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The pulvinar is the largest nucleus in the primate thalamus and has topographically organized connections with multiple cortical areas, thereby forming extensive cortico-pulvino-cortical input-output loops. Neurophysiological studies have suggested a role for these transthalamic pathways in regulating information transmission between cortical areas. However, evidence for a causal role of the pulvinar in regulating cortico-cortical interactions is sparse and it is not known whether pulvinar's influences on cortical networks are task-dependent or, alternatively, reflect more basic large-scale network properties that maintain functional connectivity across networks regardless of active task demands. In the current study, under passive viewing conditions, we conducted simultaneous electrophysiological recordings from ventral (area V4) and dorsal (lateral intraparietal area [LIP]) nodes of macaque visual system, while reversibly inactivating the dorsal part of the lateral pulvinar (dPL), which shares common anatomical connectivity with V4 and LIP, to probe a causal role of the pulvinar. Our results show a significant reduction in local field potential phase coherence between LIP and V4 in low frequencies (4-15 Hz) following muscimol injection into dPL. At the local level, no significant changes in firing rates or LFP power were observed in LIP or in V4 following dPL inactivation. Synchronization between pulvinar spikes and cortical LFP phase decreased in low frequencies (4-15 Hz) both in LIP and V4, while the low frequency synchronization between LIP spikes and pulvinar phase increased. These results indicate a causal role for pulvinar in synchronizing neural activity between interconnected cortical nodes of a large-scale network, even in the absence of an active task state., (© 2021 Wiley Periodicals LLC.)

Tonic immobility (TI) is a temporary state of profound motor inhibition associated with great danger as the attack of a predator. Previous studies carried out in our laboratory evidenced high Fos-IR in the posteroventral region of the medial nucleus of the amygdala (MEA) after induction of the TI response. Here, we investigated the effects of GABA A and GABA B of the MEA on TI duration. Intra-MEA injections of the GABA A agonist muscimol and GABA B agonist baclofen reduced TI response, while intra-MEA injections of the GABA A antagonist bicuculline and GABA B antagonist phaclofen increased the TI response. Moreover, the effects observed with muscimol and baclofen administrations into MEA were blocked by pretreatment with bicuculline and phaclofen (at ineffective doses per se). Finally, the activation of GABA A and GABA B receptors in the MEA did not alter the spontaneous motor activity in the open field test. These data support the role of the GABAergic system of the MEA in the modulation of innate fear., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The central nucleus of the amygdala (CeA) as the main output of amygdala plays an important role in memory processes. In this study we first evaluated the effects of intra-CeA administrations of different doses of a cannabinoid CB1 agonist, WIN55, 212-2, GABA(A) receptor agonist and antagonist, muscimol and bicuculline, alone on memory retention using passive avoidance learning (PAL) test in rats. Then we examined the effects of GABA(A) receptor agents on the responses induced by intra-CeA microinjection of different doses of WIN55, 212-2. We found that administration of WIN55, 212-2 (0.05, 0.1, 0.2 and 0.4μg/rat) immediately after training impaired memory retrieval in a dose-dependent fashion. Although pre-test intra-CeA administration of muscimol (125, 250 and 500ng/rat) alone had no effect on the step-through latency, its co-administration (125ng/rat) with different doses of WIN55, 212-2 potentiated the amnesic effects of any doses of WIN55, 212-2. The results also showed that pre-test intra-CeA administration of bicuculline (200, 400 and 800ng/rat) alone had no significant effect, but at dose of 200ng/rat disrupted post-training WIN55, 212-2-induced amnesia in the retention test. Furthermore, the additional effect of muscimol (125ng/rat) on memory impairment induced by WIN55, 212-2 (0.1μg/rat) was prevented by intra-CeA co-injection of bicuculline (200ng/rat). We indicated that stimulating or blocking GAGA(A) receptors in the CeA by muscimol and bicuculline interfere with WIN55, 212-2-induced deficits in memory retention in a PAL task and therefore suggests an interaction between cannabinergic and GABAergic systems of the CeA in memory process., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The aim of the present study was to investigate the existence of possible functional correlation between GABA-A and dopamine (DA) receptors of the dorsal hippocampus and the ventral tegmental area (VTA) in passive avoidance learning. Two guide cannulas were stereotaxically implanted in the CA1 region of the dorsal hippocampus and the VTA of male Wistar rats. In order to measure memory retrieval, the animals were trained in a step-through type passive avoidance task and tested 24 h after training. Post-training intra-CA1 administration of a GABA-A receptor agonist, muscimol (0.01-0.02 μg/rat) dose-dependently impaired memory retrieval. Post-training intra-VTA administration of SCH23390 (a dopamine D1 receptor antagonist; 0.1-0.8 μg/rat) or sulpiride (a D2 receptor antagonist; 0.5-1.5 μg/rat) decreased the inhibitory effect of muscimol (0.02 μg/rat, intra-CA1) on memory retrieval. Intra-VTA administration of the same doses of SCH23390, but not sulpiride, decreased the step-through latencies. On the other hand, post-training administration of muscimol (0.02 μg/rat) into the VTA inhibited memory retrieval. The administration of SCH23390 (0.01-0.2 μg/rat) or sulpiride (0.1-1 μg/rat) into the CA1 region, immediately after training, had no effect on memory retrieval. Furthermore, the amnesic effect of intra-VTA administration of muscimol was significantly decreased by intra-CA1 administration of sulpiride (0.5 and 1 μg/rat, intra-CA1), but not SCH23390. The practical conclusion is that the relationship between the hippocampus and the VTA may regulate memory formation in passive avoidance learning. Also, the correlation between the hippocampus and VTA by a dopaminergic system may be involved in mediating muscimol-induced amnesia., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

The blockade of the inhibitory mechanisms for sodium intake with GABAergic activation in the lateral parabrachial nucleus (LPBN) induces strong ingestion of water and hypertonic NaCl in satiated and normovolemic rats. A question that remains is if the activity of facilitatory mechanisms, like angiotensin II, is necessary for sodium and water intake induced by muscimol (GABA(A) receptor agonist) injected into the LPBN. Therefore, in the present study, we investigated the effects of the blockade of angiotensinergic AT(1) receptors with losartan injected i.c.v. on 0.3 M NaCl and water intake induced by muscimol injected into the LPBN in satiated and normovolemic rats. Male Holtzman rats with stainless steel cannulas implanted bilaterally into the LPBN and unilaterally into the lateral ventricle were used. Bilateral injections of muscimol (0.5 nmol/0.2 μl) into the LPBN combined with i.c.v. injection of vehicle induced 0.3 M NaCl (31.7 ± 1.8 ml/240 min, vs. saline: 0.4 ± 0.3 ml/240 min) and water intake (21.5 ± 1.9 ml/240 min, vs. saline: 0.8 ± 0.2 ml/240 min). Losartan (50 and 100 μg/1.0 μl) injected i.c.v. reduced the effects of LPBN-muscimol on 0.3 M NaCl (18.9 ± 1.9 and 9.9 ± 1.7 ml/240 min, respectively) and water intake (9.8 ± 1.7 and 5.1 ± 1.1 ml/240 min, respectively). The results suggest that the activation of central AT(1) angiotensinergic receptors is essential for hypertonic NaCl and water intake induced by the blockade of the inhibitory mechanisms with muscimol injected into the LPBN in satiated and normovolemic rats., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

We investigated the participation of gamma-aminobutyric acid (GABA) neurons of the medial septal area in eight-arm radial maze performance in rats. The intra-septal injection of muscimol, a GABA(A) agonist, caused an increase in total error and working memory error. On the other hand, no significant effect was observed with reference memory error. Donepezil and tacrine (cholinesterase inhibitors) antagonized the muscimol-induced spatial memory deficits. Histidine (1500 mg/kg, i.p.) also improved the total error and working memory error induced by muscimol. At this dose, histidine caused a significant increase in the histamine content of the cortex, hippocampus, and hypothalamus in rats. In addition, the intra-hippocampal injection of histamine also antagonized muscimol-induced spatial memory deficits. The practical conclusion is that the GABA(A) receptor of the medial septal area plays an important role in working memory, and also, the disturbance of working memory induced by muscimol is mediated not only by cholinergic but also by histaminergic systems in the spatial memory of rats.

The rostral anterior cingulate cortex (rACC) is involved in supraspinal nociceptive processing. ACC lesions relieve persistent pain, but do not affect the patient's ability to localize a noxious stimulus. Since the rACC has a high density of GABA receptors, it is possible that pain processing is influenced by these receptors in the rACC. The present experiments examined the involvement of rat rACC GABA(A) and GABA(B) receptors in regard to sensitivity to mechanical stimulation and escape/avoidance behavior in response to a noxious stimulus following L5 spinal nerve ligation. Rats were or were not afflicted with a neuropathic pain condition by an L5 spinal nerve ligation. rACC microinjection of 10 microg/microl GABA, a GABA(A) agonist (0.001 microg/microl, 0.1 microg/microl, or 0.5 microg/microl muscimol), a GABA(B) agonist (0.1 microg/microl, 1 microg/microl, or 5 microg/microl baclofen), or saline, did not alter mechanical withdrawal thresholds. Importantly, following 10 microg/microl GABA, 0.1 microg/microl, or 0.5 microg/microl muscimol microinjected into the rACC, place escape/avoidance behavior to a noxious mechanical stimulus was attenuated in injured animals. The attenuation was specific to the rACC and was blocked by a preadministered microinjection of the appropriate antagonist(s) into the rACC. In conclusion, microinjection of GABA and higher doses of muscimol did not decrease mechanical hyperalgesia but did attenuate place escape/avoidance behavior that is associated with mechanical stimulation of the ligated paw. These results provide additional support for the role of the rACC in higher order supraspinal processing of noxious events and suggest that rACC GABA(A) receptors significantly contribute to this processing.

Unlabelled: Valerian is a medicinal herb that produces anxiolytic and sedative effects. It was suggested that valerian acts via gamma-aminobutyric acid (GABA)ergic mechanisms. Previous studies showed binding of valerian extract to GABA receptors, but the functional effect of the binding has not been demonstrated. In this study we evaluated the GABAergic effect of valerian extract and one of its major constituents, valerenic acid, on brainstem neuronal activity in an in vitro neonatal rat brainstem preparation. We first observed that muscimol, a GABA(A) receptor agonist, decreased the firing rate in most brainstem neurons in a concentration-related fashion; 30 micro M produced a 38.9% +/- 3.0% (mean +/- SE) inhibition compared with control values (P < 0.01; 50% inhibitory concentration [IC(50)], 2.0 +/- 0.1 microM). This effect was antagonized by bicuculline (10 microM), a GABA(A) antagonist. Then we showed that valerian extract 3 mg/mL induced a 29.6% +/- 5.1% inhibition with an IC(50) of 240 +/- 18.7 microg/mL, whereas 100 microM valerenic acid induced a 22.2% +/- 3.4% inhibition with an IC(50) of 23 +/- 2.6 microM (both P < 0.01). Bicuculline antagonized the inhibitory effects of both the valerian extract and valerenic acid. In addition, pretreatment with valerian extract or valerenic acid decreased the brainstem inhibitory effects produced by muscimol (both P < 0.05), suggesting that these compounds play an important role in the regulation of GABAergic activity. Data from this study suggest that the pharmacological effects of valerian extract and valerenic acid are mediated through modulation of GABA(A) receptor function. Thus, valerian may potentiate the sedative effects of anesthetics and other medications that act on GABA receptors, and presurgical valerian use may cause a valerian-anesthetic interaction., Implications: Valerian is an herb used in treating anxiety and insomnia. We observed that the valerian effects are mediated through brain gamma-aminobutyric acid (GABA) receptors in a rat brainstem preparation. Thus, valerian may potentiate the effects of anesthetics that act on GABA receptors, and presurgical valerian use may cause a valerian-anesthetic interaction.

Septal gamma-aminobutyric acid (GABA) receptor activation is known to disrupt memory formation, although the mechanisms underlying this impairment remain unclear. The present study explored the possibility that high levels of septal GABA receptor activity might impair memory by down-regulating acetylcholine (ACh) function in archicortex and entorhinal cortex. To test this possibility, rats were trained on an avoidance task 15 min after receiving intra-septal infusions of vehicle or muscimol (5 nmol/0.5 microl) combined with unilateral intra-hippocampal (10 microl/1 microl) or intra-entorhinal cortex (1.875 microg/0.25 microl) infusions of vehicle or the acetylcholinesterase inhibitor physostigmine. We demonstrate that these infusions do not alter acquisition performance on a continuous multiple trial inhibitory avoidance task. However, intra-septal infusions of muscimol dramatically impair retention performance 48 h later. More importantly, infusions of physostigmine into the hippocampus or the entorhinal cortex, at doses that do not influence acquisition or retention performance when infused alone, attenuate the impairing effects of the muscimol infusions on retention. We suggest that high levels of septal GABA receptor activity might impair memory by down-regulating ACh levels in the hippocampal region, and that such memory impairments can be ameliorated by increasing ACh levels in the hippocampus or entorhinal cortex.

The interaction of 5-aminolevulinic acid (ALA) with GABA(A) receptors has been proposed to underlie the neurological dysfunctions of ALA-accumulating disorders, such as acute intermittent porphyria. The effects of ALA on [3H]muscimol binding to human and rat cerebral cortical membranes were compared. ALA (0.1-10 mM) significantly inhibited the binding of [3H]muscimol (12 nM), with a similar potency in rat and human membranes (IC50 = 199 vs. 228 microM, respectively). Kinetical analysis revealed that ALA (1 mM) significantly increased the Kd and decreased the Bmax of [3H]muscimol to both rat (100 and 50%, respectively) and human (200 and 40%, respectively) membranes, indicating a mixed-type inhibition. The similarity in the potency and mechanism of the ALA-induced inhibition of muscimol binding in rat and human membranes indicate that rat studies are useful to evaluate the neurotoxic properties of ALA towards the human GABAergic system, and may help to understand the pathophysiology of porphyria.

Background: The numbers of [3H]MUS binding sites were reported to be elevated in layers II and III, but not V or VI, in cingulate cortex of schizophrenic brains post mortem. These increases in [3H]MUS binding sites are probably due to compensatory up-regulation of GABA(A) receptors on pyramidal cells as a consequence of a selective loss of GABAergic interneurons in layer II of cingulate cortex. The number of [3H]flunitrazepam binding sites was reported to be reduced in schizophrenic cingulate cortex, and this may directly reflect the loss of GABAergic interneurons. Chronic administration of clozapine to rats was reported to significantly reduce the numbers of [3H]MUS binding sites in temporal cortex and hippocampus which may be due to selective blockade of GABA(A) receptors on GABAergic interneurons that make synaptic contact with pyramidal cells. Basket cells are GABAergic interneurons that make synaptic contact with pyramidal cells as well as other interneurons. Basket cells can also generate both theta and gamma oscillations. Clozapine increases the power of theta and gamma EEG. Schizophrenic patients show reduced EEG power at 40 Hz (gamma frequency) but not at lower frequencies during auditory stimulation. The GABA(A) receptor blocker bicuculline at 10 nM, but not 10 microM, was reported to increase the amplitude of slow oscillations (< or =1 Hz) in rat hippocampal slices. It therefore seems possible that clozapine, by selectively blocking another GABA(A) receptor, could increase the amplitude of gamma oscillations., Findings: Twenty-six compounds that inhibit [35S]TBPS binding in ways that are reversible by 10 nM R-5135 were found to increase [3H]MUS binding to membranes prepared from rat whole forebrain. In almost all cases the increases in binding were due to increases in the number of binding sites with little effect on affinity (Kd) for [3H]MUS. Concentration-response curves for the compounds revealed maximum increases in [3H]MUS (Esat) binding ranging from 140% (for meclizine) to 313% of control for honokiol. Additivity experiments showed that propofol (44% above control) and diflunisal (50% above control) were almost entirely additive, but there was also a small, but significant overlap, suggesting the existence of three groups of [3H]MUS binding sites defined by propofol and diflunisal. Meclizine was entirely additive with both propofol and diflunisal, indicating the existence of a fourth [3H]MUS binding site. Alphaxalone is also completely additive with meclizine, and has an Esat value significantly larger than that for propofol + diflunisal suggesting a fifth [3H]MUS binding site. The Esat for mefenamate is significantly greater than the Esat for alphaxalone, and mefenamate is also completely additive with meclizine, suggesting the existence of a sixth [3H]MUS binding site. The Esat for magnolol is significantly greater then the Esat, for mefenamate, and the Esat for honokiol is greater than that for magnolol, suggesting, but not proving, the existence of a seventh and an eighth group of [3H]MUS binding sites. The binding of [3H]MUS alone, without enhancers may represent a ninth group of binding sites which is probably heterogeneous as indicated by the very low pseudo Hill coefficients for bicuculline and strychnine in displacing [3H]MUS without enhancer. Altogether, our results suggest the existence of more than 9 different [3H]MUS binding sites. Clozapine was a very weak overall displacer of [3H]MUS (IC50 = 280 microM). However, 5 microM clozapine reduced [3H]MUS binding 6% (P < 0.0001, n = 10) and significantly reduced [3H]MUS binding enhanced by propofol (approximately 14%) or clotrimazole (approximately 17%) but not 17 other compounds tested., Tentative Conclusions: In the absence of enhancers [3H]MUS may bind preferentially to GABA(A) receptors on pyramidal cells and less to interneurons in cerebral cortex. Conversely, [3H]flunitrazepam may bind preferentially to GABA(A) receptors (allosterically) on interneurons and less to pyramidal cells. Clozapine appears to selectively block a small fraction (10-20%) of [3H]MUS binding sites with an IC50 value in the low micromolar range. This fraction may be preferentially located on certain GABAergic interneurons (basket cells?) that make synaptic contact with pyramidal cells. The blockade of these GABA(A) receptors by clozapine would be expected to increase the firing rate of the interneurons and the release of GABA onto pyramidal cells. Such blockade would also increase the generation of gamma oscillations by the basket cells. Some of these interneurons appear to be destroyed selectively, probably during the second trimester of gestation by a non-paralytic polio virus, in individuals who wil

Immortalized GT1-7 neurons were used to characterize the effect of muscimol, a GABAA receptor agonist, to enhance pulsatile gonadotropin-releasing hormone (GnRH) release. GT1-7 neurons were grown on Cytodex-3 beads and placed in special superfusion microchambers. The cells were superfused at a rate of 6.2 ml x h-1 with Media 199 (pH 7.35) using a commercially available perfusion system. After a pre-muscimol period of 120 min, the cells were exposed for 5 min to 0.35, 1, 5 or 10 microM muscimol or 5 microM muscimol+20 microM of the GABAA receptor antagonist, bicuculline. Following removal of the muscimol (and bicuculline, in the case of the latter experiment), the superfusion was continued for another 115 min. Sample fractions were collected at 5 min intervals throughout the perfusion. Basal GnRH release from the GT1-7 neurons was pulsatile with an average interpulse interval of 45.4+/-0.5 min and an average pulse amplitude of 191.5+/-22.6 pg x min x ml-1. Our results also demonstrated that the GABAA receptor agonist, muscimol, enhances pulsatile GnRH release from GT1-7 neurons in culture. The response to muscimol was saturable and concentration-dependent with an EC50 of 0.47 microM. The effects of 5 microM muscimol to increase GnRH pulsatility were blocked by co-exposure to the GABAA receptor antagonist, bicuculline. The average GnRH interpulse intervals were 41.7+/-1.8 min, 32.5+/-2.9 min, 30.6+/-0.7 min and 25.5+/-0.4 min in the period following exposure to 0.35, 1, 5 and 10 microM of muscimol, respectively (post-muscimol period). GnRH pulse amplitude (mean-area for each pulse) was increased during exposure to muscimol but not during the pre- or post-muscimol periods. The GABAA receptor antagonist, bicuculline, itself had no effect on pulsatile GnRH release. These results are consistent with previously published reports suggesting that activation of the GABAA receptor stimulates hypothalamic GnRH release in embryonic and neonatal animals., (Copyright 1999 Elsevier Science B.V.)

The effects of intrathecally (i.t.) administered GABA(A)-receptor antagonist picrotoxin or bicuculline on the antinociception produced by i.t. serotonin (5-HT), gamma-aminobutyric acid (GABA), muscimol--the GABA(A) agonist or baclofen--the GABA(B) agonist were investigated and compared using the tail-flick assay in rats. The results showed that 1) both i.t. picrotoxin (1.5 nmol) and i.t. bicuculline (0.5 nmol) exhibited a partial and later-emerged blockade on the antinociception produced by 5-HT (120 nmol) or GABA (1.5 nmol); 2) both i.t. picrotoxin and i.t. bicuculline, with the same dosages, completely blocked the antinociception produced by muscimol (1.0 nmol), but showed no effects on that produced by baclofen (0.3 nmol). The results suggest that GABA may mediate the 5-HT-induced antinociception at the spinal level, with the GABA(B)-receptors exhibiting the effect at the early-stage and the GABA(A)-receptors at the later stage of the 5-HT-induced antinociception.

The present study was designed to investigate the modulatory effects of blockade of spinal GABAA and GABAB receptors on antinociception induced by supraspinally administered mu- and epsilon-opioid receptor agonists. The effects of intrathecal (i.t.) injections with GABAA and GABAB receptor antagonists, SR 95531 [2-(3-carboxypropyl)-3-amino-6-(4-mehylphenyl)pyridazinium bromide] and 5-aminovaleric acid, respectively, on the antinociception induced by morphine (a mu-opioid receptor agonist) and beta-endorphin (an epsilon-opioid receptor agonist) injected intracerebroventricularly (i.c.v.) were studied. Antinociception was assayed using the tail-flick test. The i.t. injection of SR 95531 (0.04-0.16 nmol) and 5-aminovaleric acid (32.5-130 nmol), administered alone did not affect the latencies of the tail-flick response, but selectively antagonized the inhibition of the tail-flick response induced by muscimol (a GABAA receptor agonist) and baclofen (a GABAB receptor agonist), respectively. The i.t. injection of SR 95531 attenuated dose-dependently the inhibition of the tail-flick response induced by i.c.v. administered morphine, without affecting the i.c.v. administered beta-endorphin-induced response. 5-Aminovaleric acid attenuated dose-dependently the inhibition of the tail-flick response induced by beta-endorphin, without affecting the response to i.c.v. administered morphine. Our results indicate that GABAA but not GABAB receptors located at the spinal cord appears to be involved in the antinociception induced by morphine administered supraspinally whereas GABAB but not GABAA receptors located at the spinal cord may be involved in the antinociception induced by supraspinally administered beta-endorphin, supporting further the hypothesis that morphine and beta-endorphin administered supraspinally produce their antinociception via the activation of different descending pain inhibitory systems.

We have recently reported a new protocol for inducing long-term depression through activation of GABAA receptors in the hippocampal site. This long-term depression is reversed by bicuculline and potentiated by neurosteroids such as alphaxalone and 5 alpha-pregnan-3 alpha-ol-20-one. It was also shown that glutamate receptor activity is not involved in the induction of this type of long-term depression. The present study investigates the role of calcium in the induction of this novel form of long-term depression and attempts to determine the mechanism of reversal of muscimol-induced long-term depression. Extracellular recordings were made in the CA1 pyramidal cell layer of rat hippocampal slices following orthodromic stimulation of Schaffer collateral fibres in stratum radiatum (0.01 Hz). It was observed that the muscimol-induced long-term depression can be obtained in the absence of calcium in the bathing medium. In addition to this, the long-term depression was reversed by N-methyl-D-aspartate, kainic acid, high potassium medium, veratrine and the calcium ionophore A23187 but not high calcium (10 mM) medium. High potassium medium in the absence of calcium reversed the long-term depression induced by muscimol 10 microM. The results suggest that this type of glutamate-independent long-term depression can be induced in the absence of extracellular calcium. Extracellular calcium is not necessary for reversal of the long-term depression, although when intracellular calcium levels are raised, as by A23187, this is capable of inducing reversal.

Involvement of GABAergic systems in action of antidepressants was examined in the forced swim test in rats. Rats were forced to swim in a cylinder for 15 min on day 1 and for 5 min on day 2. Desipramine, mianserin and buspirone, administered after the 15-min swim session on day 1 and before the 5-min swim test on day 2, dose-dependently decreased the duration of immobility in the swim test on day 2. Baclofen attenuated the decreased duration of immobility induced by desipramine, mianserin and buspirone in the swim test, although baclofen did not affect the duration of immobility when it was injected alone. Muscimol dose-dependently decreased the duration of immobility in the swim test on day 2. Bicuculline antagonized the decreased duration of immobility induced by muscimol. However, bicuculline failed to antagonize the decreased duration of immobility induced by desipramine, mianserin and buspirone. These results suggest that GABA(B) but not GABA(A) receptor systems may be involved in action of antidepressants.

We studied the effect of ZnCl2 application on GABAA receptor-mediated 36CI- flux in microsacs prepared from whole rat hippocampus and in region-specific hippocampal slices. Slices were obtained from the dentate gyrus (DG), which contains the zinc-enriched hilar region, and from the CA1 region which contains lower levels of endogenous zinc. Muscimol (10 microM)-evoked 36Cl- flux was significantly reduced by ZnCl2 (100 microM) in hippocampal microsacs. In hippocampal slices, muscimol (50 microM)-evoked 36Cl- efflux was higher in CA1 (112.5 +/- 27.9% above basal efflux rate) than in DG slices (29.7 +/- 5.6%). In the presence of ZnCl2, the muscimol effect on efflux rate in CA1 and DG regions was decreased to 10.6 +/- 5.4% and 6.9 +/- 4.9%, respectively. Preincubation with the zinc chelator, tetrakis(2-pyridylmethyl)ethylenediamine (TPEN, 20 microM), caused a significant increase in muscimol-evoked 36Cl- efflux only in DG slices (57.2 +/- 7.0%), suggesting that GABAA receptors in the DG of rat hippocampus under physiological conditions may function under the inhibitory influence of endogenous chelatable zinc. In intracellular recordings, ZnCl2 (100 microM) application had no effect on the responses to GABA applied perisomatically or in the dendritic region of CA1 neurons. The lack of Zn2+ effect on the postsynaptic GABAA receptor-mediated responses suggests that the decreases of the 36Cl- efflux observed in the biochemical assays may be due to zinc action on neurons other than the principal pyramidal CA1 cells, and possibly the non-neuronal cell populations.

Since the development of tolerance to morphine antinociception in formalin-treated mice was delayed and diazepam normalized the delay, the involvement of GABAergic systems in the process was investigated. Gamma amino-n-butyric acid (GABA) at 10 mg/kg and the GABAA-receptor agonist muscimol at 0.05 mg/kg, i.p., 30 min before daily morphine injection at 10 mg/kg, s.c. completely reversed the delay in the development of morphine tolerance in the formalin-treated mice. The GABAA antagonist bicuculline at 1 mg/kg and the Cl(-)-channel blocker picrotoxin at 1 mg/kg extinguished the reverse effect of muscimol and GABA, respectively. In contrast, the GABAB antagonist CGP 35348 (3-aminopropane-diethoxymethyl-phosphinic acid) up to 100 mg/kg, i.p. failed to abolish the GABA effect; and baclofen, a GABAB-receptor agonist, at 0.5 and 2 mg/kg, i.p., 30 min before morphine was without effect on the delay. On the other hand, bicuculline was incapable of abolishing the reverse effects of diazepam on the delay of tolerance development; and likewise, the reverse effect of muscimol was not affected by flumazenil. No appreciable influence of these GABA-related compounds was seen on morphine antinociception itself nor the development of tolerance in normal mice. These results suggest that the benzodiazepine-GABAA-Cl- channel complex is involved in the mechanism underlying the delay of the development of morphine tolerance in formalin-treated mice; however, it is deduced that benzodiazepine-receptor and GABAergic systems are not always functionally coupled to each other in the mechanisms.

Effects of muscimol on the place learning in Morris water maze task were investigated in rats. 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 4 training trials (probe test). Compared to the saline-treated rats, the rats treated with muscimol on day 1-4 showed no modifications of place learning in the training trials and the probe test. However, in the rats treated with muscimol on day 1-3 and treated with saline on day 4, there was increased latency to reach the platform and reduced duration in the quadrant where the platform had been located on day 4. The increased latency in the training trials and reduced duration in the probe test on day 4 was blocked by bicuculline, when bicuculline and muscimol were co-administered on day 1-3, and saline was injected on day 4. Moreover, in the rats treated with muscimol on day 1-3, co-administration of bicuculline and muscimol on day 4 blocked place learning: increased latency in the training trials and reduced duration in the probe test was observed. These results suggest that muscimol induces state-dependent learning (SDL) in Morris water maze task, and that muscimol-induced SDL is mediated by GABAA receptors.

The carotid body chemoreceptor afferents have been reported to project to a discrete area located in the nucleus commissuralis of nucleus tractus solitarius [A. Vardhan et al., Am. J. Physiol., 264 (1993) R41-R50]. The afore-mentioned study was done in spontaneously breathing rats and the afferents and efferents located in the chest wall and the respiratory tract of these animals were intact. In order to exclude the role, if any, of these afferents and efferents, in the present experiments respiratory changes were monitored by recording the phrenic nerve activity instead of tracheal airflow. Experiments were carried out in pentobarbital-anesthetized, bilaterally vagotomized, paralyzed and artificially ventilated rats with a pneumothorax. The carotid body chemoreceptors were stimulated with tracheal administration of nitrogen for 7-10 s. The chemoreceptor stimulation induced an increase in the frequency and amplitude of phrenic nerve bursts. A decrease in the duration of inspiratory (T1), expiratory (TE) and total cycles (TTOT) was observed in the phrenic nerve activity. Inhibition of neuronal cell bodies by microinjections of muscimol (140 pmol/20 nl) into a discrete area in the commissural subnucleus of the nucleus tractus solitarius (coordinates in mm: 0.3 rostral to 0.5 caudal, 0 to 0.5 lateral and 0.3 to 0.5 deep with respect to the calamus scriptorius), attenuated the phrenic nerve responses to the carotid body stimulation. On the other hand, control injections of saline (0.9%) into this site did not alter the phrenic nerve response to the carotid body stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Heterogeneity of binding affinities for a variety of ligands was observed for gamma-aminobutyric acid type A (GABAA) receptors in the rat CNS, at both GABA and benzodiazepine recognition sites. Photoaffinity labeling by [3H]flunitrazepam and [3H]muscimol to affinity column-purified receptor proteins was examined by gel electrophoresis in sodium dodecyl sulfate. Anesthetic barbiturates (pentobarbital) and steroids (alphaxalone) both differentially stimulated the incorporation of [3H]flunitrazepam more so into the 51-kDa alpha 1 subunit than into the 53-kDa alpha 2 polypeptide, and incorporation of [3H]muscimol into the 55-kDa beta 2 subunit more so than the 58-kDa beta 3 polypeptide. Binding to these polypeptides was also affected differentially by other allosteric modulators and competitive inhibitors, including the benzodiazepine "type 1" selective ligand CL218,872. Heterogeneity in affinity of this drug for the single 51-kDa alpha 1 polypeptide strongly suggests that type I receptors, like type II, are heterogeneous. In brain sections, the extent of enhancement of [3H]muscimol binding showed significant regional variation, similar for both steroids and barbiturates, and the GABA analogues THIP and taurine inhibited muscimol binding with regional variations in affinity that were almost opposites of each other. Modulation of [3H]flunitrazepam binding by steroids, barbiturates, and THIP significantly varied with regions. Taken together, ligand binding heterogeneity exhibited by photoaffinity labeling and autoradiography demonstrate the existence of multiple pharmacological-binding subtypes resulting from the combination of multiple polypeptide gene products into several oligomeric isoreceptors. Comparison of the regional distribution of binding subtypes with that of different subunit gene products allows the following conclusions about possible subunit compositions of native pharmacological receptor subtypes present in the brain: Benzodiazepine pharmacology of the oligomeric receptor isoforms is dependent on the nature of alpha and subunits other than alpha, GABA-benzodiazepine coupling is dependent on the nature of the alpha subunits, GABA site pharmacology is dependent on the nature of the beta subunits, and several subunits including alpha and beta contribute to the degree of sensitivity to steroids and barbiturates. Finally, the presence of discrete subunits may be necessary but is not sufficient to postulate a defined pharmacological property.

The locomotor activity and grooming of conscious freely moving rats were recorded during a 60-min unilateral perfusion of the preoptic area with neuroactive compounds using the microdialysis technique. The GABA agonist, muscimol (10, 20 and 100 microM) induced a dose-dependent increase in locomotor activity and grooming which was attenuated by co-perfusion with the GABA antagonist, bicuculline (10 microM), and was blocked by systemic injection of haloperidol, a preferential dopamine D2 receptor antagonist (0.25 mg/kg). Muscimol-induced hyperactivity was associated with a simultaneous increase of striatal extracellular dopamine. These data suggest that the preoptic area is functionally linked with the extrapyramidal dopaminergic system possibly via GABAergic system.

1. The GABA and GABA-A receptor agonist muscimol changed the spontaneous mechanical activity of a circular layer isolated from cat terminal ileum, while the selective GABA-B receptor agonist (+/-)baclofen had no effect. 2. GABA at doses ranging from 1 microM to 2 mM elicited concentration-dependent biphasic responses which consisted of a relaxation followed by contraction, with a tonic and a phasic component. The EC50 values, calculated at 95% confidence limits (CL), were 94.9 microM (83.5-109.8 microM) and 66.0 microM (51.2-75.5 microM) for the relaxation and contractile phases, respectively. 3. The GABA-induced biphasic responses were sensitive to bicuculline and picrotoxinin and were entirely mimicked by muscimol. Bicuculline competitively antagonized the effects of GABA and gave closely similar pA2 values for both phases of these responses--inhibitory and stimulatory. Cross-desensitization occurred only between GABA and muscimol and not between (+/-)baclofen and GABA, or (+/-)baclofen and muscimol. 4. Both bicuculline-sensitive phases evoked by GABA and muscimol were abolished by tetrodotoxin or atropine, but were unaffected by guanethidine or naloxone. 5. The present results suggested that the biphasic GABA effect on the mechanical activity of the circular layer in cat terminal ileum was mediated by prejunctional GABA-A receptors, most probably through an action on the cholinergic pathway.

Anatomical and neurochemical results suggest that the cortico- and amygdalopetal cholinergic neurons of the nucleus basalis magnocellularis (NBM) may receive GABAergic inputs. The present experiments were undertaken to evaluate the possible influence of intra-NBM injections of the GABAA agonist, muscimol, on memory. In two experiments, rats were chronically implanted with guide cannulae placed bilaterally into the NBM. Rats were trained to a criterion of at least 83% correct on each component in a double Y-maze task that allowed a dissociation of working and reference memory. The task began with placement into one of the two end arms of the first Y-maze and the reference memory task was to go to the stem for food. Access to the second Y was then given and the working memory task was to go to the goal arm opposite the arm in the first maze from which that trial began. In experiment 1, pre-trained rats (n = 7) received muscimol (0.5 microliter) in doses of 0, 0.01, 0.1 and 1.0 microgram in a counterbalanced order with re-training to criterion between injections. In experiment 2, pre-trained rats (n = 8) received saline, muscimol (0.1 microgram), the GABAA antagonist, bicuculline (0.01 microgram), and muscimol + bicuculline. Results of experiment 1 revealed that intra-NBM muscimol produced a dose-dependent and differential impairment of working and reference memory. A dose of 0.1 microgram impaired working memory without significantly affecting reference memory; doses of 0.01 microgram and 1.0 microgram affected neither and both types of memory, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of tetrahydroisoxazolo-pyridin-3-ol (THIP) and muscimol on gastrointestinal transit and a possible mechanism for this effect were studied using the charcoal meal test in mice. These agents inhibited transit dose relatedly. The inhibitory effect was completely reversed by yohimbine and phentolamine. A minor degree of antagonism by bicuculline was observed. There were an additive response in morphine-pretreated animals and partial antagonism in naloxone-pretreated animals. Propranolol failed to modify the inhibitory effect. The findings suggest a predominantly alpha 2-receptor-mediated inhibitory effect on gastrointestinal transit for THIP and muscimol which possibly also utilizes opioid pathways. However, it appears that beta-adrenergic and GABAergic systems might not contribute to this effect.

The purpose of this study was to clarify further the site of action in the amygdala as well as functional characteristics of feeding in response to two GABA receptor agonists. Guide cannulae for microinjection were implanted stereotaxically in the rat just above the central nucleus of the amygdala (CNA). Microinjections of 0.05, 0.25, 0.5 or 1.0 nmol muscimol, a GABAA-selective receptor agonist, produced a dose- and time-dependent decrease of food intake in both the satiated and fasted rat. The bilateral injection of muscimol into the amygdala was more effective than a unilateral injection during the first 2 h, although the overall effects were similar. Microinjection of 0.1 nmol bicuculline methiodide, a GABAA receptor antagonist, into the CNA significantly blocked this inhibitory effect of 0.05 and 0.5 nmol muscimol again in both the satiated and fasted rat. Doses of 0.05, 0.5, 5.0 and 10.0 nmol of the selective GABAB agonist, baclofen, injected into homologous sites in the CNA did not alter food intake. These findings support the viewpoint that the amygdala and its central nucleus comprise a pivotal region involved in the mechanisms underlying the control of feeding behavior. Further, it is envisaged that hypophagic or anorexic responses are induced through the activation of GABAA receptors by the presynaptic release of GABA from neurons which form a component of the anatomical system for hunger and satiety.

Experiments were performed to characterize the antagonistic activity and binding properties of SR 95531 [2-(3' carbethoxy-2'-propyl)-3-amino-6-paramethoxy-phenyl-piridazinium bromide] in rat brain. SR 95531 and bicuculline methiodide inhibited muscimol-stimulated 36Cl- uptake in cortical synaptoneurosomes in a concentration-dependent manner. The inhibitory potency of SR 95531 for the muscimol-stimulated 36Cl- uptake was 15 times higher than that of bicuculline methiodide. Scatchard plots of binding isotherms exhibited two apparent binding sites for [3H]SR 95531 in both the frontal cortex and cerebellum. The IC50 value of SR 95531 for muscimol-stimulated 36Cl- uptake into cortical synaptoneurosomes was in close agreement with the KD value of low-affinity binding sites of [3H]SR 95531 in the frontal cortex. Pretreatment of the membranes with phospholipase A2 invariably decreased [3H]SR 95531 binding in the frontal cortex and cerebellum. On the other hand, the treatment significantly increased [3H]gamma-aminobutyric acid (GABA) binding in a concentration-dependent manner in the frontal cortex. Although lower concentrations of phospholipase A2 did not affect [3H]GABA binding in the cerebellum, treatment with higher concentrations of phospholipase A2 increased the binding in this region. Specific binding of [3H]SR 95531 was also detected in cultures rich in cerebellar granule cells. Pretreatment with phospholipase A2 affected the binding of [3H]GABA and [3H]SR 95531 in these cells, as in the case of the cerebellum. These effects of phospholipase A2 on the binding of [3H]GABA and [3H]SR 95531 were partially prevented by the addition of delipidated bovine serum albumin.(ABSTRACT TRUNCATED AT 250 WORDS)

The present study was designed to test whether serotonin (5-HT) preferentially affects the GABAA or GABAB receptor subtypes in the cerebellum using muscimol and baclofen as the selective agonists. Serotonin significantly augmented baclofen- and muscimol-elicited inhibitions with minimal effects of 5-HT on the spontaneous activity. The magnitudes of augmentation of baclofen and muscimol effects seen in the presence of 5-HT were comparable. In summary, this study further describes the neuromodulatory role of serotonin on GABAergic inhibition and based upon the present study, it can be concluded that both GABAA and GABAB receptor subtypes are modulated by 5-HT.

Preparations of rat jejunum were tested for their responsiveness to the GABAA receptor agonist, muscimol, and to the haem precursor, delta-aminolaevulinic acid (ALA). Both muscimol (1.0-30 microM) and ALA (1.0 microM-3.0 mM) elicited a concentration-dependent increase in tone. Pretreatment with the GABAA antagonist, bicuculline (10(-5) M), blocked effects of muscimol at all concentrations tested and attenuated effects of 0.3 mM ALA. However, bicuculline enhanced responsiveness of the preparations to ALA at low concentrations (0.01-0.05 microM), as also did picrotoxin (10(-5) M), eliciting a significant increase of tone. The significance of these findings is discussed. This finding of pharmacological activity by ALA at concentrations comparable with its blood levels during acute attacks of intermittent porphyria provides support for the proposal that is may play a role in the aetiology of the gastrointestinal manifestations of this disease.

These experiments examined the involvement of cholinergic influences in the effects of GABAergic drugs on 24-h retention of an inhibitory avoidance response by mice. A first set of experiments confirmed previous findings indicating that post-training injections (ip) of the GABAergic agonists muscimol (1.0 and 2.0 mg/kg) and baclofen (10.0 and 20.0 mg/kg) impaired retention, as well as previous findings indicating that injections of the cholinergic agonist oxotremorine (5.0 and 10.0 micrograms/kg) enhanced retention. The findings of a second set of experiments indicated that the memory-impairing effects of muscimol and baclofen were attenuated by concurrent injections of a low, and otherwise ineffective, dose of oxotremorine (2.5 micrograms/kg). These findings are interpreted as suggesting that GABAergic drugs affect memory storage through influences on cholinergic systems.

The involvement of GABA mediated neurotransmission in the central control of respiration was investigated by administration of the specific GABAA receptor agonist muscimol and the specific GABAA receptor antagonist biculline into the fourth cerebral ventricle of the rabbit. Cycle-triggered averaging of the phrenic nerve activity (PNA) was used to quantify drug-induced changes of the central respiratory pattern. Muscimol reduced the peak amplitude of PNA and increased the duration of the respiratory phases. High amounts of muscimol led to a long-lasting but reversible central apnea. Bicuculline very effectively blocked the effects of externally applied muscimol. Blockade of intrinsically active GABAergic neurotransmission by bicuculline resulted in a multitude of effects. Peak amplitude of PNA increased whereas the duration of both inspiration and expiration decreased. In this respect, effects of bicuculline and muscimol were complementary. Bicuculline reduced the slope of the inspiratory ramp, increased postinspiratory activity and induced an augmenting type of discharge activity in the last part of expiration resulting in a smooth transition between expiration and inspiration. In some cases the respiratory modulation was completely lost and PNA became perfectly tonic. This 'apneustic' type of respiratory pattern could be transformed into rhythmic breathing by increasing the respiratory drive. We conclude that neurotransmission via GABAA receptors is important for the maintenance of respiratory rhythm as well as the generation of normal respiratory pattern.

Direct injections of muscimol into the ventral tegmental area (VTA) or substantia nigra zona reticulata (SNR) have been used to selectively stimulate the mesolimbic and nigro-striatal dopamine pathways respectively. Such injections induced locomotor activity, rearing, sniffing and in some animals an intermittent grooming response. These responses were rapid in onset, dose-related and relatively short lasting (less than 40 min). Selective increases in dopamine turnover were seen in the nucleus accumbens and in the striatum following VTA and SNR injections of muscimol (100 ng) respectively. Haloperidol inhibited the behavioural consequences of VTA and SNR injections of muscimol with similar potency (ED50S 0.01-0.03 mg/kg IP), and fluphenazine did likewise (ED50S 0.05-0.16 mg/kg IP). However, thioridazine (ED50S VTA: 1.45-2.04 mg/kg IP, SNR 8.50-9.20 mg/kg IP) and in particular clozapine (ED50S VTA: 0.24-0.58 mg/kg IP, SNR: 6.10-9.70 mg/kg IP) were more potent at inhibiting the locomotor activity and sniffing responses due to VTA rather than SNR administered muscimol. Since dopamine D2 antagonists are believed to exert their anti-psychotic effects via an action on mesolimbic dopaminergic systems, and their ability to induce extrapyramidal side effects (EPS) is thought to be due to an action on nigro-striatal dopamine systems, these results suggest that the behavioural models described can be used to predict efficacy and side-effect liability of potential neuroleptic drugs.

The stable substance P analog, DiMeC7, increases spontaneous locomotor activity after infusion into the lateral ventricles or median raphe nucleus (MRN). The elevated locomotion observed after intracerebroventricular (ICV) infusion of DiMeC7 was attenuated, but not blocked, by bilateral 6-hydroxydopamine (6OHDA) lesions of the ventral tegmental area (VTA). In contrast, bilateral 6OHDA lesions of the nucleus accumbens (NAS) blocked the motor activity induced by ICV administration of DiMeC7. Similar lesions did not affect the increases in motor behavior observed after MRN infusions of DiMeC7. However, the hyperactivity following MRN microinjections of DiMeC7 was attenuated by intraperitoneal administration of the dopamine (DA) antagonist, haloperidol. The results suggest that ICV infusions of DiMeC7 increase locomotor activity by acting directly on neurons in the NAS and in part by influencing, directly or indirectly, the activity of DA cells in the VTA. The increased motor activity seen after MRN administration of DiMeC7 appears to depend on DA neurons but not on projections to the NAS.

Microinjection of the gamma-aminobutyric acidA agonist, muscimol, into the A10 region of the rat produced a dose-dependent increase in motor activity. This effect was antagonized by intra-A10 administration of the gamma-aminobutyric acidA antagonist, bicuculline, and by peripheral administration of haloperidol, and was associated with an increase in extracellular levels of dopamine metabolites in the nucleus accumbens. Although microinjection of the gamma-aminobutyric acidB agonist, baclofen, into the A10 region did not alter motor activity, it abolished the capacity of intra-A10 injection of mu opioid agonist, Tyr-D-Ala-Gly-MePhe-Gly(ol), or muscimol to increase motor activity. Baclofen also prevented the motor stimulant response to peripheral injection of cocaine or amphetamine, but was ineffective in blocking caffeine-induced behavioral activity. Pretreatment with baclofen prevented the capacity of a mu opioid agonist to elevate dopamine metabolite levels in the nucleus accumbens and prefrontal cortex in postmortem tissue. Baclofen also prevented the elevation of extracellular dopamine content in the nucleus accumbens produced by injection of a mu opioid agonist into the A10 region, as measured in the conscious rat with in vivo dialysis. Finally, when dopamine metabolite levels were elevated in the prefrontal cortex by mild footshock, it was shown that pretreatment with baclofen in the A10 region abolished this response. These data support electrophysiological studies suggesting that activation of gamma-aminobutyric acidB receptors on dopamine perikarya inhibits dopaminergic activity, while activation of gamma-aminobutyric acidA receptors results in an indirect disinhibition of dopaminergic function.

1. Extracellular recordings were made from Purkinje cell layer of rat cerebellar slices. Cell activity was sensitive to both magnesium and manganese ions. 2. Glutamic and aspartic acids both excited cell activity while gamma-aminobutyric acid (GABA), muscimol, taurine, beta-alanine and delta-aminolaevulinic acid all inhibited activity. 3. The sensitivity to GABA varied with depth while no such effect was observed with muscimol. 4. Bicuculline methiodide, picrotoxin and pitrazepin blocked the action of muscimol with pA2 values of 5.92, 5.97 and 5.71 respectively. 5. The benzodiazepines flurazepam and RU 32007 both potentiated the GABA inhibition and this potentiation was blocked by Ro 15-1788, a benzodiazepine antagonist.

Muscimol as well as catecholaminergic drugs reduce immobility time in the forced swimming test. In view of the fact that GABAergic drugs may facilitate some brain catecholaminergic functions, we investigated as to whether or not muscimol would reduce immobility time through activation of catecholaminergic mechanisms. The effect of muscimol (2 mg/Kg i.p.) on reduction of immobility time was prevented by intraperitoneal alpha-methyl-para-tyrosine (250 mg/Kg i.p.), which reduces brain catecholamine content, haloperidol (0.5 mg/Kg) and sulpiride (100 and 50 mg/Kg), antidopaminergic drugs, and meta-chlorphenyl-piperazine (0.6 and 1.25 mg/Kg), a serotonergic agonist, but not by clonidine (0.1 mg/Kg), an alpha2-adrenoceptor agonist, d, 1-propranolol (5 mg/Kg), an antagonist of beta-adrenergic receptors, or subcutaneous prazosin (3 mg/Kg), an alpha1-adrenolytic drug. Our findings indicate that a) muscimol reduces immobility time by stimulating dopaminergic neurons and b) activation of the serotonergic system antagonizes muscimol effect.

Several Good buffers (MOPS, ACES, BES, HEPES, ADA, and PIPES) competitively inhibited both high-affinity and low-affinity [3H]gamma-aminobutyric acid receptor binding to rat brain synaptic membranes. The most potent inhibitor was MOPS, which had Ki values of 180 nM and 79 nM for the high- and low-affinity binding sites, respectively. HEPES had Ki values of 2.25 mM and 115 microM. The buffers had no appreciable effect on sodium-dependent GABA binding or on gamma-aminobutyrate aminotransferase activity. Surprisingly, the buffers were extremely ineffectual as inhibitors of either high- or low-affinity [3H]muscimol binding. Indeed, they were of the order of 10(5) times less effective in this case than against [3H]GABA binding. These results clearly show (a) that the use of such buffers as MOPS or HEPES should be avoided in studying the interaction of GABA with its receptor, and (b) the binding sites of [3H]GABA and [3H]muscimol are not identical.

A behavioral study was carried out to clarify a relationship between the GABAergic and purinergic central system in aggressive behaviors induced by clonidine in mice. Mice administered a high dose of clonidine (20 mg/kg, i.p.) exhibited aggressive behaviors such as biting and attacking. These behaviors are inhibited by L-PIA (N6-L-phenylisopropyl adenosine) and stimulated by caffeine, which suggest that a blockade of adenosine receptors is involved in these behaviors. Muscimol (0.5-2 mg/kg, i.p.), a GABA-a receptor agonist, not only markedly potentiated clonidine (20 mg/kg i.p.)-induced aggressive behaviors but also elicited characteristic behaviors such as gnawing, reinforced irritability, and self-mutilation. Bicuculline (1, 2 mg/kg, i.p.), a GABA-a receptor antagonist, or picrotoxin (1 mg/kg, i.p.), a chloride channel blocker, did not significantly affect clonidine (20 mg/kg, i.p.)-induced aggressive behaviors. The potentiating effects of muscimol (0.5, 1 mg/kg, i.p.) on clonidine-induced aggressive behaviors were antagonized by bicuculline (1, 2 mg/kg, i.p.), but not affected significantly by picrotoxin (1 mg/kg, i.p.). L-PIA (0.2 mg/kg, i.p.) reduced clonidine-induced aggressive behaviors and also reversed the potentiating effects of muscimol. Stereotyped gnawing behaviors induced by combined treatment of muscimol (0.5, 1 mg/kg, i.p.) and clonidine (20 mg/kg, i.p.) were not affected by bicuculline (2 mg/kg, i.p.). The results suggest that the potentiating effects of muscimol on clonidine-induced aggressive behaviors may be induced via the stimulation of GABA-a receptors, although not necessarily associated with chloride channel functions, and may involve certain interactions between the stimulation of GABA-a receptors and the inhibition of adenosine receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Contraversive turning was evoked by the microinjection of GABAergic agents into the substantia nigra (SN) of the rat. Muscimol, the most potent GABA agonist, evoked contralateral turning when injected into the SN in doses of 0.005, 0.05, 0.5 and 5 microgram, whereas 0.5 microgram of muscimol applied at extranigral sites produced no turning. A shorter lived contraversive turning response was evoked by the intranigral micro-injection of imidazole acetic acid (10 or 50 microgram), ethanolamine-O-sulphate (25 or 50 microgram), or GABA (50 microgram). No increase in GABA-induced turning was produced by local pretreatment with pipecolic acid (5 microgram). When injected into the SN, neither picrotoxin, in doses of 0.1, 0.5, 1.0 or 2.0 microgram, nor bicuculline methiodide (Bm), in doses of 0.1 or 0.2 microgram, elicited a significant amount of turning. Picrotoxin, however, partially blocked the turning evoked by the intranigral injection of muscimol, both via the i.p. and intranigral routes of administration whereas Bm did not. In addition, haloperidol (1 mg/kg i.p.) antagonized the muscimol-induced turning. Hence, we feel GABA mimetic substances injected within the SN might evoke contralateral turning via activation of a heretofore undescribed neural system arising from the SN or by activating the ipsilateral dopaminergic neurons projecting from the SN.

We previously demonstrated that GABA and muscimol administered either into the cisterna magna or the fourth ventricle to chloralose-anesthetized cats cause respiratory depression, hypotension, and bradycardia. Injection of these substances into the lateral and third ventricles had no effect. In order to localize the site of action, muscimol and GABA were applied by Perspex rings to the ventral surface of the medulla. Application of muscimol (0.25-2.66 micrograms) to Schlaefke's area in 6 cats reduced minute ventilation from 443 +/- 38 to 291 +/- 52 ml/min by reducing tidal volume from 31.8 +/- 2.3 to 17.6 +/- 1.4 ml, without changing respiratory rate and duration of inspiration. Hypotension and bradycardia were also observed. Application of GABA (0.14-4.86 mg) produced similar effects on respiratory activity and arterial blood pressure. No significant effects occurred when high doses of these agents were applied to Loeschcke's and Mitchell's areas. Application of bicuculline (5-25 micrograms) to Schlaefke's area had the opposite effect of muscimol and GABA on respiratory activity and blood pressure, and reversed the respiratory and cardiovascular depressant effects of both agents. We conclude that GABA receptors are present at Schlaefke's area, and that activation of these receptors results in respiratory depression, hypotension, and bradycardia. Our results suggest that GABA may be an important inhibitory neurotransmitter in the modulation of respiratory and cardiovascular control.

Tyrosine hydroxylase (TH) contained in dopamine (DA) neurons of rat retina is activated in vivo as a consequence of photic stimulation. Experiments were conducted to test the effects of changes of membrane potential and of cyclic AMP-dependent protein phosphorylation on TH activity of these retinal neurons. Retinas were dissociated into suspensions of apparently viable cells to test the direct effects of pharmacological manipulations on TH activity in the absence of trans-synaptic influences. To test the effects of changes of membrane potential on TH activity we examined the effects of a depolarizing agent, potassium. Incubation of cell suspensions in Ringer's solution containing a depolarizing concentration of K+ (52 mM) resulted in a significant increase of TH activity, suggesting that membrane depolarization may trigger a series of molecular events that leads to TH activation. Incubation of cell suspensions in the presence of 8-bromo cyclic AMP, a cyclic AMP analog that is known to activate cyclic AMP-dependent processes following extra-cellular application, resulted in a significant activation of TH that was comparable to that produced in vitro by cyclic AMP-dependent protein phosphorylation. These data support the hypothesis that membrane potential plays a role in the regulation of TH activity, and indicate that cyclic AMP-dependent phosphorylation can activate retinal TH in situ. The apparent viability of the retinal cells in suspension suggests that this preparation may be useful for studying synaptic regulatory mechanisms.

Effects of intracisternal administration of the gamma-aminobutyric acid receptor agonist, muscimol, on recurrent laryngeal and phrenic nerve activities were assessed in the cat. Muscimol (0.1-2.4 micrograms) produced a dose-related inhibition of the respiratory activity in both of these nerves. The inhibition was seen as a decrease in the amplitude of nerve activity with no significant change in respiratory rate. The ED50 values for muscimol and its 95% confidence interval for inhibition of the inspiratory activity in the recurrent laryngeal and phrenic nerves were 0.22 micrograms (0.17-0.29) and 0.27 micrograms (0.19-0.38), respectively. The potency of muscimol for inhibition of these nerve activities was not significantly different. Expiratory activity in the recurrent laryngeal nerve was completely inhibited prior to either the inspiratory activity in this nerve or in the phrenic nerve. Intracisternal administration of the gamma-aminobutyric acid receptor antagonist, bicuculline, rapidly reversed the inhibition in both nerves. Reversal times for inhibition of the inspiratory activities in the recurrent laryngeal and phrenic nerves were not significantly different. Return of inspiratory activity in each nerve always preceded the return of expiratory activity in the recurrent laryngeal nerve. These results demonstrate that CNS gamma-aminobutyric acid receptors are involved in the regulation of respiratory motor outflow to the larynx as well as the diaphragm.

A number of clinically used benzodiazepines were tested for their effectiveness in blocking muscimol-induced myoclonic jerks in mice. Their ED50 values were determined from their dose-response curves. These data gave the following relative potencies with respect to diazepam: diazepam = 1, medazepam = 0.24, oxazepam = 1.27, flurazepam = 1.90, lorazepam = 3.01, nitrazepam = 3.93, clonazepam = 33.14, and flunitrazepam = 116.00. Because our earlier studies indicated that muscimol-induced myoclonic jerks probably originate from the spinal cord, the present method may prove to be suitable for quantitatively evaluating the effect of benzodiazepines on the spinal cord. The present results also indicate the possible value of flunitrazepam in the management of neurologic disorders in which preferential action on the spinal cord is desired.

The effects of chlordiazepoxide (10 mg/kg) were assessed in a holeboard by the reductions in head-dipping, rearing and locomotor activity; the correlations among all these measures were significant. Test-retest correlations were significant for all but the time spent head-dipping. On the basis of their behavioral responses to chlordiazepoxide six "strong" and six "weak" responders were identified and used for an in vitro electrophysiological study. There were no differences between the two groups in the extent to which flurazepam potentiated muscimol, but picrotoxin showed a greater antagonism of muscimol in slices from "strong" responders and flurazepam showed a greater reduction of picrotoxin potency. There was a significant correlation between the in vitro picrotoxin shift and the chlordiazepoxide-induced reduction in locomotor activity. The correlations between behavioral responses to chlordiazepoxide and the plasma benzodiazepine concentrations were low and only one (for locomotor activity) reached significance.

The interaction of the 'neurosteroid', pregnenolone-sulfate (PS), with the GABA/benzodiazepine/chloride ionophore receptor complex was investigated in rat brain subcellular preparations. At low micromolar concentrations PS competitively inhibited the binding of the convulsant [35S]t-butylbicyclophosphorothionate (TBPS) and antagonized pentobarbital-stimulated [3H]flunitrazepam binding to synaptosomes. In addition, PS inhibited muscimol-stimulated 36Cl-uptake in brain synaptoneurosomes, including that PS has characteristics of a relatively potent antagonist of the chloride channel coupled to the GABA receptor. Together with our previous finding that A-ring reduced metabolites of progesterone and deoxycorticosterone also interact with the GABA receptor complex but as hypnotic barbiturates, these data suggest that the regulation of GABAergic neurotransmission by various neurosteroids may be an important mechanism for controlling neuronal excitability.

In male Swiss mice, muscimol produced myoclonic jerks. A 3 mg/kg (i.p.) dose induced this response in all of the mice tested and the peak response of 73 jerks per min was observed between 27 and 45 min. Increasing the brain serotonin levels by the administration of 5-hydroxytryptophan (80-160 mg/kg) in combination with a peripheral decarboxylase inhibitor resulted in an inhibition of the muscimol effect. However, in a similar experiment l-dopa (80-160 mg/kg) was without effect. In doses of 3-10 mg/kg, the serotonin receptor agonist MK-212 caused a dose-dependent blockade of the response of muscimol. Of the benzodiazepines, clonazepam (0.1-0.3 mg/kg) was found to be several fold more potent than diazepam (0.3-3 mg/kg) in blocking the myoclonic jerks. While (-)-baclofen (1-3 mg/kg) proved to be an effective antagonist of muscimol, its (+)-isomer (5-20 mg/kg) lacked this property. Considering the fact that 5-HTP and the benzodiazepines have been found to be beneficial in the management of clinical myoclonus, the muscimol-induced myoclonus seems to be a satisfactory animal model that may prove useful for the development of new drug treatments for this condition. Our present study indicated the possible value of MK-212 and (-)-baclofen in the management of clinical myoclonus.

The central cardiovascular effects of 4 structural analogues of GABA were investigated. The drugs were injected intracerebroventricularly (i.c.v.) in cumulative doses into pentobarbital-anaesthetized normotensive rats. Muscimol (0.01-10 micrograms/kg), THIP (0.01-100 micrograms/kg), kojic amine (0.1-100 micrograms/kg) and isoguvacine (0.1-100 micrograms/kg) produced dose-dependent hypotension and bradycardia. The maximal fall in the mean blood pressure was of about 35% of the initial values. These effects appears to be of central origin since the intravenous (i.v.) injection of the same doses of the drugs did not produce any similar cardiovascular modifications. The hypotensive effects of muscimol and kojic amine were antagonized partly by i.c.v. bicuculline. The combination of bicuculline and kainic acid almost completely prevented the blood pressure lowering effects of muscimol, kojic amine and isoguvacine. THIP however was only slightly antagonized by bicuculline and kainic acid. Atropine i.v. also prevented partly the cardiovascular effects of all these drugs. Thus, the mechanisms of the central cardiovascular actions of GABA analogues appear to be more complex than expected and variable from one drug to another. The involvement of GABA receptors of the A and B types and of cholinergic mechanisms in the hypotensive effect of the drugs is discussed.

It has been shown that tetrodotoxin inhibits GABA-receptor binding of muscimol with solubilized membranes of the rat brain in a competitive manner, with the inhibition constant amounting to about 3 nM. Muscimol has been also demonstrated to competitively inhibit 3H-tetrodotoxin binding with the same drug.

Concentration-dependent depolarizations were evoked by glycine and beta-alanine 5 X 10(-4)-10(-2)M and by the gamma-aminobutyric acid (GABA) analogue, muscimol 10(-6)-10(-4)M. The maximal response to glycine was several-fold higher than that to muscimol on optic nerve but the reverse was found on the dorsal funiculus fibres in the cuneate nucleus. beta-Alanine evoked a similar maximal response to glycine on optic nerve but a considerably higher maximum than glycine in the cuneate nucleus. Strychnine was 19.5 times more potent as a glycine antagonist (pA2 = 6.58) than as a muscimol antagonist. Bicuculline was 156 times more potent as a muscimol antagonist than as a glycine antagonist. Other antagonists of muscimol, i.e. tubocurarine, picrotoxin and leptazol, and potentiators of muscimol, i.e. pentobarbitone and flurazepam, had little or no effect on responses to glycine. Responses to beta-alanine had pharmacological properties compatible with a mixed action on both GABA and glycine receptors. The rat isolated optic nerve appears to be a useful preparation for studying the pharmacology of the neuronal glycine receptor plus chloride ionophore complex.