European Journal of Pharmacology, 219 (1992) 245- 251 © 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00

245

EJP 52603

Intrahippocampal injections of benzodiazepine and muscimol impair working memory but not reference memory of rats in the three-panel runway task Masuo Ohno, Tsuneyuki Yamamoto and Shigenori Watanabe Department of Pharmacology, Faculiy of Pharmaceutical Sciences, Kyushu University, Fukuoka 812, Japan Received 9 March 1992,revised MS received 20 May 1992,accepted 2 June 1992

In a three-panel runway task, the benzodiazepine chlordiazepoxide at 3.2 and 10 mg/kg i.p. significantly increased the number of errors (attempts to pass through two incorrect panels of the three panel-gates at four choice points) in a test of working memory, but it had no effect on errors in a test of reference memory. This effect of 10 mg/kg chlordiazepoxide on working memory was blocked by the benzodiazepine receptor antagonist flumazenil at 10 mg/kg. Intrahippocampal injection of chlordiazepoxide at 10 and 32 /zg/side significantly increased the number of working memory errors. This effect of in'trahippocampal chlordiazepoxide (32/zg/side) was attenuated not only by flumazenil at 10 mg/kg but also by the y-aminobutyric acid (GABA)A receptor antagonist bicuculline at 3.2 mg/kg. Intrahippocampal injection of the GABA A receptor agonist muscimol at 100 and 320 ng/side also significantly increased working memory errors. Neither chiordiazepoxide nor muscimol affected the number of reference memory errors when injected into the hippocampus at doses up to 32 p.g/side or 320 ng/side, respectively. These results suggest that activation of the GABAA/benzodiazepine receptor complex in the hippocampus impairs working memory, but does not affect reference memory. Hippocampus; Chlordiazepoxide; Benzodiazepine; Muscimol; GABA (y-aminobutyric acid); Memory (working, reference)

1. Introduction

Benzodiazepines have been known to produce anterograde amnesia as an undesired side effect in humans (Clarke et al., 1970; Curran, 1986, 1991; Ghoneim and Mewaldt, 1975; Lister, 1985), though their main use resides in the treatment of anxiety and insomnia. It has also been shown that benzodiazepine compounds such as chlordiazepoxide and diazepam impair learning and memory in experimental animals, as assessed with the radial arm maze (Hodges and Green, 1986; Willner and Birbeck, 1986), Morris water maze (McNamara and Skelton, 1991; McNaughton and Morris, 1987), and passive avoidance tasks (Nabeshima et al., 1990a). Since the amnesic effect of benzodiazepines is reversed by the benzodiazepine receptor antagonist flumazenil in humans and animals, it is likely that this impairment is mediated by a specific benzodiazepine receptor (Dorow et al., 1987; Nabeshima et al., 1990a; O'Boyle et al., 1983). However, there have been few studies Correspondence to: M. Ohno, Department of Pharmacology,Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812, Japan.

designed to determine where in the brain benzodiazepines influence learning and memory. Animal experiments can be used to study two types of memory: working memory and reference memory (Honig, 1978; Olton et al., 1979). Working memory allows animals to remember information that is useful for a single session of an experiment b u t not for subsequent session, whereas reference memory is defined as the holding of information that is of continued value across all sessions. We have previously reported that a three-panel runway task can be used to study learning and memory in the rat (Furuya et al., 1988; Yamamoto et al., 1990b), in particular because it allows us to distinguish between working and reference memory (Ohno et al., 1991). In this study, we investigated the effects of intrahippocampal injections of chlordiazepoxide on working and reference memory, as assessed with the three-panel runway task, because the hippocampus is necessary for normal memory functions both in humans and in other animals (Becker et al., 1980; Corkin, 1984; Morris et al., 1982; Olton and Feustle, 1981; Zola-Morgan et al., 1986). We also studied the possibility that the 3,-aminobutyric acid (GABA)-ergic system is involved in the memory im-

246

pairment resulting from intrahippocampal chlordiazepoxide, since benzodiazepines are known to exert their effects by facilitating central GABAergic transmission through activation of G A B A A / b e n z o d i a z e p i n e receptor complexes (Bowery et al., 1984; Olsen, 1981).

2. Materials and methods

2.1. Subjects Eight- to ten-week-old male rats of the Wistar strain were obtained from Nippon SLC. The initial free-feeding weights were 230-250 g, but the rats were maintained at approximately 80% of these weights before the experiment. The rats were housed in groups of four per cage at a constant temperature (23 + 2°C), with a 12-h light-dark cycle (light period: 07:00-19:00 h), and with water freely available.

2.2. Apparatus Working memory and reference memory were assessed with a three-panel runway apparatus (Furuya et al., 1988; Ohno et al., 1991; Yamamoto et al., 1990b). In brief, this apparatus (175 x 36 x 25 cm) was composed of a start box, a goal box and four consecutive choice points intervening between them. Each choice point consisted of a gate with three panels (12 x 25 cm). The rats were prevented from passing through two of the three panels in the gate by front stoppers, and were prevented from returning to the start box or to a previous choice point by rear stoppers affixed to each of the panels of the gates. When the rats reached the goal box, they received two food pellets (about 50 mg each; Muromachi Kikai) as positive reinforcement.

2.3. Acquisition training Initially, all the front stoppers were removed so that a rat could pass through any one of the three panelgates at each choice point. The rats were made to run the task repeatedly until the time that elapsed from leaving the start box to reaching the goal box was consistently below 20 s. Once this time was reached, the rats were given six consecutive trials (defined as one session) per day with the removal of the front stopper of only one of the three panel-gates (the correct panel-gate) at each choice point. Trials were run at 2-min intervals. Water was freely available between trials in the home cage. Separate groups of animals were used for the working and reference memory experiments. In the test of working memory, the locations of the correct panelgates were kept constant within a session, but were changed from one session to the next. Twelve different

patterns of correct panel-gate locations were used, as described previously (Furuya et al., 1988; Yamamoto et al., 1990b). In the test of reference memory, the correct panel-gate locations were kept constant, both within a session and in succeeding sessions. The number of times an animal attempted to pass through an incorrect panel-gate (defined as an error) and the time required for the animal to obtain food pellets (defined as latency) were recorded for each rat in each trial of a session. The criterion for learning was fewer than 12 and fewer than six errors summed across the six trials of a session in the tests of working and reference memory, respectively. A rat was used in the experiment if it achieved this criterion throughout three consecutive sessions.

2. 4. Surgery and experimental procedures After achieving the criterion of learning, the rats underwent bilateral chronic cannula implantation for microinjection of drugs into the dorsal hippocampus. Each rat was anesthetized with sodium pentobarbital (40 m g / k g i.p.) and was fixed in a stereotaxic instrument. A stainless steel guide cannula (external diameter: 0.7 mm) was placed 1.0 mm above the dorsal hippocampus (3.8 mm posterior to the bregma, 2.2 mm lateral to the midline, 3.2 mm ventral to the surface of the skull measured at the bregma), according to the atlas of Paxinos and Watson (1982). The cannula was fixed to the skull with three screws and dental acrylic cement. The rats were allowed at least 5 days to recover from surgery before the runway session was resumed. The rats were tested after they had achieved the criterion of learning in the tests of working and reference memory after surgical manipulations. A stainless steel injection cannula (external diameter: 0.35 mm) was used to infuse the drugs. The injection cannula was connected to a 5-/xl Hamilton syringe via a polyethylene tube. Two microliters of drug solution or vehicle were injected bilaterally into the dorsal hippocampus through the injection cannula, the tip of which protruded 1.0 mm below the tip of the guide cannula. The rate of injection was 0.5 /~1 per minute. The injection cannula was left in place for 1 min after completion of the injection, to facilitate the diffusion of the drug. Microinjections were never made more than five times into a single rat, and a minimum of 3 days was allowed between microinjections. Performance in the three-panel runway task during noninjected sessions was not affected by repeated intrahippocampal injections, and met the criterion of learning.

2.5. Drugs The drugs used in this study were chlordiazepoxide hydrochloride (Hoffman-La Roche), flumazenil (Ro

247 15-1788; Yamanouchi Pharmaceutical Co., Ltd.), muscimol (Sigma Chemical Co.) and ( - ) - b i c u c u l l i n e methiodide (Sigma Chemical Co.). Flumazenil was suspended in saline solution with a few drops of Tween 80. Other drugs were dissolved in saline. The runway test was given 20 and 10 rain after i.p. and intrahippocampal injections of drugs, respectively. Rats were given fiumazenil or bicuculline 10 and 15 min before the i.p. and intrahippocampal injections, respectively.

session since this test was used to evaluate the ability of the rats to retain the constant location of correct panel-gates. The presence of a significant difference between the groups was determined by a one-way analysis of variance (ANOVA), followed by Dunnett's test when F ratios reached significance (P < 0.05).

2.6. Histology

In the three-panel runway task, the random performance level was four errors per trial, or 24 errors per session. In the test of working memory, the number of errors made from the second to the sixth trial (working memory errors) markedly decreased with repeated training, whereas the errors in the first trial remained constant at approximately four. About 15-20 training sessions were required for the rats to reach the criterion of less than 12 errors summed across the six trials of a session. The latency also decreased as the sessions were repeated, and it was stable from the 10th session on. In the test of reference memory, the number of errors and the latency in all six trials of a session decreased with repeated training. The rats could run the task within the six-error criterion summed across six trials after they had had 5 - 1 0 training sessions. Chlordiazepoxide at 1.0-10 m g / k g given i.p. before testing dose dependently increased the number of errors in the working memory task (F(3,28) = 16.80, P < 0.01), an effect that reached significance for the 3.2 and 10 m g / k g doses, while it had no effect on the number of errors made in the first trial (table 1). The latency recorded in the first trial (F(3,28)= 9.86, P < 0.01) and that recorded from the second to the sixth trial (F(3,28) = 58.67, P < 0.01) were prolonged when rats were given 10 m g / k g of chlordiazepoxide. Flumazenil at 10 m g / k g did not affect the number of errors or the latency. The increase in the number of working memory errors that resulted from 10 m g / k g of chlordiazepoxide was attenuated by pretreatment with

After completion of the experiment, the rats given intrahippocampal injections of drugs were anesthetized with ether and their brains were perfused with 10% formalin solution through the left cardiac ventricle. The brain was then removed, frozen and sliced to a thickness of 50 ~m. Sliced sections were stained with cresyl violet. The placement of the cannula was verified histologically. The stained sections showed that the tips of the injection cannulas were located in the dorsal hippocampus. Several animals were injected with 2 / z l of cresyl violet dye in order to estimate the extent of diffusion. Ten minutes after this microinjection, the rats were anesthetized and prepared for cardiac perfusion. After the brain had been removed and sliced, the diffusion of dye was confirmed by visual inspection. The dye injections were confined to the dorsal hippocampus, in the region ranging from 2.8 to 4.8 mm posterior to the bregma.

2. Z Data analysis In the test of working memory, the number of errors and the latency summed from the second to the sixth trial of a session were used to evaluate the ability of the rats to remember new correct panel-gate locations, and are thus presented separately from those recorded in the first trial. In the test of reference memory, both parameters were summed across all six trials of a

3. Results

TABLE 1 Increases in working memory errors and latency to reach the goal box induced by i.p. injection of chlordiazepoxide, and their reversal by flumazenil. The runway test was held 20 min after chlordiazepoxide was given. Flumazenil was injected 10 min before chlordiazepoxide. Values are means-+S.E, of errors and latencies recorded in the first trial, and those summed from the second to the sixth trial of a session. The significance of the differences from the saline-treated group (a p < 0.05, b p < 0.01) and from the 10 mg/kg chlordiazepoxide-treated group (c p < 0.05, d p < 0.01) was determined by a one-wayANOVA followed by Dunnett's test. Drug

mg/kg

N

Number of errors Trial 1

Trial 2-6

Trial 1

8 8 8 8 5

4.1 +0.2 3.6 :t:0.3 3.8 + 0.2 4.4 + 0.5 3.85=0.6

4.8_+1.1 5.4 _+1.2 9.9 -+1.8 a 17.0-+1.0 b 4.4 -+1.3

11.8_+0.5 10.3_+1.1 9.6 + 1.0 29.35:5.8 b 9.65:1.3

Trial 2-6 28.4+ 1.0 25.4 + 1.2 30.15=1.7 55.0-+2.7 b 28.4 -+4.3

8

4.1 -+0.4

4.8 5:1.3 d

13.1_+1.4 c

29.3 -+1,5 a

(i.p.) Saline Chlordiazepoxide Chlordiazepoxide Chlordiazepoxide Flumazenil Chlordiazepoxide + flumazenil

1.0 3.2 I0.0 10.0 10.0 10.0

Latency(s)

248 TABLE 2

Reference memory

Working memory

Effect of i.p. injection of chlordiazepoxide on reference memory errors and latency to reach the goal box. T h e runway test was given 20 min after chlordiazepoxide. Values are m e a n s ± S.E. of errors and latencies s u m m e d across all six trials of a session. Drug

mg/kg (i.p.)

N

N u m b e r of errors trial 1 - 6

Latency (s) trial 1 - 6

Saline Chlordiazepoxide Chlordiazepoxide

3.2 10.O

5 5 5

2.0±0.5 1.8 ± 0.9 2.6 ± 0.8

37.6±1.7 33.6 ± 1.2 39.4 ± 3.7

~

5

4

4

-;3

,6 z

.m O

"6

.0 2 E -! Z

---o L4..

1

10 m g / k g of flumazenil (F(1,14) = 68.57, P < 0.01). Flumazenil at 10 m g / k g also significantly attenuated the increased latency in the first trial (F(1,14)= 7.37, P < 0.05), and in the second to sixth trials (F(1,14)= 55.47, P < 0.01) in rats given 10 m g / k g chlordiazepox±de. In the reference memory task, chlordiazepoxide at doses up to 10 m g / k g did not affect the number of errors or the latency across all six trials of a session (table 2). Chlordiazepoxide at 3 . 2 - 3 2 / ~ g / s i d e , given bilaterally into the dorsal hippocampus, caused a dose-dependent increase in the number of working memory errors (F(3,20) = 8.89, P < 0.01), an effect that was significant for the 10 and 32 ~ g / s i d e doses, while it did not affect the number of errors made in the first trial (fig. 1 and table 3). The latency recorded from the second to the sixth trial of a session (F(3,20)= 3.49, P < 0.05) was significantly prolonged only when rats were given intrahippocampal chlordiazepoxide at 32 /zg/side. The increase in working memory errors induced by intrahippocampal chlordiazepoxide at 32 /zg/side was significantly attenuated by flumazenil at 10 m g / k g (F(1,10) = 10.82, P < 0.01) and by bicuculline at 3.2 m g / k g (F(1,10)= 12.39, P < 0.01). Bicuculline at 3.2

0

0 I

I

I

I

I

I

1

2

3

4

5

6

Trials

1

2

3 4 Trials

5

6

Fig. 1. Effects of intrahippocampal injections of chlordiazepoxide on the n u m b e r of errors in tests of working and reference memory. Cannulas were implanted in rats that had achieved the criterion of learning. T h e rats were allowed at least 5 days to recover from surgery before being used for drug tests. T h e runway test was given 10 min after chlordiazepoxide was administered ( o : saline, e: 3.2 izg/side, A : 1 0 / z g / s i d e , • : 32 izg/side). Each point represents the m e a n ± S.E. of errors for six animals recorded in each trial of a session.

m g / k g by itself had no significant effect on the number of errors or on the latency (N = 5; data not shown). The prolongation in latency in the rats given intrahippocampal chlordiazepoxide at 32 ~ g / s i d e was also attenuated by these doses of flumazenil (F(1,10) = 5.82, P < 0.05) and bicuculline (F(1,10) = 9.27, P < 0.05). Intrahippocampal injection of muscimol at 100 and 320 ng/side significantly increased the number of working memory errors (F(2,15)= 21.07, P < 0.01), without affecting errors in the first trial. Intrahippocampal muscimol also significantly increased the latency both in the first trial (F(2,15) = 4.00, P < 0.05) and in the second to sixth trials (F(2,15) = 10.16, P < 0.01).

TABLE 3 Increases in working m e m o r y errors and latency to reach the goal box induced by intrahippocampal injections of chlordiazepoxide and muscimol, and their reversal by flumazenil and hicuculline. T h e runway test was held 10 min after intrahippocampal chlordiazepoxide and muscimol. Flumazenil and bicuculline were given i.p. 15 min before the intrahippocampal injections. Values are m e a n s + S.E. of errors and lateneies recorded in the first trial, and those s u m m e d from the second to the sixth trial of a session. T h e significance of the differences from the saline-treated group (a p < 0.05, b p < 0.01) and from the 32/.~g/side chlordiazepoxide-treated group (c p < 0.05, d p < 0.01) was determined by a one-way A N O V A followed by D u n n e t t ' s test. Drug

Saline Chlordiazepoxide Chiordiazepoxide Chlordiazepoxide Chlordiazepoxide + flumazenil 10 m g / k g Chlordiazepoxide + bicuculline 3.2 m g / k g Muscimol Muscimol

/~g/side

N

N u m b e r of errors

Latency(s)

Trial 1

Trial 2 - 6

Trial 1

Trial 2 - 6

3.2 10.0 32.0 32.0

6 6 6 6 6

3.85:0.2 4.05:0.6 3.3 ± 0.3 4.5 ± 0.5 4.0 ± 0.3

4.85:0.8 7.75:1.1 14.0 5:2.5 b 17.7 ± 2.7 b 8.2 + 1.1 d

14.0+ 1.1 15.25:1.9 14.7 ± 2.2 17.2 5:2.0 15.0 5:0.6

31.35:2.2 31.75:2.9 46.5 5 : 9 . 2 52.7 ± 5.8 a 38.0 ± 1.8 c

32.0

6

4.0 5:0.4

7.8 ± 0.8 d

13.3 5:1.5

33.2 5 : 2 . 7 c

6 6

4.2 ± 0.3 4.8±0.4

14.7 5:1.6 b 18.05:1.9 b

30.3 ± 6.5 a 31.8±5.5 a

66.5 5:11.8 b 82.55:7.7 b

0.1 0.32

249 TABLE 4 Effects of intrahippocampal injections of chlordiazepoxideand muscimol on reference memory errors and latency to reach the goal box. The runway test was held 10 min after the drugs were given. Values are means+ S.E. of errors and latencies summed across all six trials of a session. Drug

p.g/side

Saline Chlordiazepoxide 10.0 Chlordiazepoxide 32.0 Muscimol 0.1 Muscimol 0.32

N Numberof errors trial 1-6 6 2.7 + 0.7 6 3.0 + 0.9 6 1.8 + 0.6 6 1.7___0.5 6 3.0 + 0.7

Latency(s) trial 1-6 46.7 + 3.2 48.3 + 7.4 47.2+ 7.4 39.5+ 3.5 44.2+ 3.0

In the reference memory task, intrahippocampal injection of chlordiazepoxide at doses up to 32/.Lg/side, or muscimol up to 320 ng/side, had no effect on the number of errors or on the latency across all six trials of a session (fig. 1 and table 4).

4. Discussion In this study, i.p. administration of the benzodiazepine chlordiazepoxide impaired working memory, but did not affect reference memory in the three-panel runway task. The impairment of working memory induced by chlordiazepoxide was significantly attenuated by the benzodiazepine receptor antagonist flumazenil. These findings suggest that activation of benzodiazepine receptors results in impairment of working memory, i.e. acquisition of new and variable information, but does not affect reference memory, i.e. retention and retrieval of constant information. Hodges and Green (1986) used an eight-arm radial maze with four food-baited arms and found that the rats given chlordiazepoxide remembered the constant non-rewarded arms, but exhibited an increase in re-entries into rewarded arms from which food had already been taken. This pattern of errors after chlordiazepoxide administration may reflect a deficit in working memory that is not accompanied by an impairment of reference memory. Benzodiazepines have recently been found to impair acquisition but not retention or retrieval of a passive avoidance response (Nabeshima et al., 1990a) and to impair place learning in a water maze (McNamara and Skelton, 1991). These findings, coupled with the present results, are consistent with the clinical observations that benzodiazepine compounds cause anterograde amnesia in humans. The major effects of these compounds are on the acquisition and consolidation of new information, but not on the retrieval of information learned before the drugs are given (Clarke et al., 1970; Curran, 1986, 1991; Dorow et al., 1987; Ghoneim and Mewaldt, 1975; Lister, 1985).

We have previously demonstrated that rats trained for the three-panel runway task preoperatively and then subjected to dorsal hippocampal lesions exhibit a marked impairment of working memory, while they have normal retention of reference memory (Kitajima et al., 1992). It has also been shown that lesions of the hippocampus and fimbria-fornix produce a severe deficit in working memory, but do not have much i n f u e n c e on reference memory, as assessed with the radial maze with only half of the arms baited (Jarrard, 1980; Olton and Papas, 1979). Similar results have been obtained with rats with hippocampal neuronal damage following transient forebrain ischemia (Davis et al., 1986, 1987). The results of these lesioning studies suggest that the hippocampus plays a crucial role in working memory, but not in reference memory. Thus, the selective impairment of working memory after chlordiazepoxide is consistent with the observations in the water maze task that diazepam impairs spatial learning, which is dependent on hippocampal function, without affecting visual discrimination, which is resistant to hippocampal damage (Brioni and Arolfo, 1992; McNamara and Skelton, 1991; Morris et al., 1982). These findings prompted us to investigate the effects of intrahippocampal chlordiazepoxide in order to determine the site in the brain of this drug's deleterious action on working memory. The present study clearly showed that chlordiazepoxide, injected into the dorsal hippocampus, caused a significant impairment of working memory, and that this effect was reversed by flumazenil. Thus, it is conceivable that activation of hippocampal benzodiazepine receptors impairs working memory performance. It has been proposed that there are at least two pharmacologically, biochemically and functionally distinct subtypes of benzopiazepine receptors, which can be differentiated by their affinities for the triazolopyridazine derivative CL 218,872 and for/3-carboline esters (Braestrup and Nielsen, 1981; Dubnick et ~al., 1983). The high-affinity sites for CL 218,872 and/3-carbolines are designated benzodiazepine type-1 receptors, which are not coupled to G A B A receptors, whereas the lowaffinity sites are designated type-2 receptors, which are coupled to G A B A A receptors and chloride ionophores (Klepner et al., 1979; Taguchi and Kuriyama, 1987). The relative proportions of these two subtypes differ in different brain regions, and the hippocampus contains both benzodiazepine type-1 and type-2 receptors (Braestrup and Nielsen, 1981; Young et al., 1981). In this study, intrahippocampal chlordiazepoxide-induced impairment of working memory was reversed by the G A B A A receptor antagonist bicuculline as well as by flumazenil. The G A B A A receptor agonist muscimol also impaired working memory in the three-panel runway task when injected into the hippocampus. These results indicate that activation of the GABAA/

250

benzodiazepine type-2 receptor complex in the hippocampus, which may facilitate GABAergic inhibition of hippocampal neurons, can account for the deleterious effect of chlordiazepoxide on working memory, i.e. acquisition of new and variable information. In contrast, reference memory performance was not affected by intrahippocampal injections of chlordiazepoxide or muscimol. Thus, activation of hippocampal G A B A A / benzodiazepine receptors does not modulate reference memory. This is also true of hippocampal lesions (Kitajima et al., 1992). The hippocampus is known to receive cholinergic innervation from the medial septum via the fimbriafornix (Mesulam et al., 1983). Using the three-panel runway task, we previously found that intrahippocampal administration of the muscarinic receptor antagonist scopolamine impaired working memory without affecting reference memory (Ohno et al., 1992), and that the cholinesterase inhibitors physostigmine and tetrahydroaminoacridine alleviated the impairment of working memory in hippocampal-lesioned rats (Kitajima et al., 1992; Yamamoto et al., 1990a). These findings suggest that the septohippocampal cholinergic system has an important role in working memory performance. There is evidence that this system is under an inhibitory GABAergic regulation in the septum. Intraseptal administration of muscimol reduces the turnover rate of acetylcholine and also reduces high-affinity choline uptake in the rat hippocampus (Costa et al., 1983; Richter and Gormley, 1986; Wood, 1986). Recently, it has been reported that muscimol, injected into the medial septum, impairs working memory, as tested with the radial maze task (Chrobak et al., 1989) and the T-maze alternation task (Givens and Olton, 1990). We cannot therefore exclude the possibility that systemic benzodiazepines impair working memory by affecting the septohippocampal cholinergic system via septal GABAA/benzodiazepine receptors. This is supported by the finding that physostigmine partially attenuates the disruptive effect of chlordiazepoxide on a passive avoidance response (Nabeshima et al., 1990a, b). In conclusion, the present study provides evidence that the facilitation of GABAergic transmission through activation of the GABAA/benzodiazepine receptor complex in the hippocampus leads to an impartment of working memory without affecting reference memory. Acknowledgements We wish to express our appreciation to Yamanouchi Pharmaceutical Co., Ltd. for providing us with the generous supply of flumazenil. This research was supported in part by the Mochida Memorial Foundation for Medical and Pharmaceutical Research and by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.

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Intrahippocampal injections of benzodiazepine and muscimol impair working memory but not reference memory of rats in the three-panel runway task.

In a three-panel runway task, the benzodiazepine chlordiazepoxide at 3.2 and 10 mg/kg i.p. significantly increased the number of errors (attempts to p...
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