes of ethanol availability

(Figure 6.0). A longer lever-pressing period may reflect a larger degree of

uncertainty or surprise regarding the timing of ethanol acquisition, which could be

encoded by the transient dopamine response. This finding may account for

the striking variability in dopamine activity observed during the initial 5 minutes of

access. However, because the range of operant response times was not evenly

distributed, a more rigorous test is needed to substantiate this hypothesis, one in

which the lever-press period is systematically varied between animals.

In any case, these results provide strong evidence that post-ingestion action of

ethanol does not stimulate extracellular dopamine levels pharmacologically in a

limited-access model of operant self-administration. Rather, it appears that

increased dopamine activity is a response to some aspect of ethanol acquisition

during reinforcement. However, this conclusion does not eliminate the possibility

that meaningful changes in phasic dopamine activity could be occurring during

ethanol consumption but on a time scale and level undetectable with

microdialysis. Electrophysiological studies provide some evidence that ethanol

can activate ventral tegmental dopamine neurons in vitro (Brodie et al., 1999;

Appel et al., 2003) and in paralyzed animals (Gessa et al., 1985). Recent

findings also indicate that acetaldehyde, a product of ethanol metabolism, can

also increase the excitability of these cells in anesthetized animals (Foddai et al.,

2004). Future work using techniques that are capable of measuring phasic

191

dopamine activity during ethanol-reinforced behavior, such as in vivo

voltammetry (Roitman et al., 2004), could further clarify this issue. The data

presented in Chapters 2-4 represent the first studies of accumbal dopamine

activity during different stages in the development of ethanol reinforcement. We

initially determined that a small rise in dopamine concentrations occurs within 5

minutes of access to 10% ethanol in rats with established patterns of ethanol-

reinforced behavior (over 40 days of training). The increase was brief and

dopamine levels returned to baseline thereafter. To test the hypothesis that this

effect was related to low pharmacological levels of ethanol or dopaminergic

tolerance due to the long-term self-administration procedure, we designed a

short-term training protocol in which the rats were habituated to 10% ethanol

(plus 10% sucrose) over a 7 day period. The presence of sucrose in the drinking

solution had the desired effect of increasing intake over a relatively brief period.

Despite these manipulations, the dopamine response was remarkably similar to

that observed under conditions of long-term ethanol reinforcement.

Based on the idea that the development of reinforcement is dopamine

dependent, we hypothesized that alterations in dopamine release, distinct from

those in well-trained animals, may be occurring during ethanol related learning at

an earlier stage. Examination of the effects of an initial exposure to ethanol in

naïve rats showed that changes in dopamine activity were absent during limited-

access drinking. These animals self-administered a novel solution of 10%

192

sucrose with 5 or 10% ethanol during the experiment after being trained to

respond for 10% sucrose. However, the new solution appeared to have aversive

properties based on intake levels, which were low. Therefore, the interpretation

of these data was limited because it was unknown whether the absence of

dopamine activity was simply due to insufficient pharmacological levels of

ethanol. Nonetheless, these findings collectively indicate that the transient

increase in accumbal dopamine concentration during ethanol access is only

observable in rats that have developed ethanol-reinforced behavior. These

results suggest that plasticity, or long-term changes in synaptic function, within

the mesolimbic system may be occurring during the transition to motivated

ethanol self-administration.

We next tested the hypothesis that κ-opioid transmission inhibits dopamine

activity and ethanol drinking behavior during reinforcement, which had not been

explored previously. Prior studies showed that ethanol administration can

enhance tissue levels of dynorphin, the endogenous κ-opioid ligand (Lindholm et

al., 2000; Marinelli et al., 2005) and that activation of the κ-opioid receptor inhibits

mesolimbic dopamine activity (Di Chiara and Imperato, 1988; Heijna et al., 1990;

Spanagel et al., 1992; Devine et al., 1993; Xi et al., 1998; Margolis et al., 2003).

However, blockade of the κ-opioid receptor with the long-acting antagonist, nor-

binaltorphimine has not been shown to alter measures of ethanol-reinforced

behavior (Williams and Woods, 1998; Holter et al., 2000). These negative data

193

may be due to the low doses of the antagonist (3-5 mg/kg) used in these

experiments or possibly because brain ethanol concentrations did not reach

pharmacological levels.

Our work extended these studies by demonstrating that blockade of the κ-opioid

receptor with a high-end dose of nor-binaltorphimine (20 mg/kg) did not

significantly affect ethanol reinforcement, including operant responding and

intake. Despite the lack of behavioral changes, the dopamine time course under

nor-binaltorphimine blockade differed from controls. The transient dopamine

response within the first 5 minutes of drinking was absent, but a latent increase in

dopamine concentration occurred 15 minutes later. During this period, dopamine

and ethanol concentrations displayed a positive correlation, which is consistent

with a pharmacological relationship. This suggests that the blockade of κ-opioid

receptors temporarily uncovered dopamine activity that is normally inhibited by

ethanol-induced dynorphin release following self-administration, which supports

our original hypothesis. This possibility is supported by work from Zapata et al.

(personal communication), which showed that an equivalent dose of nor-

binaltorphimine potentiated the dopamine response to i.p. ethanol administration.

Although the small changes in dopamine activity did not correspond to

modifications in ethanol-reinforced behavior, it is possible that the behavioral

response to κ-opioid blockade is not manifested until the animal has experienced

further ethanol reinforcement under nor-binaltorphimine conditions. 194

However, the effects of kappa blockade on dopamine activity in the present study

were subtle and not as robust as expected. Nor-binaltorphimine is highly

selective for the κ-opioid receptor compared to the µ and δ receptors

(Portoghese et al., 1987; Marki et al., 2000), which was supported by its ability to

block the inhibition of basal dopamine levels mediated by the kappa agonist,

U50488. However, it is still possible that the dopamine activity during self-

administration was influenced by nor-binaltorphimine action at non-opioid binding

sites. While no direct evidence for this is found in the literature, nor-

binaltorphimine is a large bivalent molecule that contains a particular nitrogen

group (N17’) that is thought to interact with a glutamate residue (Glu297) on

transmembrane domain 6 of the kappa receptor, which determines its selectivity

(Portoghese et al., 1994; Hjorth et al., 1995). Therefore, nor-binaltorphimine

could interact, in theory, with any protein involved in signal transduction of such

composition, potentially altering its function. Non-specific binding can amount to

40% of total binding with radiolabeled nor-binaltorphimine in vitro (Marki et al.,

2000). Although this idea is unlikely and difficult to test, it cannot be discounted.

6.1 Future directions

The present findings raise intriguing questions, which subsequent studies could

potentially address. For example, at what stage in the development of ethanol

reinforcement does neuroadaptation within the mesolimbic mesolimbic system

occur with regard to the transient dopamine response during drinking? Clearly,

195

there is a fundamental change in neurochemical activity between the initial

ethanol exposure period and the seventh day of habituation in the short-term

training procedure. To determine the timing of this event, dialysate dopamine

concentrations could be measured across successive self-administration

sessions beginning at the second day of exposure. Rather than gradually

increasing the concentration of ethanol in the drinking solution, as we typically

do, a single concentration would be used over the sessions to avoid changes in

stimulus strength that could also alter the dopamine response or behavior.

A more precise determination of the timing involved in the transient dopamine

response within the self-administration session is also needed. For example, is

the increase sudden, occurring during the initial acquisition of the solution, or is it

an additive effect over the 5-minute sampling period? To test this idea using

microdialysis, the sampling interval would have to be reduced to 1-min.

However, this question is best answered with electrochemical detection methods,

such as fast-scan cyclic voltammetry, which permit the analysis of dopamine

release on a subsecond time scale.

Additionally, the regression tests suggested that the transient dopamine activity

is related to the duration of the operant response due to uncertainty or

unpredictability in the timing of ethanol acquisition. To assess this hypothesis

one experiment would involve systematically varying the lever-pressing time. We

196

previously utilized “time outs,” or periodic access to the lever, to lengthen the

time associated with responding for microdialysis sampling. Therefore, this same

procedure could be modified for this type of experiment.

In terms of the effects of κ-opioid blockade on dopamine activity during ethanol

self-administration, it remains unknown whether nor-binaltorphimine affects

dopamine concentrations during sucrose self-administration. Because the

ethanol drinking solutions also contained sucrose, a complete interpretation of

the ethanol data cannot be made without these results. These experiments

would simply involve administering nor-binaltorphimine to rats trained to respond

for sucrose rather than ethanol plus sucrose. In a related issue, the possible

ventral tegmental or accumbal mechanisms involved in the effects of kappa

blockade on dopamine during ethanol reinforcement are uncertain. For example,

to test the hypothesis that accumbal kappa receptors modulate the latent

dopaminergic response after ethanol self-administration, nor-binaltorphimine

could be administered within this site using microdialysis and a pretreatment time

similar to that used in the systemic experiments.

197

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VITA

William Maurice Doyon was born in Holloman A.F.B., New Mexico on January

19, 1974, the son of William Maurice Doyon Sr. and Maria Esther Doyon.

William completed studies at New Mexico State University in Las Cruces, New

Mexico in May 1999 where he received a Bachelor of Science in Biology and

Bachelor of Arts in Psychology. He began graduate studies in the Pharmacology

and Toxicology program at the University of Texas in August 2000.

Permanent address: 3113 Shawnee Trail, Alamogordo, New Mexico 88310

This dissertation was typed by the author.

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