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substantia innominata complex
Neuroscience Letters, 124 (1991) 246250 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100055E
246
NSL 07647
Motor activity and the GABAA-receptor in the ventral pallidum/ substantia innominata complex R u u d van den Bos a n d A l e x a n d e r R. Cools Psychoneuropharmacology Research Unit, Catholic University Nijmegen, Nijrnegen ( The Netherlands) Received 21 June 1990; Revised version received I 0 December 1990; Accepted 7 January 1991)
Key words: Motor activity; Muscimol; Bicuculline; Antagonism; Ventral pallidum; Substantia innominata; Rat The present study deals with the role of the 7-aminobutyric acid-A (GABAA) receptor in motor activity in the rostral part of the ventral pallidum/ substantia innominata complex. Both the specific GABAA antagonist bicuculline (25-100 ng/0.5/tl) and the GABAA agonist muscimol (25 ng/0.5 Ftl) enhanced motor activity. It was moreover found that bicuculline (50-100 ng) dose-dependently attenuated the activity induced by muscimol (25 ng). Conversely, muscimol (25 ng) attenuated the biculline (25-50 ng) induced activity. These data thus show that both stimulation and blockade of GABAA receptors within the area under study enhance motor activity.
The subpallidal area, i.e. the ventral pallidum/substantia innominata complex (VP/SI complex) is one of the major projection areas of the nucleus accumbens and olfactory tubercle [9, 11]. Biochemical [17], electrophysiological [18] and behavioural [4, 10] experiments have suggested that the accumbens-VP/SI pathway contains 7-aminobutyric acid (GABA) as neurotransmitter. It has been shown that blocking the GABA-activity with GABA antagonists in the VP/SI complex enhances motor activity [1, 10, 13, 16]. No studies have thus far shown that the specific GABAA agonist muscimol [6, 14] is able to attenuate spontaneous activity (see however Fig. 1 in ref. 2). Instead, muscimol has in fact been found to enhance motor activity [2, 5, 13, 15]. Although this is a rather controversial finding, relatively few studies have looked deeper into the latter phenomenon. It was decided therefore to study the role of the GABAA receptor in the regulation of motor activity further using the GABAA-selective agonist muscimol and antagonist bicuculline [6, 14] as tools. Male Wistar rats (n = 69), weighing 180-200 g at the time of the operation, were anaesthetized with pentobarbital (60 mg/kg; Narcovet) and bilaterally equipped with cannulae (total length 9.0 mm; i.d. 0.2 mm; o.d. 0.65 mm) aimed at the rostral areas of the VP/SI complex (A 7.4, H 3.1, L + / - 2 . 5 mm stereotactic coordinates; see refs. 4, 5). The cannulae were inserted under a lateral anCorrespondence." A.R. Cools, Psychoneuropharmacology Research Unit, Catholic University Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
gle of a 5 ° (tip of the cannulae 4.0 mm below the surface of the skull), and secured onto the skull with dental acrylic cement (Paladur) and stainless-steel retaining screws. After the operation the rats were individually housed in macrolon cages in a temperature-controlled room (T=21 + I ° C ) on a standard day-night cycle (18.0006.00 h lights off). Water and food were available as required. Rats were given at least one week of recovery from the operation before the experiments started. Hand-held rats were bilaterally injected with the solvent or the appropriate drugs with a Hamilton syringe extending 4.0 mm under the tip of the cannulae. A volume of 0.5 /tl was delivered over a 5 s period after which the needle was kept in place for another 10 s. Distilled water (AD; PBNI, The Netherlands) was used as solvent for the drugs and as control throughout the study. Both muscimol (Serva, F.R.G.) and bicucullinemethiodide (Sigma, U.S.A.) were dissolved in AD. Bicuculline was freshly prepared each time. Muscimol was dissolved and stored in small aliquots (1000 /tl) at -20°C until use. In case it was intended to antagonize drug-induced effects, drugs were mixed in one solution and co-injected. All experiments were performed between 10.00 and 15.00 h. After the experiments the rats were sacrificed by an overdose of pentobarbital and perfused transcardially with 4% formalin. The brains were removed and stored on 4% formalin until sectioning. Brain slices (20-30/zm) were cut on a freezing microtome (Bright, U.K.) and injection sites were determined according to the atlas of Paxinos and Watson [12].
247
Rats were habituated to the motor cages for 60 minutes after which AD was injected in all the rats used in this experiment. Activity was measured for 180 minutes. The day after (i.e. the experimental day) the rats were rehabituated to the motor cages for 60 minutes and subsequently injected with AD or the appropriate drugs or their combination. Activity was again measured for 180 minutes. Using the present paradigm the rats were thus habituated to both the injection (-procedure) and the
experimental cages. Rats were only used for one experiment. Motor activity was measured in motor cages (36 x 2 4 x 25 cm) equipped with three photo-electric cells; two along the long, one along the short end of the cages, 2 cm above the grid-floor (see ref. 7). The motor cages were individually placed in diffusely illuminated sound-proof chambers. The number of beam interruptions was automatically registered. Data were trans-
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Fig. 1. Representative series of injection sites within the rostral VP/SI complex. Planes were taken from the atlas of Paxinos and Watson [I 2] and represent the following anteriorities (ram from bregma, A-E): -0.26, -0.30, -0.40, - 0 . 8 0 and -0.92, respectively. The shaded area represents the ventral pallidum, the stippled area the globus pallidus, and the hatched area the substantia innominata according to the atlas of Paxinos and Watson [12]. Abbreviations are also according to this atlas (CPU, caudatoputamen complex).
248 Counts / 10 rain
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Fig. 2. Effect of bicuculline (bic; 25-100 ng/0.5/4; A), muscimol (msm; 25 ng/0.5/d; B) and their combination (C) on motor activity in habituated rats. Horizontal axis, time after injection; vertical axis, counts per 10 min. Shown are means_S.E.M.'s of 7-9 rats per group. A: O - O AD 0.5/4; 0 - 0 bic 25 ng/0.5/4; & - A bic 50 ng/0.5/4; A - A bic, 100 ng/0.5 #1; B: O - O AD 0.5 #1; 0 - 0 msm 25 ng/0.5/A; C: O - O msm 25 ng/0.5/4; 0 - 0 msm 2 5 + b i c 25 ng/0.5 #1; ~ - , 5 msm 2 5 + b i c 50 ng/0.5 #1; A - A msm 2 5 + b i c 100 ng/0.5 #1
ferred from a temporary buffer to a PDP-11 computer (Digital, The Netherlands). Means + S.E.M.'s were subsequently calculated per block of 10 min. Statistics were performed using a two-way analysis of variance (ANOVA) with doses or drugs and time (unit= 10 min) as factors, followed by a Student-Newman-Keuls (SNK) test when appropriate (SAS Institute, U.S.A.). Cell-entries were scores per rat per block of 10 min. In cases in which single periods of 10 min were analysed t-tests were used. Only the scores of the experimental day, i.e. the second day, were statistically analysed (two-way ANOVA and t-test for independent measurements). The injection sites were confined to the rostral part of the VP/SI complex (Fig. 1). Three rats were discarded for inappropriate placement of their cannulae. Bicuculline enhanced motor activity (Fig. 2A; ANOVA: F3,558= 37.04, P~<0.01). A bell-shaped curve seemed to exist however: whereas the doses of 25 and 50 ng clearly enhanced activity, the dose of 100 ng only slightly enhanced it. This was confirmed by a post-hoc SNK test: the results of the 100 ng dose were not significantly different from AD, whereas the results of the 25 and 50 ng doses were (P ~<0.05). Furthermore the dose of 50 ng was significantly (P~<0.05) different from the dose of 25 ng. As can be seen in Fig. 2B, muscimol (25 ng) enhanced motor activity (ANOVA: Fi,270=240.34, P~<0.01). When the above-mentioned doses of bicuculline were combined with 25 ng muscimol a dose-dependent attenuation of the muscimol induced activity was found (Fig. 2C; ANOVA: F3,486=4.98, P~<0.01). Moreover a significant interaction term was found (F51,486=2.02, P~<0.01). It is clear that the effect was most marked in the first 60 min, which appears to agree with the shortterm effect of bicuculline (cf. Fig. 2A). Analysing accordingly the first 60 rain it turned out that both 50 and 100 ng bicuculline were effective in attenuating (P~<0.05, SNK test) the muscimol induced activity (ANOVA: /73,162 = 7.98, P ~<0.01). Furthermore the curves showed a parallel shift; accordingly there was no significant interaction-term (F15,162= 1.49, P~<0.12). When the remainder of the experimental period was analysed, i.e. 70-180 min after injection, it turned out that the rats treated with muscimol 25 ng and bicuculline 100 ng were significantly more active (P~<0.05, SNK-test) than those in any of the other groups (ANOVA: F3,324=4.20, P<~0.01). Muscimol also significantly attenuated the bicuculline induced activity in the first 10 min period (compare Fig. 2C with A): msm 25+bic 25 vs bic 25 (mean+S.E.M.): 111.1+96.0 vs 521.5+79, t=3.300 d f = 15, P~<0.01; msm 25+bic 50 vs bic 50:78.0+9.7 vs 819.0+147.8, t=5.001 df=14, P~<0.01 (two-tailed ttests).Muscimol also antagonized the bicuculline
249
induced activity in the second 10 min period: msm 25+bic 50 vs bic 5 0 : 1 7 8 + 1 3 5 . 0 vs 836.1+182.4, t = 2.900 d f = 14, P ~<0.05 two-tailed t-test). Bicuculline dose-dependently enhanced motor activity (cf. ref. 1), which was attenuated by the specific GABAA agonist muscimol [6, 14], allowing the conclusion that the effect was mediated via GABAA receptors. A new finding is the observation that bicuculline had a dual effect: at lower doses it markedly enhanced motor activity whereas at higher doses it produced only a weak effect. This phenomenon needs to be studied in more detail in future studies. Muscimol enhanced motor activity (cf. refs. 2, 5, 13, 15). The specific GABAA antagonist bicuculline [6, 14] dose-dependently attenuated this muscimolinduced activity. Moreover the muscimol induced activity returned to baseline values at the moment that the efficacy of bicuculline began to wear off. From these data it can be concluded that both stimulation of GABAA receptors and blockade of GABAA receptors lead to an increase in motor activity. At this stage it cannot be decided whether these effects are mediated via GABAA receptors located on the same populations of neurons or via GABAA-receptors located on different (sub)populations of neurons. In the latter case differences in the basal GABA activity within each subpopulation may determine the differential sensitivity to the agonist and the antagonist respectively (cf. ref. 8 and references cited therein). Although Scheel-Krtiger observed that the area from which muscimol induced motor activity differs slightly from the area from which the indirect GABAA antagonist picrotoxin induced motor activity [13], such a distinction was not found in the present study: the injection-sites for bicuculline and muscimol were located in the same area (Fig. 1). From the graphs it is clear that differences between the bicuculline and muscimol induced activity existed. First, the activity induced by bicuculline was short-lasting in contrast to the activity of muscimol, which was longlasting. Second, the onset of the bicuculline induced activity was faster than the onset of the muscimol induced activity. In these respects the bicuculline induced motor activity resembled the motor activity induced by Damphetamine in the nucleus accumbens [3] or dopamine in the olfactory tubercle [7]. Until now no clear counterpart for a muscimol type of activity has been found in the nucleus accumbens or olfactory tubercle. Thus future experiments should be directed at elucidating the behavioural differences between the activity induced by muscimol and bicuculline. At present it is not clear whether these differences were due to differences in pharmacokinetic or physicochemical properties of the drugs, differences in interaction with GABAA receptors, or whether they reflected interactions with differentially localized
GABAA receptors. It should be noted that previous experiments have shown that neither bicuculline nor muscimol was effective in increasing motor activity after injection into the caudal areas of the VP/SI complex [5]: instead, they affect tonic electromyographic activity in the triceps muscle of the forelimb [4]. These data not only exclude the posssibility that the - differential - effects observed after injection of muscimol and bicuculline in the present study were due to - differential spread of these drugs to caudal areas but also underline the earlier reported heterogeneity of the VP/SI complex [5]. In conclusion, the main findings of this study are that both bicuculline and muscimol enhanced motor activity when injected into the rostral part of the VP/SI complex and that these effects could be attenuated by muscimol and bicuculline, respectively. This study thus not only confirms but also extends the findings of previous experiments that muscimol [2, 5, 13, 15] and bicuculline [1] enhance motor activity. Since muscimol and bicuculline are a GABAA-specific agonist and antagonist, respectively [6, 14], it seems likely that the effect of each single drug was mediated via GABAA receptors. I Austin, M.C. and Kalivas, P.W., Enkephalinergic and GABAergic modulation of motor activity in the ventral pallidum, J. Pharmacol. Exp. Ther., 252 (1990) 1370-1377. 2 Baud, P., Mayo, W., Le Moal, M. and Simon H., Locomotor hyperactivity in the rat after infusion of muscimol and [o-Ala]Metenkephalin into the nucleus basalis magnocellularis. Possible interaction with cortical cholinergic projections, Brain Res., 452 (1988) 203-21 I. 3 Bos, van den R., Cools, A.R. and 0gren, S.-O., Differential effects of the selective D2-antagonist raclopride in the nucleus accumbens of the rat on spontaneous and D-amphetamine induced activity, Psychopharmacology, 95 (1988) 447-451. 4 Bos, van den R. and Cools, A.R., Involvement of the substantia innominata/ventral pallidum complex in transmitting forelimb muscular rigidity evoked from the nucleus accumbens in rats, Neurosci. Lett., 103 (1989) 303-308. 5 Bos, van den R, and Cools, A.R., The ventral pallidum/substantia innominata complex: further evidence for heterogeneity as determined by the effects of GABA-ergic drugs, Behav. Pharmacol., 1 (Suppl. 1) (1989) 29. 6 Bowery, N.G., Hill, D.R. and Hudson, A.L., Characteristics of GABAB-receptor binding sites on rat whole brain synaptic membranes, Br. J. Pharmacol., 78 (1983) 191-206. 7 Cools, A.R., Mesolimbic dopamine and its control of locomotor activity in rats: differences in pharmacology and light/dark periodicity between the olfactory tubercle and the nucleus accumbens, Psychopharmacology, 88 (1986) 451-459. 8 Cools, A.R., Brachten, R., Heeren, D., Willemen, A. and Ellenbroek, B., Search after the neurobiological profile of individualspecific features of Wistar rats, Brain Res. Bull., 24 (1990) 49-69. 9 Heimer, L. and Wilson, G.D., The subcortical projections of the allocortex: similarities in the neural associations of the hippocampus, the piriform cortex and the neocortex. In M. Santini (Ed.) Golgi Centennial Symposium, Raven, New York, 1975, pp. 177193.
250 10 Jones, D.L. and Mogenson, G.J., Nucleus accumbens to globus pallidus GABA projection subserving ambulatory activity, Am. J. Physiol., 238 (1980) R65-R69. 11 Mogenson, G.J., Swanson, L.W. and Wu, M., Neural projections from nucleus accumbens to globus pallidus, substantia innominata, and lateral preoptic-lateral hypothalamic area: an anatomical and electrophysiological investigation in the rat, J. Neurosci., 3 (1983) 189-202. 12 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd ed., Academic Press, New York, 1986. 13 Scheel-Kriiger, J., The GABA receptor and animal behavior. In S.J. Enna (Ed.) The GABA Receptors, Humana Press, Clifton, N J, pp. 215-256. 14 Simmonds, M.A., Multiple GABA-receptors and associated regulatory sites, Trends Neurosci., 6 (1983) 279-281.
15 Swerdlow, N.R. and Koob, G.F. The neural substrates of apomorphine-stimulated locomotor activity following denervation of the nucleus accumbens, Life Sci., 35 (1984) 2537-2544. 16 Swerdlow, N.R. and Koob, G.F., Lesions of the dorsomedial nucleus of the thalamus, medial prefrontal cortex and pedunculopontine nucleus: effects on motor activity mediated by nucleus accumbens-ventral pallidum circuitry, Brain Res., 412 (1987) 233-243. 17 Walaas, I. and Fonnum, F., The distribution and origin of glutamate decarboxylase and choline acetyltransferase in ventral pallidum and other basal forebrain regions, Brain Res., 177 (1979) 325336. 18 Yang, C.R. and Mogenson, G.J., Ventral pallidum neuronal responses to dopamine receptor stimulation in the nucleus accumbens, Brains Res., 489 (1989) 237-246.
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Motor activity and the GABAA-receptor in the ventral pallidum/ substantia innominata complex R u u d van den Bos a n d A l e x a n d e r R. Cools Psychoneuropharmacology Research Unit, Catholic University Nijmegen, Nijrnegen ( The Netherlands) Received 21 June 1990; Revised version received I 0 December 1990; Accepted 7 January 1991)
Key words: Motor activity; Muscimol; Bicuculline; Antagonism; Ventral pallidum; Substantia innominata; Rat The present study deals with the role of the 7-aminobutyric acid-A (GABAA) receptor in motor activity in the rostral part of the ventral pallidum/ substantia innominata complex. Both the specific GABAA antagonist bicuculline (25-100 ng/0.5/tl) and the GABAA agonist muscimol (25 ng/0.5 Ftl) enhanced motor activity. It was moreover found that bicuculline (50-100 ng) dose-dependently attenuated the activity induced by muscimol (25 ng). Conversely, muscimol (25 ng) attenuated the biculline (25-50 ng) induced activity. These data thus show that both stimulation and blockade of GABAA receptors within the area under study enhance motor activity.
The subpallidal area, i.e. the ventral pallidum/substantia innominata complex (VP/SI complex) is one of the major projection areas of the nucleus accumbens and olfactory tubercle [9, 11]. Biochemical [17], electrophysiological [18] and behavioural [4, 10] experiments have suggested that the accumbens-VP/SI pathway contains 7-aminobutyric acid (GABA) as neurotransmitter. It has been shown that blocking the GABA-activity with GABA antagonists in the VP/SI complex enhances motor activity [1, 10, 13, 16]. No studies have thus far shown that the specific GABAA agonist muscimol [6, 14] is able to attenuate spontaneous activity (see however Fig. 1 in ref. 2). Instead, muscimol has in fact been found to enhance motor activity [2, 5, 13, 15]. Although this is a rather controversial finding, relatively few studies have looked deeper into the latter phenomenon. It was decided therefore to study the role of the GABAA receptor in the regulation of motor activity further using the GABAA-selective agonist muscimol and antagonist bicuculline [6, 14] as tools. Male Wistar rats (n = 69), weighing 180-200 g at the time of the operation, were anaesthetized with pentobarbital (60 mg/kg; Narcovet) and bilaterally equipped with cannulae (total length 9.0 mm; i.d. 0.2 mm; o.d. 0.65 mm) aimed at the rostral areas of the VP/SI complex (A 7.4, H 3.1, L + / - 2 . 5 mm stereotactic coordinates; see refs. 4, 5). The cannulae were inserted under a lateral anCorrespondence." A.R. Cools, Psychoneuropharmacology Research Unit, Catholic University Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
gle of a 5 ° (tip of the cannulae 4.0 mm below the surface of the skull), and secured onto the skull with dental acrylic cement (Paladur) and stainless-steel retaining screws. After the operation the rats were individually housed in macrolon cages in a temperature-controlled room (T=21 + I ° C ) on a standard day-night cycle (18.0006.00 h lights off). Water and food were available as required. Rats were given at least one week of recovery from the operation before the experiments started. Hand-held rats were bilaterally injected with the solvent or the appropriate drugs with a Hamilton syringe extending 4.0 mm under the tip of the cannulae. A volume of 0.5 /tl was delivered over a 5 s period after which the needle was kept in place for another 10 s. Distilled water (AD; PBNI, The Netherlands) was used as solvent for the drugs and as control throughout the study. Both muscimol (Serva, F.R.G.) and bicucullinemethiodide (Sigma, U.S.A.) were dissolved in AD. Bicuculline was freshly prepared each time. Muscimol was dissolved and stored in small aliquots (1000 /tl) at -20°C until use. In case it was intended to antagonize drug-induced effects, drugs were mixed in one solution and co-injected. All experiments were performed between 10.00 and 15.00 h. After the experiments the rats were sacrificed by an overdose of pentobarbital and perfused transcardially with 4% formalin. The brains were removed and stored on 4% formalin until sectioning. Brain slices (20-30/zm) were cut on a freezing microtome (Bright, U.K.) and injection sites were determined according to the atlas of Paxinos and Watson [12].
247
Rats were habituated to the motor cages for 60 minutes after which AD was injected in all the rats used in this experiment. Activity was measured for 180 minutes. The day after (i.e. the experimental day) the rats were rehabituated to the motor cages for 60 minutes and subsequently injected with AD or the appropriate drugs or their combination. Activity was again measured for 180 minutes. Using the present paradigm the rats were thus habituated to both the injection (-procedure) and the
experimental cages. Rats were only used for one experiment. Motor activity was measured in motor cages (36 x 2 4 x 25 cm) equipped with three photo-electric cells; two along the long, one along the short end of the cages, 2 cm above the grid-floor (see ref. 7). The motor cages were individually placed in diffusely illuminated sound-proof chambers. The number of beam interruptions was automatically registered. Data were trans-
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Fig. 1. Representative series of injection sites within the rostral VP/SI complex. Planes were taken from the atlas of Paxinos and Watson [I 2] and represent the following anteriorities (ram from bregma, A-E): -0.26, -0.30, -0.40, - 0 . 8 0 and -0.92, respectively. The shaded area represents the ventral pallidum, the stippled area the globus pallidus, and the hatched area the substantia innominata according to the atlas of Paxinos and Watson [12]. Abbreviations are also according to this atlas (CPU, caudatoputamen complex).
248 Counts / 10 rain
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Fig. 2. Effect of bicuculline (bic; 25-100 ng/0.5/4; A), muscimol (msm; 25 ng/0.5/d; B) and their combination (C) on motor activity in habituated rats. Horizontal axis, time after injection; vertical axis, counts per 10 min. Shown are means_S.E.M.'s of 7-9 rats per group. A: O - O AD 0.5/4; 0 - 0 bic 25 ng/0.5/4; & - A bic 50 ng/0.5/4; A - A bic, 100 ng/0.5 #1; B: O - O AD 0.5 #1; 0 - 0 msm 25 ng/0.5/A; C: O - O msm 25 ng/0.5/4; 0 - 0 msm 2 5 + b i c 25 ng/0.5 #1; ~ - , 5 msm 2 5 + b i c 50 ng/0.5 #1; A - A msm 2 5 + b i c 100 ng/0.5 #1
ferred from a temporary buffer to a PDP-11 computer (Digital, The Netherlands). Means + S.E.M.'s were subsequently calculated per block of 10 min. Statistics were performed using a two-way analysis of variance (ANOVA) with doses or drugs and time (unit= 10 min) as factors, followed by a Student-Newman-Keuls (SNK) test when appropriate (SAS Institute, U.S.A.). Cell-entries were scores per rat per block of 10 min. In cases in which single periods of 10 min were analysed t-tests were used. Only the scores of the experimental day, i.e. the second day, were statistically analysed (two-way ANOVA and t-test for independent measurements). The injection sites were confined to the rostral part of the VP/SI complex (Fig. 1). Three rats were discarded for inappropriate placement of their cannulae. Bicuculline enhanced motor activity (Fig. 2A; ANOVA: F3,558= 37.04, P~<0.01). A bell-shaped curve seemed to exist however: whereas the doses of 25 and 50 ng clearly enhanced activity, the dose of 100 ng only slightly enhanced it. This was confirmed by a post-hoc SNK test: the results of the 100 ng dose were not significantly different from AD, whereas the results of the 25 and 50 ng doses were (P ~<0.05). Furthermore the dose of 50 ng was significantly (P~<0.05) different from the dose of 25 ng. As can be seen in Fig. 2B, muscimol (25 ng) enhanced motor activity (ANOVA: Fi,270=240.34, P~<0.01). When the above-mentioned doses of bicuculline were combined with 25 ng muscimol a dose-dependent attenuation of the muscimol induced activity was found (Fig. 2C; ANOVA: F3,486=4.98, P~<0.01). Moreover a significant interaction term was found (F51,486=2.02, P~<0.01). It is clear that the effect was most marked in the first 60 min, which appears to agree with the shortterm effect of bicuculline (cf. Fig. 2A). Analysing accordingly the first 60 rain it turned out that both 50 and 100 ng bicuculline were effective in attenuating (P~<0.05, SNK test) the muscimol induced activity (ANOVA: /73,162 = 7.98, P ~<0.01). Furthermore the curves showed a parallel shift; accordingly there was no significant interaction-term (F15,162= 1.49, P~<0.12). When the remainder of the experimental period was analysed, i.e. 70-180 min after injection, it turned out that the rats treated with muscimol 25 ng and bicuculline 100 ng were significantly more active (P~<0.05, SNK-test) than those in any of the other groups (ANOVA: F3,324=4.20, P<~0.01). Muscimol also significantly attenuated the bicuculline induced activity in the first 10 min period (compare Fig. 2C with A): msm 25+bic 25 vs bic 25 (mean+S.E.M.): 111.1+96.0 vs 521.5+79, t=3.300 d f = 15, P~<0.01; msm 25+bic 50 vs bic 50:78.0+9.7 vs 819.0+147.8, t=5.001 df=14, P~<0.01 (two-tailed ttests).Muscimol also antagonized the bicuculline
249
induced activity in the second 10 min period: msm 25+bic 50 vs bic 5 0 : 1 7 8 + 1 3 5 . 0 vs 836.1+182.4, t = 2.900 d f = 14, P ~<0.05 two-tailed t-test). Bicuculline dose-dependently enhanced motor activity (cf. ref. 1), which was attenuated by the specific GABAA agonist muscimol [6, 14], allowing the conclusion that the effect was mediated via GABAA receptors. A new finding is the observation that bicuculline had a dual effect: at lower doses it markedly enhanced motor activity whereas at higher doses it produced only a weak effect. This phenomenon needs to be studied in more detail in future studies. Muscimol enhanced motor activity (cf. refs. 2, 5, 13, 15). The specific GABAA antagonist bicuculline [6, 14] dose-dependently attenuated this muscimolinduced activity. Moreover the muscimol induced activity returned to baseline values at the moment that the efficacy of bicuculline began to wear off. From these data it can be concluded that both stimulation of GABAA receptors and blockade of GABAA receptors lead to an increase in motor activity. At this stage it cannot be decided whether these effects are mediated via GABAA receptors located on the same populations of neurons or via GABAA-receptors located on different (sub)populations of neurons. In the latter case differences in the basal GABA activity within each subpopulation may determine the differential sensitivity to the agonist and the antagonist respectively (cf. ref. 8 and references cited therein). Although Scheel-Krtiger observed that the area from which muscimol induced motor activity differs slightly from the area from which the indirect GABAA antagonist picrotoxin induced motor activity [13], such a distinction was not found in the present study: the injection-sites for bicuculline and muscimol were located in the same area (Fig. 1). From the graphs it is clear that differences between the bicuculline and muscimol induced activity existed. First, the activity induced by bicuculline was short-lasting in contrast to the activity of muscimol, which was longlasting. Second, the onset of the bicuculline induced activity was faster than the onset of the muscimol induced activity. In these respects the bicuculline induced motor activity resembled the motor activity induced by Damphetamine in the nucleus accumbens [3] or dopamine in the olfactory tubercle [7]. Until now no clear counterpart for a muscimol type of activity has been found in the nucleus accumbens or olfactory tubercle. Thus future experiments should be directed at elucidating the behavioural differences between the activity induced by muscimol and bicuculline. At present it is not clear whether these differences were due to differences in pharmacokinetic or physicochemical properties of the drugs, differences in interaction with GABAA receptors, or whether they reflected interactions with differentially localized
GABAA receptors. It should be noted that previous experiments have shown that neither bicuculline nor muscimol was effective in increasing motor activity after injection into the caudal areas of the VP/SI complex [5]: instead, they affect tonic electromyographic activity in the triceps muscle of the forelimb [4]. These data not only exclude the posssibility that the - differential - effects observed after injection of muscimol and bicuculline in the present study were due to - differential spread of these drugs to caudal areas but also underline the earlier reported heterogeneity of the VP/SI complex [5]. In conclusion, the main findings of this study are that both bicuculline and muscimol enhanced motor activity when injected into the rostral part of the VP/SI complex and that these effects could be attenuated by muscimol and bicuculline, respectively. This study thus not only confirms but also extends the findings of previous experiments that muscimol [2, 5, 13, 15] and bicuculline [1] enhance motor activity. Since muscimol and bicuculline are a GABAA-specific agonist and antagonist, respectively [6, 14], it seems likely that the effect of each single drug was mediated via GABAA receptors. I Austin, M.C. and Kalivas, P.W., Enkephalinergic and GABAergic modulation of motor activity in the ventral pallidum, J. Pharmacol. Exp. Ther., 252 (1990) 1370-1377. 2 Baud, P., Mayo, W., Le Moal, M. and Simon H., Locomotor hyperactivity in the rat after infusion of muscimol and [o-Ala]Metenkephalin into the nucleus basalis magnocellularis. Possible interaction with cortical cholinergic projections, Brain Res., 452 (1988) 203-21 I. 3 Bos, van den R., Cools, A.R. and 0gren, S.-O., Differential effects of the selective D2-antagonist raclopride in the nucleus accumbens of the rat on spontaneous and D-amphetamine induced activity, Psychopharmacology, 95 (1988) 447-451. 4 Bos, van den R. and Cools, A.R., Involvement of the substantia innominata/ventral pallidum complex in transmitting forelimb muscular rigidity evoked from the nucleus accumbens in rats, Neurosci. Lett., 103 (1989) 303-308. 5 Bos, van den R, and Cools, A.R., The ventral pallidum/substantia innominata complex: further evidence for heterogeneity as determined by the effects of GABA-ergic drugs, Behav. Pharmacol., 1 (Suppl. 1) (1989) 29. 6 Bowery, N.G., Hill, D.R. and Hudson, A.L., Characteristics of GABAB-receptor binding sites on rat whole brain synaptic membranes, Br. J. Pharmacol., 78 (1983) 191-206. 7 Cools, A.R., Mesolimbic dopamine and its control of locomotor activity in rats: differences in pharmacology and light/dark periodicity between the olfactory tubercle and the nucleus accumbens, Psychopharmacology, 88 (1986) 451-459. 8 Cools, A.R., Brachten, R., Heeren, D., Willemen, A. and Ellenbroek, B., Search after the neurobiological profile of individualspecific features of Wistar rats, Brain Res. Bull., 24 (1990) 49-69. 9 Heimer, L. and Wilson, G.D., The subcortical projections of the allocortex: similarities in the neural associations of the hippocampus, the piriform cortex and the neocortex. In M. Santini (Ed.) Golgi Centennial Symposium, Raven, New York, 1975, pp. 177193.
250 10 Jones, D.L. and Mogenson, G.J., Nucleus accumbens to globus pallidus GABA projection subserving ambulatory activity, Am. J. Physiol., 238 (1980) R65-R69. 11 Mogenson, G.J., Swanson, L.W. and Wu, M., Neural projections from nucleus accumbens to globus pallidus, substantia innominata, and lateral preoptic-lateral hypothalamic area: an anatomical and electrophysiological investigation in the rat, J. Neurosci., 3 (1983) 189-202. 12 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd ed., Academic Press, New York, 1986. 13 Scheel-Kriiger, J., The GABA receptor and animal behavior. In S.J. Enna (Ed.) The GABA Receptors, Humana Press, Clifton, N J, pp. 215-256. 14 Simmonds, M.A., Multiple GABA-receptors and associated regulatory sites, Trends Neurosci., 6 (1983) 279-281.
15 Swerdlow, N.R. and Koob, G.F. The neural substrates of apomorphine-stimulated locomotor activity following denervation of the nucleus accumbens, Life Sci., 35 (1984) 2537-2544. 16 Swerdlow, N.R. and Koob, G.F., Lesions of the dorsomedial nucleus of the thalamus, medial prefrontal cortex and pedunculopontine nucleus: effects on motor activity mediated by nucleus accumbens-ventral pallidum circuitry, Brain Res., 412 (1987) 233-243. 17 Walaas, I. and Fonnum, F., The distribution and origin of glutamate decarboxylase and choline acetyltransferase in ventral pallidum and other basal forebrain regions, Brain Res., 177 (1979) 325336. 18 Yang, C.R. and Mogenson, G.J., Ventral pallidum neuronal responses to dopamine receptor stimulation in the nucleus accumbens, Brains Res., 489 (1989) 237-246.