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JPET #76968 1 The Selective 7 Nicotinic Acetylcholine Receptor Agonist PNU-282987 Enhances GABAergic Synaptic Activity in Brain Slices and Restores Auditory Gating Deficits in Anesthetized Rats M. Hajós, R. S. Hurst, W. E. Hoffmann, M. Krause 1 , T. M. Wall 2 , N. R. Higdon 2 , and V. E. Groppi Department of Neuroscience (MH, RSH, MK, TMW, NRH) and CNS Molecular Sciences (VEG), Pfizer Global Research & Development, Pfizer Inc. Groton, CT and Ann Arbor, MI, USA JPET Fast Forward. Published on November 2, 2004 as DOI:10.1124/jpet.104.076968 Copyright 2004 by the American Society for Pharmacology and Experimental Therapeutics.

Transcript of The Selective 7 Nicotinic Acetylcholine Receptor Agonist ... · This deficit leads to disrupted...

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The Selective α7 Nicotinic Acetylcholine Receptor Agonist PNU-282987

Enhances GABAergic Synaptic Activity in Brain Slices and

Restores Auditory Gating Deficits in Anesthetized Rats

M. Hajós, R. S. Hurst, W. E. Hoffmann, M. Krause1, T. M. Wall2, N. R. Higdon2, and

V. E. Groppi

Department of Neuroscience (MH, RSH, MK, TMW, NRH) and

CNS Molecular Sciences (VEG),

Pfizer Global Research & Development, Pfizer Inc. Groton, CT and Ann Arbor, MI, USA

JPET Fast Forward. Published on November 2, 2004 as DOI:10.1124/jpet.104.076968

Copyright 2004 by the American Society for Pharmacology and Experimental Therapeutics.

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Running title: PNU-282987, a novel α7 nAChR agonist

Corresponding author: Mihály Hajós, PharmD, Ph.D.

Department of Neuroscience

Pfizer Global Research and Development

Eastern Point Road

Groton, CT 06340, USA

Telephone: (860) 686-6967

Fax: (860) 715-2349

E-mail: [email protected]

Number of text pages: 34

Number of tables: 0

Number of figures: 8

Number of words in the abstract: 219

Number of words in the introduction: 593

Number of words in the discussion: 1098

Number of References: 39

Recommended Section: Neuropharmacology

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Abbreviations:

BPS, Phosphate Buffer Saline; C, conditioning; CHRNA7, α7 nicotinic acetylcholine receptor

subunit gene; CNQX, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate 6-

cyano-7-nitroquinoxaline-2,3-dione; EEG, electroencephalograph; GABA, gamma-aminobutyric

acid; GTS-21, 3-[(2,4-Dimethoxy)benzylidene]-anabaseine dihydrochloride (DMXBA); MLA,

methyllycaconitine; nAChR, nicotinic acetylcholine receptor; N40, auditory evoked potential,

negative deflection at 40 ms latency; nRT, reticular thalamic nucleus; P20, auditory evoked

potential, positive deflection at 20 ms latency; P50, auditory evoked potential, positive deflection

at 50 ms latency; PNU-282987, (N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide

hydrochloride; PSTH, peristimulus time histograms; TTX, tetrodotoxin

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ABSTRACT Schizophrenic patients are thought to have an impaired ability to process sensory information.

This deficit leads to disrupted auditory gating measured electrophysiologically as a reduced

suppression of the second of paired auditory-evoked responses (P50), and is proposed to be

associated with decreased function and/or expression of the homomeric α7 nicotinic

acetylcholine receptor (nAChR). Here we provide evidence that N-[(3R)-1-azabicyclo[2.2.2]oct-

3-yl]-4-chlorobenzamide hydrochloride (PNU-282987), a novel selective agonist of the α7

nAChR, evoked whole-cell currents from cultured rat hippocampal neurons that were sensitive

to the selective α7 nAChR antagonist methyllycaconitine (MLA), and enhanced GABAergic

synaptic activity when applied to hippocampal slices. Amphetamine-induced sensory gating

deficit, determined by auditory evoked potentials in hippocampal CA3 region, was restored by

systemic administration of PNU-282987 in chloral hydrate anaesthetized rats. Auditory gating

of rat reticular thalamic neurons was also disrupted by amphetamine, however PNU-282987

normalized gating deficit only in a subset of tested neurons (6 out of 11). Furthermore, PNU-

282987 improved the inherent hippocampal gating deficit occurring in a subpopulation of

anaesthetized rats, and enhanced amphetamine-induced hippocampal theta oscillation. We

propose, that the α7 nAChR agonist PNU-282987, via modulating/enhancing hippocampal

GABAergic neurotransmission, improves auditory gating and enhances hippocampal oscillatory

activity. These results provide further support for the concept that drugs that selectively activate

α7 nAChRs may offer a novel, potential pharmacotherapy in treatment of schizophrenia.

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INTRODUCTION

It is recognized that development of schizophrenia is genetically influenced, and a subset of

genes are predisposing to the illness. Among a number of genetic linkage sites, the homomeric,

α7 nicotinic acetylcholine receptor (α7 nAChR) subunit gene, CHRNA7 has been implicated in

schizophrenia (Stassen et al., 2000; Gault et al., 2003). Thus, a genetic linkage of the 15q13-15

region of chromosome 15 containing CHRNA7 has been established to impaired auditory gating,

a presumed indicator of dysfunctional sensory processing in schizophrenia (Freedman et al.,

1997; Leonard et al., 2002). Deficiency in auditory (P50) gating has been regarded as a

manifestation of an impaired sensory filtering mechanism leading to inefficient sensory

processing and disturbed perception in schizophrenic patients (Light and Braff, 1999; Freedman

et al., 2003; Thoma et al., 2003). Based on the previous clinical observation that nicotine

transiently improves auditory gating in schizophrenics (Adler et al., 1993) and the association

between α7 nAChRs and auditory gating in preclinical models (e.g. Stevens et al., 1998), it has

been proposed that activation of α7 nAChRs would improve sensory processing and thus provide

benefit for positive and/or negative symptoms, or impaired cognitive function in schizophrenic

patients (Stevens et al., 1998; Bodnar et al., 2004; Hajos et al., 2003b; Martin et al., 2004).

We have recently described PNU-282987 (N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-

chlorobenzamide hydrochloride) as a potent and selective α7 nAChR agonist (Bodnar et al.,

2004). This compound showed high affinity for the rat α7 nAChR (Ki = 26 nM) and activity at

the α7-5-HT3 chimera (EC50 = 128 nM) and showed a negligible block of α1β1γδ and α3β4

nAChRs (> 60 µM). In addition, PNU-282987 was found to be inactive at all tested monoamine,

muscarine, glutamate and GABA receptors at 1 µM concentration, except 5-HT3 receptors (Ki =

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930 nM; Bodnar et al., 2004). In the present study, we further evaluated its in vitro

pharmacological characteristics and its action on auditory gating processes. PNU-282987 was

compared to reference α7 nAChR agonists for functional activity using cultured rat hippocampal

neurons and for the ability to modulate GABAergic synaptic activity in isolated rat hippocampal

slices. In order to evaluate the in vivo activity of PNU-282987, auditory gating experiments

were carried out in anaesthetized rats. Physiological gating in the hippocampal CA3 region or

reticular thalamic nucleus (nRT) was disrupted by systemic administration of amphetamine

(Stevens et al., 1996; Krause et al., 2003), and the efficacy of PNU-282987 to reverse the

amphetamine-induced gating deficit was determined. The efficacy of the partial α7 nAChR

agonist GTS-21 (Briggs et al., 1997) was also evaluated in our hippocampal gating model since

GTS-21 has been shown previously to improve the inherent auditory gating deficits in DBA mice

(Stevens et al., 1998) or in isolation reared rats (O’Neill et al., 2003).

It is known that enhanced catecholamine neurotransmission in the hippocampal formation leads

to synchronized activity, i.e. theta oscillation in the hippocampus (Berridge and Foote, 1991;

Hajos et al., 2003a), and pronounced hippocampal theta activity has been demonstrated after

systemic administration of amphetamine (Krause et al., 2003). Since hippocampal theta activity

is thought to be associated with synaptic plasticity and hippocampal-dependent cognitive

processes (Buzsaki 2002; Seager et al., 2002), and cognitive-enhancing compounds have been

shown to augment evoked theta activity (Kinney et al., 1999), possible modulations of

amphetamine-induced hippocampal theta activity by PNU-282987 were also analyzed.

Interestingly, a subset of rats used in the present study (approximately 5%) showed consistent

impairment in hippocampal auditory gating at control measurements. These animals were not

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treated with amphetamine, instead the ability of PNU-282987 and GTS-21 was tested to

normalize their inherent gating deficit.

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METHODS

Cell Isolation and Culture Conditions: Sprague-Dawley rats (postnatal day 3) were killed by

decapitation and their brains were removed and placed in ice cold Hibernate-A medium.

Hippocampal regions were gently removed, cut into small pieces and placed in Hibernate-A

medium with 1 mg/ml papain for 60 min at 35°C. After digestion, the tissues were washed

several times in Hibernate-A media and transferred to a 50 ml conical tube containing 6 ml

Hibernate-A medium with B27 supplement (2%). Neurons were dissociated by gentle trituration

through a series of three 9 inch Pasteur pipettes with decreasing tip diameters. Cells were

purified over a Nycoprep gradient according to the methods of Brewer (1997). Cells were plated

onto poly-D-lysine/laminin coated coverslips at a density of 300 – 700 cells/mm2, allowed to

adhere for 1 hour at room temperature and then transferred to 24-well tissue culture plates

containing warmed culture medium composed of Neurobasal-A medium, B27 supplement (2%),

L-glutamine (0.5 mM), 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.25 mg/ml

Fungizone. Cells were maintained in a humidified incubator at 37°C and 6% CO2 for 1 – 2

weeks. The medium was changed after 24 hours and then approximately every three days

thereafter.

Brain slice isolation: Spague-Dawley rats ranging from postnatal day 16 to 21 were anesthetized

with Halothane, decapitated, and the brains were removed and blocked. The region containing

the hippocampus was sectioned into 350 micron slices (Microslicer, DSK model 1500E) under

ice-cold slicing buffer composed of (in mM): NaCl (130), NaHCO3 (26), NaH2PO4 (1.25), KCl

(3), CaCl2 (0.5), MgCl2 (10), glucose (10), ascorbic acid (0.4), lidocaine (0.2) continuously

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bubbled with a mixture of O2/CO2 (95:5). Slices were warmed slowly to room temperature in the

same bath solution as above but with 1 mM Ca2+ and no lidocaine; the slices were allowed to

recover for at least 1 hour before recording.

Patch-Clamp Electrophysiology: Whole cell currents were recorded using an Axopatch 200B

amplifier (Axon Instruments, Union City, CA). Analog signals were filtered at 1/5 the sampling

frequency, digitized, stored, and measured using pCLAMP software (Axon Instruments). Patch

pipettes were pulled from borosilicate capillary glass using a Flaming/Brown micropipette puller

(P97, Sutter Instrument, Novato, CA) and filled with an internal pipette solution composed of (in

mM): CsCH3SO3 (126), CsCl (10), NaCl (4), MgCl2 (1), CaCl2 (0.5), EGTA (5), HEPES (10),

ATP-Mg (3), GTP-Na (0.3), phosphocreatin (4), pH 7.2. QX314 (4 mM) was included in the

pipette solution for experiments measuring synaptic activity in brain slices. The resistances of

the patch pipettes when filled with internal solution ranged between 3 – 6 MΩ. All experiments

were conducted at room temperature. Cultured cells were continuously superfused with an

external bath solution containing (in mM): NaCl (140), KCl (5), CaCl2 (2), MgCl2 (1), HEPES

(10), glucose (10), pH 7.4. Bicuculline (10 µM), CNQX (5 µM) and tetrodotoxin (TTX, 0.5 µM)

were included in the bath solution to diminish spontaneous synaptic activity. Compounds were

delivered via a multibarrel fast perfusion exchange system (Warner Instrument, Hamden, CT).

For experiments with brain slices, tissue was transferred to a recording chamber superfused with

a recording buffer composed of (in mM): NaCl (130), NaHCO3 (26), NaH2PO4 (1.25), KCl (3),

CaCl2 (2), MgCl2 (1), glucose (10), ascorbic acid (0.4), AP-5 (0.01), CNQX (0.005), saturated

with O2/CO2 (95:5). The recording chamber was mounted on the stage of a Zeiss Axioscope

microscope with IR-DIC optics and water immersion objectives. Slices were continuously

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superfused with the recording buffer at 3 to 4 ml per minute. Either PNU-282987 or DMSO was

applied by bath application; solution exchange was achieved in < 2 min. All data are reported as

mean ± SEM. Statistical analysis was performed with a two-tailed Students t-test for populations

of unequal variance.

Animals and Surgical Procedures: Experiments were performed on male Sprague-Dawley rats

(weighing 250–300 g) in chloral hydrate anesthesia (400 mg/kg, IP), under an approved animal

use protocol and were in compliance with the Animal Welfare Act Regulations (9 CFR parts 1,

2, and 3) and with the Guide for the Care and Use of Laboratory Animals, National Institutes of

Health guidelines. The femoral artery and vein were cannulated for monitoring arterial blood

pressure and administration of test compounds or additional doses of anesthetic, respectively.

The anesthetized rat was placed in a Kopf stereotaxic frame, and unilateral craniotomy was

performed above the regions of the reticular thalamus or CA3 region of the hippocampus. Body

temperature of the rat was maintained at 37o C by means of an isothermal (37o C) heating pad

(Braintree Scientific, Brain-tree, MA). After conclusion of experiments, animals were

euthanized with an IV bolus of chloral hydrate; brains were removed, blocked and frozen for

histological verification of electrode placement.

Hippocampal EEG recordings: Field potentials (electroencephalogram, EEG) were recorded

from the CA3 region of the left hippocampus, 3.8 mm ventral, 3.5 mm posterior, and 3.0 mm

lateral from bregma (Paxinos and Watson 1986), using a monopolar, stainless steel

macroelectrode (Rhodes Medical Instruments, Woodland Hills, CA). Data were digitized and

stored using the Spike2 software package (Cambridge Electronic Design, Cambridge, UK).

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Rhythmic synchronized (theta) and large-amplitude irregular hippocampal activities were

distinguished in the EEG; quantitative EEG analysis was performed by means of Fast Fourier

transformation (Hajos et al., 2003a). Power spectrum density of EEG was calculated between 0

and 12 Hz, and determined in periods of 10 minutes prior or after drug treatment. Theta peak

was defined as the highest power between 3 and 6 Hz. Auditory evoked potentials were

determined by measuring the potential difference between the positive and the negative

deflection 20 and 40 ms after stimulation (P20 and N40), respectively. For quantification, 50

sweeps were averaged, and the amplitude was determined and the ratio of the response after the

second stimulus (test, T) and the first stimulus (conditioning, C) was calculated. This T/C ratio

is used as a measure of sensory (auditory) gating. Statistical significance was determined by

means of two-tailed paired Student’s t-test.

Single unit recordings from reticular thalamic nucleus: Glass microelectrodes filled with 2

mol/L NaCl and 2% pontamine sky blue (impedance 4-10 MΩ) were lowered 5.2–5.6 mm into

the left nRT (3.0 mm posterior and 3.6 mm lateral with respect to bregma), using a hydraulic

microdrive (Kopf Instruments, Tujunga, CA). In order to identify neurons in the auditory sector

of the reticular thalamus, continuous auditory stimulation was presented during electrode

descent. Spontaneously active nRT neurons were recorded extracellularly, and only those

neurons that responded with activation to auditory stimuli were selected for our studies (Krause

et al., 2003). Extracellular signals were amplified, low-pass filtered, and action potentials

discriminated on-line (Neurolog System, Hertfordshire, UK). At the end of each experiment, dye

was deposited iontophoretically from the recording electrode, and location of the electrode tip

was verified by microscopic inspection of slide-mounted and cresyl violet–stained sections.

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Data were digitized, stored, and analyzed using the Spike3 software package. Firing rates and

interspike time interval histograms were determined at baseline and after drug administration.

Raster displays and peristimulus time histograms (PSTH) were constructed from the evoked

responses to auditory stimulation on-line. The number of events (i.e. extracellularly recorded

action potentials) before auditory stimulation and after the conditioning and test stimuli were

determined using PSTHs. The number of events after the test stimulus divided by the number of

events after the conditioning stimulus was called the T/C ratio. Statistical significance was

determined by means of two-tailed paired Student’s t-test.

Auditory Stimulation: Auditory stimulation consisted of two consecutive tone bursts 10 ms

duration at a frequency of 5 kHz. The sound pressure level was 95 dB between the ear bars as

determined with a sound level meter (RadioShack, Fort Worth, TX). Tones were delivered

through hollow earbars. Recording hippocampal auditory gating, delay between the first

“conditioning” stimulus and second “test” stimulus was 0.5 s. Due to the long-lasting activation

of nRT neurons to auditory stimulus, gating of nRT neurons was tested by paired tones with 1 s

interval between conditioning and test stimuli. The time interval between tone-pairs was 10 s for

both hippocampal and nRT recordings.

Experimental design and Drug treatment: Baseline auditory gating was determined by an

average of 50 sequential evoked potentials (hippocampal CA3 region) or PSTH (nRT neurons) in

response to conditioning and test stimuli. Amphetamine (D-amphetamine sulfate, 1 mg/kg, IV)

was administered in order to disrupt sensory gating. Recordings of evoke potentials or PSTHs

commenced 5 min after amphetamine administration, and 4 blocks of 25 evoked potentials were

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computed. Disruption in auditory gating was affirmed if the mean of the last 50 evoked

potentials showed gating deficit equal or exceeding a 0.2 increase in T/C ratio. Auditory gating

measurements started 5 min after IV administration of the drug or vehicle. Levels of auditory

gating (T/C rations) have been determined from means of 50 subsequent evoked potentials at

time intervals between 5 and 15 minutes, as well as between 15 and 30 minutes after drug or

vehicle administration. In addition, auditory gating was calculated from all 100 evoked

potentials after drug or vehicle treatment.

Materials: Cell culture reagents were purchased from Life Technologies. (-)-Nicotine tartrate

salt, papain, bicuculline methiodide, CNQX, D-amphetamine sulfate and terodotoxin (TTX) with

citrate buffer were purchased from Sigma. MLA was purchased from Research Biochemicals.

PNU 282987 (N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride) and

GTS-21 (DMXBA; 3-[(2,4-Dimethoxy)benzylidene]-anabaseine dihydrochloride) were obtained

from the Medicinal Chemistry Department, Pfizer, Inc. (Kalamazoo, MI). For auditory gating

experiments, compounds were dissolved in Phosphate Buffer Saline (BPS) based upon their salt

weights and the concentrations were adjusted so that injection volumes equaled 1ml/kg body

weight. Control animals received PBS.

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RESULTS

Activation of α7 nAChRs on cultured rat hippocampal neurons by PNU-282987 and

reference agonists.

Examples of whole-cell currents evoked by the α7 nAChR agonists nicotine, GTS-21 and PNU-

282987 are shown in Fig. 1A. Agonists were applied for 1 s once every 30 s at a series of

concentrations. Because hippocampal neurons express varying levels of functional α7 nAChRs,

the nonselective agonist (-)-nicotine (100 µM) was applied to each cell to normalize the data for

comparisons between cells. In addition, because multiple nicotinic receptor subtypes are

expressed by these neurons (e.g., Alkondon and Albuquerque, 1993), nicotine-evoked currents

were recorded in the absence and presence of the selective α7 nAChR antagonist

methyllycaconitine (MLA). To minimize the influence of other receptor subtypes, cells were

included in this study only if the current evoked by nicotine was inhibited >90% by 10 nM

MLA. As illustrated in Fig. 1B, some cells did express a small but measurable amount of

nicotine-evoked current that was resistant to MLA, reflecting the fraction of current mediated by

non-α7 nAChRs (traces shown in Fig. 1B were recorded from the same cell as those shown in

the third row in Fig. 1A; peak nicotine-evoked currents were –289 pA and -19 pA in the absence

and presence of MLA, respectively). In contrast, the current evoked by PNU-282987 was

completely inhibited by 10 nM MLA in every cell tested, even those that had a MLA-resistant

component to the nicotine response (e.g., Fig. 1B). These results suggest that PNU-282987

activated only MLA-sensitive or α7-containing nAChRs on the cell soma and/or proximal

dendrites. The concentration-response of the three compounds are shown in Fig. 1C normalized

to the peak current evoked by 100 µM nicotine.

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PNU-282987 elevates spontaneous GABAergic synaptic activity in hippocampal slices.

Previous work has demonstrated that within the rat hippocampus, nAChRs are predominantly

expressed on GABAergic interneurons and that activation of those receptors modulates

GABAergic synaptic activity (Alkondon et al., 1997; Jones and Yakel, 1997; Köfalvi et al.,

2000; Ji and Dani, 2000). We therefore evaluated the ability of PNU-282987 to modulate

GABAergic synaptic activity in acutely isolated rat hippocampal slices. Spontaneous

GABAergic synaptic events were recorded from CA1 pyramidal neurons for 3 to 10 min. under

baseline conditions and then for an additional 10 min. in the presence of either vehicle (0.1%

DMSO) or PNU-282987. Bath application of 30 nM and 300 nM PNU-282987 more than

doubled frequency of synaptic activity in about half the cells tested (3 of 6 cells and 5 of 11 cells

for 30 nM and 300 nM, respectively), but the average change in frequency was significantly

different from the vehicle control only for cells treated with 300 nM PNU-282987 (p = 0.002,

Fig. 2). No clear effect was observed with 1 µM PNU-282987, possibly reflecting the

desensitization of α7 nAChRs during the 10 min. treatment.

Effects of nAChR agonists on auditory gating in anaesthetized rats

Auditory Gating in the Hippocampus

Hippocampal field potential recordings revealed evoked responses to auditory stimulation in

anesthetized rats. Auditory gating, expressed as the ratio between evoked potentials to paired

conditioning (C) and testing (T) stimuli was determined at baseline by an average of 50

subsequently evoked potentials. Systemic administration of amphetamine (1 mg/kg, IV)

disrupted auditory gating in the majority of the treated rats, as indicated by a significant increase

of the T/C ratio (Figs. 3 & 4A, B). The increase in T/C ratio was due both to an increase in

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amplitude in response to the test stimuli and a decrease in amplitude in response to the

conditioning stimuli (Fig. 3 & 4C). Because the absolute level of disruption induced by

amphetamine was somewhat variable, only rats showing ≥ 0.2 increase in the T/C ratio

(approximately 70% of tested animals) were used for subsequent evaluation of α7 nAChR

agonists or vehicle. In addition, rats with appropriate auditory gating displayed average

amplitudes of conditioning evoked potential over 100 µV, providing excellent signal/noise ratio

for evaluating parallel changes in amplitudes of evoked potentials to conditioning and test

stimuli induced by drug treatments.

Administration of vehicle (PBS, 1 ml/kg, IV, n=6) did not alter amphetamine-induced gating

deficit as determined from the average of 50 evoked potentials measured between 5 to 15

minutes after vehicle application (Fig 4A). Disrupted auditory gating prevailed for at least 30

minutes following amphetamine administration, as indicated by a significant increase in T/C

ratio calculated over this time period from 100 evoked potentials (T/C; 0.59 + 0.11; p < 0.02 vs.

control). In contrast, administration of PNU-282987 (1 mg/kg, IV, n=6) significantly restored

auditory gating (Fig. 4B; determined from the average of 50 evoked potentials), by reversing the

action of amphetamine on the amplitude of evoked potentials, particularly on test stimuli (Fig.

4C). Significant drug action was also established when the degree of auditory gating was

calculated from 100 evoked potentials (T/C; 0.37 + 0.07; p < 0.03 vs. amphetamine). The partial

α7 nAChR agonist GTS-21 also reversed amphetamine-induced gating deficit: T/C values were

0.14 + 0.04 at baseline, 0.48 + 0.03 after amphetamine (1 mg/kg, IV; p < 0.005) and 0.09 + 0.05

after subsequent administration of GTS-21 (1 mg/kg, IV; p < 0.005, vs. amphetamine, n=4).

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In agreement with our previous observations (Krause et al., 2003), administration of

amphetamine resulted in synchronization of hippocampal EEG (Fig. 5). Quantitative EEG

analysis showed a significant increase in EEG power resulting in a peak frequency of 4.4 + 0.1

Hz (baseline value: 1.6 + 0.2 Hz; p < 0.0001; n=12), thereby indicating an increased synchrony

in the theta frequency range (Figs. 5 & 6). Interestingly, amphetamine elicited pronounced

hippocampal theta activity irrespective of its effect on auditory gating, indicating different

mechanisms involved in these two pharmacological responses. Subsequent administration of

vehicle (PBS, 1 ml/kg, IV) or PNU-282987 (1 mg/k, IV) did not alter peak frequency of

hippocampal EEG (data not shown) however PNU-282987 significantly enhanced theta power

(Fig. 6).

Although most of chloral hydrate anaesthetized rats showed normal auditory gating

(characterized by a T/C ration lower than 0.2), approximately 5% of rats displayed persistent

auditory gating deficits (monitored by blocks of subsequent averages of 25 evoked potentials)

with a T/C ratio > 0.5 under baseline conditions. Administration of the α7 nAChR partial

agonist GTS 21 (1 mg/kg, IV, n=4) or the α7 nAChR agonist PNU-282987 (1 mg/kg, IV, n=4)

significantly improved auditory gating in these rats (Fig. 7).

Auditory Gating in the Thalamic Reticular Nucleus

Reticular thalamic neurons responded to auditory stimuli with a typical discharge of bursts of

action potentials (n =11 neurons from 11 rats; Fig. 8). Auditory evoked activity of reticular

thalamic neurons showed oscillations at 7–12 Hz, with each burst comprising ~6 action

potentials, as it has been described previously (Krause et al., 2003). The number of evoked

potentials was determined in 800 ms post-stimulus interval after conditioning and test stimuli,

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and the ratio between the number of spikes after test stimuli and the number of spikes after the

conditioning stimuli represented auditory gating (Krause et al., 2003). Administration of

amphetamine (1 mg/kg, IV, n=11) disrupted auditory gating in each tested neuron (Fig. 8).

Subsequent administration of the α7 nAChR agonist PNU-282987 (1 mg/kg, IV) restored

auditory gating in about half of reticular thalamic neurons (n=6 out of 11; Fig 8B,C). As has

been reported previously (Krause et al., 2003), amphetamine changed the firing pattern of

reticular thalamic neurons from burst firing to single-spike firing mode (Fig. 8A).

Administration of PNU-282987 did not reverse amphetamine-induced firing pattern change in

reticular thalamic neurons (n=11).

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DISCUSSION

Previous studies have shown that within the rat hippocampus α7 nAChRs are expressed

predominantly on GABAergic interneurons where they function to modulate inhibitory synaptic

transmission (Alkondon et al., 1997; Jones and Yakel, 1997; Köfalvi et al., 2000; Ji and Dani,

2000). Impaired function of these interneurons, due in part to decreased expression of α7

nAChRs, has been proposed to contribute to the neuropathology of schizophrenia (Freedman et

al., 2000). Thus, activation of α7 nAChRs by selective agonists could provide an effective

therapy for treating the cognitive deficits of schizophrenia (e.g. Levin and Rezvani, 2002). We

recently reported that PNU-282987 is a potent and selective agonist of human and rat α7

nAChRs (Bodnar et al., 2004). When applied to cultured rat hippocampal neurons, PNU-282987

evoked MLA-sensitive currents that were readily detectable when briefly applied at

concentrations ≥ 300 nM or approximately 30-fold lower than that required for either nicotine or

GTS-21 (Fig. 1). It should be noted however that while these results provide good evidence that

PNU-282987 selectively activated α7-containing nAChRs on the cell body and/or proximal

dendrites, they do no exclude the possibility that PNU-282987 activated MLA-resistant currents

in the axon terminals. The effects of prolonged application of PNU-282987 on GABAergic

synaptic transmission was evaluated in acutely isolated rat hippocampal slices. In agreement

with the reported role of the α7 nAChR in the hippocampus, bath application of 30 nM and 300

nM PNU-282987 increased the frequency of synaptic events by >2-fold in about half the cells

tested although the average effect was significant only for the 300 nM group, and no clear effect

was observed at the highest tested concentration of 1 µM. These results suggest that 300 nM

PNU-282987 activated a sufficient number of receptors to produce a measurable change in

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synaptic activity and that a balance was achieved between receptor activation and receptor

desensitization that allowed for a relatively long lasting response. The high cell to cell

variability observed with 30 nM and 300 nM PNU-282987 likely reflects both inhibitory and

disinhibitory actions produced by excitation of multiple hippocampal interneurons within the

circuit influencing the recorded pyramidal cell (Ji and Dani, 2000).

In order to analyze in vivo activity of α7 nAChR agonists, auditory gating experiments were

carried out in anaesthetized rats. Physiological auditory gating was disrupted by amphetamine

since impaired hippocampal gating is well demonstrated following systemic administration of

amphetamine (Stevens et al., 1996; Krause et al., 2003). Impairment of gating was apparent as a

result of a significant decrease in amplitude of evoked potentials to conditioning stimuli, and a

significant increase in amplitude of evoked potentials to test stimuli, leading to an increased T/C

ratio. Since dopamine D2 receptor antagonists reverse the amphetamine-induced gating deficit,

it has been proposed that enhanced dopamine neurotransmission results in disrupted gating

(Stevens et al., 1996; Krause et al., 2003). Furthermore, enhanced catecholamine

neurotransmission by amphetamine (Light et al., 1999) or cocaine (Adler et al., 2001) leads to

impaired gating in humans. Interestingly, it has been demonstrated that amphetamine- or

cocaine-induced gating deficit can be reversed not only with D2 antagonists, but with nicotine or

nicotinic agonists as well (Stevens et al., 1995; Stevens et al., 1999; Adler et al., 2001),

presumably interacting with inhibitory neuronal circuitry involved in gating, i.e. GABAergic

interneurons in the hippocampus (Stevens et al., 1999; Freedman et al., 2000). Subsequent

experiments indicated a key role for the α7 nAChR in nicotine-induced improvement in auditory

gating and in gating mechanisms in general. It was shown that α7 nAChR stimulation

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normalizes chronic cocaine-induced loss of hippocampal sensory inhibition in C3H mice

(Stevens et al., 1999). Furthermore, inherently impaired auditory gating in DBA/2 mice was

normalized by GTS-21 (1 to 10 mg/kg, SC.; Stevens et al., 1998). Our current findings provide

further evidence that α7 nAChR agonists can normalize abnormal auditory sensory gating, since

we confirmed the efficacy of the partial agonist GTS-21, and demonstrated that the structurally

distinct, highly selective and potent PNU-282987 reversed amphetamine-induced gating deficit.

Interestingly, a subset of rats in our experiments showed a gating deficit at baseline

measurements. Although it is unclear what mechanisms contributed to this pathological gating,

it was normalized by both GTS-21 and PNU-282987. Recently it has been reported that social

isolation (O’Neill et al., 2003) or early maternal deprivation (Ellenbroek et al., 2004) can also

impair sensory gating in adult rats suggesting that early life events such as stress could contribute

to gating abnormality. Similar to our current finings, GTS-21 can normalize auditory gating

deficits in isolation-reared rats (O’Neill et al., 2003).

Systemic administration of amphetamine disrupted auditory gating in nRT neurons as we

reported previously (Krause et al., 2003). Although PNU-282987 reversed hippocampal gating

deficit in all amphetamine-treated rats, it reversed gating deficit only in half of the tested nRT

neurons. The reason for the heterogeneous response of the nRT neurons is presently unknown,

but could reflect heterogeneity in expression of α7 nAChRs by nRT neurons, or a disparity in the

synaptic input/circuit connectivity of nRT neurons. Although within the human thalamus the

highest alpha-bungarotoxin binding, reflecting α7 nAChR expression has been localized in nRT

(Spurden et al., 1997), recent publication on rat brain nAChRs indicates a predominant presence

of heteromeric nAChRs (labeled with epibatidine) in the thalamus, including nRT (Tribollet et

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al., 2004). In addition, PNU-282987 did not modify amphetamine-induced changes in firing

pattern characteristics, in contrast to the D2 antagonists, haloperidol (Krause et al, 2003).

In line with our previous findings, amphetamine not only disrupted auditory gating in

anaesthetized rats, but also induced a slow rhythmic activity in the hippocampal EEG, with a

significant increase in theta power and frequency (Krause et al, 2003). Subsequent

administration of the selective α7 nAChR agonist PNU-282987 further enhanced hippocampal

rhythmic activity as revealed by a significant increase in theta power. In contrast, administration

of vehicle, (or the D2 antagonist haloperidol, Krause et al., 2003) did not heighten theta activity,

although haloperidol normalized amphetamine-induced gating deficit. The fact that PNU-

282987 further synchronized hippocampal activity, and significantly augmented theta power

could be a contributing mechanism to pro-cognitive actions of α7 nAChR agonists described

recently both in animal models (Van Kampen et al., 2004; Young et al., 2004) and humans

(Kitagawa et al., 2003).

In conclusion, the highly selective and potent α7 nAChR agonist PNU-282987 enhances

GABAergic synaptic activity in the hippocampus in vitro, and reverses amphetamine-induced

auditory gating deficit in anaesthetized rats. In addition, PNU-282987 improves the inherent

gating deficit observed in a subset of rats, and enhances amphetamine-induced hippocampal

theta activity. These results support the concept that α7 nAChR agonists represent a novel,

potential pharmacotherapy in treatment of schizophrenia.

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FOOTNOTES

Address correspondence to: Mihály Hajós, PharmD, PhD, Neuroscience Department, Pfizer

Global Research and Development, Eastern Point Road, MS 8220-4083, Groton, CT 06340,

USA. E-mail: [email protected]

1 Current address: Baylor College of Medicine, Division of Neuroscience, Houston, TX 77030 2 Current address: MPI-CardIon Laboratories, Kalamazoo, MI 49008

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LEGENDS FOR FIGURES

Figure 1

Agonist-activation of nAChRs on cultured rat hippocampal neurons. A. Whole-cell currents

evoked by 1 s applications of nicotine (100 µM) and three concentrations of either nicotine

(upper row), GTS-21 (middle row) and PNU-282987 (lower row). Sequential agonist challenges

were separated by a 30 s wash-out period. Traces shown on any row were all recorded from the

same cell. B. Example of currents evoked by nicotine (100 µM) and PNU-282987 (30 µM) in

the presence of 10 nM MLA. Both traces were recorded from the same cell as the traces shown

in the third row of panel A. C. Concentration-response relationships for nicotine, GTS-21 and

PNU-282987. Data points represent the peak current evoked by the indicated concentration of

the test compound normalized to the peak current evoked by 100 µM nicotine from the same cell.

Figure 2 PNU-282987 produces a long-lasting enhancement of GABAergic synaptic activity in

hippocampal slices. A. Example of synaptic events recorded under baseline conditions (left)

and in the presence of 300 nM PNU-282987 (right). B. Summary of change in frequency of

spontaneous synaptic activity relative to baseline for 0.1% DMSO (vehicle) and PNU-282987

(30, 300, 1000 nM). The mean change in synaptic activity was evaluated by comparing the

activity measured during a 3 – 10 min. baseline period to the activity measured during 10 min.

treatement with the vehile or PNU-282987 from the same cell. The mean change in frequency

was as follows: -8% ± 9% (n=10) for 0.1% DMSO, 143 % ± 65% (n=6) for 30 nM PNU-282987,

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103% ± 28% (n=11) for 300 nM PNU-282987, and –13% ± 30% (n=6) for 1000 nM PNU-

282987.

Figure 3

Typical hippocampal auditory evoked potentials (summation of 50 subsequent evoked potentials)

in response to conditioning and test stimuli (intertone interval 0.5 s) in control conditions, after

administration of Amphetamine (AMP, 1.0 mg/kg, IV) and following a subsequent

administration of (A) phosphate buffered saline (PBS) or (B) PNU-282987 (1 mg/kg, IV).

Figure 4

Hippocampal auditory gating expressed as a ratio between evoke potential amplitudes (n=50) to

test and conditioning stimuli (T/C ratio). Administration of amphetamine (1.0 mg/kg, IV)

disrupted auditory gating indicated by an increase in T/C ration. Following a subsequent

administration of vehicle (PBS, 1 ml/kg, IV, n=6) auditory gating remained disrupted (A).

Administration of PNU-282987 (1 mg/kg, IV) restored auditory gating (n = 6; p < 0.01; B).

Amplitudes of hippocampal evoked potentials: Amphetamine-induced decrease in the amplitude

of the conditioning response and an increase in the amplitude of the test response were reversed

by PNU-282987 (C).

Figure 5

Effects of amphetamine and PNU-282987 on rhythmic activity in the hippocampal

electroencephalogram (EEG). Hippocampal EEG (left) and power spectra (right) under control

conditions, after administration of amphetamine (1.0 mg/kg, IV), and after subsequent

administration of PNU-282987 (1 mg/kg, IV). Amphetamine induced a slow rhythmic activity

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in the hippocampal EEG in the theta frequency range, indicated by an increase in power between

3 and 6 Hz. The power of the rhythmic theta activity was enhanced after administration of PNU-

282987.

Figure 6

Summary graph showing changes in EEG power at peak theta frequency after amphetamine and

a subsequent administration of either vehicle (PBS, 1 ml/kg, IV, n=7) or PNU-282987 (1 mg/kg,

IV, n=5).

Figure 7

Effects of the α7 nAChR partial agonist GTS-21 and full agonist PNU-282987 on auditory

gating in rats showing inherent auditory gating deficit. Both compounds improved gating as

indicated by a significant reduction in T/C ratio.

Figure 8

A: Typical recordings from a single unit in the reticular nucleus of the thalamus showing

auditory gating.

Control: Distribution of spikes over a period of 24 stimulations (upper panel) and post stimulus

time histogram (lower panel, bin size 2 ms) after conditioning I and test pulse (T) of a single unit

recorded in the reticular nucleus of the thalamus.

Amphetamine: Administration of amphetamine (1 mg/kg, IV) reduces the number of spikes

after the conditioning stimulus and increases the number of spikes after the test stimulus. At the

same time burst firing is abolished and the unit fires in a phasic fashion

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Amphetamine + PNU-282987: Subsequent administration of the α7 nAChR agonist PNU-

282987 (1.0 mg/kg, IV) restores amphetamine-induced gating deficit. Note that amphetamine-

induced tonic-firing mode is still prevailing.

B: Summary graphs for auditory gating in single units in the reticular thalamic nucleus when

PNU-282987 restored amphetamine-induced gating deficit (n = 6 animals). Gating is expressed

as a ratio of number of spikes after test pulse and conditioning pulse. Averaged T/C ratios after

administration of amphetamine (AMP, 1.0 mg/kg, IV) were significantly higher from control

condition; PNU-282987 (PNU282, 1.0 mg/kg, IV) significantly reversed this effect.

C: Summary graphs for auditory gating in single units in the reticular thalamic nucleus when

PNU-282987 failed to restore amphetamine-induced gating deficit (n = 5 animals). Gating is

expressed as a ratio of number of spikes after test pulse and conditioning pulse. Averaged T/C

ratios after administration of amphetamine (AMP, 1.0 mg/kg, IV) or amphetamine + PNU-

282987 (1 mg/kg, IV) were significantly different from control condition.

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0

30

60

90

120

150

180

0.1 1 10 100Concentration (µM)

Res

po

nse

(%

Nic

oti

ne) PNU-282987

Nicotine

GTS-21

PNU-282987 (0.3, 3, 30 µM)

Nicotine (10, 30, 100 µM)

GTS-21 (1, 10, 100 µM)

A.

B.

NIC (100 µM)

NIC (100 µM)

NIC (100 µM)

C.

Nicotine (100 µM) PNU-282987 (30 µM)

+ MLA (10 nM)

20 pA

1 s

1 s

100 pA

1 s

100 pA

1 s

100 pA

Fig.1.

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Baseline PNU-282987 (300 nM)

2 s

50 pA

b0725

Syn

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(% c

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om b

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DM

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)

PNU-282987 (nM)

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B.

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100

200

300

400

30 300 1000

Fig.2.

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100 ms

100 µ

V

Control AMP (1mg/kg) AMP & 282987 (1mg/kg)

Conditioning Response

100 ms

100 µ

V10

0 µV

Conditioning Response

B

100 ms

100 µ

VControl AMP (1mg/kg) AMP & PBS (1ml/kg)

Conditioning Response

100 ms

100 µ

V

Conditioning Response

A

Fig. 3

Test Response

Test Response

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0.00

0.20

0.40

0.60

0.80T

/C R

atio

ControlAMPAMP & PBS

**

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A

0.00

0.20

0.40

0.60

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Rat

io

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#

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# p < 0.01 vs. AMP

B

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100

150

200

250

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* p < 0.05 vs. Control

# p < 0.05 vs. AMP

*

*

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ConditioningTest

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Control

Amphetamine

Amphetamine + PNU-282987

12

8

4

12

8

4

Pow

er, µ

V2 x

103

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12

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Fig. 5

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0

5

10

15

20

25

PBS (1ml/kg, n=7) 282987 (1 mg/kg, n=5)

The

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* p < 0.05 vs. Control

# p < 0.05 vs. AMP

*

*

*

* #

Fig. 6

Page 41: The Selective 7 Nicotinic Acetylcholine Receptor Agonist ... · This deficit leads to disrupted auditory gating measured electrophysiologically as a reduced suppression of the second

0

0.2

0.4

0.6

0.8

T/C

Rat

io

*

*

Control

Drug (1.0 mg/kg, IV)

* p < 0.05 vs. Control

GTS-21 PNU-282987

Fig. 7

Page 42: The Selective 7 Nicotinic Acetylcholine Receptor Agonist ... · This deficit leads to disrupted auditory gating measured electrophysiologically as a reduced suppression of the second

20100

5

00 1 2 3

# S

pike

s#

Stim

ulat

ions

C T#

Spi

kes

20100

5

00 1 2 3

# S

timul

atio

ns

AControl

20100

5

00 1 2 3

# S

pike

s#

Stim

ulat

ions

Amphetamine

Amphetamine + PNU-282987

Time [s]

C

0.3

0.6

0.9

1.2

1.5

T/C

0.3

0.6

0.9

1.2

1.5

Control AMP PNU282

p < 0.05

B

T/C

Control PNU282AMP

p < 0.050.05 p 0.01p 0.01

0.3

0.6

0.9

1.2

1.5

0

<

Fig. 8