Dopaminergic Agonists Have Both Presynaptic and Postsynaptic Effects on the Guinea-pig's Medial Vestibular Nucleus Neurons

A number of studies have indicated a possible interaction between dopamine and the vestibular system. Using intracellular recordings in brainstem slices, we have tested the effects of dopamine and other dopaminergic compounds on guinea-pig medial vestibular nucleus (MVN) neurons. In normal medium, MVN neurons were depolarized by dopamine as well as by (-)quinpirole and piribedil, which are selective D₂ dopaminergic agonists. The dependence of this effect on the presence of D₂ receptors was confirmed by using (-)sulpiride, a D₂ antagonist which blocked the depolarizing effect of dopamine. Dopaminergic D₁ receptors were apparently not involved in this effect since a selective D₁ agonist, SKF-38393, had no effect on MVN neurons and the D₁ antagonist (+) SCH-23390 could not block the effect of dopamine. These depolarizing responses to dopamine must be due to a presynaptic action on terminals that normally release GABA spontaneously on MVN neurons, and tonically maintain them in a state of hyperpolarization. Indeed, such a spontaneous release was demonstrated to occur in the slice since application of bicuculline, a GABA_A antagonist, depolarized MVNneurons in normal saline, but not in a high Mg²⁺/low Ca²⁺ solution known to block synaptic transmission. When dopamine was applied in conditions in which no GABA_A-dependent transmission could occur (either in the presence of bicuculline or in a high Mg²⁺/low Ca²⁺ solution) only a hyperpolarizing, most probably postsynaptic, effect occurred. These results indicate that dopamine might exert in vivo a significant modulatory action on the vestibular system, either by a direct action on the vestibular neurons or by modulation of GABAergic transmission.     

Inhibitory action of CGS 21680 on cerebral cortical neurons is antagonized by bicuculline and picrotoxin—is GABA involved?

The possibility of an involvement of endogenously released GABA in the inhibitory actions of A₁ and A_2a adenosine receptor agonists on rat cerebral cortical neurons discharges was examined using the GABA_A antagonists bicuculline and picrotoxin. The A₁ agonist N⁶-cyclopentyladenosine (CPA), the A_2a agonist CGS 21680 and the non-selective receptor agonist, adenosine, depressed neuronal firing. Applications of bicuculline or picrotoxin enhanced the spontaneous firing rate of cortical neurons, indicating the presence of ongoing GABA-ergic inhibition. Antagonism of GABA_A receptors blocked the depressant effects of CGS 21680 on neuronal firing; was without effect on CPA-evoked inhibitions and tended to reduce the duration of adenosine-evoked inhibitions. These results suggest that the depressant effects of A_2a receptor activation are due to an increase in GABA-ergic inhibition, likely as a consequence of increased GABA release. GABA does not appear to be involved in adenosine A₁ receptor-mediated inhibition of neuronal firing.     

The role of GABA in NMDA-dependent long term depression (LTD) of rat medial vestibular nuclei

The role of GABA in NMDA-dependent long term depression (LTD) in the medial vestibular nuclei (MVN) was studied on rat brainstem slices. High frequency stimulation (HFS) of the primary vestibular afferents induces a long lasting reduction of the polysynaptic (N2) component of the field potentials recorded in the dorsal portion of the MVN. The induction but not the maintenance of this depression was abolished by AP5, a specific blocking agent for glutamate NMDA receptors. The involvement of GABA in mediating the depression was checked by applying the GABA_A and GABA_B receptor antagonists, bicuculline and saclofen, before and after HFS. Under bicuculline and saclofen perfusion, HFS provoked a slight potentiation of the N2 wave, while the N2 depression clearly emerged after drug wash-out. this indicates that GABA is not involved in inducing the long term effect, but it is necessary for its expression. Similarly, the LTD reversed and a slight potentiation appeared when both drugs were administered after its induction. Most of these effects were due to the bicuculline, suggesting that GABA_A receptors contribute to LTD more than GABA_B do. According to our results, it is unlikely that the long lasting vestibular depression is the result of a homosynaptic LTD. On the contrary, our findings suggest that the depression is due to an enhancement of the GABA inhibitory effect, caused by an HFS dependent increase in gabaergic interneuron activity, which resets vestibular neuron excitability at a lower level.     

Synaptic inhibition in the isolated respiratory network of neonatal rats

Gramicidin-perforated patch-clamp recording revealed phasic Cl^–-mediated hyperpolarizations in respiratory neurons of the brainstem–spinal cord preparation from newborn rats. The in vitro respiratory rhythm persisted after block of γ-aminobutyric acid (GABA), i.e. GABA_A, receptor-mediated inhibitory postsynaptic potentials (IPSPs) with bicuculline and/or glycinergic IPSPs with strychnine. In one class of expiratory neurons, bicuculline unmasked inspiration-related excitatory postsynaptic potentials (EPSPs), leading to spike discharge. Bicuculline also blocked hyperpolarizations and respiratory arrest due to bath-applied muscimol, whereas strychnine antagonized similar responses to glycine. The reversal potential of respiration-related IPSPs and responses to GABA, muscimol or glycine was not affected by CO₂/HCO₃^–-free solutions, but shifted from about ?65 mV to values more positive than ?20 mV upon dialysis of the cells with 144 instead of 4 mm Cl^–. Impairment of GABA uptake with nipecotic acid or glycine uptake with sarcosine evoked a bicuculline- or strychnine-sensitive decrease of respiratory frequency which could lead to respiratory arrest. Also, the GABA_B receptor agonist baclofen led to reversible suppression of respiratory rhythm. This in vitro apnoea was accompanied by a K⁺ channel-mediated hyperpolarization (reversal potential ?88 mV) of tonic cells, whereas membrane potential of neighbouring respiratory neurons remained almost unaffected. Both baclofen-induced hyperpolarization and respiratory depression were antagonised by 2-OH-saclofen, which did not affect respiration-related IPSPs per se. The results show that synaptic inhibition is not essential for rhythmogenesis in the isolated neonatal respiratory network, although (endogenous) GABA and glycine have a strong modulatory action. Hyperpolarizing IPSPs mediated by GABA_A and glycine receptors provide a characteristic pattern of membrane potential oscillations in respiratory neurons, whereas GABA_B receptors rather appear to be a feature of non-respiratory neurons, possibly providing excitatory drive to the network.     

GABAergic inhibition gates excitatory LTP in perirhinal cortex

The perirhinal cortex (PRh) is a key region downstream of auditory cortex (ACx) that processes familiarity linked mnemonic signaling. In gerbils, ACx‐driven EPSPs recorded in PRh neurons are largely shunted by GABAergic inhibition (Kotak et al., 2015, Frontiers in Neural Circuits, 9). To determine whether inhibitory shunting prevents the induction of excitatory long‐term potentiation (e‐LTP), we stimulated ACx‐recipient PRh in a brain slice preparation using theta burst stimulation (TBS). Under control conditions, without GABA blockers, the majority of PRh neurons exhibited long‐term depression. A very low concentration of bicuculline increased EPSP amplitude, but under this condition TBS did not significantly increase e‐LTP induction. Since PRh synaptic inhibition included a GABA_B receptor‐mediated component, we added a GABA_B receptor antagonist. When both GABA_A and GABA_B receptors were blocked, TBS reliably induced e‐LTP in a majority of PRh neurons. We conclude that GABAergic transmission is a vital mechanism regulating e‐LTP induction in the PRh, and may be associated with auditory learning.     

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.     

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.     

Early postnatal maturation of GABAA-mediated inhibition in the brainstem respiratory rhythm-generating network of the mouse

It is well established that GABA_A‐mediated postsynaptic potentials are excitatory in many brain regions during embryonic and early postnatal life. The pre‐Bötzinger complex (PBC) in the brainstem is an essential component of the respiratory rhythm‐generating network, where GABA_A‐mediated inhibition plays a critical role in generating a stable respiratory rhythm in adult animals. In the present study, using the perforated patch technique, we investigated the maturation of GABA_A receptor‐mediated effects on rhythmically active PBC neurons and on the motor output in slice preparations from P0–15 neonatal mice. The reversal potential of GABA_A receptor‐mediated current (E_GABA‐A) switched from depolarizing to hyperpolarizing within the first postnatal week. E_GABA‐A was ?13.7 ± 9.8 mV at P0, then it changed to ?44.8 ± 7.0 mV at P2 and ?71.5 ± 6.8 mV at P4. Perfusion of bicarbonate‐free saline has no detectable influence on E_GABA‐A, indicating that a lack of Cl^– extrusion during P0–3 is mainly responsible for early GABA_A‐ergic excitation. At the network level, blockade of GABA_A receptors with bicuculline did not significantly change the frequency of rhythmic bursts recorded from hypoglossal nerve roots before P3, whereas it increased the coefficient of variation. After P3, bicuculline increased burst frequency with little effect on the coefficient of variation. Thus, chloride‐mediated inhibition, which appears in PBC neurons after P3, coincides with the appearance of GABA_A‐mediated modulation of the respiratory rhythm. GABA_A receptor‐activated inhibition may therefore be necessary for frequency modulation in the respiratory network beginning on the fourth postnatal day in the mouse brainstem.     

GABA and valproate modulate trigeminovascular nociceptive transmission in the thalamus

Objective: To study the role of GABA receptors in thalamic relay neurons in the ventroposteromedial (VPM) nucleus of the rat activated by a trigeminovascular nociceptive stimulus in relationship to migraine, and the potential modulation of nociceptive transmission by GABA acting anti-convulsants.Methods: Trigeminovascular nociceptive afferents were identified in the VPM by electrical stimulation of the superior sagittal sinus (SSS), and cell bodies identified by activation with l-glutamate. The effect of GABA, valproate and gabapentin ejection during SSS stimulation and microiontophoresis of l-glutamate was studied. GABA responses were characterized with the selective GABA_A and GABA_B agonists muscimol and baclofen, respectively, and the antagonists bicuculline (GABA_A) and hydroxysaclofen (GABA_B).Results: GABA inhibited the response to SSS stimulation and l-glutamate ejection. Both the selective GABA_A receptor agonist muscimol, and the GABA_B agonist baclofen strongly inhibited the post-synaptic response to l-glutamate. This inhibition could be antagonised by co-ejection of the appropriate antagonist. The post-synaptic inhibitory action of GABA on the cell bodies of third order neurons could be partially antagonised by co-ejection of bicuculline but not by hydroxysaclofen. Valproate inhibited the responses to SSS stimulation and l-glutamate ejection. Bicuculline, but not hydroxysaclofen, was able to antagonise the effect of valproate on both responses to l-glutamate and SSS stimulation. Gabapentin did not alter the responses to l-glutamate and SSS stimulation.Interpretation: These results indicate that GABA_A and GABA_B receptors on thalamic neurons can modulate trigeminovascular nociceptive transmission in the VPM nucleus. Sodium valproate can inhibit trigeminovascular nociception at the level of VPM through GABA_A receptor mechanisms, whereas gabapentin does not alter trigeminovascular nociception.     

Cholinergic cell expression in the developing rat medial septal nucleus in vitro is differentially controlled by GABAA and GABAB receptors

The early appearance and relative abundance of GABAergic neurons in basal forebrain cholinergic nuclei like the medial septum suggest that the maturation of the later developing cholinergic neurons in these nuclei may be controlled by GABA. To examine this possibility, the effects of both exogenous GABA and specific GABA receptor agonists, as well as that of endogenous GABA on the phenotypic expression and survival of the cholinergic neurons in primary cultures from the fetal rat medial septum, were studied. Treatment of these cultures for six days with GABA significantly decreased the enzymatic activity of choline acetyltransferase (EC 2.3.1.6) (ChAT) in a dose-dependent manner. This response to exogenous GABA was blocked by bicuculline, mimicked by muscimol and slightly potentiated by saclofen. Consistent with this latter observation, the GABA_B receptor agonist, baclofen, dose-dependently increased septal ChAT activity. However, while the effect of baclofen on cholinergic expression was lost in the absence of glia, the suppressive effects of GABA or muscimol were more marked. Acetylcholinesterase (EC 3.1.1.7) (AChE) expression in mixed neuronal–glial cultures, was, like ChAT activity, increased or decreased in intensity with the inclusion of baclofen or muscimol, respectively. Although the number of AChE positive neurons in muscimol-treated cultures was significantly lower than that in controls, no changes in neither neuronal nor general cell viability were noted. Finally, as GABA_A or GABA_B receptor antagonists bicuculline and picrotoxin or saclofen, when applied alone to mixed cultures, increased or decreased ChAT activity, respectively, it appears that endogenous GABA, tonically released in the developing septum, may, via specific receptor types, differentially control the biochemical maturation of the cholinergic neurons.     

Neuronal regulation of astrocyte morphology in vitro is mediated by GABAergic signaling

The addition of isolated neurons to monolayers of cultured astrocytes induced a morphological change in the astrocytes that came into contact with the added neuronal cell bodies or neurites. The change, which included an increase in the complexity of cell shape, took at least 3 days to become detectable and was enhanced in proportion to the number of attached neurons. Astrocytes that did not make contact with any neurons had a less complex contour, comparable to those in control cultures with no neurons added. Treatment of neuron-astrocyte cocultures with a sodium channel blocker, tetrodotoxin, suppressed the neuron-induced morphological changes in astrocytes. A GABA_A-receptor antagonist, bicuculline, mimicked the inhibitory effect of tetrodotoxin. In cultures without added neurons, morphological alteration of astrocytes was also observed when cultures were incubated for 1 or more days with exogenous GABA together with a GABA-uptake inhibitor, 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol. The effect of exogenous GABA was mimicked by treatment with a GABA_A-receptor agonist, muscimol, and blocked by bicuculline treatment. These results suggest that GABA released from neurons with their activity serves as a signal from neurons to astrocytes that triggers the morphological change in astrocytes through the activation of GABA_A receptors. GLIA 20:1-9, 1997. © 1997 Wiley-Liss, Inc.     

GABA induces GABAergic MSCs in cultured embryonic rat thalamic neurons

Application of 0.1–10 μM GABA in the vicinity of cultured embryonic rat thalamic neurons recorded with patch pipettes in the presence of 2 μM TTX induced or increased the frequency of miniature synaptic currents (MSCs) that reversed polarity at the Cl⁻ equilibrium potential. These MSCs were blocked by the GABA_A receptor antagonist bicuculline and exhibited exponential decay kinetics that closely paralleled those estimated from fluctuation analysis of Cl⁻ channels activated pharmacologically by applying 1–10 μM GABA to the same cells. We conclude that the MSCs are mediated by GABA. Application of the GABA_A receptor agonist muscimol activated Cl⁻ current but failed to induce GABAergic MSCs while submicromolar concentrations of GABA evoked GABAergic MSCs but did not activate Cl⁻ channels. The GABA_B receptor agonist (-)baclofen did not mimic GABA in inducing MSCs. Induction of GABAergic MSCs by GABA required extracellular Ca²⁺. Verapamil and Co²⁺, which block voltage-dependent calcium channels, completely blocked GABA-induced MSCs independent of their effects on the direct activation of a Cl⁻ current response. The results indicate that GABA can trigger GABAergic Cl⁻-dependent MSCs in a Ca_o²⁺-dependent manner. The mechanism may involve a novel receptor and/or signal transduction pathway. Synapse 25:15–23, 1997. © 1997 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Effects of GABA on neurons of the gustatory and visceral zones of the parabrachial nucleus in rats

Response characteristics of neurons in the gustatory and visceral zone of the parabrachial nucleus (PBN) to γ-aminobutyric acid (GABA) were examined using whole cell recordings in brain slices of the rat. Based on the recording site, neurons were divided into three groups: neurons in the dorsolateral quadrant of the PBN (DL-neurons), neurons in the dorsomedial quadrant of the PBN (DM-neurons) and neurons in the ventromedial quadrant of the PBN (VM-neurons). Recordings were made from 44 DL-, 43 DM-, 39 VM-neurons. Superfusion of GABA resulted in a concentration-dependent reduction in input resistance in 67.5% of the neurons in the PBN (73.1% of the DL-, 62.5% of the DM-, 66.7% of the VM-neurons). No obvious difference of the concentration–response curve was found among three groups. The mean reversal potential of the GABA effect was about −74 mV and no significant differences were observed among three groups of neurons. The GABA response was partly or completely blocked by the GABA_A antagonist bicuculline in all neurons tested. Superfusion of the GABA_A agonist muscimol resulted in a decrease of the input resistance in all neurons tested. It was concluded that GABA functions as an inhibitory neurotransmitter in both gustatory and visceral part of the PBN, mediated in part, by GABA_A receptors.     

Cytotoxic action of lindane in neocortical GABAergic neurons is primarily mediated by interaction with flunitrazepam-sensitive GABAA receptors

The cytotoxic action of the γ-isomer of hexachlorocyclohexane (γ-HCH; lindane) was studied in cultured mouse neocortical neurons by measurements of the reduction in mitochondrial function using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) test. The cells were exposed to 30-300 μM lindane in the culture medium for different periods of time and lindane cytotoxicity was found to be time- and concentration-dependent. Lindane cytotoxicity could be ameliorated by addition of gamma aminobutyric acid (GABA) in a concentration-dependent manner but this effect of GABA was not blocked by bicuculline or picrotoxinin (PTX). Lindane induced cytotoxicity was also reduced by the GABA_A receptor agonists muscimol and THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol). This effect was enhanced by the simultaneous presence of flunitrazepam but only at the highest lindane concentrations studied (200 and 300 μM). Flunitrazepam by itself had no effect on lindane-induced cytotoxicity. The protective effect of GABA plus flunitrazepam was blocked by the benzodiazepine receptor antagonist flumazenil and by the GABA_A antagonist bicuculline, suggesting the involvement of central benzodiazepine receptors allosterically coupled to the GABA recognition site at the GABA_A receptor. When 100 μM PTX was used to suppress the protective effect of GABA and flunitrazepam, a significant effect of PTX was observed only at 300 μM lindane. The GABA_B receptor agonist, baclophen, only marginally reduced the cytotoxic effect induced by the highest lindane concentrations. It is concluded that the cytotoxic action of lindane in neocortical neurons in culture is mediated primarily through an interaction with allosterically coupled GABA-benzodiazepine recognition sites at the GABA_A receptor. J. Neurosci. Res. 52:276–285, 1998. © 1998 Wiley-Liss, Inc.     

Bicuculline‐ and neurosteroid‐sensitive tonic chloride current in rat hypoglossal motoneurons and atypical dual effect of SR95531

Hypoglossal motoneurons (HMs) are known to be under ‘permanent’ bicuculline‐sensitive inhibition and to show ‘transient’ synaptic γ‐aminobutyric acid (GABA)_A and glycine inhibitory responses. The present paper describes a permanent bicuculline‐sensitive current that should contribute to their tonic inhibition. This current was recorded in brainstem slices superfused without any exogenous agonist and remained detectable with tetrodotoxin. It could also be blocked by the other GABA_A antagonists picrotoxin (PTX) and 2‐(3‐carboxypropyl)‐3‐amino‐6‐(4 methoxyphenyl)pyridazinium bromide) (SR95531; gabazine), but persisted in the presence of a specific blocker of α5‐containing GABA_A receptors. Addition of 2 μm 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol hydrochloride (THIP), known to preferentially activate GABA_A receptors devoid of a γ‐subunit, induced a sustained anionic current that could be further enhanced by neurosteroids such as allopregnanolone (100 nm ). Thus, HMs show a tonic inhibitory current carried by extrasynaptic γ‐free GABA_A receptors, highly sensitive to neurosteroids. A second result was obtained by using SR95531 at concentrations sufficiently high to rapidly block the tonic current above the chloride equilibrium potential (E_Cl). Surprisingly, below E_Cl, SR95531 (10–40 μm ) activated a sustained inward current, associated with a conductance increase, and resistant to bicuculline or PTX (100 μm ). Similarly, after blockade of the bicuculline‐sensitive current, SR95531 activated an outward current above E_Cl. The bicuculline‐resistant anionic current activated by SR95531 could be blocked by a GABA_C receptor antagonist. Thus, two types of inhibitory GABA receptors, belonging to the GABA_A and GABA_C families, are able to show a sustained activity in HMs and provide promising targets for neuroprotection under overexcitatory situations known to easily damage these particularly fragile neurons.     

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.     

Functional GABAA receptors on human glioma cells

Glioma cells in acute slices and in primary culture, and glioma-derived human cell lines were screened for the presence of functional GABA_A receptors. Currents were measured in whole-cell voltage clamp in response to γ-aminobutyric acid (GABA). While cells from the most malignant glioma, the glioblastoma multiforme, did not respond to GABA, an inward current (under our experimental conditions with high Cl^? concentration in the pipette) was induced in gliomas of lower grades, namely in 71% of oligodendroglioma cells and in 62% of the astrocytoma cells. Glioma cell lines did not express functional GABA_A receptors, irrespective of the malignancy of the tumour they originate from. The currents elicited by application of GABA were due to activation of GABA_A receptors; the specific agonist muscimol mimicked the response, the antagonists bicuculline and picrotoxin blocked the GABA-activated current and the benzodiazepine receptor agonist flunitrazepam augmented the GABA-induced current and the benzodiazepine inverse agonist DMCM decreased the GABA current. Cells were heterogeneous with respect to the direction of the current flow as tested in gramicidin perforated patches: in some cells GABA hyperpolarized the membrane, while in the majority it triggered a depolarization. Moreover, GABA triggered an increase in [Ca²⁺]_i in the majority of the tumour cells due to the activation of Ca²⁺ channels. Our results suggest a link between the expression of GABA receptors and the growth of glioma cells as the disappearance of functional GABA_A receptors parallels unlimited growth typical for malignant tumours and immortal cell lines.     

Effects of the conformationally restricted GABA analogues,Cis‐and Trans‐4‐aminocrotonic acid,on GABA neurotransmission in primary neuronal cultures

The effects of the GABA analogues, cis‐ and trans‐4‐aminocrotonic acid (ACA) on GABA_A receptor function and GABA uptake, together with the presence of ρ‐1 subunit mRNA and putative GABA_C receptors, were studied in primary cultures of neocortical neurons and cerebellar granule cells. Both isomers induced a Cl^‐ influx, which was inhibited by bicuculline, t‐butylbicyclophosphorothionate (TBPS), picrotoxinin (PTX), and γ‐hexachlorocyclohexane (γ‐HCH or lindane). [³H]‐flunitrazepam binding was also increased by both isomers and this increase was inhibited by bicuculline. In neocortical neurons, the trans‐isomer completely inhibited the [³H]GABA uptake, whereas the cis‐isomer produced only a 25% inhibition at the highest concentration used. The possible presence of GABA_C receptors was investigated only in neocortical cultures by using RT‐PCR in order to detect the presence of the mRNA encoding the ρ‐1 subunit which assembles to form homooligomeric Cl^‐ channels. The results presented here show that ρ‐1 subunits, and thus GABA_C receptors, may represent a very minor population of GABA receptors in these neuronal preparations. We conclude that both GABA analogues may act as agonists at the GABA_A receptors, although with very different potencies. J. Neurosci. Res. 57:95–105, 1999. © 1999 Wiley‐Liss, Inc.     

GABA induces a unique rise of [CA]i in cultured rat hippocampal neurons

GABA evoked a reversible rise of free intracellular calcium concentration ([Ca]i) in cultured rat hippocampal neurons, detected with Fluo-3 fluorescence in a confocal laser scanning microscope. The GABA-evoked change of [Ca]i was mimicked by muscimol and not by baclofen, but was only minimally affected by picrotoxin or bicuculline, indicating that this effect of GABA is not likely to be mediated by activation of GABA_B receptor or by a conventional chloride-linked GABA_A receptor. GABA-evoked rise of [Ca]i expressed a marked desensitization; only 10–20 minutes after a previous exposure to GABA was the response to a subsequent application fully expressed. This desensitization was not seen in electrophysiological responses to GABA or in [Ca]i changes evoked by NMDA in the same neurons. The GABA response appeared to be developmentally regulated and was seen in 1–7-day-old more than in 21–28-day-old cells. It is suggested that GABA evokes a unique change of [Ca]i in young hippocampal neurons.     

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.