Concentration-dependent effects of muscimol to enhance pulsatile GnRH release from GT1-7 neurons in vitro

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.     

GABAA receptor modulation of temperature sensitive neurons in the diagonal band of Broca in vitro

Several regions of the brain, including the horizontal limb of the diagonal band of Broca (HDB), contain neurons that are responsive to changes in local temperature. These neurons are hypothesized to participate in thermoregulation and sleep–wake control. The HDB contains a large number of γ-aminobutyric acid (GABA) terminals, and it has many neurons that utilize GABA as a neurotransmitter. Therefore, in this study we characterized the in vitro effects of the GABA_A receptor agonist muscimol (0.5, 0.25, 0.1 and 0.0625 μM doses) and the GABA_A receptor antagonist bicuculline (3.0 and 1.0 μM doses) on the firing rate and thermosensitivity of HDB neurons. Of the 51 neurons recorded in a submerged slice chamber, 53% were warm sensitive, 45% were temperature insensitive and 2% were cold sensitive. All neurons exposed to bath applied muscimol exhibited reductions in both firing rate and thermosensitivity. Muscimol induced reductions were maintained for at least 20 min after washout. Neurons exposed to bicuculline had no change in firing rate or thermosensitivity. However, after bicuculline washout there were reductions in both firing rate and thermosensitivity. These findings support the hypothesis that GABA_A receptor induced inhibition of HDB thermosensitive neurons can modulate both thermoregulation and sleep–wake control.     

Duality of Glutamatergic and Gabaergic Control of PulsatileGnRH Secretion by Rat Hypothalamic Explants: II. ReducedNR2C- and GABAA-Receptor-Mediated Inhibition atInitiation of Sexual Maturation

N-methyl-D-aspartate (NMDA) receptors and γ-aminobutyric acid (GABA) receptors are involved in the mechanism of pulsatile gonadotrophin-releasing hormone (GnRH) secretion. The aim of this study was to elucidate the role of those receptors in the acceleration of pulsatile GnRH secretion seen at onset of puberty. Using hypothalamic explants from prepubertal (15 days), early pubertal (25 days) and adult (50 days) male rats, we studied the effects of pharmacological antagonists and antisense oligodeoxynucleotides on GnRH release evoked by NMDA and GABA receptor agonists as well as the interval between spontaneous GnRH secretory pulses. At the three studied ages, the muscimol-evoked release of GnRH is similarly inhibited by the GABA_A receptor antagonist bicuculline. In contrast, the frequency of pulsatility is stimulated by bicuculline as indicated by a reduction of the mean GnRH interpulse interval from 60 to 40 min and such an effect is seen at 15 days only. The GnRH interpulse interval is also reduced by GABA_A receptor antisense oligodeoxynucleotides at 15 days while no effects are seen at 25 days. At the three studied ages, the NMDA-evoked release of GnRH and the GnRH interpulse interval are similarly inhibited by 100 or 500 μM of the NMDA receptor antagonist 7-chlorokynurenic acid (7CK). These effects are consistent with the increase of GnRH interpulse interval caused by NR2A antisense oligodeoxynucleotides at 15 days (86 vs 64 min in controls) as well as 25 days (44 vs 36 min). A low (5 μM) concentration of 7CK does not result in any effect except a reduction of GnRH interpulse interval which is seen at 15 days only. A similar reduction of GnRH interpulse interval is obtained using NR2C antisense oligodeoxynucleotides at 15 days (50 vs 64 min in controls) while no effects are seen at 25 days (35 vs 36 min). At 25 days, muscimol can prevent the developmental increase in frequency of pulsatile GnRH secretion. In summary, pulsatile GnRH secretion by the prepubertal hypothalamus characteristically involves an inhibition mediated through GABA_A receptors and the NR2C subunit of NMDA receptors. Based on these data, we propose a model for the mechanism of the onset of puberty which involves the disappearance or inactivation of GABAergic neurons located in the retrochiasmatic hypothalamus and expressing the NR2C subtype of NMDA receptors.     

The maturation of GABAA receptor-mediated control of luteinizing hormone secretion in immature male rats

In male rats, a GABA_A receptor antagonist, bicuculline methiodide or an agonist, muscimol, was infused intravenously for 30 min into the male rat before (postnatal days 16–17 and days 30–31) and after (over day 45) the sexual maturation. Neither bicuculline nor muscimol infusion significantly altered serum LH concentrations at days 16–17. At the days 30–31 and after day 45, bicuculline infusion significantly increased and muscimol infusion significantly decreased serum LH. In conclusion, in male rats, the functional GABA_A receptor system to inhibit the release of LH seems to be established before the sexual maturation, between 18 and 29 days of age.     

Acute effects of γ-vinyl GABA (vigabatrin) on hippocampal GABAergic inhibition in vitro

The acute effects of γ-vinyl-GABA (GVG) on GABAergic inhibition were investigated in the hippocampal slice preparation using the paired-pulse test of inhibition during extracellular recordings. Superfusion of GVG (100–500 μM) for 60 min resulted in a concentration-dependent decrease in GABAergic inhibition. Slices superfused with higher concentrations of GVG (0.5–1 mM) were hyperexcitable as demonstrated by the appearance of multiple spikes. Binding studies showed that GVG (1 mM) had no effect on the binding of [³H]flunitrazepam or [³H]TBOB and displaced no more than 15% of specific [³H]GABA binding, which indicates that GVG-induced disinhibition is not mediated through an action at the GABA_A receptor complex. Consistent with this suggestion is the finding that GVG (500 μM) had little effect on the inhibition of the orthodromically evoked CA1 population spike produced by the GABA_A receptor agonist muscimol (10 μM), whereas this inhibition was considerably attenuated by the GABA_A receptor antagonist, bicuculline methiodide (5 μM). The results of this study suggest that the acute actions of GVG on the GABAergic neurotransmitter system are not involved in its anticonvulsant effect.     

γ-Aminobutyric acid receptor subtype antagonists differentially alter opioid-induced feeding in the shell region of the nucleus accumbens in rats

Food intake is significantly increased by administration of μ-selective opioid agonists into the nucleus accumbens, particularly its shell region. Pretreatment with either opioid (μ, δ₁, δ₂ or κ₁) or dopaminergic (D₁) receptor antagonists in the nucleus accumbens shell reduce μ opioid agonist-induced feeding. Selective GABA_A (muscimol) and GABA_B (baclofen) agonists administered into the nucleus accumbens shell each stimulate feeding which is respectively and selectively blocked by GABA_A (bicuculline) and GABA_B (saclofen) antagonists. The present study investigated whether feeding elicited by the μ-selective opioid agonist, [ -Ala²,NMe⁴,Gly-ol⁵]-enkephalin in the nucleus accumbens shell was decreased by intra-accumbens pretreatment with an equimolar dose range of either GABA_A or GABA_B antagonists, and further, whether general opioid or selective GABA antagonists decreased feeding elicited by GABA_A or GABA_B agonists in the nucleus accumbens shell. Feeding elicited by the μ-selective opioid agonist was dose-dependently increased following intra-accumbens pretreatment with GABA_A (bicuculline) antagonism; this enhancement was significantly blocked by pretreatment with general or μ-selective opioid antagonists. In contrast, μ opioid agonist-induced feeding elicited from the nucleus accumbens shell was dose-dependently decreased by GABA_B (saclofen) antagonism. Neither bicuculline nor saclofen in the nucleus accumbens shell altered baseline food intake. Whereas muscimol-induced feeding elicited from the nucleus accumbens shell was reduced by bicuculline and naltrexone, but not saclofen pretreatment, baclofen-induced feeding elicited from the nucleus accumbens shell was reduced by saclofen, but not by bicuculline or naltrexone. These data indicate that GABA_A and GABA_B receptor subtype antagonists differentially affect feeding elicited by μ opioid receptor agonists within the nucleus accumbens shell in rats.     

GABAA and GABAB receptors modulating basal and footshock-induced nitric oxide releases in rat prefrontal cortex

Using an in vivo brain microdialysis technique, we measured extracellular levels of nitric oxide (NO) metabolites (NO_x⁻) in the medial prefrontal cortex (mPFC) upon perfusion of γ-aminobutyric acid (GABA) receptor antagonists as well as agonists, and also examined the effects of GABA receptor agonists on mild intermittent footshock-induced NO releases in the mPFC in conscious rats. Perfusion of either bicuculline methiodide, a GABA_A receptor antagonist, or saclofen, a GABA_B receptor antagonist, through a microdialysis probe resulted in dose-dependent increases in NO_x⁻ levels. Higher-dose perfusion of either muscimol (50 μM), a GABA_A receptor agonist, or baclofen (250 μM), a GABA_B receptor agonist resulted in a significant decrease in NO_x⁻ levels. The elevated levels of NO_x⁻ after mild intermittent footshock were attenuated by perfusion of either muscimol (10 μM) or baclofen (50 μM), either of which alone did not affect basal NO_x⁻ levels. These findings are likely to provide helpful clues to our understanding of the inhibitory modulation of basal and footshock-induced NO metabolites releases by GABA_A and GABA_B receptors in the mPFC.     

Partial characterization of K-induced increase in [Ca]cyt and GnRH release in GT1-7 neurons

Secretion of pituitary gonadotropins is regulated centrally by the hypothalamic decapeptide gonadotropin releasing hormone (GnRH). Using the immortalized hypothalamic GT1-7 neuron, we characterized pharmacologically the dynamics of cytosolic Ca²⁺ and GnRH release in response to K⁺-induced depolarization of GT1-7 neurons. Our results showed that K⁺ concentrations from 7.5 to 60 mM increased [Ca²⁺]_cyt in a concentration-dependent manner. Resting [Ca²⁺]_cyt in GT1-7 cells was determined to be 69.7 ± 4.0 nM (mean ± S.E.M.; N = 69). K⁺-induced increases in [Ca²⁺]_cyt ranged from 58.2 nM at 7.5 mM [K⁺] to 347 nM at 60 mM [K⁺]. K⁺-induced GnRH release ranged from about 10 pg/ml at 7.5 mM [K⁺] to about 60 pg/ml at 45 mM [K⁺]. K⁺-induced increases in [Ca²⁺]_cyt and GnRH release were enhanced by 1 μM BayK 8644, an L-type Ca²⁺ channel agonist. The BayK enhancement was completely inhibited by 1 μM nimodipine, an L-type Ca²⁺ channel antagonist. Nimodipine (1 μM) alone partially inhibited K⁺-induced increases in [Ca²⁺]_cyt and GnRH release. Conotoxin (1 μM) alone had no effect on K⁺-induced GnRH release or [Ca²⁺]_cyt, but the combination of conotoxin (1 μM) and nimodipine (1 μM) inhibited K⁺-induced increase in [Ca²⁺]_cyt significantly more (p < 0.02) than nimodipine alone, suggesting that N-type Ca²⁺ channels exist in GT1-7 neurons and may be part of the response to K⁺. The response of [Ca²⁺]_cyt to K⁺ was linear with increasing [K⁺] whereas the response of GnRH release to increasing [K⁺] appeared to be saturable. K⁺-induced increase in [Ca²⁺]_cyt and GnRH release required extracellular [Ca²⁺]. These experiments suggest that voltage dependent N- and L-type Ca²⁺ channels are present in immortalized GT1-7 neurons and that GnRH release is, at least in part, dependent on these channels for release of GnRH.     

Fluorescence imaging of GABAA receptor-mediated intracellular [Cl] in P19-N cells reveals unique pharmacological properties

This study describes the pharmacological properties of GABA_A receptors expressed in P19-N cells using fluorescence imaging of intracellular chloride with 6-methoxy-N-ethylquinolinium iodide (MEQ). We show that application of the GABA agonist, muscimol (10–200 μM), produces time- and concentration-dependent increases in intracellular [Cl⁻] that are blocked by bicuculline. Diazepam (10 μM) and pentobarbital (1 mM) potentiate muscimol-stimulation. These receptors exhibit novel pharmacological properties. The neurosteroid, 3α-hydroxy-5α-pregnane-20-one (1–10 μM) exhibited weak potency in enhancement of muscimol-stimulation. Ethanol (50 and 100 mM) exhibited high efficacy on muscimol responses, a 4- to 5-fold potentiation, respectively, of muscimol (10 μM) alone. GABA and muscimol allosterically modulated specific binding of [³H]flunitrazepam to differentiated P19 cells. Modulation of GABA_A receptor mediated increases in intracellular [Cl⁻] demonstrated stability in response magnitude from 7 to 15 days following removal of retinoic acid. In concert, GABA_A receptor subunit mRNA and protein expression patterns in these neuron-like cells were stable over the same period. Using RT-PCR we determined that differentiated P19 cells lack γ1, γ2L, α6 and δ subunit mRNAs while expressing α1, α2, α3, α4, α5, β1, β2, β3, γ2S and γ3. Furthermore, subunit specific antibody immunocytochemical labeling of cells with a neuronal morphology indicated the presence of α1, α2, α4, and γ2 subunits (the only subunits tested). Therefore, P19-N cells should prove useful to researchers in need of a model cell culture system in which to study function and regulation of neuronal GABA_A receptors.     

Involvement of GABAA and GABAB receptors in afterdischarge generation in rat hippocampal slices

It has been hypothesized that a disruption of γ-aminobutyric acid (GABA) receptor-mediated processes may be involved in the pathophysiology of focal epilepsy. This disinhibition hypothesis has been postulated from the results of in vitro experiments of the interictal activity of focal epilepsy. Less is known, however, about how disinhibition may be involved in the production of the ictal activity. We therefore examined the pharmacological effects of selective agonists and antagonists of GABA_A and GABA_B receptors on ictal-like afterdischarges (ADs) induced following repetitive high-frequency electrical stimulation in the CA1 region of rat hippocampal slices. The GABA_A receptor antagonist bicuculline (5 μM) fully blocked AD generation, as did the GABA_A receptor agonist muscimol (2 μM), which is thought to produce a tonic inhibition during application. However, the benzodiazepine receptor agonist diazepam (5 μM), which enhances the inhibitory postsynaptic potential induced by synaptically released GABA, increased the number of spikes in the AD to 148.3% of the control value. On the other hand, the GABA_B receptor antagonist phaclofen (1 mM) increased the number of spikes in the AD to 234.7% of the control value, while the GABA_B receptor agonist baclofen (5 μM) reduced it to 46.9%. We therefore conclude that synaptic, but not tonic, activation of GABA_A receptors appears to be necessary for ictal-like AD generation, while GABA_B receptor activation plays a protective role. We therefore propose a modification to the simple disinhibition hypothesis.     

The role of dorsal striatal GABAA receptors in dopamine agonist-induced behavior and neuropeptide gene expression

The purpose of this study was to investigate whether GABA_A receptors in the dorsal striatum regulate basal or stimulant-induced behaviors. Correspondingly, the question of possible GABA_A receptor control of neuropeptide mRNA expression in nigrostriatal neurons was addressed. The GABA_A receptor antagonist, bicuculline, was unilaterally or bilaterally microinjected into the dorsal striatum of rats in a series of 3 studies. In the first study, unilateral administration of 10–50 ng/μl of bicuculline did not alter behavior. However, 250 ng/μl bicuculline produced motor dyskinesias and/or seizures. In the second study, 100 ng/μl bicuculline administered unilaterally prior to saline or amphetamine treatment, produced mild twitching in 61% of rats but did not affect amphetamine (2.5 mg/kg, i.p.)-induced behavioral activity, specifically rearing and sniffing. In the third study, 75 ng/μl of bicuculline was administered unilaterally or bilaterally into the striatum in two separate experiments. Administration of bicuculline either unilaterally or bilaterally produced mild transient twitching of the forelimbs but did not affect behaviors induced by the selective D₁ receptor agonist SKF-82958 (0.5 mg/kg, s.c.). Three hours after unilateral bicuculline administration, the brains were removed and processed for quantitative in situ hybridization. Bicuculline did not significantly affect the basal or SKF-82958-induced increase in preprodynorphin or substance P mRNA expression in striatonigral neurons on the side of injection. These data suggest that blockade of GABA_A receptors in the dorsal striatum does not affect dopamine agonist-stimulated behaviors or neuropeptide mRNA expression in striatonigral neurons in the rat striatum.     

Zinc modulation of GABAA receptor-mediated chloride flux in rat hippocampal slices

We studied the effect of ZnCl₂ application on GABA_A receptor-mediated ³⁶Cl⁻ 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 μM)-evoked ³⁶Cl⁻ flux was significantly reduced by ZnCl₂ (100 μM) in hippocampal microsacs. In hippocampal slices, muscimol (50 μM)-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 μM), caused a significant increase in muscimol-evoked ³⁶Cl⁻ efflux only in DG slices (57.2 ± 7.0%), suggesting that GABA_A 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 μM) 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 GABA_A 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.     

Effects of taurine on depolarization-evoked release of amino acids from rat cortical synaptosomes

Effects of taurine on endogenous aspartic acid (Asp), glutamic acid (Glu) and γ-aminobutyric acid (GABA) release has been investigated using synaptosomes prepared from rat cerebral cortex. Although basal release of these amino acids was not affected, taurine inhibited KCl (30 mM)-evoked overflow of Asp, Glu and GABA in a concentration-dependent manner with potencies (IC₅₀) of 1 μM, 0.8 μM and 5 nM, respectively. Taurine (10 μM) maximally inhibited K⁺-evoked Asp, Glu and GABA overflow by 28, 37 and 65%, respectively. Phaclofen (10 μM, a GABA_B receptor antagonist), but not bicuculline (10 μM, a GABA_A receptor antagonist), counteracted the inhibition of GABA overflow, although the inhibition of Asp and Glu overflow was not attenuated. These data suggest that taurine may inhibit GABA release through the activation of presynaptic GABA_B autoreceptors and, at high concentration, also act on Asp- and Glu-nerve terminals to regulate release of excitatory amino acids in rat cortex.     

Waglerin-1 inhibits GABAA current of neurons in the nucleus accumbens of neonatal rats

The effect of Waglerin-1, a 22-amino acid peptide purified from the venom of Wagler's pit viper on the whole cell current response (I_GABA) to γ-aminobutyric acid (GABA) was examined for neurons freshly isolated from the nucleus accumbens of 3- to 7-day-old rats. Waglerin-1 depressed I_GABA induced by subsaturating concentrations of GABA; the IC₅₀ for I_GABA induced by 10 μM GABA was 2.5 μM Waglerin-1. This concentration of Waglerin-1 shifted the GABA concentration–response curve to the right in a parallel manner, increasing the GABA EC₅₀ from 12±3 to 27±5 μM. The depressant effect of Waglerin-1 was greater at negative holding potentials. Zn²⁺ also inhibited I_GABA with an IC₅₀ of 0.3 μM. Phosphorylation state appeared to modulate GABA_A receptor sensitivity to the inhibitory effect of Waglerin-1 since dialysis of neurons with N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide HCl (H-89), an inhibitor of protein kinase A, prevented inhibition. The data are discussed in terms of developmental influences on the subunit composition of GABA_A receptors in neurons of the nucleus accumbens.     

Clathrin-coated vesicles from bovine brain contain uncoupled GABAA receptors

Clathrin-coated vesicles are thought to be a vehicle for the sequestration of GABA_A receptors. For coated vesicles from bovine cerebrum, we examined the binding properties of [³H]muscimol, a GABA_A-specific agonist, [³H]flunitrazepam, a benzodiazepine agonist, and [³⁵S]t-butylbiocyclophosphorthionate (TBPS), a ligand for GABA_A receptor channels. Under standard conditions, the binding level of [³H]muscimol, [³H]flunitrazepam, and [³⁵S]TBPS to coated vesicles represented 12.3±1.8%, 7.9±1%, and 10.2±1.8%, respectively, of that in crude synaptic membranes. Coated vesicles showed a single [³H]flunitrazepam binding site with a K_D value (12 nM) which was 9-fold that for synaptic membranes. The allosteric coupling between binding sites was measured by the addition of GABA to [³H]flunitrazepam and [³⁵S]TBPS binding assays. For [³H]flunitrazepam binding to synaptic membranes, GABA gave an EC₅₀=2.0 μM and at saturation (100 μM) an enhancement of 122%. This stimulation was completely blocked by the GABA antagonist SR95531. In contrast, neither GABA nor SR95531 had a significant effect on [³H]flunitrazepam binding to CCVs, indicating that the allosteric interaction between GABA and benzodiazepine binding sites is abolished. Likewise, GABA displaced nearly all of the [³⁵S]TBPS binding to synaptic membranes but had no effect on binding to coated vesicles, indicating that coupling between the GABA binding sites and chloride channel is also impaired. Thus GABA_A receptors appear to be uncoupled during normal intracellular trafficking via coated vesicles. The presence of major GABA_A receptor subunits on these particles was verified by quantitative immunoblotting. Relative to the levels in synaptic membranes, CCVs contained 110±14% and 29.5±3.8%, respectively, of the immunoreactivity for GABA_A receptor β2 and α1 subunits. Thus, in comparison to GABA_A receptors on synaptic membranes, those on CCVs have a reduced α1/β2-subunit ratio. It may be suggested that a selective decline in the content of α1 subunits in coated vesicles could in part account for GABA_A receptor uncoupling.     

Inhibitory effect of 17β-estradiol in the parabrachial nucleus is mediated by GABA

In the present investigation, electrophysiological recordings of thalamic relay neurons were used to investigate the role of estrogen as a modulator of visceral afferent information through the PBN to forebrain structures. Experiments were done in anaesthetized (sodium thiobutabarbitol; 100 mg/kg) male and ovariectomized female rats supplemented for 7 days prior with either 17β-estradiol (OVX-E₂) or saline (OVX-S). A portion of the right cervical vagus was isolated for the electrical activation (0.8 Hz, 2 ms duration) of visceral afferents. The evoked single and multi-unit activity was recorded via a recording electrode in the ventrobasal thalamus. Exogenous microinjection of 17β-estradiol (0.1, 0.25 and 0.5 μM; 200 nl) into the parabrachial nucleus (PBN) produced a significant, dose-dependent attenuation in the magnitude of visceral afferent activation-evoked responses of neurons recorded in the thalamus in both male and OVX-E₂ groups. No effect on evoked thalamic activity was observed following injection of estrogen into the PBN of OVX-S animals. Co-injection of estrogen with the GABA_A receptor antagonist, bicuculine (0.1 μM; 200 nl) but not phaclofen (GABA_B; 0.1, 0.5 or 1 μM; 200 nl) resulted in an increase in the evoked thalamic response in males (55±11%) and OVX-E₂ female (68±15%) rats. These studies suggest that estrogen inhibits neurotransmission in the PBN via an interaction with the GABA_A receptor to modulate the flow of visceral information to the thalamus.     

Nucleus accumbens dopamine release modulation by mesolimbic GABAA receptors—an in vivo electrochemical study

The role of GABA receptors in regulating the mesolimbic dopamine (DA) system and drug reinforced behaviors has not been well characterized. Using fast-cyclic voltammetry, the effects of specific GABA receptor modulation on DA release in the nucleus accumbens (NAcc) and heroin self-administration (SA) behavior was investigated. The GABA_A agonist muscimol, administered either intravenously or directly into the ventral tegmental area (VTA), significantly increased DA release in the NAcc in 7 of the 10 rats tested. DA release decreased in the remaining three rats; both effects were blocked by pretreatment with the GABA_A receptor antagonist bicuculline. In contrast, the GABA_B agonist baclofen decreased, while 2-OH-saclofen (a GABA_B antagonist) increased DA release in the NAcc. However, when VTA GABA_B receptors were previously activated or inactivated by microinjections of baclofen or 2-OH-saclofen, systemic injections of muscimol caused an inhibition of NAcc DA release. These results suggest that GABA_A receptors may be co-localized on both DA neurons and non-DA (GABAergic) interneurons in the VTA, with the effects of GABA_A determined by the net effect of both direct inhibition and indirect disinhibition of DA neurons. Finally, although a DA releaser, muscimol was neither self-administered in drug naive rats, nor did it substitute for heroin in rats previously trained to self-administer heroin, suggesting that GABA_A receptors appear to play a complex role in mediating drug reinforcement, depending upon the dynamic functional state of GABA_A receptors on both tegmental DA and non-DA neurons.     

In vivo neurochemical evidence that stimulation of accumbal GABAA and GABAB receptors each reduce acetylcholine efflux without affecting dopamine efflux in the nucleus accumbens of freely moving rats

Cholinergic neurons in the nucleus accumbens contain GABA_A and GABA_B receptors that are thought to inhibit neural activity. We analyzed the roles of GABA_A and GABA_B receptors in regulating accumbal acetylcholine efflux of freely moving rats using in vivo microdialysis. The effects of GABA receptor ligands on the accumbal dopamine efflux were also analyzed because accumbal cholinergic and dopaminergic neurons could mutually interact. Drugs were applied intracerebrally through the dialysis probe. Doses of compounds indicate total amount administered (mol) during 30–60 min infusions. To monitor basal acetylcholine, a low concentration of physostigmine (50 nM) was added to the perfusate. GABA_A receptor agonist muscimol (3 and 30 pmol) induced a dose‐related decrease in accumbal acetylcholine. GABA_B receptor agonist baclofen (30 and 300 pmol) also produced a dose‐related decrease in acetylcholine. GABA_A receptor antagonist bicuculline (60 pmol) which failed to alter baseline acetylcholine counteracted the muscimol (30 pmol)‐induced decrease in acetylcholine. GABA_B receptor antagonist 2‐hydroxysaclofen (12 nmol) which failed to change baseline acetylcholine, counteracted the baclofen (300 pmol)‐induced decrease in acetylcholine. Neither muscimol (30 pmol) nor baclofen (300 pmol) which reduced accumbal acetylcholine altered baseline accumbal dopamine. Neither bicuculline (60 pmol) nor 2‐hydroxysaclofen (12 nmol) also affected the baseline dopamine. These results show that GABA_A and GABA_B receptors each exert inhibitory roles in the regulation of accumbal cholinergic neural activity. The present results also provides in vivo neurochemical evidence that stimulation of GABA_A and GABA_B receptors each reduce acetylcholine efflux without affecting dopamine efflux in the nucleus accumbens of freely moving rats.     

GABA-ergic control of α-Melanocyte-Stimulating Hormone (α-MSH) release by frog neurointermediate lobe in vitro

Measurement of glutamate decarboxylase (GAD) activity in the intermediate lobe of the frog pituitary and brain showed that neurointermediate lobe extracts represented 12% of the GAD activity detected in the whole brain. No significant activity was measured in distal lobe extracts. Immunocytochemical studies revealed GAD-containing fibers among the parenchyma! cells of the pars intermedia. The localization of GAD-like material in the intermediate lobe of the frog pituitary suggested a possible role of γ-aminobutyric acid (GABA) in the regulation of melanotropic cell secretion. Administration of GAB A (10⁻⁶ to 10⁻⁴ M), to perifused neurointermediate lobes caused a brief stimulation of alpha-melanocyte stimulating hormone (α-MSH) release followed by an inhibition. Picrotoxin (10⁻⁴ M), a Cl⁻ channel blocker, abolished only the stimulatory effect of GAB A (10⁻⁴ M), whereas bicuculline (10⁻⁴ M), a specific antagonist of GABA_A receptors, totally inhibited the effects of GABA (both stimulatory and inhibitory phases). Bicuculline induced by itself a slight stimulation of α-MSH release, suggesting that GABA-ergic nerve fibers present in the intermediate lobe are functionally active in vitro. The GABA_A agonist muscimol (10⁻⁷ to 10⁻⁴ M) mimicked the biphasic effect of GABA on α-MSH release. Administration of baclofen, a specific GABA_BB agonist (10⁻⁷ to 10⁻⁴ M) induced a dose-dependent inhibition of α-MSH secretion. In contrast to GABA or muscimol, baclofen did not cause any stimulatory effect whatever the dose. Taken together these result suggested that GABA_A and GABa_b receptors were present on frog melanotrophs. Since bicuculline totally inhibited GABA effects (stimulation and inhibition) on α-MSH release, it appears however that the effect of GABA is mainly achieved through activation of G AB A_A receptors.     

Guanosine phosphate analogs modulate ethanol potentiation of GABAA-mediated synaptic currents in hippocampal CA1 neurons

Recent studies have demonstrated that ethanol potentiation of GABA_A receptor function can be regulated by second-messenger-dependent processes. As a preliminary step to further characterize this interaction, we used the whole-cell patch-clamp technique to study the effects of guanosine phosphate analogs on ethanol potentiation of GABA_A-mediated synaptic transmission, in rat hippocampal CA1 neurons. Intracellular dialysis with 400 μM GDPβS, an analog that inhibits G-protein coupled events, significantly reduced ethanol, but not pentobarbital, potentiation of IPSCs. In contrast, dialysing neurons with 100 μM GTPγS, an irreversible G-protein activator, selectively facilitated ethanol potentiation of GABA_A IPSCs. These results suggest that one or more G-protein coupled events regulate the ethanol sensitivity of synaptic GABA_A receptors.