Evidence for GABA involvement in stress-induced inhibition of male amphibian sexual behavior

The behavioral effects of GABA analogs were investigated to determine whether GABAergic neurotransmission is involved in the stress-induced inhibition of masculine sexual behaviors in rough-skinned newts (Taricha granulosa). Injections of bicuculline, a GABA antagonist, stimulated male sexual behaviors in a dose-dependent fashion, and the minimum effective dose was 40-fold less when administered centrally rather than systemically, suggesting a central nervous system site of action. Injections of muscimol, a GABA agonist, suppressed reproductive behaviors in male newts, and this inhibition lasted at least 5 hr and was proportional to the dose of muscimol administered. The inhibitory effects of muscimol on newt sexual behaviors could be reversed by a single 100-microgram ip injection of arginine vasotocin. The inhibitory effects of confinement stress or corticosterone (CS) injections on newt sexual behaviors were blocked by pretreatment of newts with mercaptopropionic acid, a blocker of GABA synthesis. As well, bicuculline prevented the inhibition of sexual behavior induced by CS injection. These results support the conclusion that, in a male amphibian, the GABAergic system is involved in the inhibitory mechanisms regulating sexual behaviors and that CS mediates the stress-induced inhibition of sexual behaviors through the GABAergic system.     

GABA and the behavioral effects of anxiolytic drugs

Much recent research has shown that benzodiazepine binding sites in the central nervous system are associated with GABA receptors. It is therefore possible that the pharmacological and therapeutic effects of benzodiazepines and drugs with similar profiles are mediated through GABAergic mechanisms. In this paper the evidence is considered for a possible involvement of GABA in the behavioral effects of anxiolytic drugs. There are a number of reports that the behavioral actions of anxiolytics can be antagonised by GABA antagonists such as bicuculline or picrotoxin but there are many contradictory findings and these drugs are difficult to use effectively in behavioral studies. In general, GABA agonists do not exert anxiolytic-like behavioral effects after systemic injection but intracerebral administration of muscimol has been shown to produce benzodiazepine-like actions. Although a number of questions remain unanswered, current evidence does not provide strong support for a role for GABA in the behavioral effects of anxiolytic drugs.     

MUSCIMOL UPTAKE, RELEASE AND BINDING IN RAT BRAIN SLICES

Abstract— The GABA analogue, muscimol, was taken up relatively inefficiently compared to GABA by slices of rat cerebral cortex at 37 C. Muscimol uptake followed saturation kinetics (K_m ImM. V_m 0.1 μmol g mini and showed an absolute dependence on sodium ions. The relative susceptibilities of muscimol uptake and GABA high affinity uptake to a variety of inhibitors, including (-)-nipecotic acid. (+)-2.4-diaminobutyric acid and arecaidine, and the stimulation of muscimol efflux by 50μM-GABA, suggest that muscimol and GABA share some common transport carriers. Since L-histidine inhibited muscimol uptake hut not GABA high affinity uptake, at least part of the observed muscimol uptake may be mediated by the 'small basic'amino acid transport system. Muscimol appeared to he taken up into nerve terminals, since uptake was inhibited by the neuronal uptake inhibitor cis -3-aminocyclohexanecarboxylic acid but not by the glial uptake inhibitor β-alanine. Muscimol efflux was stimulated in a calcium-dependent manner by an increased potassium ion concentration.
Sodium-independent binding of muscimol was observed in slices of rat cerebral cortex at 4 C. Binding could be inhibited by a variety of substances. including GABA, isoguvacine and (+)-bicuculline methochloride, which are known to inhibit the binding of muscimol to putative GABA receptors associated with synaptic membranes purified from rat brain.     

Sleep is differently modulated by basal forebrain GABA(A) and GABA(B) receptors

There is evidence that GABA plays a major role in sleep regulation. GABA(A) receptor agonists and different compounds interacting with the GABA(A) receptor complex, such as barbiturates and benzodiazepines, can interfere with the sleep/wake cycle. On the other hand, there is very little information about the possible role of GABA(B) receptors in sleep modulation. The nucleus basalis of Meynert (NBM), a cholinergic area in the basal forebrain, plays a pivotal role in the modulation of sleep and wakefulness, and both GABA(A) and GABA(B) receptors have been described within the NBM. This study used unilateral infusions in the NBM to determine the effects of 3-hydroxy-5-aminomethylisoxazole hydrobromide (muscimol hydrobromide, a GABA(A) receptor subtype agonist) and beta-(aminomethyl)-4-chlorobenzenepropanoic acid (baclofen, a GABA(B) receptor subtype agonist) on sleep parameters in freely moving rats by means of polygraphic recordings. Muscimol (0.5 nmol) and baclofen (0.7 nmol) induced an increase in slow-wave sleep and an inhibition of wakefulness. Muscimol, but not baclofen, also caused a decrease in desynchronized sleep parameters. The results reported here indicate that 1) the NBM activation of both GABA(A) and GABA(B) receptors influences the sleep/wake cycle, and 2) GABA(A) but not GABA(B) receptors are important for desynchronized sleep modulation, suggesting that the two GABAergic receptors play different roles in sleep modulation.     

Effect of the GABAA agonist muscimol on prolactin secretion from human prolactin-secreting adenomas and GH3 rat pituitary tumour cells.

The effect of muscimol, a specific potent GABAA receptor agonist, on prolactin release from human prolactin-secreting tissue was investigated using a perifusion system. Perifusion studies on normal rat anterior pituitary tissue, which has identical GABA receptors to those found in normal human pituitary glands, show that muscimol has a specific biphasic effect on prolactin release. This is characterized by an initial transient stimulation (222.3 +/- 21.6% of basal) lasting for 5-10 min followed by a more prolonged inhibitory phase (63.9 +/- 3.1% inhibition of basal). Five human prolactin-secreting adenomas were studied, and in none of the tumours could a biphasic response be demonstrated. One of the prolactin-secreting adenomas had a blunted inhibitory response, but the other 4 showed no inhibitory effect of muscimol on prolactin release. Muscimol had no significant effect on basal or thyrotropin-releasing-hormone (TRH)-stimulated prolactin secretion from GH3 rat pituitary tumour cells. These studies suggest that the GABAergic effect on prolactin secretion is absent or altered in both rat and human prolactin-secreting tumour cells.     

Effects of muscimol and flurazepam on the sleep EEG in the rat

In order to assess the possible role of GABA receptor function in the hypnotic property of benzodiazepines, we have examined the sleep EEG in rats given the GABA agonist muscimol, alone and in combination with flurazepam. Muscimol 0.05 and 0.1 mg/kg IP failed to alter sleep latency or total sleep time, and did not interact with the sleep-enhancing properties of flurazepam 20 mg/kg IP. These observations, in conjunction with a previous study of bicuculline, suggest that the hypnotic property of benzodiazepines may not be mediated by alteration of GABAergic activity.     

Muscimol and γ-hydroxybutyrate: Similar interactions with convulsant agents

Muscimol has been shown to be a potent GABA agonist in several preparations. After systemic administration, muscimol is rapidly metabolized in the periphery and little, if any, unchanged muscimol gains access to the brain. A major metabolite of muscimol may be structurally analogous to γ-hydroxybutyric acid. In this study it is shown that both muscimol and γ-hydroxybutyrate antagonize convulsions induced by 3-mercaptopropionate, an inhibitor of GABA synthesis, and strychnine, a glycinergic antagonist, while potentiating convulsions induced by bicuculline, a putative GABA antagonist, and pentylenetetrazol, a generalized excitant and possible GABA antagonist. Although these results apparently contradict previously reported data, it is proposed that these anomalies reflect differences dependent upon varying dose regimens of muscimol. The differential effects of low vs. high doses of muscimol may reflect differences in the accessibility to, or affinity of, morphologically distinct GABA receptors that mediate different pharmacological functions.     

GABA in the entopeduncular nucleus and the subthalamic nucleus participates in mediating dopaminergic striatal output functions

The bilateral intracerebral injection of the specific GABA agonists muscimol (25, 100 ng) and THIP (500 ng) into the pallido-entopeduncular nucleus (EP) and the subthalamic nucleus (STN) of rats induced a behavioural stimulation closely resembling the syndrome evoked by direct stimulation of dopamine receptors in the striatum or by the systemic injection of dopamine agonists. The rats showed strong locomotor and rearing activity followed by characteristic stereotyped behaviour consisting of sniffing and gnawing activity. The stimulation induced by muscimol (25 ng) was found independent of dopamine, since the dopamine antagonist haloperidol (1 mg/kg s.c.) induced no blockade. Injection of the GABA antogonist picrotoxin (100 ng) into the EP or STN induced sedation and catalepsy. The unilateral injection of muscimol and picrotoxin provoked contraversive and ipsiversive postural changes. Related behavioral effects were induced by GABAergic drugs injected in substantia nigra, zona reticulata (SNR). These data provide support for the new hypothesis that GABA in the EP, SNR and STN is important for the expression of behavior related to stimulation of dopamine receptors in the striatum. The effects may be induced by a dopamine activation of the descending striato-EP, striato-SNR GABAergic pathways and possibly also the pallido-STN GABAergic pathway. The findings suggest that in addition to a pathology of the dopamine system there may also be a GABAergic dysfunction in the efferent system of the basal ganglia localized to the EP, SNR and STN in diseases, such as parkinsonism, Huntington's chorea and possibly schizophrenia.     

Pharmacological evidence for GABAergic regulation of specific behaviors in Drosophila melanogaster.

We have identified several GABAergic-modulated behaviors in Drosophila melanogaster by employing a pharmacological approach to disrupt GABA transporter function in vivo. Systemic treatment of adult female flies with the GABA transport inhibitors DL-2,4-diaminobutyric acid (DABA) or R,S-nipecotic acid (NipA), resulted in diminished locomotor activity, deficits in geotaxis, and the induction of convulsive behaviors with a secondary loss of the righting reflex. Pharmacological evidence suggested that the observed behavioral phenotypes were specific to disruption of GABA transporter function and GABAergic activity. The effects of GABA reuptake inhibitors on locomotor activity were dose dependent, pharmacologically distinct, and paralleled their known effects in mammalian systems. Recovery of normal locomotor activity and the righting reflex in DABA- and NipA-treated flies was achieved by coadministration of bicuculline (BIC), a GABA receptor antagonist that supresses GABAergic activity in mammals. Recovery of these behaviors was also achieved by coadministration of gabapentin, an anticonvulsant agent that interacts with mammalian GABAergic systems. Finally, behavioral effects were selective because other specific behaviors such as feeding activity and female sexual receptivity were not affected. Related pharmacological analyses performed in vitro on isolated Drosophila synaptic plasma membrane vesicles demonstrated high affinity, saturable uptake mechanisms for [3H]-GABA; further competitive inhibition studies with DABA and NipA demonstrated their ability to inhibit [3H]-GABA transport. The existence of experimentally accessible GABA transporters in Drosophila that share conserved pharmacological properties with their mammalian counterparts has resulted in the identification of specific behaviors that are modulated by GABA.     

The influence of gabaergic drugs upon LH secretion from the pituitary of the common carp: an in vitro study

In the present study, the perifusions of whole pituitary glands of spermiating male common carp were performed in the presence of several GABAergic drugs. Muscimol (agonist of GABA(A) receptors) and bicuculline (the antagonist of the same type of GABA receptors) did not modify basal LH release. LH basal secretion was not modified when pituitaries were perifused with baclofen--an agonist of GABAB receptors. On the other hand, baclofen at doses of 10(-8) and 10(-4) M significantly decreased GnRH-A-induced LH release to about 86% and 88% of LH levels in control group, respectively. In our previous study we have shown that GABA decreased basal and GnRH-A-stimulated in vivo and in vitro LH release. In conclusion, it can be suggested that in the mature male carp GABA exerts an inhibitory influence on GnRH-stimulated LH release, probably through the inhibition of the GnRH action on gonadotropes. This inhibition seems to be mediated by the B type of GABA receptors.     

Intra-accumbens baclofen, but not muscimol, increases second order instrumental responding for food reward in rats

Stimulation of either GABA(A) or GABA(B) receptors within the nucleus accumbens shell strongly enhances food intake in rats. However the effects of subtype-selective stimulation of GABA receptors on instrumental responses for food reward are less well characterized. Here we contrast the effects of the GABA(A) receptor agonist muscimol and GABA(B) receptor agonist baclofen on instrumental responding for food using a second order reinforcement schedule. Bilateral intra-accumbens administration of baclofen (220-440 pmol) stimulated responding but a higher dose (660 pmol) induced stereotyped oral behaviour that interfered with responding. Baclofen (220-660 pmol) also stimulated intake of freely available chow. Muscimol (220-660 pmol) was without effect on responding for food on this schedule but did stimulate intake of freely available chow. Unilateral administration of either baclofen or muscimol (220 pmol) induced similar patterns of c-fos immunoreactivity in several hypothalamic sites but differed in its induction in the central nucleus of the amygdala. We conclude that stimulation of GABA(A) or GABA(B) receptors in the nucleus accumbens shell of rats produces clearly distinguishable effects on operant responding for food.     

Enhanced depressor effect of muscimol in the DOCA/NaCl hypertensive rat: evidence for altered GABAergic activity in brain

To elucidate the role of the central gamma-aminobutyric acid (GABA) system in the maintenance of deoxycorticosterone (DOCA)NaCl hypertension, the responses of mean arterial pressure (MAP), plasma norepinephrine (NE), and epinephrine (EP) to intracerebroventricular (ICV) administration of muscimol, a GABA agonist, and the responses of MAP to bicuculline, a GABA antagonist, and to clonidine, an alpha 2-adrenoceptor agonist known to lower blood pressure by inhibiting sympathetic tone, were examined in conscious, unrestrained 4 week DOCA/NaCl hypertensive rats and age-matched uninephrectomized control rats. Muscimol (50-1000 ng/300 g, ICV) caused dose-dependent decreases in MAP which were greater in DOCA/NaCl rats than in controls. Basal plasma NE and EP were significantly higher in DOCA/NaCl rats than in controls. Muscimol (1000 ng/300 g, ICV) induced decreases in plasma EP which were greater in DOCA/NaCl rats than in controls without changing NE levels in either group. Bicuculline (3 micrograms/300 g, ICV) caused increases in MAP which were the same in both groups. The depressor response to clonidine (5 micrograms/300 g) was greater in DOCA/NaCl rats than in controls. These results suggest that the activity of the central GABAergic system is altered in the rat with established DOCA/NaCl hypertension and that the alteration in central GABAergic function may be related to the increased sympathoadrenal activity and the maintenance of hypertension in this model.     

STRUCTURE-ACTIVITY STUDIES ON THE INHIBITION OF GABA BINDING TO RAT BRAIN MEMBRANES BY MUSCIMOL AND RELATED COMPOUNDS

Abstract— A series of compounds structurally related to muscimol (5-aminomethyl-3-isoxazolol) was tested as inhibitors of the sodium-independent binding of GABA to membranes from rat brain. Muscimol, 5-(l-aminoethyl)-3-isoxazolol, 5-(2-aminoethyl)-3-isoxazolol (homomuscimol), and the bicyclic derivative 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) were relatively potent inhibitors of GABA binding. THIP is an analogue of muscimol locked in a folded conformation. The structurally related compound 1,2,3,6-tetrahydropyridine-4-carboxylic acid (isoguvacine), a semirigid analogue of trans-4-aminocrotonic acid, was also a potent inhibitor of GABA binding. Apart from muscimol, these inhibitors of GABA binding did not influence the sodium-dependent,'high-affinity' uptake of GABA in rat brain slices, whereas the potent GABA uptake inhibitors guvacine and nipecotic acid did not influence GABA binding. The present results support previous findings that different conformational modes of GABA interact with GABA postsynaptic receptors and the neuronal GABA transport system in rat brain, and indicate that the 'active conformation' of GABA with respect to the receptors is partially folded and almost planar. Based on a comparison of the present results with previous in vivo studies the structural requirements for GABA-like activity in rat cerebral cortex and cat spinal cord seem to be somewhat different.     

Pharmacological evidence for GABAergic regulation of specific behaviors in Drosophila melanogaster

We have identified several GABAergic‐modulated behaviors in Drosophila melanogaster by employing a pharmacological approach to disrupt GABA transporter function in vivo. Systemic treatment of adult female flies with the GABA transport inhibitors DL ‐2,4‐diaminobutyric acid (DABA) or R,S‐nipecotic acid (NipA), resulted in diminished locomotor activity, deficits in geotaxis, and the induction of convulsive behaviors with a secondary loss of the righting reflex. Pharmacological evidence suggested that the observed behavioral phenotypes were specific to disruption of GABA transporter function and GABAergic activity. The effects of GABA reuptake inhibitors on locomotor activity were dose dependent, pharmacologically distinct, and paralleled their known effects in mammalian systems. Recovery of normal locomotor activity and the righting reflex in DABA‐ and NipA‐treated flies was achieved by coadministration of bicuculline (BIC), a GABA receptor antagonist that supresses GABAergic activity in mammals. Recovery of these behaviors was also achieved by coadministration of gabapentin, an anticonvulsant agent that interacts with mammalian GABAergic systems. Finally, behavioral effects were selective because other specific behaviors such as feeding activity and female sexual receptivity were not affected. Related pharmacological analyses performed in vitro on isolated Drosophila synaptic plasma membrane vesicles demonstrated high affinity, saturable uptake mechanisms for [³H]‐GABA; further competitive inhibition studies with DABA and NipA demonstrated their ability to inhibit [³H]‐GABA transport. The existence of experimentally accessible GABA transporters in Drosophila that share conserved pharmacological properties with their mammalian counterparts has resulted in the identification of specific behaviors that are modulated by GABA. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 245–261, 2002; DOI 10.1002/neu.10030     

Gamma-aminobutyric acid and alcohol actions: neurochemical studies of long sleep and short sleep mice

Effects of ethanol and pentobarbital on the GABA receptorchloride channel complex were evaluated in mice selected for differential sensitivity to the hypnotic effects of ethanol (long sleep and short sleep lines). 36Cl- influx, [35S]tbutylbicyclophosphorothionate (TBPS) and [3H]muscimol binding were measured in a membrane vesicle suspension (microsacs) from cerebellum or forebrain. Muscimol was found to be a more potent stimulator of 36Cl- flux in the LS cerebellum, as compared to the SS cerebellum, but a similar maximal level of uptake was achieved in the two lines. Muscimol displaced [35S]TBPS (a ligand for the convulsant site) from cerebellar microsacs, and LS mice were also more sensitive than SS mice to this action of muscimol. However, the number or affinity of high affinity [3H]muscimol binding sites did not differ between the lines. Physiologically relevant concentrations of ethanol (15-50 mM) potentiated muscimol stimulation of 36Cl- uptake in LS cerebellum but had no effect in SS cerebellum. Ethanol failed to alter stimulated chloride flux hippocampal microsacs from either line. Both the LS and SS lines responded similarly to pentobarbital potentiation of muscimol stimulated chloride uptake regardless of brain region. The demonstrated difference between the LS and SS mice in muscimol stimulated chloride uptake as well as in muscimol displacement of [35S]TBPS binding offers a biochemical explanation for the line differences in behavioral responses to GABAergic agents. Moreover, the findings suggest that genetic differences in ethanol hypnosis are related to differences in the sensitivity of GABA-operated chloride channels to ethanol.     

Studies of the role of gamma-aminobutyric acid in the hypothalamic control of feed intake in sheep

Fourteen sheep were used to study the role of gamma-aminobutyric acid (GABA) on the hypothalamic control of feed intake. Injections (1 microL) of pentobarbital (262 nmol) into preoptic and paraventricular areas induced feeding in satiated sheep. Injections of GABA into the same loci gave variable results, probably because the neuronal and glial uptake of GABA limits its effects. Muscimol, a GABA agonist with a higher affinity for postsynaptic GABA receptors than GABA, injected at doses from 0 to 0.750 nmol, gave a cubic dose-response curve; the highest feed intake was measured at 0.5 nmol. The response induced by muscimol was blocked by preinjections of two GABA antagonists, picrotoxin and bicuculline, with picrotoxin being more effective than bicuculline. Muscimol responsive loci were identified mainly in the preoptic, paraventricular, and anterior hypothalamus. The data suggests that neurons sensitive to gamma-aminobutyric acid may be implicated in the control of feed intake in sheep.     

Effect of gamma-aminobutyric acid and muscimol on corticosterone secretion in rats.

The effect of gamma-aminobutyric acid-receptor agonists, GABA and muscimol on the pituitary-adrenocortical activity, measured indirectly through corticosterone secretion, and the receptors involved were investigated in conscious rats. GABA given ip induced a dual effect, in lower dose (10 mg/kg) it significantly decreased the resting serum corticosterone levels while in higher doses (100-500 mg/kg) it considerably raised that level. Muscimol (0.5 mg/kg ip) also increased the corticosterone concentration. Both GABA and muscimol given intracerebroventricularly (icv) induced a significant, dose-related increase in serum corticosterone levels. Bicuculline, a GABAA-receptor antagonist, totally abolished the corticosterone response to GABA but did not influence the response to muscimol. Pretreatment with atropine did not affect the corticosterone response to GABA but significantly diminished the response to muscimol. These results suggest that GABA moderately inhibits the pituitary-adrenal axis at the pituitary level but significantly stimulates it at the hypothalamic level. The stimulatory effect of GABA, but not muscimol, is mediated by hypothalamic GABAA-receptors, and in the effect of muscimol hypothalamic cholinergic, muscarinic receptors are involved to a significant extent.     

Ventrobulbular hypotensive action of muscimol]

Muscimol, a rigid analogue of GABA has been injected in the CNS of urethane anesthetized, normotensive cats. Injected either intracisternally (1 or 2 microgram/kg, 0.05 ml) or directly by microinjection in a restricted ventrolateral region of the brain stem (0.5 or 1 microgram/kg, 0.5 microliter), muscimol induced hypotension and bradycardia. These central cardiovascular effects of muscimol were antagonized by bicuculline, a "specific" GABA antagonist agent. These data emphasize the involvement of gabergic mechanisms in the central cardiovascular control, at least in the ventrolateral part of the medulla oblongata.     

Neuropharmacological spectrum of muscimol

Muscimol was tested in comparison with a series of reference compounds in a variety of situations in which GABA-related drugs are known to have an effect. Muscimol blocked the convulsions and/or lethality due to picrotoxinin, strychnine and a low dose of bicuculline. It was inactive against higher dosis of bicuculline, metrazole or electroshock convulsions. Muscimol reduced both the basal and the picrotoxin-induced multi-unit activity of the neurons of the dorsal Deiters' nucleus ; although active at low doses, the maximum effect of muscimol was relatively weak. Muscimol potentiated neuroleptic-induced catalepsy, and this effect was bicuculline sensitive ; it did not induce catalypsy in the presence of sulpiride. At high doses muscimol blocked apomorphine-induced stereotyped behaviour. It is proposed that muscimol is a GABA agonist of high affinity but of relatively low efficacy as based on its spectrum of neuropharmacological activities “in vivo”.     

Effect of GABAergic substances on firing rate and thermal coefficient of hypothalamic neurons in the juvenile chicken

The goal of the study is to investigate the GABAergic action on firing rate (FR) and temperature coefficient (TC) on hypothalamic neurons in the juvenile chicken. Extracellular recordings were obtained from 37 warm-sensitive, 32 cold-sensitive and 56 temperature-insensitive neurons in brain slices to determine the effect of GABA(A)-receptor agonist muscimol, GABA(A)-receptor antagonist bicuculline, GABA(B)-receptor agonist baclofen and GABA(B)-receptor antagonist CGP 35348. Muscimol and baclofen in equimolar concentrations (1 microM) significantly inhibited FR of the neurons, regardless of their type of thermosensitivity. In contrast, bicuculline, as well as CGP 35348 (10 microM) increased FR of the majority of the neurons. The TC of most chick hypothalamic neurons could not be estimated during muscimol application because FR was completely inhibited. GABA(B)-receptor agonist specifically increased TC. This effect was restricted to cold-sensitive neurons, which were determined in a high number. The TC was significantly increased (p<0.05) by baclofen and significantly decreased (p<0.05) by CGP 35348. The effects of muscimol and baclofen on FR and TC were prevented by co-perfusion of the appropriate antagonists bicuculline and CGP 35348. The results suggest that the fundamental mechanisms of GABAergic influence on temperature sensitive and insensitive neurons in the chicken PO/AH are conserved during evolution of amniotes.