Topographic studies of the effects of microinjections of muscimol on the hypothalamic control of feed intake in sheep

Ten sheep were used to define the anatomical basis for the feeding systems sensitive to gamma-aminobutyric acid, by using intrahypothalamic microinjections of the gamma-aminobutyric acid agonist, muscimol. In satiated sheep, 1 microL of muscimol (0.5 nmol/microL) elicited feeding when injected into paraventricular, ventromedial, and anterior hypothalamic areas. Similar injections into 39 sites tested in 6-h fasted sheep failed to decrease feed intake. The data suggest that neurons sensitive to gamma-aminobutyric acid in medial hypothalamus may be involved in the initiation of feeding.     

Neuropeptide Y-induced feeding is dependent on GABAA receptors in neonatal chicks

In mammals and birds, neuropeptide Y (NPY) and gamma-aminobutyric acid (GABA) are found in brain areas known to be involved in the control of ingestive behavior and act to increase voluntary food intake. In rats, significant evidence suggest a functional and behavioral interaction between NPY and GABA mediated transmission in various brain regions, including the arcuate and paraventricular nuclei of the hypothalamus which can be important in the regulation of feeding behavior. In the present study, the effect of intracerebroventricular (ICV) administration of NPY and GABA receptor antagonists on food intake was examined in neonatal chicks. The ICV injection of NPY strongly stimulated food intake while co-administration of NPY and picrotoxin, a GABA_A antagonist, (but not CGP54626, a GABA_B antagonist) weakened food intake induced by NPY. These results suggest that central NPY stimulates food intake in neonatal chicks by interaction with the GABAergic system via GABA_A receptors.     

Effects of intrahypothalamic injections of GABA, muscimol, pentobarbital, and L-glutamic acid on feed intake of satiated sheep

Five wethers were surgically prepared with cranial implants to study the role of gabaminergic neural pathways on the hypothalamic control of feeding behaviour in ruminants. In the first experiment, the animals were injected (1 microL) with a physiological Tyrode (0.95%) solution, muscimol (0.5 and 1.0 nmol), GABA (0.5 and 1.0 nmol), and L-glutamic acid (0.5 and 1.0 nmol). Feed intake following injections of muscimol (1.0 nmol) and L-glutamic acid (0.5 and 1.0 nmol) was twice as large as that following the Tyrode solution, at 60-min postinjections. These results, however, were not statistically significant (p = 0.12-0.15). In the second experiment, the animals were injected (1 microL) with saline, muscimol (0.8 nmol), L-glutamic acid (0.8 nmol), and pentobarbital (0.26 mumol). Fifteen minutes after the injections, pentobarbital had induced a significant feeding response when compared with control values (p less than 0.01), whereas the effect of L-glutamic acid was not significant. However, 30 min after the injections, feed intake of sheep having received L-glutamic acid was higher than that obtained with the control injections (p less than 0.01). The response to pentobarbital was stronger than that to either muscimol or L-glutamic acid. Histological analyses of brain tissue indicated that injections were performed in the ventromedial hypothalamus of four sheep and in the dorsomedial hypothalamus of the other. The data indicate that L-glutamic acid stimulates feed intake by acting either as a precursor of GABA or by a direct stimulation of glutaminergic neural pathways involved in the control of feed intake.     

Evidence that GABA in the nucleus dorsalis raphé induces stimulation of locomotor activity and eating behavior

Recent electrophysiological studies have provided evidence that GABA controls inhibitorily the activity of the serotonin containing cell bodies in nucleus dorsalis raphé (NDR). The present investigation shows that local injection of baclofen or the GABA agonist muscimol (25–100 ng) into the NDR strongly increased locomotor activity and stimulated eating in satiated rats. These effects are antagonized by the GABA antagonists bicuculline or picrotoxin given systemically or locally. Muscimol injected in NDR also decreased serotonin and 5-hydroxyindole acetic acid in hypothalamus but not in striatum. These findings support a transmitter role of GABA in NDR and may be interpreted related to a decreased activity of serotonin.     

Influence of the GABAergic system on eating behavior induced by the tail pinching technic in rats

The present experiment was undertaken to investigate the possible involvement of GABAergic neurons in the regulation of the feeding behavior induced by tail pinching. Injections before the tail pinching of gamma-aminobutyric acid (GABA) agonist (Muscimol: 570 micrograms.kg-1 i.p. 20 min before testing), GABA antagonist (Picrotoxine: 1,5 mg.kg-1 i.p. 30 min before testing) and a GABA transaminase inhibitor (Acetate dipropyle: 200 mg.kg-1 i.p. 40 min before testing) have no effects on the ingestion of sweet milk. These preliminary results provide support for the hypothesis that the feeding behavior induced by TP is not mediated through GABAergic system.     

Comparison between pentobarbital- and muscimol-induced feeding in satiated sheep

Twenty sheep were used to study the mechanisms by which the intracerebral administration of pentobarbital and of muscimol induces feeding in ruminants. Injections of 1 mumol calcium induced a weak feeding response at 1 h postinjection compared with control values (108 vs. 63 g, p less than 0.05). Injections of 78 mumol pentobarbital and of 100 nmol muscimol elicited strong feeding responses (p less than 0.01). A preinjection of 1 mumol calcium reduced the response to pentobarbital by about 40% but did not affect the response to muscimol. Administration of 1.1 mmol sodium chloride reduced the effect to pentobarbital by about 60% but only partially decreased the effect to muscimol. Administration of picrotoxin, a GABA antagonist, slightly decreased the feeding response to pentobarbital and to muscimol. Administration of gamma-vinyl GABA, an inhibitor of the enzyme GABA transaminase, did not affect feeding behavior of sheep at any of the doses tested (0-10 mumol). Injections of gamma-vinyl GABA followed by equimolar injections of GABA failed to provoke any feeding response. The data suggest that pentobarbital and muscimol may induce feeding by acting on a similar hypothalamic receptor complex but by different mechanisms. The lack of effect of GABA itself remains unexplained.     

A pharmacological analysis of the GABA-ergic system of the primordial hippocampus in the frog Rana temporaria]

The data on evoked potentials (EP) in the frog primordial hippocampus during the surface application of gamma-aminobutyric acid (GABA), beta-alanine and aminooxyacetic acid in the region of EP recording are presented. These results in the aggregate with the earlier described effects of picrotoxin, bicuculline, muscimol, baclofen (on EP) and GABA (on intracellular potentials) permit to suggest the presence of GABA-ergic inhibitory system and two types of GABA receptors in the frog primordial hippocampus.     

Regional Selectivity of a γ-Aminobutyric Acid-Induced [3H]Acetylcholine Release Sensitive to Inhibitors of γ-Aminobutyric Acid Uptake

The effects of gamma-aminobutyric acid (GABA) on the release of [3H]acetylcholine ([3H]ACh) were studied in synaptosomes prepared from rat hippocampus, cerebral cortex, hypothalamus, and striatum and prelabelled with [3H]choline. When synaptosomes were exposed in superfusion to exogenous GABA (0.01-0.3 mM) the basal release of newly synthesized [3H]ACh was increased in a concentration-dependent way in hippocampus, cortex, and hypothalamus nerve endings. In contrast, the release of [3H]ACh was not significantly affected by GABA in striatal synaptosomes. The effect of GABA was not antagonized significantly by bicuculline or picrotoxin. Muscimol caused only a slight not significant increase of [3H]ACh release when tested at 0.3 mM whereas, at this concentration, (-)-baclofen was totally inactive. The GABA-induced release of [3H]ACh was counteracted by SKF 89976A, SKF 100561, and SKF 100330A, three strong and selective GABA uptake inhibitors. The data suggest that, in selective areas of the rat brain, GABA causes release of [3H]ACh following penetration into cholinergic nerve terminals through a GABA transport system.     

Actions of avermectin B1a on the gamma-aminobutyric acidA receptor and chloride channels in rat brain

The interaction of avermectin B1a (AVM) with the gamma-aminobutyric acid (GABA) receptor of rat brain was studied using radioactive ligand binding and tracer ion flux assays. Avermectin potentiated the binding of [3H]flunitrazepam and inhibited the binding of both [3H]muscimol and [35S]t-butylbicyclophosphorothionate to the GABAA receptor. Inhibition of muscimol binding by AVM suggested competitive displacement. Two kinds of 36chloride (Cl) flux were studied. The 36Cl efflux from preloaded microsacs was potentiated by AVM and was highly inhibited by the Cl-channel blocker 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS). However, it was not potentiated by GABA nor was it sensitive to the convulsants picrotoxin or bicuculline. On the other hand, 36Cl-influx measurement in a different microsac preparation of rat brain was very sensitive to GABA and other GABA-ergic drugs. Avermectin induced 36Cl influx into these microsacs in a dose-dependent manner, but to only 35% of the maximal influx induced by GABA. The AVM-induced 36Cl influx was totally blocked by bicuculline. It is suggested that AVM opens the GABAA-receptor Cl channel by binding to the GABA recognition site and acting as a partial receptor agonist, and also opens a voltage-dependent Cl channel which is totally insensitive to GABA but is very sensitive to DIDS.     

Restoration of the luteinizing hormone surge in middle-aged female rats by altering the balance of GABA and glutamate transmission in the medial preoptic area

Hypothalamic glutamate and gamma-aminobutyric acid (GABA) neurotransmission are involved in the ovarian hormone-induced GnRH-LH surge in rodents. We previously reported that middle-aged rats have significantly less glutamate release in the medial preoptic area than young rats on the day of the LH surge. The present study tested the hypothesis that the delayed and attenuated LH surge in ovariohysterectomized middle-aged rats primed with ovarian steroids results from reduced hypothalamic glutamate and increased GABA(A) neurotransmission. Microdialysis results show that middle-aged rats with attenuated LH surges had reduced extracellular glutamate and increased extracellular GABA levels in the medial preoptic area compared with young rats. Blocking GABA(A) receptors with bicuculline or inhibiting synaptic glutamate reuptake with L-trans-pyrrolidine-2,4-dicarboxylic acid increased extracellular Glu in the medial preoptic area and partially restored LH surge amplitude in middle-aged rats without altering LH surge onset. Complete recovery of LH surge amplitude was observed in middle-aged rats treated with the combination of bicuculline and L-trans-pyrrolidine-2,4-dicarboxylic acid. This treatment also restored the extracellular glutamate:GABA ratio in the medial preoptic area of middle-aged rats to the level of young rats. Immunoblot analysis revealed that estradiol and progesterone treatment reduced SLC32A1(formerly known as vesicular GABA transporter) levels and increased SLC17A6 (formerly known as vesicular glutamate transporter 2) levels in the anterior hypothalamus of ovariohysterectomized young but not middle-aged rats. These data suggest that both reduced availability of glutamate and increased activation of GABA(A) receptors under estrogen-positive feedback conditions contribute to the age-related delay in onset and attenuated amplitude of the LH surge.     

γ-Aminobutyric Acid-Activated 36Cl- Influx: A Functional In Vitro Assay for CNS γ-Aminobutyric Acid Receptors of Insects

The effects of gamma-aminobutyric acid (GABA) on the uptake of 36Cl- into a membrane microsac preparation from isolated nerve cords of the cockroach Periplaneta americana was studied. On addition of 1 microM GABA (after 4-s incubation, then rapid quenching) the influx of 36Cl- was stimulated to a level 75% above that of the control value. This stimulation was reduced by picrotoxin (100 microM), but was not significantly affected by bicuculline (100 microM). Results of 36Cl- influx experiments are in agreement with data obtained from radiolabelled ligand binding assays and electrophysiological investigations on the same tissue. The method described represents a functional in vitro assay for CNS GABA receptors of insects.     

The gamma-aminobutyric acid receptor in catfish brain

When the binding of [3H]gamma-aminobutyric acid (GABA) to its receptor in catfish synaptic membranes was studied, a high affinity (Kd = 8.4 nM) and a low affinity (Kd = 65 nM) binding component was observed. Muscimol, thiomuscimol, tetrahydroisoxazole-5,4-c-pyridin-3-ol, imidazole acetic acid and bicuculline each competitively inhibited both high affinity and low affinity [3H]GABA binding. The potency of these inhibitors was similar to that reported for the GABA receptor from mammalian brain. It is concluded that the GABA receptor from catfish brain has very similar properties to the receptor from mammalian central nervous system and consequently has not undergone any obvious evolutionary changes.     

An ionotropic GABA receptor with novel pharmacology at bullfrog cone photoreceptor terminals

Characteristics of ionotropic gamma-aminobutyric acid (GABA) receptors at bullfrog cone terminals were studied by patch clamp techniques in isolated cell and retinal slice preparations. GABA-induced inward currents from isolated cones reversed in polarity at a potential, very close to the chloride equilibrium potential, and they were completely suppressed by picrotoxin. Unexpectedly, the GABA current was dose-dependently potentiated by the well-known GABA(A) receptor antagonist bicuculline (BIC), but was suppressed by gabazine, another GABA(A) antagonist, and imidazole-4-acetic acid (I4AA), a GABA(C) receptor antagonist. Similarly, currents induced by both GABA(A) agonist muscimol and GABA(C) agonist cis-4-aminocrotonic acid (CACA) were also potentiated by BIC. Furthermore, currents induced from cones by GABA and kainate-caused depolarization of horizontal cells in retinal slice preparations were both potentiated by BIC. All these results suggest that the ionotropic GABA receptor at the bullfrog cone terminal exhibits novel pharmacology, distinct from both traditional GABA(A) and GABA(C) receptors.     

Dopaminergic Regulation of Cone Retinomotor Movement in Isolated Teleost Retinas: II. Modulation by γ-Aminobutyric Acid and Serotonin

In the accompanying paper we reported that 3,4-dihydroxyphenylethylamine (dopamine) induced light-adaptive retinomotor movements in teleost photoreceptors and that this effect was mediated by D2 dopamine receptors located on the photoreceptors themselves. In this study, we investigated the effects on cone retinomotor movement of three agents that have been reported by others to modulate retinal dopamine release: gamma-aminobutyric acid (GABA), 5-hydroxytryptamine (5-HT, serotonin), and melatonin. We report here that the GABA antagonists bicuculline and picrotoxin induced light-adaptive cone contraction in dark-adapted green sunfish retinas cultured in constant darkness; thus they mimic the effect of light or exogenously applied dopamine. Since their effects were blocked by either the D2 dopamine antagonist sulpiride or by Co2+, it seems likely that these agents act by enhancing retinal dopamine release. The GABA agonist muscimol produced effects opposite to those of GABA antagonists. Muscimol inhibited light-induced cone contraction in previously dark-adapted retinas and induced dark-adaptive cone elongation in light-adapted retinas. These results suggest that in green sunfish retinas, as has been reported for other retinas, GABA inhibits dopamine release. 5-HT induced light-adaptive cone contraction in dark-adapted retinas; thus 5-HT also mimics the effect of light or exogenously applied dopamine. The effect of 5-HT was blocked by sulpiride, Co2+, or the 5-HT antagonist mianserin. These results suggest that 5-HT induces cone contraction by stimulating dopamine release. Melatonin neither inhibited dopamine-induced cone contraction in retinas cultured in the dark nor induced cone elongation in retinas cultured in the light. Our results suggest that both GABA and 5-HT (but not melatonin) affect cone retinomotor movements in green sunfish by modulating dopamine release: GABA by inhibiting and 5-HT by stimulating dopamine release. We report in the companion paper that dopamine induced contraction in isolated cone fragments. Together these observations strongly suggest that dopamine serves as the final extracellular messenger directly inducing light-adaptive cone retinomotor movement, and that GABA and 5-HT affect these movements by modulating dopamine release.     

GABA-mediated inhibition of breathing in the late gestation sheep fetus

The effects of the GABA antagonist picrotoxin, and the GABA agonist muscimol, have been studied in chronically instrumented unanaesthetized fetal sheep of 115-132 days gestation. Picrotoxin (300-400 micrograms/kg intravenous bolus injection) induced a period of stimulated breathing (40-112 min) which was associated with high voltage electrocortical activity, but inhibited by hypoxia. Muscimol (4 mg infused) had the opposite effect and caused a prolonged period of apnoea (85-418 mins) which was followed by a rebound period of increased breathing. These observations suggest that the GABA-ergic system may be involved in the apnoea of high voltage sleep states in the late gestation fetal sheep, but not in the apnoea associated with hypoxaemia in the fetus.     

Regulation of Dopamine Release from Interplexiform Cell Processes in the Outer Plexiform Layer of the Carp Retina

The gamma-aminobutyric acid (GABA) antagonists bicuculline and picrotoxin stimulate a four- to fivefold increase in endogenous dopamine release from isolated intact carp retina. The release evoked by these agents is Ca2+ dependent, a finding suggesting a vesicular release. Using light microscopic autoradiography, we have localized the sites of dopamine release to the dopaminergic interplexiform cell processes of the outer plexiform layer, which synapse onto horizontal cells. Our findings support previous suggestions that the dopaminergic interplexiform cells receive GABAergic inhibitory input and that the effects of GABA antagonists on horizontal cells are mediated by dopamine release from the interplexiform cells.     

Antagonist actions of bicuculline methiodide and picrotoxin on extrasynaptic gamma-aminobutyric acid receptors

The effects of picrotoxin and bicuculline methiodide to block depolarizing responses of extrasynaptic receptors for gamma-aminobutyric acid (GABA) are compared using excitability testing of myelinated axons in amphibian peripheral nerve. The actions of the antagonists appear both complex and dissimilar. Picrotoxin (10-1000 microM) produces large reversible depressions of the maximal response to GABA (0.01-10mM) and increases the EC50 from 0.33 to 12.6 mM. With high concentrations of agonist and antagonist an insensitive component is apparent. The action of picrotoxin is not classically noncompetitive: it may represent a mixed antagonism (competitive and noncompetitive) or a noncompetitive one, masked by the presence of receptor reserve and (or) secondary depolarizing influences (e.g., GABA-evoked [K+] o accumulation). Bicuculline methiodide (10-200 microM) shifts the GABA concentration-response curve to the right; maximal responses persist and are even enhanced. The impression that bicuculline methiodide has a competitive action is supported by analysis of its inhibition of responses to low concentrations of the agonist. It is suggested that the enhancement of GABA responses by bicuculline methiodide and their apparent resistance to block by picrotoxin may be due to a common secondary effect of the antagonists such as a decrease in membrane conductance to K+ and (or) block of transmitter uptake.     

Reduced endogenous GABA-mediated inhibition in the PVN on renal nerve discharge in rats with heart failure

One characteristic of heart failure (HF) is increased sympathetic activation. The paraventricular nucleus (PVN) of the hypothalamus (involved in control of sympathetic outflow) has been shown to have increased neuronal activation during HF. This study examined the influence of endogenous GABA input (inhibitory in nature) into the PVN on renal sympathetic nerve discharge (RSND), arterial blood pressure (BP), and heart rate (HR) in rats with HF induced by coronary artery ligation. In alpha-chloralose- and urethane-anesthetized rats, microinjection of bicuculline (a GABA antagonist) into the PVN produced a dose-dependent increase in RSND, BP, and HR in both sham-operated control and HF rats. Bicuculline attenuated the increase in RSND and BP in HF rats compared with control rats. Alternatively, microinjection of the GABA agonist muscimol produced a dose-dependent decrease in RSND, BP, and HR in both control and HF rats. Muscimol was also less effective in decreasing RSND, BP, and HR in HF rats than in control rats. These results suggest that endogenous GABA-mediated input into the PVN of rats with HF is less effective in suppressing RSND and BP compared with control rats. This is partly due to the post-release actions of GABA, possibly caused by altered function of post-synaptic GABA receptors in the PVN of rats with HF. Reduced GABA-mediated inhibition in the PVN may contribute to increased sympathetic outflow, which is commonly observed during HF.     

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.     

GABA receptors on the cell-body membrane of an identified insect motor neuron

The pharmacology of a gamma-aminobutyric acid (GABA) receptor on the cell body of an identified motor neuron of the cockroach (Periplaneta americana) was investigated by current-clamp and voltage-clamp methods. Iontophoretic application of GABA increased membrane conductance to chloride ions, and prolonged application resulted in desensitization. Hill coefficients, determined from dose-response data, indicated that binding of at least two GABA molecules was required to activate the chloride channel. Differences between vertebrate GABAA receptors and insect neuronal GABA receptors were detected. For the GABA receptor of motor neuron Df, the following rank order of potency was observed: isoguvacine greater than muscimol greater than or equal to GABA greater than 3-aminopropanesulphonic acid. The GABAB receptor agonist baclofen was inactive. Of the potent vertebrate GABA receptor antagonists (bicuculline, pitrazepin, RU5135 and picrotoxin), only picrotoxin (10(-7) M) produced a potent, reversible block of the response to GABA of motor neuron Df. Both picrotoxinin and picrotin also blocked GABA-induced currents. Bicuculline hydrochloride (10(-4) M) and bicuculline methiodide (10(-4) M) were both ineffective when applied at resting membrane potential (-65 mV), although at hyperpolarized levels partial block of GABA-induced current was sometimes observed. Pitrazepin (10(-4) M) caused a partial, voltage-independent block of GABA-induced current. The steroid derivative RU5135 was inactive at 10(-5) M. In contrast to the potent competitive blockade of vertebrate GABAA receptors by bicuculline, pitrazepin and RU5135, none of the weak antagonism caused by these drugs on the insect GABA receptor was competitive. Flunitrazepam (10(-6) M) potentiated GABA responses, providing evidence for a benzodiazepine site on an insect GABA-receptor-chloride-channel complex.