Gen. Pharmac. Vol. 29, No. 3, pp. 453-456, 1997 Copyright 0 1997 Elsevier Science Inc. Printed in the USA.

ELSEVIER

ISSN 0306-3623197 $17.00 + .OO PI1 SO306-3623(96)00483-l

All rights reserved

Effects of Tyr-D-Arg-Phe-P-Ala-NH2, a Novel Dermorphin Analog, on Elevated Plus-Maze Learning

and Spontaneous Alternation Performance in Mice

Makoto Ukai,‘” Jun Monrna,’ Norihiro Shinkai,’ Yusuke Susaki2 and Tsutomu Kumeyumul

‘DEPARTMENT OF CHEMICAL PHARMACOLOGY, FACULTY OF PHARMACEUTICAL SCIENCES, MEIJO UNIVERSITY, NAGOYA 468, JAPAN AND ‘DEPARTMENT OF BIOCHEMISTRY, TOHOKU

COLLEGE OF PHARMACY, SENDAI 981, JAPAN [TEL: +81-52-832-1781; FAX: +81-52-834-87801

ABSTRACT. 1. The effects of intracerebroventricular administration of Tyr-o-Arg-Phe-B-Ala-NH2

(TAPA), a novel dermorphin analog, on plus-maze learning and spontaneous alternation performance

were investigated in mice. 2. The pre- or posttraining or preretention administration of TAPA (0.3-3.0 ng) alone failed to

affect transfer latency of plus-maze learning, whereas TAPA (3 ng) produced a significant decrease in

percent alternation without affecting total arm entries.

3. B-Funaltrexamine (5 Pg) almost completely reversed the TAPA (3 ng)-induced decrease in per-

centage of alternation.

4. These results suggest that stimulation of P-opioid receptors disrupts spontaneous alternation

performance associated with spatial working memory. GEN PHARMAC 29;3:453456, 1997. 0 1997

Elsevier Science Inc.

KEY WORDS. Try-o,Arg-Phe-B-Ala-NH2, k,-opioid receptor, spontaneous alternation performance,

elevated plus-maze learning, mouse

INTRODUCTION

It has been reported that opioid neuronal systems are involved in

memory processes. For example, 8-endorphin and enkephalins im-

pair memory processes,, whereas naloxone facilitates it (Castellano

and Pavone, 1985; Izquierdo, 1980; Izquierdo and Netto, 1985; Iz-

quierdo et al., 1985; Rigter et al., 1979).

Spontaneous alternation performance, a natural tendency to ex-

plore a less recently visited arm in a Y-maze, should be based on

working memory and is impaired by drugs having amnesic properties

such as scopolamine, morphine and MK-801 (Parada-Turska and

Turski, 1990; Sarter et al., 1988; Stone et al., 1991). Itoh et al.

(1994) have reported that the k,-opioid receptor agonist [n-Ala2,

NMePhe4,Gly-ollenkephalin (DAMGO) impairs spontaneous alter- nation performance. Mbreover, Tyr-n-Arg-Phe-B-Ala-NH* (TAPA),

a recently introduced novel dermorphin analog, has much higher se-

lectivity and affinity for p=opioid receptors than does DAMGO (Sa-

saki et al., 1991), indicating that TAPA is one of the rigorous phar- macological tools available for determining the role of k.-opioid

receptors in the brain. The pre- or posttraining administration of

TAPA (0.3 and/or 3 ng) has been demonstrated to impair retention

performance 24 hr after training of a passive avoidance response

(Ukai et al., 199310, 1995a). The impairing effects of TAPA on a

passive avoidance response are reversed by the F.-selective opioid re-

ceptor antagonist B-funaltrexamine. However, to date, the effects of TAPA on memory have not been evaluated in detail in learning

tasks other than passive avoidance response.

Consequently, the present study was designed to examine the ef- fects of intracerebroventricular injection of TAPA on spontaneous

*To whom correspondence should be addressed. Received 10 April 1946; revised September 1996; accepted 10 October

1996.

alternation performance and elevated plus-maze learning in which

the transfer latency on retention trial is a measure of learning and

memory in mice (Itoh et al., 1990).

METHODS

Animals

Male ddY mice (Nihon SLC Co. Ltd., Japan), weighing between 30

and 35 g, were used. The animals were housed in standard plastic

cages in a temperature-controlled room (23~ 1°C). Food and water

were freely available and a 12-hr light-dark cycle was set. The mice

were kept at least 5 days in home cages before starting experiments.

The behavioral experiments were conducted between 13:00 and

17:00 in a sound-attenuated room.

Drugs

TAPA, which was synthesized by a solid-phase method (Sasaki et

al., 1991), and B-funaltrexamine (Research Biochemicals Inc., Na-

tick, MA, USA) were used. TAPA was dissolved in sterile isotonic

saline in polypropylene containers. The injection was made with a

4-mm long needle (30 gauge) attached to a 50-~1 microsyringe

(Hamilton Co., Reno, NV, USA), according to the method of Ha-

ley and McCormick (1957).

Elevated plus-me

1. Apparatus. The plus-maze was made of plywood and consisted of

two open arms (25 ~8 cm) and two enclosed arms (25 X8X 20

cm). The arms extended from a central platform (8 X 8 cm). The

plus-maze was elevated to a height of 50 cm above the floor. The

open arms and the central platform were painted white and

454

the enclosed arms were painted black. A white fine line was drawn in the middle of the floor of each enclosed arm.

2. Procedure. The procedure of the plus-maze test was identical with that described in a previous report (Itoh et al., 1990). On the first

trial (training), a mouse was placed at the end of one open arm facing away from the central platform, and the time it took for the mouse to move from the open arm to either of the enclosed

arms (transfer latency) was recorded. If the mouse did not enter

the enclosed arm within 90 set, it was pushed gently on the back

into the enclosed arm and transfer latency was assigned to 90 sec.

Then the mouse was gently taken out of plus-maze 10 set after it entered the enclosed arm and was returned to its home cage.

Twenty-four hours later, the second trial (retention test) was

performed. The mice were again put into the plus-maze, and transfer latency was recorded up to a maximum of 90 sec.

Spontaneous alternation

1.

2.

Apparatus. A black painted Y-maze made of plywood was used. Each arm was 40 cm long, 12 cm high, 3 cm wide at the bottom

and 10 cm wide at the top and positioned at an equal angle.

Procedure. The testing procedure was based upon that of Sarter

et al. (1988). Each mouse was placed at the end of one arm and allowed to freely move through the maze for an 8-min test ses-

sion. The maximum number of alternations was then the total

number of arms entered minus two, and the percent alternation

was calculated as (actual alternations/maximum alternations) X 100. For example, if the three arms were called A, B and C and

a mouse consecutively entered arms in the sequence ACBA- BACBAB, its performance would exhibit five alternations

(ABC, CBA, BAC, ACB and CBA) out of eight (10-2) possible

alternations, resulting in a percent alternation of 62.5. In the

present study, mice that entered arms less than eight times during

the test were eliminated, because the data obtained from these

mice were not considered to reflect precise alternation.

Statistical analysis

All of the results were expressed as the means?SEM. All of the data

were analyzed by Kruskal-Wallis analysis of variance by ranks. If there were significant H values, post hoc comparisons were made by

using nonparametric Bonferroni’s multiple comparison test (two-

tailed). The criterion for statistical significance was PcO.05 in all

statistical evaluations.

RESULTS Effects of TAPA on plus-me learning

TAPA (0.3-3.0 ng) had no effects on transfer latency of plus-maze learning when administered 15 min before training (Kruskal-Wallis

analysis: H=4.47, P>O.OS; Fig. lA), immediately after training (Kruskal-Wallis analysis: H=4.62, P~0.05; Fig. 1B) or 15 min be- fore retention (Kruskal-Wallis analysis: H=2.44, P>O.O5; Fig. 1C).

Effects of TAPA un spontaneous alternation performance

TAPA (0.3 and 1.0 ng) did not affect percent alternation, but a higher dose (3 ng) of the peptide significantly depressed percent al- ternation (Kruskal-Wallis analysis: H=12.83, P<O.Ol; Fig. 2). In contrast, TAPA (0.3-3.0 ng) failed to produce any effects on total arm entries (Kruskal-Wallis analysis: H=8.79, P>O.OS; Fig. 2).

A

B

C

0

Control 0.3 1 3

TAPA (ng/mouse)

FIGURE 1. Effect of TAPA on transfer latency to enclosed arm of elevated plus-maze in mice. Each value represents the mean?SEM. TAPA was given to mice at different points in time: (A) 15 min before training; (B) immediately after training; (C) 15 min before retention. The number of mice used is given in paren- theses.

Efjects of f3+maltrexamine

TAPA (3 ng) again decreased percent alternation, whereas B-funal- trexamine (5 p_g) alone had no significant effects on percent alter- nation (Fig. 3). B-Funaltrexamine (5 pg) almost completely re- versed the effects of TAPA (3 ng; Kruskal-Wallis analysis: H= 14.44, PcO.05; Fig. 3). There were no significant changes in to- tal arm entries (Kruskal-Wallis analysis: Hc4.67, P>O.OS; Fig. 3).

DISCUSSION

Cholinergic neurons projecting from the nucleus basalis of Meynert (nbM) to cerebral cortex degenerate in the brain of Alzheimer dis- ease patients, whereas p,-opioid-receptor binding has been reported to decrease in the brain of Alzheimer disease patients (Hiller et al., 1987). Moreover, lesioning of the nbM produces a significant de- crease in p-opioid-receptor binding of the rat cerebral cortex re- sulting from excessive stimulation of CL-opioid receptors (downregu- lation)(Ofri et al., 1992) and elicits amnesia (Ukai et al., 1993a). It thus appears that the disorder of p,-opioid neuronal systems is in- volved in cognitive malfunctions.

Although spontaneous alternation performance may be con- cerned with some psychological factors, such as perseveration, atten-

CL-Opioid Receptor Agonist and Memory

40

30

*E 20

5

E b

z IO

E

0 Control 0.3

TAPA (ng/mouse)

FIGURE 2. Effect of TAPA on spontaneous alternation perfor- mance in mice. Each value represents the mean+SEM. TAPA

was given to mice 15 min before measurement. The number of

mice used is given in parentheses. **P<O.Ol versus control.

tion and decision-making, the performance has also been used for

assessing cognitive function associated with spatial working mem-

ory, because it is impaired by drugs with amnesic properties, such as

scopolamine, morphine and MK-80 1 (Parada-Turska and Turski, 1990; Sarter et al., 1988; Stone et al., 1991). Itoh et nl. (1994) have

recently reported that DAMGO impairs spontaneous alternation

performance without affecting total arm entries. The effects of DAMGO are reversed,by 6-funaltrexamine, a k-opioid receptor an-

tagonist. Therefore, it is possible that p-opioid receptor agonists

elicit amnesia. TAPA has much higher selectivity and affinity for

p,-opioid receptors than does DAMGO, indicating that TAPA is a rigorous tool for assessing the functional role of p,-opioid receptors.

TAPA is active in a passive avoidance response (Ukai et al., 1993b,

1995a). For example, TAPA (0.3 and 3.0 ng) shortens step-down latency of a passive avoidance response when administered before or

immediately after training. The effective doses (0.3 and 3.0 ng) of

TAPA are lower than those (10 and 30 ng) of DAMGO. @-Funal-

trexamine (5 p,g) antagonizes the effects of TAPA on this passive avoidance response, indicating that the effects of TAPA are medi- ated by CL-opioid receptors. However, TAPA (0.3-3.0 ng) was inac-

80 -

.g 70 -

iij E aJ

% 80-

E

8 5

a 50-

40 -

30

8 ‘C

F

20

!ii

3 P 10

0

i

i (16)

Control

TAPA (ng/mouse) 0 5 5

f3-FNA @g/mouse)

FIGURE 3. Effects of TAPA and its combination with p-funal-

trexamine (R-FNA) on spontaneous alternation performance in mice. Each value represents the mean?SEM. TAPA and P-FNA

were given to mice 15 min and 24 hr before measurement, respec-

tively. The number of mice used is given in parentheses. *P<O.O5 versus controls; ##P<O.Ol versus TAPA alone.

tive in the elevated plus-maze learning tests when administered at

different points in time relative to the learning and retention trials.

Additionally, the doses of TAPA used seem to be appropriate, be-

cause doses larger than 3 ng should affect different behavioral re-

sponses (Ukai et al., 199315, 1995a). Although the passive avoidance

response and elevated plus-maze learning are considered to be based on long- rather than short-term memory (Itoh et al., 1993), the ef-

fects of TAPA may depend on the behavioral tests used. Similar findings have been observed in the effects of galanin, which evokes

memory dysfunction in a passive avoidance response, but not in ele-

vated plus-maze learning (Ukai et al., 199513). Moreover, the exact

reason why TAPA did not affect memory in elevated plus-maze tests remains undetermined.

Although low doses of TAPA (0.3 and 1.0 ng) were inactive, in-

creasing its dose to 3 ng inhibited spontaneous alternation perfor-

mance without affecting total arm entries (an index of locomotor

activity). The inhibitory effects of TAPA were almost completely

antagonized by p-funaltrexamine, indicating that the effects of TAPA are mediated by k-opioid receptors. In addition, the effective

dose (3 ng) of TAPA in spontaneous alternation performance was

456

almost identical with those in passive avoidance response. It has been reported that a 3-ng dose of TAPA fails to affect nociceptive responses (Ukai et al., 1993b). Th ere ore, f these results provide fur- ther support for the reports that p.-opioid receptor agonists lead to amnesia related to spatial working memory without affecting other behavioral responses, such as nociceptive responses or locomotor ac- tivity (Itoh et al., 1994; Ukai et al., 1993b, 1995a).

It is likely that spontaneous alternation performance associated with short-term memory affects long-term memory assessed by ele- vated plus-maze learning. However, such interaction between short- and long-term memory did not exist in the present study, further suggesting that the memory depends on learning tasks used.

Spatial alternation performance is associated with septo-hippo- campal cholinergic activity (Givens and Olton, 1990). DAMGO reportedly inhibits the high K+-induced release of acetylcholine from slices of the nucleus accumbens (Heijna et al., 1990, 1992) and hippocampus (Lapchak et al., 1989). The DAMGO-induced impair- ment of alternation performance is significantly improved by sys- temic injection of physostigmine (Itoh et al., 1994). Therefore, the TAPA-induced impairment of spontaneous alternation performance may be due to the inhibition of hippocampal cholinergic activity through the stimulation of p-opioid receptors, despite the lack of experimental evidence.

SUMMARY

The effects of intracerebroventricular administration of TAPA, a novel dermorphin analog, on plus-maze learning and spontaneous alternation performance were investigated in mice. The pre- or posttraining or preretention administration of TAPA (0.3-3.0 ng) alone failed to affect transfer latency of plus-maze learning, whereas TAPA (3 ng) produced a significant decrease in percent alternation without affecting total arm entries. P-Funaltrexamine (5 kg) almost completely reversed the TAPA (3 ng)-induced decrease in percent alternation. These results suggest that stimulation of p,,-opioid re- ceptors disrupts spontaneous alternation performance associated with spatial working memory.

This research evas supported in part b Grant-in-Aid for Scientific Research from rhe Minisrry of Education, Science, Sports and Cukure, Japan.

References Castellano C. and Pavane F. (1985) D ose- and strain-dependent effects of

dermorphin and [o-Ala’-o-Leu’lenkephahn on passive avoidance behav- ior in mice. Behau. Neurosci. 99, 1120-1127.

Givens B. S. and Olton D. S. (1990) Cholinergic and GABAergic modula- tion of medial septal area: effect on working memory. Behuu. Neurosci. 104, 849-855.

Haley T. J. and McCormick W. G. (1957) Ph armacological effects produced by intracerebral injection of drugs in the conscious mouse. Br. .J. Phar- mat. 12, 12-15.

Heijna M. H., Hogenboom F., Mulder A. H. and Schoffelmeer A. N. M. (1992) Opioid receptor-mediated inhibition of ‘H-dopamine and 14C- acetylcholine release from rat nucleus accumbens slices: a study on the possible involvement of K+ channel and adenylate cyclase. Naunyn- Schmiedeberg’s Arch. Phannac. 345, ‘627-632.

Heijna M. H., Padt M., Hogenboom F., Portoghese P. S., Mulder A. H. and Schoffelmeer A . . N. M. (1990) onioid receotor-mediated inhibition of

M. Ukai et al.

dopamine and acetylcholine release from slices of rat nucleus accumbens, olfactory tubercle and frontal cortex. Eur. J. Pharmac. 181, 267-278.

Hiller J. M., Itzhak Y. and Simon E. J. (1987) Selective changes in )I-, 6- and K-opioid receptor binding in certain limbic regions of the brain in Alz- heimer’s disease patients. Brain Res. 406, 17-23.

Itoh J., Nabeshima T. and Kameyama T. (1990) Utility of an elevated plus- maze for the evaluation of memory in mice: effects of nootropics, scopol- amine and electroconvulsive shock. Psychopharmacology 101, 27-33.

ltoh J., Ukai M. and Kameyama T. (1993) Dynorphin A-(1-13) potently prevents memory dysfunctions induced by transient cerebral ischemia in mice. Eur. J. Pharmac. 234, 9-15.

Itoh J., Ukai M. and Kameyama T. (1994) Dynorphin A-(1-13) potently im- proves the impairment of spontaneous alternation performance induced by the CL-selective opioid receptor agonist DAMGO in mice. J. Phannac. Exp. Ther. 269, 15-21.

Izquierdo I. (1980) Effects of p-endorphin and naloxone on aquisition, mem- ory, and retrieval of shuttle avoidance and habituation learning in rats. Psychopharmacology 69, 11 l-l 15.

Izquierdo I., De Almeida M. A. M. R. and Emiliano V. R. (1985) Unlike P-endorphin, dynorphinl-li does not cause retrograde amnesia for shut- tle avoidance or inhibitory avoidance learning in rats. Psychopha-ol- ogy 87, 216-218.

Izquierdo I. and Netto C. A. (1985) Role of p-endorphin in behavioral regu- lation. Ann. N. Y. Acad. Sci. 444, 162-177.

Lapchak P. A., Araujo D. M. and Collier B. (1989) Regulation of endoge- nous acetylcholine release from mammalian brain slices by opiate recep- tors: hippocampus, striatum and cerebral cortex of guinea pig and rat. Neuroscience 31, 313-325.

Ofri D., Fan L.-Q., Simon E. J. and Holler J. M. (1992) Lesioning of the nu- cleus basalis of Meynert has differential effects on I*-, 6- and K-opioid re- ceptor binding in rat brain: a quantitative autoradiographic study. Brain Res. 581, 252-260.

Parada-Turska J. and Turski W. A. (1990) Excitatory amino acid antagonists and memory: effects of drugs acting at N-methyl-o-aspartate receptors in learning and memory tasks. Neurophartnacology 29, 111 l-l 116.

Rigter H., Hannan T. J., Messing R. B., Martinez Jr. J. L., Vasquez B. J., Jen- sen R. A., Veliquette J. and McGaugh J. L. (1979) Enkephalins interfere with acquisition of an active avoidance response. Life Sci. 26, 337-345.

Sarter M., Bodewitz G. and Stephens D. (1988) Attenuation of scopol- amine-induced impairment of spontaneous alternation behaviour by an- tagonist but not inverse agonist and agonist p-carbolines. Psychophanna- cology 94, 491-495.

Sasaki Y., Ambo A. and Suzuki K. (1991) St u les on analgesic oligopeptides d‘ VII: solid phase synthesis and biological properties of Tyr-n-Arg-Phe-P- Ala-NH1 and its fluorinated aromatic acid derivatives. Chem. Pharm. Bull. 39, 2316-2318.

Stone W. S., Walser B., Gold S. D. and Gold P. E. (1991) Scopolamine- and morphine-induced impairment of spontaneous alternation performance in mice: reversal with glucose and with cholinergic and adrenergic ago- nists. Behav. Neurosci. 105, 264-271.

Ukai M., Kobayashi T. and Kameyama T. (1993a) Dynorphin A-( I-13) at- tenuates basal forebrain-lesion-induced amnesia in rats. Brain Res. 625, 355-356.

Ukai M., Kobayashi T., Mori K., Shinkai N., Sasaki Y. and Kameyama T. (1995a) Attenuation of memory with Tyr-n-Arg-Phe-P-Ala-NH2, a novel dermorphin analog with high affinity for p-opioid receptors. Eur. J. Pharmac. 287, 245-249.

Ukai M., Miura M. and Kameyama T. (1995b) Effects of galanin on passive avoidance response, elevated plus-maze learning and spontaneous alter- nation performance in mice. Peptides 16, 1283-1286.

Ukai M., Mori K., Hashimoto S., Kobayashi T., Sasaki Y. and Kameyama T. (1993b) Tyr-o-Arg-Phe-P-Ala-NH2, a novel dermorphin analog, impairs memory consolidation in mice. Eur. J. Pharmac. 239, 237-240.