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Involvement of cholinergic systems in the deficit of place learning in Morris water maze task induced by baclofen in rats
BRAIN RESEARCH Brain Research 683 (1995) 209-214
ELSEVIER
Research report
Involvement of cholinergic systems in the deficit of place learning in Morris water maze task induced by baclofen in rats Yutaka Nakagawa *, Yoshinori Ishibashi, Toshio Yoshii, Eijiro Tagashira Tsukuba Research Laboratories, Experimental Biomedical Research Inc. (Jisseiken), 8-5-1, Chuo, Ami-machi, Inashiki-gun, lbaraki 300-03, Japan Accepted 22 Febuari 1995
Abstract
Effects of oxotremorine on the deficit of place learning in the Morris water maze task induced by baclofen and scopolamine were examined to determine the i~wolvement of brain cholinergic systems in the deficit of learning induced by baclofen. Rats were given 4 training trials per day with the submerged platform at a fixed location in the maze for 4 days. On day 4, rats were required to swim in the pool without the platform after the 4th training trial (probe test). Baclofen as well as scopolamine dose-dependently increased the escape latency in the training trials. In the probe test, baclofen as well as scopolamine dose-dependently reduced the duration in the quadrant where the platform had been originally located. Increased latency in the training trials and reduced duration in the probe test induced by scopolamine were dose-dependently attenuated by oxotremorine. Increased latency and reduced duration in the baclofen-treated rats were improved by oxotremorine as well as 2-hydroxysaclofen. Baclofen but not scopolamine induced motor incoordination in the rotarod test. Oxotremorine failed to improve motor incoordination induced by baclofen. These results suggest that cholinergic systems may be involved in the deficit of place learning induced by baclofen, and that the ameliorative effects of oxotremorine may not be due to improvement of motor incoordination.
Keywords: Place learning; Morris water maze task; Baclofen; 2-Hydroxysaclofen; Oxotremorine; Motor incoordination
1. Introduction
GABA is the main inhibitory neurotransmitter in the brain. There is evidence that there are two different GABA receptors in the brain: GABA A and GABA B receptors [3,19]. GABA A receptors are coupled with benzodiazepine receptors and C1- channels [14,27]. On the other hand, GABA B receptors are coopled with G protein [15]. The activation of GABA B receptors decreases the amplitude of Ca 2÷ currents [13] and increases the K ÷ conductance [25]. GABA A and GABA B receptors have somewhat different physiological actions. GABA A receptors are mainly involved in anxiety and convulsion [19]. In contrast, GABA B receptors are mainly related to depression and analgesia [18,31]. Howeve:r, GABA A as well as GABA B receptors play an important role in learning and memory [4-6,11,16,17,21,32,34,36]
* Corresponding author. Fax: (81) (298) 87-9065. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All fights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 5 ) 0 0 3 0 2 - 9
We previously reported that pre-training injections of baclofen (GABA u agonist) as well as muscimol (GABA A agonist) had detrimental effects on passive avoidance learning in rats when the test session was conducted 24 h later [24]. In that study, we also found that muscimol-reduced latency disappeared when muscimol was re-injected before the test session: muscimol induced state-dependent learning. Alternatively, the rats injected with baclofen before the training and test sessions showed reduced latency: baclofen failed to induce state-dependent learning. These findings provide the direct evidence that GABA A and GABA B receptors may influence learning and memory in a different manner. Cholinergic systems in the brain play an important role in learning and memory [35]. Especially, dementia of the Alzheimer type (DAT) has been shown to be closely associated with deficit in the central cholinergic systems [8]. Recently, GABA B receptor systems are also found to be abnormal in DAT: scatchard analysis and competition studies showed significant decrement in receptor density (Bmax) for GABA B receptors [7]. It is reported that GABA B
Y. Nakagawa et al. /Brain Research 683 (1995) 209-214
210
systems may interact with cholinergic systems [6,10,30,32]. Based on these results, we designed the experiment to examine the involvement of cholinergic systems in the deficits of learning and memory induced by baclofen. In the present study, the Morris water maze task was chosen because the step-through passive avoidance task which was used in the previous study [24] appeared to be influenced by muscle relaxation induced by baclofen. In that study, 2 to 4 of 10 rats given baclofen were moved to the dark compartment by the experimenter in the training session because of muscle relaxation [24]. A rotarod test was also conducted to examine the relationship between motor incoordination and the deficit of performance in the Morris water maze task.
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2. Materials and methods
Cl
2.1. Animals
10
Male Wistar rats (Charles River J a p a n ) , weighing 260 g at the beginning of the experiment, were used. were housed in groups in an air-and light-controlled (temperature; 24 + 2 ° C, light phase; 08.00-20.00 h). and water were given ad libitum.
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0.1
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SCO (0.3 mg/kg, i.p.) Fig. 2. Effects of oxotremorine on the deficit of place learning in the Morris water maze task induced by scopolamine. Upper panel: scopolamine (SCO, 0.3 mg/kg) and oxotremorine(OXO) were injected i.p. 30
2.2. Apparatus and procedure
sco(0.3)
SCO(1,0)
10 O" 1
2
3
4
Days 40 O v
0.01
30-
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and 15 rain before the training trials, respectively.Numbers in parentheses: drug dose in mg/kg. * P <0.05, * * P<0.01 versus SA+SA; # P < 0.05, ## P < 0.01 versus SCO + SA. lower panel: duration in the quadrant where the platform had been located. See Fig. 1 for further information.
60
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SA
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._o 20-
10"
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0.3
1.0
SCO (mg/kg, i.p.) Fig. 1. Effects of scopolamine on place learning in the Morris water maze task. Upper panel: mean latency to reach the platform in the training trials. Scopolamine (SCO) was injected i.p. 30 min before the training trials. Ten rats were used in each group. Numbers in parentheses: drug dose in m g / k g . SA, saline; * * P < 0.01 versus saline control. Lower panel: duration in the quadrant where the platform had been located. A 60-s probe test was conducted after the 4th training trial on day 4. Data are expressed as mean with S.E.M.
Morris water maze task A circular pool (150 cm in diameter, 35 cm high) was filled 24 cm deep with clear water (21 + 1° C). A clear platform (13 cm in diameter) was submerged 1 cm below the water surface. The pool was conceptually divided into qua&ants: northeast (NE), northwest (NW), southeast (SE) and southwest (SW). The platform was located in the center of NW quadrant throughout the training trials except the probe test which was conducted on day 4. Rats were given 4 training trials each day for 4 consecutive days. For each training trial, the rats were placed in the water facing the pool wall at one of 4 positions (north, south, east or west poles) in a different order each day, and allowed to swim until they reached the platform. Latency to reach the platform was recorded up to 60 s. On the platform they remained for 10 s before removal. A rat which failed to reach the platform within 60 s was guided to it by the experimenter and the maximum latency was scored. After each training trial, the rat was dried with a towel and allowed to remain in a cage for the intertrial interval (approximately 3 min).
Y. Nakagawa et aL /Brain Research 683 (1995) 209-214
On day 4, a 60-s probe test without the platform was conducted after the 4th training trial. The time spent in the NW quadrant was recorded. Rotarod test Motor incoordination was tested for 3 min using a rotarod performance apparatus (9 cm in diameter, 5 rpm; Natsume Seisakusho Co., Tokyo, Japan). The day before the test, each rat had been trained to run on a rotarod until it could remain there for 3 min without falling.
211
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I
1
2
2.3. Drugs
40
Baclofen (RBI), 2-hydroxysaclofen (RBI), scopolamine hydrobromide (Sigma), oxotremorine (Sigma) were used. 2-Hydroxysaclofen was first dissolved in 0.16 M NaOH, then the solution was neutralized with equimolar HC1 [1] and diluted with saline to final concentration of 25/zg/2.5 ~1. 2-Hydroxysaclofen was, injected i.c.v, in a volume of 2.5 /~l/rat. Other drugs were dissolved in saline, and injected i.p. in a volume of 1 m l / k g except for baclofen which was injected in a volume of 4 ml/kg. 2-Hydroxysaclofen was injected 35 and 5 min before the training trials. Baclofen, scopolamine and oxotremorine were injected 30, 30 and 15 rain before the training trials, respectively. The dosage of scopolamine was expressed as the salt.
3
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Fig. 4. Effects of 2-hydroxysaclofen on the deficit of place learning in the Morris water maze task induced by baciofen. Upper panel: mean latency to reach the platform in the training trials. Baclofen (BAC, 4 mg/kg) was injected i.p. 30 rain before the training trials. 2-Hydroxysaclofen (2OHSAC, 25 p,g/2.5 /zl/rat) was injected i.c.v. 35 and 5 rain before the training trials. * * P < 0.01 versus S A + S A ; # # P < 0.01 versus BAC+ SA. Lower panel: duration in the quadrant where the platform had been located. See Fig. 1 for further information.
60 ~-, 501 o m t~ 40.
2.4. Statistics
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Between-group comparisons were assessed by Dunnett test following one-way ANOVA for each training trial and probe test.
20 BAC(2) BAC(4)
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2
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4
3. Results
40 O
3. I. Effects of oxotremorine on scopolamine-induced deficit of place learning in the Morris water maze task
-T
to 3 0 t-
._o 2o 10 SA
1
2
4
BAC (mg/kg, i.p.) Fig. 3. Effects of baclofen on place learning in the Morris water maze task. Upper panel: mean latency to reach the platform in the training trials. Baclofen (BAC) was injected i.p. 30 rain before the training trials. Numbers in parentheses: drug dose in mg/kg. * * P < 0.01 versus saline control. Lower panel: duration in the quadrant where the platform had been located. See Fig. 1 for further information.
Upper panels of Figs. 1 and 2 show the latency to reach the platform in the training trials. Scopolamine dose-dependently increased the latency. Oxotremorine dose-dependently attenuated the increase in latency induced by scopolamine (0.3 mg/kg). On day 4, a 60-s probe test without the platform was conducted after the 4th training trial. Scopolamine dosedependently reduced the duration in the quadrant where the platform had been originally located (Fig. 1). The rats given oxotremorine showed dose-dependent improvement of scopolamine (0.3 mg/kg)-reduced duration in the probe test (Fig. 2).
212
Y. Nakagawa et al. / Brain Research 683 (1995) 209-214 60 ¸ o 50 (D 09 4 0
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...1 10
Y. Nakagawa et al. /Brain Research 683 (1995) 209-214
suggest that place learning may be 'probed' rather in the probe test than in the training trials. We found here that badofen (GABA B agonist) as well as scopolamine induced the deficit of place learning. Baclofen dose-dependently increased the latency in the training trials, and reduced theduration in the probe test. 2-Hydroxysaclofen (GAB.A s antagonis0, injected i.c.v., blocked the deficit of place learning induced by baclofen, confirming that baclofen-induced deficit of place learning observed here is mediated by GABA a receptors. Our present results are consistent with the previous reports that baclofen induced the deficit of learning and memory in animals [4,6,11,24,34,36] and humans [30]. Moreover, GABA B antagonists suclh as 2-hydroxysaclofen, phaclofen, CGP35348 and CGP36742 improved the deficits of learning and memory in rodents and monkeys [5,21], and enhanced long-term potentiation in vitro [9,22,26]. These findings support that GABAB receptor systems are related to learning and memory. The present study showed that oxotremorine improved the deficit of place learning induced by baclofen. Oxotremorine attenuated the increased latency in the training trials and the reduced duration during the probe test in the baclofen-treated rats (Fig. 5). Taking into consideration that oxotremorine also improved the scopolamine-induced deficit of place learning (Fig. 2), it is concluded that cholinergic systems may play an important role in the deficit of place learning induced by baclofen. Previously, baclofen has been reported to exacerbate the deficit in the radial arm maze task in the scopolamine-treated rats [32], and to decrease the release of acetylcholine in rat brain [10]. Additionally, Castellano and McGaugh [6] found that oxotremorine attenuated the baclofen-impaired passive avoidance learning. These findings suggest that cholinergic systems may interact with GABA a receptor systems in the brain. It is possible that GABA a receptor systems may modulate learning and memory through influences on cholinergic systems. We examined the effects of oxotremorine on the rotarod performance in the baclofen-treated rats in order to determine whether the ameliorative effects of oxotremorine on baclofen-impaired place learning were due to improvement of motor incoordination. Oxotremorine at a dose of 0.1 m g / k g failed to improve the motor incoordination induced by baclofen, while oxotremorine at this dose improved the baclofen-impaired place learning. Thus, it is concluded that ameliorative effects of oxotremorine on the deficit of learning and memory induced by baclofen may not be due to the improvement of motor incoordination. Animals trained on a task under some drugs often show a failure of learning performance when they are tested in the absence of the drugs. The failure, however, is ameliorated when the drugs are re-introduced. These phenomena are termed state-dependent learning [28]. In state-dependent learning (SDL), it is hypothesized that acquisition of a task under a drug may require the same or similar drug
213
state for recall, because the drug serves as a relevant internal cue [2]. Previously, we reported that muscimol, a GABA A agonist, induced SDL in the passive avoidance task in rats: pre-training injection of muscimol reduced step-through latency in the test session, but muscimol-reduced latency disappeared when muscimol was re-injected before the test session [24]. Alternatively, the rats injected with baclofen before the training and/or test sessions showed reduced latency: baclofen failed to induce SDL [24], suggesting that baclofen induced deficits of learning and memory. We found in the present study that baclofen also induced deficit of place learning in Morris water maze task. Additionally, muscimol also induced SDL in Morris water maze task [23]. Thus it is concluded that GABA A and GABA B receptors may influence learning and memory in a different manner: activation of G A l A A receptors may induce SDL, whereas GABA a receptor stimulus may impair learning and memory.
References [1] Akema, T. and Kimura, F., 2-Hydroxysaclofen, a potent GABA B receptor antagonist, stimulates luteinizing hormone secretion in female rats, Brain Res., 546 (1991) 143-145. [2] Bliss, D.IC, Theoretical explanations of drug-dissociated behaviors, Fed. Proc., 33 (1974) 1787-1796. [3] Bowery, N., GABA n receptors and their significance in mammalian pharmacology, Trends Pharmacol. Sci., 10 (1989) 401-407. [4] Castellano, C., Brioni, J.D., Nagahara, A.H. and McGaugh, J.L., Post-training systemic and intra-amygdala administration of the GABA n agnnist baclofen impairs retention, Behav. Neural. Biol., 52 (1989) 170-179. [5] Castellano, C., Cestari, V., Cabib, S. and Puglisi-Allegra, S., Straindependent effects of post-training GABA receptor agonists and antagonists on memory storage in mice, Psychopharmacology, 111 (1993) 134-138. [6] Castellano, C. and McGaugh, J.L., Oxotremorine attenuates retrograde amnesia induced by post-training administration of the GABAergic agonists muscimol and baclofen, Behav. Neural. Biol., 56 (1991) 25-31. [7] Chu, D.C.M., Penney Jr., J.B. and Young, A.B., Cortical GABA B and GABAA receptors in Alzheimer's disease: a quantitative autoradiographic study, Neurology, 37 (1987) 1454-1459. [8] Collerton, D., Cholinergic function and intellectual decline in Alzheimer's disease, Neuroscience, 19 (1986) 1-28. [9] Davies, C.H., Starkey, S.J., Pozza, M.F. and Collingridge, G.L., GABA a autoreceptors regulate the induction of LTP, Nature, 349 (1991) 609-611. [10] DeBoer, P. and Westerink, B.H.C., GABAergic modulation of striatal cholinergic interactions: an in vivo microdialysis study, 3". Neurochem., 62 (1994) 70-75. [11] DeSousa, N.J., Beninger, R.J., Jhamandas, K. and Boegman, R.J., Stimulation of GABAa receptors in the basal forebrain selectively impairs working memory of rats in the double Y-maze, Brain Res., 641 (1994) 29-38. [12] Dokla, C.P.J. and Rydelek-Fitzgerald, L., Comparison of tetrahydroaminoacridine and physostigmine on scopolamine-induced free swim behavior in the rat, Psychopharmacology, 103 (1991) 240-243. [13] Dunlap, IC and Fischbach, G.D., Neurotransmitters decrease the calcium conductance activated by depolarization of embryonic chick sensory neurones, J. Physiol., 317 (1981) 519-535.
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[14] Guidotti, A., Corda, M.G., Wise, B.C., Vaccarino, W.F. and Costa, E., GABAergic synapses: supramolecular organization and biochemical regulation, Neuropharmacology, 22 (1983) 1471-1479. [15] Hill, D.R., Bowery, N.G. and Hudson, A.L., Inhibition of GABA n receptor binding by gnanyl nucleotides, J. Neurochem., 42 (1984) 652-657. [16] Ishihara, S., Hiramatsu, M., Kameyama, T. and Nabeshima, T., Development of tolerance to amnesic effects of chlordiazepoxide in relation to GABAergic and cholinerglc neuronal systems, Eur. J. Pharmacol., 230 (1993) 313-320. [17] Jerusalinsky, D., Quillfeldt, J.A., Walz, R., Da Silva, R.C., e Silva, M.B., Bianchin, M., Schmitz, P., Zanatta, M.S., Ruschel, A.C., Paczko, N., Medina, J.H. and Izquierdo, I., Effect of the infusion of the GABAA receptor agonist, muscimol, on the role of the entorhinal cortex, amygdala, and hippocampus in memory processes, Behay. Neural. Biol., 61 (1994) 132-138. [18] Lloyd, K.G., Thuret, F. and Pilc, A., Upregnlation of gamma-aminobutyric acid (GABA) B binding sites in rat frontal cortex: a common action of repeated administration of different classes of antidepressants and electroshock, J. Pharmacol. Exp. Ther., 235 (1985) 191-199. [19] Matsumoto, R.R., GABA receptors: are cellular differences reflected in function?, Brain Res. Rev., 14 (1989) 203-225. [20] McNamara, R.K. and Skelton, R.W., Assessment of a cholinergic contribution to chlordiazepoxide-induced deficits of place learning in the Morris water maze, Pharmacol. Biochem. Behav., 41 (1992) 529-538. [21] Mondadofi, C., Jaekel, J. and Preiswerk, G., CGP36742: the first orally active GABA n blocker improves the cognitive performance of mice, rats, and rhesus monkeys, Behav. Neural. Biol., 60 (1993) 62-68. [22] Mott, D.D. and Lewis, D.V., Facilitation of the induction of longterm potentiation by GABA n receptors, Science, 252 (1991) 17181720. [23] Nakagawa, Y., Ishibashi, Y., Yoshii, T. and Tagashira, E., Muscitool induces state-dependent learning in Morris water maze task in rats, Brain Res., 681 (1995) 126-130. [24] Nakagawa, Y., Iwasaki, T., Ishima, T. and Kimura, K., Interaction between benzodiazepine and GABAA receptors in state-dependent learning, Life Sci., 52 (1993) 1935-1945.
[25] Nicoll, R.A. and Newberry, N.R., A possible postsynaptic inhibitory action for GABA n receptors on hippocampal pyramidal cells, Neuropharmacology, 78 (1984) 849-850. [26] Olpe, H.-R. and Karlsson, G., The effects of baclofen and two GABAn-receptor antagonists on long-term potentiation, Psychopharmacology, 342 (1990) 194-197. [27] Olsen, R.W., GABA-benzodiazepine-barbiturate receptor interactions, J. Neurochem., 37 (1981) 1-13. [28] Overton, D.A., State-dependent or 'dissociated' learning produced with pentobarbital, J. Comp. Physiol. Psychol., 57 (1964) 3-12. [29] Paylor, R. and Rudy, J.W., Chofinergic receptor blockade can impair the rat's performance on both the place learning and cued versions of the Morris water task: the role of age and pool wall brightness, Behav. Brain Res., 36 (1990) 79-90. [30] Sandyk, R. and Gillman, M.A., Baclofen-induced memory impairment, Clin. Neuropharmacol., 8 (1985) 294-295. [31] Sawynok, J., GABA B receptors and analgesia. In N.G. Bowery, H. Bittiger and H.-R. Olpe (Eds.), GABA B receptors in mammalian function, John Willey and Sons, West Sussex, England, 1990, pp. 369-383. [32] Sidel, E.S., Tilson, H.A., McLamb, R.L., Wilson, W.A. and Swartzwelder, H.S., Potential interactions between GABAb and cholinergic systems: baclofen augments scopolamine-induced performance deficits in the eight-ann radial maze, Psychopharmacology, 96 (1988) 116-120. [33] Smith, C.P.S., Hunter, A.J. and Bennett, G.W., Effects of (R)alpha-methylhistamine and scopolamine on spatial learning in the rat assessed using a water maze, Psychopharmacology, 114 (1994) 651-656. [34] Stackman, R.W. and Walsh, T.J., Baclofen produces dose-related working memory impairments after intraseptal injection, Behav. Neural Biol., 61 (1994) 181-185. [35] Sunderland, T., Tariot, P.N. and Newhouse, P.A., Differential responsivity of mood, behavior, and cognition to cholinergic agents in elderly neuropsychiatric populations, Brain Res. Rev., 13 (1988) 371-389. [36] Swartzwelder, H.S., Tilson, H.A., McLamb, R.L. and Wilson, W.A., Baclofen disrupts passive avoidance retention in rats, Psychopharmacology, 92 (1987) 398-401.
ELSEVIER
Research report
Involvement of cholinergic systems in the deficit of place learning in Morris water maze task induced by baclofen in rats Yutaka Nakagawa *, Yoshinori Ishibashi, Toshio Yoshii, Eijiro Tagashira Tsukuba Research Laboratories, Experimental Biomedical Research Inc. (Jisseiken), 8-5-1, Chuo, Ami-machi, Inashiki-gun, lbaraki 300-03, Japan Accepted 22 Febuari 1995
Abstract
Effects of oxotremorine on the deficit of place learning in the Morris water maze task induced by baclofen and scopolamine were examined to determine the i~wolvement of brain cholinergic systems in the deficit of learning induced by baclofen. Rats were given 4 training trials per day with the submerged platform at a fixed location in the maze for 4 days. On day 4, rats were required to swim in the pool without the platform after the 4th training trial (probe test). Baclofen as well as scopolamine dose-dependently increased the escape latency in the training trials. In the probe test, baclofen as well as scopolamine dose-dependently reduced the duration in the quadrant where the platform had been originally located. Increased latency in the training trials and reduced duration in the probe test induced by scopolamine were dose-dependently attenuated by oxotremorine. Increased latency and reduced duration in the baclofen-treated rats were improved by oxotremorine as well as 2-hydroxysaclofen. Baclofen but not scopolamine induced motor incoordination in the rotarod test. Oxotremorine failed to improve motor incoordination induced by baclofen. These results suggest that cholinergic systems may be involved in the deficit of place learning induced by baclofen, and that the ameliorative effects of oxotremorine may not be due to improvement of motor incoordination.
Keywords: Place learning; Morris water maze task; Baclofen; 2-Hydroxysaclofen; Oxotremorine; Motor incoordination
1. Introduction
GABA is the main inhibitory neurotransmitter in the brain. There is evidence that there are two different GABA receptors in the brain: GABA A and GABA B receptors [3,19]. GABA A receptors are coupled with benzodiazepine receptors and C1- channels [14,27]. On the other hand, GABA B receptors are coopled with G protein [15]. The activation of GABA B receptors decreases the amplitude of Ca 2÷ currents [13] and increases the K ÷ conductance [25]. GABA A and GABA B receptors have somewhat different physiological actions. GABA A receptors are mainly involved in anxiety and convulsion [19]. In contrast, GABA B receptors are mainly related to depression and analgesia [18,31]. Howeve:r, GABA A as well as GABA B receptors play an important role in learning and memory [4-6,11,16,17,21,32,34,36]
* Corresponding author. Fax: (81) (298) 87-9065. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All fights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 5 ) 0 0 3 0 2 - 9
We previously reported that pre-training injections of baclofen (GABA u agonist) as well as muscimol (GABA A agonist) had detrimental effects on passive avoidance learning in rats when the test session was conducted 24 h later [24]. In that study, we also found that muscimol-reduced latency disappeared when muscimol was re-injected before the test session: muscimol induced state-dependent learning. Alternatively, the rats injected with baclofen before the training and test sessions showed reduced latency: baclofen failed to induce state-dependent learning. These findings provide the direct evidence that GABA A and GABA B receptors may influence learning and memory in a different manner. Cholinergic systems in the brain play an important role in learning and memory [35]. Especially, dementia of the Alzheimer type (DAT) has been shown to be closely associated with deficit in the central cholinergic systems [8]. Recently, GABA B receptor systems are also found to be abnormal in DAT: scatchard analysis and competition studies showed significant decrement in receptor density (Bmax) for GABA B receptors [7]. It is reported that GABA B
Y. Nakagawa et al. /Brain Research 683 (1995) 209-214
210
systems may interact with cholinergic systems [6,10,30,32]. Based on these results, we designed the experiment to examine the involvement of cholinergic systems in the deficits of learning and memory induced by baclofen. In the present study, the Morris water maze task was chosen because the step-through passive avoidance task which was used in the previous study [24] appeared to be influenced by muscle relaxation induced by baclofen. In that study, 2 to 4 of 10 rats given baclofen were moved to the dark compartment by the experimenter in the training session because of muscle relaxation [24]. A rotarod test was also conducted to examine the relationship between motor incoordination and the deficit of performance in the Morris water maze task.
60 "-" o 50 o) 4 0 •
#
#
v
>" 3 0 o E
SA+SA
~
~
~
##
- - o - sco~sA ""~__~'~ SCO÷OXO(0.01) = SCO+OXO(0,03) .J 1020
~
##
SCO+OXO(0.1)
0 1
2
3
4
Days •*
##
40-
o Q (o 30"
I
-I-
(-
.9 ~ 20
2. Materials and methods
Cl
2.1. Animals
10
Male Wistar rats (Charles River J a p a n ) , weighing 260 g at the beginning of the experiment, were used. were housed in groups in an air-and light-controlled (temperature; 24 + 2 ° C, light phase; 08.00-20.00 h). and water were given ad libitum.
200They room Food
o 50
o E
40
o ~
**~ ~,-.
0.03
0.1
OXO (mg/kg, i.p.)
SA
SCO (0.3 mg/kg, i.p.) Fig. 2. Effects of oxotremorine on the deficit of place learning in the Morris water maze task induced by scopolamine. Upper panel: scopolamine (SCO, 0.3 mg/kg) and oxotremorine(OXO) were injected i.p. 30
2.2. Apparatus and procedure
sco(0.3)
SCO(1,0)
10 O" 1
2
3
4
Days 40 O v
0.01
30-
2 0 ----o-sco(0.1)
(D
SA
and 15 rain before the training trials, respectively.Numbers in parentheses: drug dose in mg/kg. * P <0.05, * * P<0.01 versus SA+SA; # P < 0.05, ## P < 0.01 versus SCO + SA. lower panel: duration in the quadrant where the platform had been located. See Fig. 1 for further information.
60
v
SA
-I30
e-.
._o 20-
10"
SA
0.1
0.3
1.0
SCO (mg/kg, i.p.) Fig. 1. Effects of scopolamine on place learning in the Morris water maze task. Upper panel: mean latency to reach the platform in the training trials. Scopolamine (SCO) was injected i.p. 30 min before the training trials. Ten rats were used in each group. Numbers in parentheses: drug dose in m g / k g . SA, saline; * * P < 0.01 versus saline control. Lower panel: duration in the quadrant where the platform had been located. A 60-s probe test was conducted after the 4th training trial on day 4. Data are expressed as mean with S.E.M.
Morris water maze task A circular pool (150 cm in diameter, 35 cm high) was filled 24 cm deep with clear water (21 + 1° C). A clear platform (13 cm in diameter) was submerged 1 cm below the water surface. The pool was conceptually divided into qua&ants: northeast (NE), northwest (NW), southeast (SE) and southwest (SW). The platform was located in the center of NW quadrant throughout the training trials except the probe test which was conducted on day 4. Rats were given 4 training trials each day for 4 consecutive days. For each training trial, the rats were placed in the water facing the pool wall at one of 4 positions (north, south, east or west poles) in a different order each day, and allowed to swim until they reached the platform. Latency to reach the platform was recorded up to 60 s. On the platform they remained for 10 s before removal. A rat which failed to reach the platform within 60 s was guided to it by the experimenter and the maximum latency was scored. After each training trial, the rat was dried with a towel and allowed to remain in a cage for the intertrial interval (approximately 3 min).
Y. Nakagawa et aL /Brain Research 683 (1995) 209-214
On day 4, a 60-s probe test without the platform was conducted after the 4th training trial. The time spent in the NW quadrant was recorded. Rotarod test Motor incoordination was tested for 3 min using a rotarod performance apparatus (9 cm in diameter, 5 rpm; Natsume Seisakusho Co., Tokyo, Japan). The day before the test, each rat had been trained to run on a rotarod until it could remain there for 3 min without falling.
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60
o 50 O t~ 40 e--
30 20
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I
1
2
2.3. Drugs
40
Baclofen (RBI), 2-hydroxysaclofen (RBI), scopolamine hydrobromide (Sigma), oxotremorine (Sigma) were used. 2-Hydroxysaclofen was first dissolved in 0.16 M NaOH, then the solution was neutralized with equimolar HC1 [1] and diluted with saline to final concentration of 25/zg/2.5 ~1. 2-Hydroxysaclofen was, injected i.c.v, in a volume of 2.5 /~l/rat. Other drugs were dissolved in saline, and injected i.p. in a volume of 1 m l / k g except for baclofen which was injected in a volume of 4 ml/kg. 2-Hydroxysaclofen was injected 35 and 5 min before the training trials. Baclofen, scopolamine and oxotremorine were injected 30, 30 and 15 rain before the training trials, respectively. The dosage of scopolamine was expressed as the salt.
3
4
Days
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##
o --I-
(o 30 --4 tO
.m
20
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SA
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Fig. 4. Effects of 2-hydroxysaclofen on the deficit of place learning in the Morris water maze task induced by baciofen. Upper panel: mean latency to reach the platform in the training trials. Baclofen (BAC, 4 mg/kg) was injected i.p. 30 rain before the training trials. 2-Hydroxysaclofen (2OHSAC, 25 p,g/2.5 /zl/rat) was injected i.c.v. 35 and 5 rain before the training trials. * * P < 0.01 versus S A + S A ; # # P < 0.01 versus BAC+ SA. Lower panel: duration in the quadrant where the platform had been located. See Fig. 1 for further information.
60 ~-, 501 o m t~ 40.
2.4. Statistics
>" 30. tO
Between-group comparisons were assessed by Dunnett test following one-way ANOVA for each training trial and probe test.
20 BAC(2) BAC(4)
._1 10 ~
1
2
Days
3
4
3. Results
40 O
3. I. Effects of oxotremorine on scopolamine-induced deficit of place learning in the Morris water maze task
-T
to 3 0 t-
._o 2o 10 SA
1
2
4
BAC (mg/kg, i.p.) Fig. 3. Effects of baclofen on place learning in the Morris water maze task. Upper panel: mean latency to reach the platform in the training trials. Baclofen (BAC) was injected i.p. 30 rain before the training trials. Numbers in parentheses: drug dose in mg/kg. * * P < 0.01 versus saline control. Lower panel: duration in the quadrant where the platform had been located. See Fig. 1 for further information.
Upper panels of Figs. 1 and 2 show the latency to reach the platform in the training trials. Scopolamine dose-dependently increased the latency. Oxotremorine dose-dependently attenuated the increase in latency induced by scopolamine (0.3 mg/kg). On day 4, a 60-s probe test without the platform was conducted after the 4th training trial. Scopolamine dosedependently reduced the duration in the quadrant where the platform had been originally located (Fig. 1). The rats given oxotremorine showed dose-dependent improvement of scopolamine (0.3 mg/kg)-reduced duration in the probe test (Fig. 2).
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Y. Nakagawa et al. / Brain Research 683 (1995) 209-214 60 ¸ o 50 (D 09 4 0
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...1 10
Y. Nakagawa et al. /Brain Research 683 (1995) 209-214
suggest that place learning may be 'probed' rather in the probe test than in the training trials. We found here that badofen (GABA B agonist) as well as scopolamine induced the deficit of place learning. Baclofen dose-dependently increased the latency in the training trials, and reduced theduration in the probe test. 2-Hydroxysaclofen (GAB.A s antagonis0, injected i.c.v., blocked the deficit of place learning induced by baclofen, confirming that baclofen-induced deficit of place learning observed here is mediated by GABA a receptors. Our present results are consistent with the previous reports that baclofen induced the deficit of learning and memory in animals [4,6,11,24,34,36] and humans [30]. Moreover, GABA B antagonists suclh as 2-hydroxysaclofen, phaclofen, CGP35348 and CGP36742 improved the deficits of learning and memory in rodents and monkeys [5,21], and enhanced long-term potentiation in vitro [9,22,26]. These findings support that GABAB receptor systems are related to learning and memory. The present study showed that oxotremorine improved the deficit of place learning induced by baclofen. Oxotremorine attenuated the increased latency in the training trials and the reduced duration during the probe test in the baclofen-treated rats (Fig. 5). Taking into consideration that oxotremorine also improved the scopolamine-induced deficit of place learning (Fig. 2), it is concluded that cholinergic systems may play an important role in the deficit of place learning induced by baclofen. Previously, baclofen has been reported to exacerbate the deficit in the radial arm maze task in the scopolamine-treated rats [32], and to decrease the release of acetylcholine in rat brain [10]. Additionally, Castellano and McGaugh [6] found that oxotremorine attenuated the baclofen-impaired passive avoidance learning. These findings suggest that cholinergic systems may interact with GABA a receptor systems in the brain. It is possible that GABA a receptor systems may modulate learning and memory through influences on cholinergic systems. We examined the effects of oxotremorine on the rotarod performance in the baclofen-treated rats in order to determine whether the ameliorative effects of oxotremorine on baclofen-impaired place learning were due to improvement of motor incoordination. Oxotremorine at a dose of 0.1 m g / k g failed to improve the motor incoordination induced by baclofen, while oxotremorine at this dose improved the baclofen-impaired place learning. Thus, it is concluded that ameliorative effects of oxotremorine on the deficit of learning and memory induced by baclofen may not be due to the improvement of motor incoordination. Animals trained on a task under some drugs often show a failure of learning performance when they are tested in the absence of the drugs. The failure, however, is ameliorated when the drugs are re-introduced. These phenomena are termed state-dependent learning [28]. In state-dependent learning (SDL), it is hypothesized that acquisition of a task under a drug may require the same or similar drug
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state for recall, because the drug serves as a relevant internal cue [2]. Previously, we reported that muscimol, a GABA A agonist, induced SDL in the passive avoidance task in rats: pre-training injection of muscimol reduced step-through latency in the test session, but muscimol-reduced latency disappeared when muscimol was re-injected before the test session [24]. Alternatively, the rats injected with baclofen before the training and/or test sessions showed reduced latency: baclofen failed to induce SDL [24], suggesting that baclofen induced deficits of learning and memory. We found in the present study that baclofen also induced deficit of place learning in Morris water maze task. Additionally, muscimol also induced SDL in Morris water maze task [23]. Thus it is concluded that GABA A and GABA B receptors may influence learning and memory in a different manner: activation of G A l A A receptors may induce SDL, whereas GABA a receptor stimulus may impair learning and memory.
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