An extrasynaptic GABAA receptor mediates tonic inhibition in thalamic VB neurons

Whole cell patch-clamp recordings were obtained from thalamic ventrobasal (VB) and reticular (RTN) neurons in mouse brain slices. A bicuculline-sensitive tonic current was observed in VB, but not in RTN, neurons; this current was increased by the GABA(A) receptor agonist 4,5,6,7-tetrahydroisothiazolo-[5,4-c]pyridine-3-ol (THIP; 0.1 microM) and decreased by Zn(2+) (50 microM) but was unaffected by zolpidem (0.3 microM) or midazolam (0.2 microM). The pharmacological profile of the tonic current is consistent with its generation by activation of GABA(A) receptors that do not contain the alpha(1) or gamma(2) subunits. GABA(A) receptors expressed in HEK 293 cells that contained alpha(4)beta(2)delta subunits showed higher sensitivity to THIP (gaboxadol) and GABA than did receptors made up from alpha(1)beta(2)delta, alpha(4)beta(2)gamma(2s,) or alpha(1)beta(2)gamma(2s) subunits. Western blot analysis revealed that there is little, if any, alpha(3) or alpha(5) subunit protein in VB. In addition, co-immunoprecipitation studies showed that antibodies to the delta subunit could precipitate alpha(4), but not alpha(1) subunit protein. Confocal microscopy of thalamic neurons grown in culture confirmed that alpha(4) and delta subunits are extensively co-localized with one another and are found predominantly, but not exclusively, at extrasynaptic sites. We conclude that thalamic VB neurons express extrasynaptic GABA(A) receptors that are highly sensitive to GABA and THIP and that these receptors are most likely made up of alpha(4)beta(2)delta subunits. In view of the critical role of thalamic neurons in the generation of oscillatory activity associated with sleep, these receptors may represent a principal site of action for the novel hypnotic agent gaboxadol.     

Nanomolar allopregnanolone potentiates rat cerebellar GABAA receptors

The ionophore function of gamma-aminobutyric acid A (GABA(A)) receptors was studied by whole-cell patch clamp electrophysiology in primary cultures of rat cerebellar cortex. Chloride currents elicited by 1 microM GABA were potentiated by allopregnanolone with a plateau of high affinity (EC(50) = 14 nM) and a peak of potentiation around 1 microM allopregnanolone. Furosemide (0.1 mM) eliminated the high affinity phase and increased the EC(50) to 685 nM. GABA(A) receptors of rat cerebellar synaptosomal membranes were labelled with [(3)H]ethynylbicycloorthobenzoate (EBOB). Allopregnanolone displaced [(3)H]EBOB binding with IC(50) = 320 nM. The displacing potency of allopregnanolone was strongly enhanced (IC(50) = 39 nM) in the presence of 400 nM GABA and 60 nM SR 95531. Nanomolar potentiation by allopregnanolone can be associated with cerebellar GABA(A) receptors containing alpha(6), beta(2-3) and delta subunits. This might be suitable for physiological modulation of tonic inhibitory neurotransmission via extrasynaptic GABA(A) receptors in cerebellar granule cells by neurosteroids.     

Pharmacology of GABA(A) receptors of retinal dopaminergic neurons

When the vertebrate retina is stimulated by light, a class of amacrine or interplexiform cells release dopamine, a modulator responsible for neural adaptation to light. In the intact retina, dopamine release can be pharmacologically manipulated with agonists and antagonists at GABA(A) receptors, and dopaminergic (DA) cells receive input from GABAergic amacrines. Because there are only 450 DA cells in each mouse retina and they cannot be distinguished in the living state from other cells on the basis of their morphology, we used transgenic technology to label DA cells with human placental alkaline phosphatase, an enzyme that resides on the outer surface of the cell membrane. We could therefore identify DA cells in vitro after dissociation of the retina and investigate their activity with whole cell voltage clamp. We describe here the pharmacological properties of the GABA(A) receptors of solitary DA cells. GABA application induces a large inward current carried by chloride ions. The receptors are of the GABA(A) type because the GABA-evoked current is blocked by bicuculline. Their affinity for GABA is very high with an EC(50) value of 7.4 microM. Co-application of benzodiazepine receptor ligands causes a strong increase in the peak current induced by GABA (maximal enhancement: CL-218872 220%; flunitrazepam 214%; zolpidem 348%) proving that DA cells express a type I benzodiazepine-receptor (BZ1). GABA-evoked currents are inhibited by Zn(2+) with an IC(50) of 58.9 +/- 8.9 microM. Furthermore, these receptors are strongly potentiated by the modulator alphaxalone with an EC(50) of 340 +/- 4 nM. The allosteric modulator loreclezole increases GABA receptor currents by 43% (1 microM) and by 107% (10 microM). Using outside-out patches, we measured in single-channel recordings a main conductance (29 pS) and two subconductance (20 and 9 pS) states. We have previously shown by single-cell RT-PCR and immunocytochemistry that DA cells express seven different GABA(A) receptor subunits (alpha1, alpha3, alpha4, beta1, beta3, gamma1, gamma2(S), and gamma2(L)) and by immunocytochemistry that all subunits are expressed in the intact retina. We show here that at least alpha1, beta3 and gamma2 subunits are assembled into functional receptors.     

Changes in GABA(A) receptor subunit expression in the midbrain during the oestrous cycle in Wistar rats

In women, the late luteal phase or "premenstrual" period is commonly associated with psychological disturbances, which include mood changes and increased aggression. The underlying cause is unknown but one possibility is that fluctuations in levels of neuroactive steroids precipitate changes in expression of GABA(A) receptor subunits that result in functional changes in inhibitory control systems. The present study investigated the levels of expression of alpha4, beta1 and delta GABA(A) receptor subunits in the periaqueductal gray matter (PAG) in rats and whether plasticity occurs during the oestrous cycle in females. In male rats alpha4, beta1 and delta subunit immunoreactive neurones were present throughout the PAG in similar numbers. In female rats in proestrus, oestrus and early dioestrus, the density of alpha4, beta1 and delta subunit immunoreactive cells was similar to males. However, in late dioestrus, the numbers increased significantly, especially in the dorsolateral PAG, a region which is particularly rich in GABAergic interneurones. These parallel changes may reflect an increase in expression of the alpha4beta1delta GABA(A) receptor subtype. Recombinant alpha4beta1delta receptors, expressed in Xenopus oocytes, exhibited and EC(50) for GABA an order of magnitude lower (2.02+/-0.33 microM; mean+/-S.E.M.) than that found for the most ubiquitous alpha1beta2gamma2 GABA(A) receptor (32.8+/-2.5 microM). Increased expression of alpha4beta1delta GABA(A) receptors in the interneurones of the PAG could render the panic circuitry abnormally excitable by disinhibiting the ongoing GABAergic inhibition. Similar changes in neuronal excitability within the PAG in women consequent to falling steroid levels in the late luteal phase of the menstrual cycle could contribute to the development of pre-menstrual dysphoria.     

Effect of extracellular pH on GABA-activated current in rat recombinant receptors and thin hypothalamic slices.

We studied the effects of extracellular pH (pHo) on gamma-aminobutyric acid (GABA)-mediated Cl- current in rat hypothalamic neurons and recombinant type-A GABA (GABA(A)) receptors stably expressed in human embryonic kidney cells (HEK 293), using whole cell and outside-out patch-clamp recordings. In alpha3beta2gamma2s receptors, acidic pH decreased, whereas alkaline pH increased the response to GABA in a reversible and concentration-dependent manner. Acidification shifted the GABA concentration-response curve to the right, significantly increasing the EC50 for GABA without appreciably changing the slope or maximal current induced by GABA. We obtained similar effects of pH in alpha1beta2gamma2 receptors and in GABA-activated currents recorded from thin hypothalamic brain slices. In outside-out patches recorded from alpha3beta2gamma2 recombinant receptors, membrane patches were exposed to 5 microM GABA at control (7.3), acidic (6.4), or alkaline (8.4) pH. GABA activated main and subconductance states of 24 and 16 pS, respectively, in alpha3beta2gamma2 receptors. Alkaline pH(o) increased channel opening frequency and decreased the duration of the long closed state, resulting in an increase in open probability (from 0.0801 +/- 0.015 in pH 7.3 to 0.138 +/- 0.02 in pH 8.4). Exposure of the channels to acidic pH(o) had the opposite effects on open probability (decreased to 0.006 +/- 0.0001). Taken together, our results indicate that the function of GABA(A) receptors is modulated by extracellular pH. The proton effect is similar in recombinant and native receptors and is dependent on GABA concentration. In addition, the effect appears to be independent of the alpha-subunit isoform, and is due to the ability of H+ to alter the frequency of channel opening. Our findings indicate that GABAergic signaling in the CNS may be significantly altered during conditions that increase or decrease pH.     

Selective potentiation of GABA-mediated Cl- current by lanthanum ion in subtypes of cloned GABAA receptors.

The effect of lanthanum ion (La3+) on gamma-aminobutyric acid (GABA)-mediated Cl- currents was examined in the alpha 1 beta 2 or alpha 1 beta 2 gamma 2 subtype of GABAA receptors expressed in a human kidney cell line (A293), using a whole-cell configuration of patch-clamp techniques. La3+ dose-dependently stimulated the Cl- currents in the alpha 1 beta 2 gamma 2 subtype with an EC50 of 21.3 +/- 3.5 microM with a maximal potentiation of 240 +/- 16% as normalized to the GABA response at 5 microM. In the alpha 1 beta 2 subtype, however, the ion marginally potentiated GABA response, a maximal stimulation being less than 70% with an EC50 for La3+ near 200 microM. The stimulation of GABA response by La3+ in the alpha 1 beta 2 gamma 2 subtype was due to a decrease in the half maximal concentration for GABA and was more pronounced at the negative membrane potentials. This selectivity of La3+ toward the subtypes of GABAA receptors contrasts to that of Zn2+ which inhibits the currents in the alpha 1 beta 2, but not in the alpha 1 beta 2 gamma 2 subtype (Neuron, 5: (1990) 781-788). It appears that these polyvalent cations are useful in understanding the molecular basis for the functional diversity and in characterizing the molecular organization of native GABAA receptors.     

Functional GABAA receptor heterogeneity of acutely dissociated hippocampal CA1 pyramidal cells

CA1 pyramidal cells were voltage clamped, and GABA was applied to individual cells with a modified U-tube, rapid drug application system. With Vh = -50 mV, inward currents elicited by 10 microM GABA were inhibited by GABAA receptor (GABAR) antagonists and were baclofen insensitive, suggesting that GABA actions on isolated CA1 pyramidal cells were GABAR mediated. GABA concentration-response curves averaged from all cells were fitted best with a two-site equation, indicating the presence of at least two GABA binding sites, a higher-affinity site (EC50-1 = 11.0 microM) and a lower-affinity site (EC50-2 = 334.2 microM), on two or more populations of cells. The effects of GABAR allosteric modulators on peak concentration-dependent GABAR currents were complex and included monophasic (loreclezole) or multiphasic (diazepam) enhancement, mixed enhancement/inhibition (DMCM, zolpidem) or multiphasic inhibition (zinc). Monophasic (70% of cells) or biphasic (30% of cells) enhancement of GABAR currents by diazepam suggested three different sites on GABARs (EC50-1 =1.8 nM; EC50-2 = 75.8 nM; EC50-3 = 275.9 nM) revealing GABAR heterogeneity. The imidazopyridine zolpidem enhanced GABAR currents in 70% of cells with an EC50 = 222.5 nM, suggesting a predominance of moderate affinity alpha2 (or alpha3-) subtype-containing BZ Type IIA receptors. A small fraction of cells (10%) had a high affinity for zolpidem, something that is suggestive of alpha1 subtype-containing BZ Type I receptors. The remaining 30% of cells were insensitive to or inhibited by zolpidem, suggesting the presence of alpha5 subtype-containing BZ Type IIB receptors. Whether BZ Type I and Type II receptors coexist could not be determined. The beta-carboline methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) inhibited GABAR currents in all cells at midnanomolar concentrations, but in addition, potentiated GABAR currents in some cells at low nanomolar concentrations, characterizing two groups of cells, the latter likely due to functional assembly of alpha5betaxgamma2GABARs. In all cells, GABAR currents were moderately sensitive (EC50 = 9 microM) to loreclezole, consistent with a relatively greater beta3 subtype, than beta1 subtype, subunit mRNA expression. Two populations of cells were identified based on their sensitivities to zinc(IC50 = 28 and 182 microM), suggesting the presence of at least two GABAR isoforms including alpha5beta3gamma2 GABARs. Consistent with the heterogeneity of expression of GABAR subunit mRNA and protein in the hippocampus and based on their differential responses to GABA and to allosteric modulators, distinct populations of CA1 pyramidal cells likely express multiple, functional GABAR isoforms.     

Redox modulation of recombinant human GABA(A) receptors

We previously reported that GABA-evoked currents of rat retinal ganglion cells were modulated by redox agents. In this study, we further characterized the effects of redox modulation on GABA receptors using recombinant human subunits in the Xenopus oocyte expression system with two-electrode voltage-clamp recording. GABA receptors composed of subunits alpha(1-3), beta(1-3), gamma(1), gamma(2S,) and rho(1) were expressed. The sulfhydryl reducing agent dithiothreitol reversibly potentiated the responses of various combinations of functional recombinant GABA(A) subunits, whether expressed as triplets (alpha(1)beta(1-3)gamma(1,2S)), pairs (alpha(1-3)beta(1-3); beta(1-3)gamma(1,2S)), or singly (beta(2)). These effects of dithiothreitol were rapidly reversible, and the oxidizing agent 5-5'-dithiobis-2-nitrobenzoic acid exerted the opposite effect. In contrast to these effects on GABA(A) receptors, dithiothreitol had no effect on the responses of homomeric GABA rho(1) (GABA(C)) receptors. The degree of dithiothreitol potentiation of GABA(A) receptor responses depended on subunit composition. Co-expression of gamma(2S) with alpha(1)beta(1-3) subunits resulted in markedly less dithiothreitol potentiation of GABA-evoked currents than that observed for alpha(1-3)beta(1-3) subunits in the absence of gamma(2S). None the less, the magnitude of dithiothreitol potentiation could be restored by using a combination of lower GABA concentrations (5-10 microM) and higher dithiothreitol concentrations (5-20mM). N,N,N', N'-tetrakis(2-pyridyl-methyl)ethylenediamine, a high-affinity Zn(2+) chelator, also potentiated GABA(A) receptor currents. However, the potentiation produced by 10mM dithiothreitol was larger than that produced by saturating concentrations of N,N,N', N'-tetrakis(2-pyridyl-methyl)ethylenediamine (100 microM), implying that at least part of the effect of dithiothreitol was due to redox modulation rather than Zn(2+) chelation. Dithiothreitol also potentiated the spontaneous current of homomeric GABA(A) receptors composed of beta subunits. Mutation of a single cysteine residue in the M3 domain, yielding homomeric beta(3)(C313A) receptors, abrogated dithiothreitol potentiation of the spontaneous current.In summary, this study further characterizes the modulatory effects of redox agents on recombinant GABA(A) receptors. The degree of redox modulation of GABA(A) receptors depended on subunit composition. In contrast to their effect on GABA(A) receptors, redox agents were not found to modulate GABA(C) receptors composed of homomeric rho(1) subunits. Using site-directed mutagenesis, a cysteine residue was located in the beta(3) subunit which may comprise one of the redox-active sites that underlies the modulation of heteromeric GABA(A) receptors by reducing and oxidizing agents.     

Proton modulation of alpha 1 beta 3 delta GABAA receptor channel gating and desensitization

Alphabetagamma GABA(A) receptor currents are phasic and desensitizing, whereas alphabetadelta GABA(A) receptor currents are tonic and have no fast desensitization. alphabetagamma receptors are subsynaptic and mediate phasic inhibition, whereas alphabetadelta receptors are extra- or perisynaptic and mediate tonic inhibition. Given the different roles of these GABA(A) receptor isoforms and the fact that GABA(A) receptors are allosterically regulated by extracellular pH in a subunit-dependent manner, we compared the effects of changing pH on rat delta or gamma2L subunit-containing GABA(A) receptor currents. Human embryonic kidney cells (HEK293T) were transfected with cDNAs encoding rat alpha1, beta3, gamma2L, or delta GABA(A) receptor subunits in several binary and ternary combinations, and whole cell and single channel patch-clamp recordings were obtained. Lowering pH substantially enhanced alpha1beta3 receptor currents. This effect was significantly more pronounced for ternary alpha1beta3delta receptors, whereas ternary alpha1beta3gamma2L receptors were relatively insensitive to lowered pH. Lowering pH did not affect the extent of desensitization of alpha1beta3 and alpha1beta3gamma2L receptor currents, but significantly increased the extent of desensitization of alpha1beta3delta receptor currents. Lowering pH prolonged deactivation of alpha1beta3 and alpha1beta3delta receptor currents and enhanced the "steady-state" currents of alpha1beta3delta receptors evoked by long-duration (28 s) GABA applications. Lowering pH significantly increased mean open duration of alpha1beta3delta steady-state single channel currents due to introduction of a longer-duration open state, suggesting that low pH enhances alpha1beta3delta receptor steady-state currents by modifying GABA(A) receptor gating properties.     

Expression of GABA(A) receptor subunits in rat brainstem auditory pathways: cochlear nuclei, superior olivary complex and nucleus of the lateral lemniscus

Inhibition by GABA is important for auditory processing, but any adaptations of the ionotropic type A receptors are unknown. Here we describe, using in situ hybridization, the subunit expression patterns of GABA(A) receptors in the rat cochlear nucleus, superior olivary complex, and dorsal and ventral nuclei of the lateral lemniscus. All neurons express the beta3 and gamma2L subunit messenger RNAs, but use different alpha subunits. In the dorsal cochlear nucleus, fusiform (pyramidal) and giant cells express alpha1, alpha3, beta3 and gamma2L. Dorsal cochlear nucleus interneurons, particularly vertical or tuberculoventral cells and cartwheel cells, express alpha3, beta3 and gamma2L. In the ventral cochlear nucleus, octopus cells express alpha1, beta3, gamma2L and delta. Spherical cells express alpha1, alpha3, alpha5, beta3 and gamma2L. In the superior olivary complex, the expression profile is alpha3, alpha5, beta3 and gamma2L. Both dorsal and ventral cochlear nucleus granule cells express alpha1, alpha6, beta3 and gamma2L; unlike their cerebellar granule cell counterparts, they do not express beta2, gamma2S or the delta subunit genes. The delta subunit's absence from cochlear nucleus granule cells may mean that tonic inhibition mediated by extrasynaptic GABA(A) receptors is less important for this cell type. In both the dorsal and ventral nuclei of the lateral lemniscus, alpha1, beta3 and gamma2L are the main subunit messenger RNAs; the ventral nucleus also expresses the delta subunit. We have mapped, using in situ hybridization, the subunit expression patterns of the GABA(A) receptor in the auditory brainstem nuclei. In contrast to many brain regions, the beta2 subunit gene and gamma2S splice forms are not highly expressed in auditory brainstem nuclei. GABA(A) receptors containing beta3 and gamma2L may be particularly well suited to auditory processing, possibly because of the unique phosphorylation profile of this subunit combination.     

Inhibition of type A GABA receptors by L-type calcium channel blockers

Modulation of type A GABA receptors (GABAA) by L-type Ca++ channel blockers was investigated. The dihydropyridines nifedipine and nitrendipine, and the phenylalkylamine verapamil inhibited recombinant rat alpha1beta2gamma2 receptors recorded from human embryonic kidney (HEK) 293 cells; nifedipine at low concentrations also elicited modest stimulatory effects on GABA-gated current. The IC50 for GABA current inhibition was lowest for nitrendipine (17.3 +/- 1.3 microM), so subsequent studies were focused on further exploring its mechanism and possible site of action. When co-applied with GABA, nitrendipine had minimal effects on initial current amplitude, but significantly enhanced current decay rate. Nitrendipine-mediated inhibition was subunit-selective, as its IC50 was 10-fold lower in alpha1beta2 receptors. Nitrendipine's effect in recombinant human alpha1beta2gamma2 receptors was similar (IC50=23.0 +/- 1.3 microM) to that observed in rat receptors of the same configuration, indicating the site of action is conserved in the two species. The inhibitory effects were dependent on channel gating, were independent of transmembrane voltage, and were also observed in GABAA receptors recorded from hypothalamic brain slices. The pharmacologic mechanism of inhibition by nitrendipine was non-competitive, indicating it does not act at the GABA binding site. Nitrendipine block was retained in the presence of the benzodiazepine antagonist flumazenil, indicating it does not interact at the benzodiazepine site. The actions of nitrendipine were not affected by a mutation (beta2T246F) that confers resistance to the channel blocker picrotoxin, and they were not altered in the presence of the picrotoxin site antagonist alpha-isopropyl-alpha-methyl-gamma-butyrolactone, demonstrating nitrendipine does not act at the picrotoxin site of the GABAA receptor. Possible interaction of nitrendipine with the Zn++ site was also eliminated, as mutation of beta2 H267 to A, which confers resistance to Zn++, had no effect on nitrendipine-mediated inhibition. Our data suggest some of the central effects of dihydropyridines may be due to actions at GABAA receptors. Moreover, the effects may be mediated through interaction with a novel modulatory site on the GABAA receptor.     

Altered receptor subtypes in the forebrain of GABA(A) receptor delta subunit-deficient mice: recruitment of gamma 2 subunits

A GABA(A) receptor delta subunit-deficient mouse line was created by homologous recombination in embryonic stem cells to investigate the role of the subunit in the brain GABA(A) receptors. High-affinity [(3)H]muscimol binding to GABA sites as studied by ligand autoradiography was reduced in various brain regions of delta(-/-) animals. [(3)H]Ro 15-4513 binding to benzodiazepine sites was increased in delta(-/-) animals, partly due to an increment of diazepam-insensitive receptors, indicating an augmented forebrain assembly of gamma 2 subunits with alpha 4 subunits. In the western blots of forebrain membranes of delta(-/-) animals, the level of gamma 2 subunit was increased and that of alpha 4 decreased, while the level of alpha1 subunits remained unchanged. In the delta(-/-) forebrains, the remaining alpha 4 subunits were associated more often with gamma 2 subunits, since there was an increase in the alpha 4 subunit level immunoprecipitated by the gamma 2 subunit antibody. The pharmacological properties of t-butylbicyclophosphoro[(35)S]thionate binding to the integral ion-channel sites were slightly altered in the forebrain and cerebellum, consistent with elevated levels of alpha 4 gamma 2 and alpha 6 gamma 2 subunit-containing receptors, respectively.The altered pharmacology of forebrain GABA(A) receptors and the decrease of the alpha 4 subunit level in delta subunit-deficient mice suggest that the delta subunit preferentially assembles with the alpha 4 subunit. The delta subunit seems to interfere with the co-assembly of alpha 4 and gamma 2 subunits and, therefore, in its absence, the gamma 2 subunit is recruited into a larger population of alpha 4 subunit-containing functional receptors. These results support the idea of subunit competition during the assembly of native GABA(A) receptors.     

GABA(A) receptors: immunocytochemical distribution of 13 subunits in the adult rat brain

GABA(A) receptors are ligand-operated chloride channels assembled from five subunits in a heteropentameric manner. Using immunocytochemistry, we investigated the distribution of GABA(A) receptor subunits deriving from 13 different genes (alpha1-alpha6, beta1-beta3, gamma1-gamma3 and delta) in the adult rat brain. Subunit alpha1-, beta1-, beta2-, beta3- and gamma2-immunoreactivities were found throughout the brain, although differences in their distribution were observed. Subunit alpha2-, alpha3-, alpha4-, alpha5-, alpha6-, gamma1- and delta-immunoreactivities were more confined to certain brain areas. Thus, alpha2-subunit-immunoreactivity was preferentially located in forebrain areas and the cerebellum. Subunit alpha6-immunoreactivity was only present in granule cells of the cerebellum and the cochlear nucleus, and subunit gamma1-immunoreactivity was preferentially located in the central and medial amygdaloid nuclei, in pallidal areas, the substantia nigra pars reticulata and the inferior olive. The alpha5-subunit-immunoreactivity was strongest in Ammon's horn, the olfactory bulb and hypothalamus. In contrast, alpha4-subunit-immunoreactivity was detected in the thalamus, dentate gyrus, olfactory tubercle and basal ganglia. Subunit alpha3-immunoreactivity was observed in the glomerular and external plexiform layers of the olfactory bulb, in the inner layers of the cerebral cortex, the reticular thalamic nucleus, the zonal and superficial layers of the superior colliculus, the amygdala and cranial nerve nuclei. Only faint subunit gamma3-immunoreactivity was detected in most areas; it was darkest in midbrain and pontine nuclei. Subunit delta-immunoreactivity was frequently co-distributed with alpha4 subunit-immunoreactivity, e.g. in the thalamus, striatum, outer layers of the cortex and dentate molecular layer. Striking examples of complementary distribution of certain subunit-immunoreactivities were observed. Thus, subunit alpha2-, alpha4-, beta1-, beta3- and delta-immunoreactivities were considerably more concentrated in the neostriatum than in the pallidum and entopeduncular nucleus. In contrast, labeling for the alpha1-, beta2-, gamma1- and gamma2-subunits prevailed in the pallidum compared to the striatum. With the exception of the reticular thalamic nucleus, which was prominently stained for subunits alpha3, beta1, beta3 and gamma2, most thalamic nuclei were rich in alpha1-, alpha4-, beta2- and delta-immunoreactivities. Whereas the dorsal lateral geniculate nucleus was strongly immunoreactive for subunits alpha4, beta2 and delta, the ventral lateral geniculate nucleus was predominantly labeled for subunits alpha2, alpha3, beta1, beta3 and gamma2; subunit alpha1- and alpha5-immunoreactivities were about equally distributed in both areas. In most hypothalamic areas, immunoreactivities for subunits alpha1, alpha2, beta1, beta2 and beta3 were observed. In the supraoptic nucleus, staining of conspicuous dendritic networks with subunit alpha1, alpha2, beta2, and gamma2 antibodies was contrasted by perykarya labeled for alpha5-, beta1- and delta-immunoreactivities. Among all brain regions, the median emminence was most heavily labeled for subunit beta2-immunoreactivity. In most pontine and cranial nerve nuclei and in the medulla, only subunit alpha1-, beta2- and gamma2-immunoreactivities were strong, whereas the inferior olive was significantly labeled only for subunits beta1, gamma1 and gamma2. In this study, a highly heterogeneous distribution of 13 different GABA(A) receptor subunit-immunoreactivities was observed. This distribution and the apparently typical patterns of co-distribution of these GABA(A) receptor subunits support the assumption of multiple, differently assembled GABA(A) receptor subtypes and their heterogeneous distribution within the adult rat brain.     

Modulation of extrasynaptic THIP conductances by GABAA-receptor modulators in mouse neocortex

THIP is a hypnotic drug, which displays a unique pharmacological profile, because it activates a subset of extrasynaptic gamma-aminobutyric acid type A (GABA(A)) receptors containing delta-subunits. It is important to study the physiology and pharmacology of these extrasynaptic receptors and to determine how THIP interacts with other hypnotics and anesthetics. Here, we study the modulation of the extrasynaptic response to THIP using three classes of GABA(A)-receptor ligands. In whole cell recordings from mouse neocortical layer 2/3 pyramidal cells, THIP induced an extrasynaptic tonic current of 44 +/- 5 pA. The benzodiazepine site agonist and hypnotic zolpidem (500 nM), which displays selectivity for alpha(1/2/3)- and gamma(2)-containing receptors, did not alter the tonic current induced by THIP. The anesthetic etomidate (1 microM), which shows selectivity for beta(2)- and beta(3)-containing GABA(A) receptors, potentiated the THIP current by 126%. Etomidate also induced a small tonic GABA(A) current per se. The anesthetic propofol (1 microM), which displays broad-spectrum modulatory effects on several GABA(A)-receptor subtypes, enhanced the tonic THIP current by 117%. Finally, all three compounds modulated the function of intrasynaptic receptors activated by synaptically released GABA. Our study shows that the extrasynaptic GABA(A) receptors responsible for the tonic THIP conductance likely do not contain alpha(1)-, alpha(2)-, alpha(3)-, and gamma(2)-subunits. Thus the tonic GABAergic conductance in the neocortex is presumably mediated by alpha(4)beta(2/3)delta receptors, which are likely to play a major role for neocortical excitability. Furthermore, our study has deepened the knowledge about the cellular actions of THIP as well as THIP's interactions with other hypnotics and anesthetics.     

Differential benzodiazepine pharmacology of mammalian recombinant GABAA receptors

We compared gamma-aminobutyric acid (GABA)-activated currents and their modulation by benzodiazepines in cultured human cells transfected with complementary desoxyribonucleic acid (cDNA) encoding different GABAA receptor subunits. Flunitrazepam, a benzodiazepine agonist which potentiates GABA responses in both neurons and astrocytes was only effective in receptors containing the gamma 2 subunit (alpha 1 beta 1 gamma 2 and alpha 5 beta 1 gamma 2). The beta-carboline methyl-4-ethyl-6,7-dimethoxy-beta-carboline-3-carboxylate (DMCM) decreased GABA-activated currents in receptors composed of alpha 1 beta 1 gamma 1 and alpha 1 beta 1 gamma 2 subunits but increased GABA-activated currents in receptors containing the alpha 5 subunit (alpha 5 beta 1 gamma 1 and alpha 5 beta 1 gamma 2). These results strongly suggest that flunitrazepam and DMCM do not act on isosteric sites and that differences in the responsiveness of GABAA receptors to these compounds are based on different subunit compositions of GABAA receptors.     

The expression of GABAA beta subunit isoforms in synaptic and extrasynaptic receptor populations of mouse dentate gyrus granule cells

The subunit composition of GABA(A) receptors influences their biophysical and pharmacological properties, dictates neuronal location and the interaction with associated proteins, and markedly influences the impact of intracellular biochemistry. The focus has been on alpha and gamma subunits, with little attention given to beta subunits. Dentate gyrus granule cells (DGGCs) express all three beta subunit isoforms and exhibit both synaptic and extrasynaptic receptors that mediate 'phasic' and 'tonic' transmission, respectively. To investigate the subcellular distribution of the beta subunits we have utilized the patch-clamp technique to compare the properties of 'tonic' and miniature inhibitory postsynaptic currents (mIPSCs) recorded from DGGCs of hippocampal slices of P20-26 wild-type (WT), beta(2)(-/-), beta(2N265S) (etomidate-insensitive), alpha(1)(-/-) and delta(-/-) mice. Deletion of either the beta(2) or the delta subunit produced a significant reduction of the tonic current and attenuated the increase of this current induced by the delta subunit-preferring agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP). By contrast, mIPSCs were not influenced by deletion of these genes. Enhancement of the tonic current by the beta(2/3) subunit-selective agent etomidate was significantly reduced for DGGCs derived from beta(2N265S) mice, whereas this manipulation had no effect on the prolongation of mIPSCs produced by this anaesthetic. Collectively, these observations, together with previous studies on alpha(4)(-/-) mice, identify a population of extrasynaptic alpha(4)beta(2)delta receptors, whereas synaptic GABA(A) receptors appear to primarily incorporate the beta(3) subunit. A component of the tonic current is diazepam sensitive and is mediated by extrasynaptic receptors incorporating alpha(5) and gamma(2) subunits. Deletion of the beta(2) subunit had no effect on the diazepam-induced current and therefore these extrasynaptic receptors do not contain this subunit. The unambiguous identification of these distinct pools of synaptic and extrasynaptic GABA(A) receptors should aid our understanding of how they act in harmony, to regulate hippocampal signalling in health and disease.     

Anabolic steroids induce region- and subunit-specific rapid modulation of GABA(A) receptor-mediated currents in the rat forebrain

Anabolic-androgenic steroids (AAS) have become significant drugs of abuse in recent years with the highest increase reported in adolescent girls. In spite of the increased use of AAS, the CNS effects of these steroids are poorly understood. We report that in prepubertal female rats, three commonly abused AAS, 17alpha-methyltestosterone, stanozolol, and nandrolone, induced rapid and reversible modulation of GABAergic currents in neurons of two brain regions known to be critical for the expression of reproductive behaviors: the ventromedial nucleus of the hypothalamus (VMN) and the medial preoptic area (mPOA). All three AAS significantly enhanced peak synaptic current amplitudes and prolonged synaptic current decays in neurons of the VMN. Conversely all three AAS significantly diminished peak current amplitudes of synaptic currents from neurons of the mPOA. The endogenous neuroactive steroids, 3alpha-hydroxy-5alpha-pregnan-20-one and 5alpha-androstane-3alpha,17beta-diol, potentiated currents in the VMN as did the AAS. In contrast to the negative modulation induced by AAS in the mPOA, the endogenous steroids potentiated responses in this region. To determine the concentration response relationships, modulation by the AAS, 17alpha-methyltestosterone (17alpha-meT), was assessed for currents evoked by ultrafast perfusion of brief pulses of GABA to acutely isolated neurons. Half-maximal effects on currents elicited by 1 mM GABA were elicited by submicromolar concentrations of AAS for neurons from both brain regions. In addition, the efficacy of 10(-5) to 10(-2) M GABA was significantly increased by 1 microM 17alpha-meT. Previous studies have demonstrated a striking dichotomy in receptor composition between the VMN and the mPOA with regard to gamma subunit expression. To determine if the preferential expression of gamma(2) subunit-containing receptors in the VMN and of gamma(1) subunit-containing receptors in the mPOA could account for the region-specific effects of AAS in the two regions, responses elicited by ultrafast perfusion of GABA to human embryonic kidney 293 cells transfected with alpha(2), beta(3), and gamma(2) or alpha(2), beta(3), and gamma(1) subunit cDNAs were analyzed. As with native VMN neurons, positive modulation of GABA responses was elicited for alpha(2)beta(3)gamma(2) recombinant receptors, while negative modulation was induced at alpha(2)beta(3)gamma(1) receptors as in the mPOA. Our data demonstrate that AAS in doses believed to occur in steroid abusers can induce significant modulation of GABAergic transmission in brain regions essential for neuroendocrine function. In addition, the effects of these steroids can vary significantly between brain regions in a manner that appears to depend on the subunit composition of GABA(A) receptors expressed.     

Perinatal alcohol exposure leads to prolonged upregulation of hypothalamic GABA A receptors and increases behavioral sensitivity to gaboxadol

Prenatal alcohol exposure (AE) is associated with lasting abnormalities of sleep and motor development, but the underlying mechanisms are unknown. We hypothesized that AE alters development of GABAergic signaling in the hypothalamic regions important for the control of sleep and motor activity. Alcohol (5.25 g/(kg day)) was administered intragastrically to male rats on postnatal days (PD) 4-9, a period of brain development equivalent to the human third trimester (AE group). Control pups were sham-intubated (S group). Motor activity was monitored on PD27 and 28. On PD29 and 30, GABA A receptor subunit mRNA levels and alpha4 and delta subunit proteins were quantified by RT-PCR and immunoblotting, respectively, in the wake- and motor activity-promoting perifornical (PF) region of the posterior hypothalamus and the sleep-promoting ventrolateral preoptic (VLPO) region of the anterior hypothalamus. Then, in 47-52-day-old rats, motor activity was quantified following administration of GABA A receptor agonist, gaboxadol (5 mg/kg s.c.). In the PF region, mRNA and protein levels for the alpha4 and delta subunits were significantly higher and beta3 and gamma2 subunit mRNAs were also increased in the AE group. In the VLPO region, only the delta subunit mRNA was increased. Spontaneous motor activity was lower and suppressed more by gaboxadol in the AE than S group, and the latency to a transient total loss of activity after gaboxadol was shorter in the AE group. Thus, perinatal AE leads to GABA A receptor overexpression in the vigilance- and motor activity-promoting hypothalamic PF region, with the neurochemical and functional outcomes lasting long beyond the period of the insult.     

Central-type benzodiazepines inhibit release of alpha-melanocyte-stimulating hormone from the rat hypothalamus.

In a previous work, we have shown that GABA inhibits the release of alpha-melanocyte-stimulating hormone (alpha-melanotropin) from hypothalamic neurons through activation of GABAA receptors [Delbende et al. (1989) Brain Res. 497, 86-93]. Since GABA-gated channel activity can be allosterically modulated by a variety of compounds including benzodiazepines, we have investigated the effect of benzodiazepines in the control of alpha-melanotropin release by the rat basal hypothalamus. This study was conducted in vitro using perifused rat hypothalamic slices and the amount of alpha-melanotropin release was monitored with a sensitive and highly specific radioimmunoassay. Infusion of clonazepam (50 microM), a selective agonist for central-type benzodiazepine binding sites, induced an inhibition of KCl (50 mM)-evoked alpha-melanotropin release. The inhibitory effect of clonazepam was rapid and reversible. Administration of Ro 15-1788 (100 microM), a specific antagonist for central-type benzodiazepine receptors or SR 95531, a GABAA receptor antagonist, completely reversed the inhibitory effect of clonazepam. In addition, Ro 15-1788 and SR 95531 both enhanced the amplitude of the response observed during prolonged KCl infusion on alpha-melanotropin neurons, suggesting the existence of a tonic inhibitory effect of endogenous GABA and/or benzodiazepines in the release of alpha-melanotropin by hypothalamic neurons. To investigate further the effect of benzodiazepines in the regulation of alpha-melanotropin neurons, rats were treated in vivo with clonazepam (5 mg/kg) or the non-selective benzodiazepine receptor agonist diazepam (3 mg/kg). Both compounds caused a significant increase in the content of alpha-melanotropin and beta-endorphin in the rat hypothalamus within 3 h.(ABSTRACT TRUNCATED AT 250 WORDS)