Functional GABA(A)-receptor-mediated inhibition in the neonatal dorsal horn

Neonatal nociceptive circuits and dorsal horn cells are characterized by an apparent lack of inhibitory control: receptive fields are large and thresholds low in the first weeks of life. It has been suggested that this may reflect immature GABA(A)-receptor (GABA(A)R) signaling whereby an early developmental shift in transmembrane anion gradient is followed by a longer period of low Cl- extrusion capacity. To investigate whether functional GABA(A)R-mediated inhibition does indeed undergo postnatal regulation at the level of dorsal horn circuits, we applied the selective GABA(A)R antagonist gabazine to the spinal cord in anesthetized rat pups [postnatal day (P) 3 or 21] while recording spike activity in single lumbar dorsal horn cells in vivo. At both ages, blockade of GABA(A)R activity resulted in enlarged hind paw receptive field areas and increased activity evoked by low- and high-intensity cutaneous stimulation, revealing comparable inhibition of dorsal horn cell firing by spinal GABA(A)Rs at P3 and P21. This inhibition did not require descending pathways to the spinal cord because perforated patch-clamp recordings of deep dorsal horn neurons in P3 spinal cord slices also showed an increase in evoked spike activity after application of gabazine. We conclude that spinal GABAergic inhibitory transmission onto single dorsal horn cells "in vivo" is functional at P3 and that low Cl- extrusion capacity does not restrict GABAergic function over the normal range of evoked sensory activity. The excitability of neonatal spinal sensory circuits could reflect immaturity in other intrinsic or descending inhibitory networks rather than weak spinal GABAergic inhibition.     

Morphological effects of isoflavones (daidzein and genistein) on hypothalamic oxytocin neurons in the neonatal mouse brain slice cultures

We investigated the immunohistochemical localization of immunoreactive (ir) cell bodies and fibers of neuropeptide Y (NPY) and galanin (GAL), and the anatomical relations between these neurons in the brain of the Siberian sturgeon Acipenser baeri to clarify the interactions between these neuropeptides. Furthermore, the anatomic relations between NPY and gonadotropin-releasing hormone (GnRH) in the brain were also examined. NPY-ir cell bodies were observed in the ventral part of the ventral telencephalon (Vv). NPY-ir fibers were observed throughout the brain, primarily in the ventral telencephalon, hypothalamus, optic tectum, and midbrain. GAL-ir cell bodies were observed in the Vv, nucleus anterioris tuberis (NAT), nucleus lateralis tuberis (NLT), and nucleus recessus posterioris (NRP). GAL-ir fibers were also observed throughout the brain. Neither NPY-ir fibers nor GAL-ir fibers were detected in the pituitary. Dual-label immunohistochemistry revealed that some GAL-ir fibers were in close contact with NPY-ir cell bodies in the Vv, and some NPY-ir fibers were in close contact with GAL-ir cell bodies in the NAT. Furthermore, some NPY-ir fibers were in close contact with GnRH-ir cell bodies in the preoptic area, and some GnRH-ir fibers were in close contact with NPY-ir cell bodies in the Vv. These findings suggest that reciprocal connections exist between the NPY and GAL neurons and between the NPY and GnRH neurons in the brain of the Siberian sturgeon.     

Regulation of synaptic input to hypothalamic presympathetic neurons by GABA(B) receptors

The hypothalamic paraventricular (PVN) neurons projecting to the spinal cord and brainstem play an important role in the control of homeostasis and the sympathetic nervous system. Although GABA(B) receptors are present in the PVN, their function in the control of synaptic inputs to PVN presympathetic neurons is not clear. Using retrograde tracing and whole-cell patch-clamp recordings in rat brain slices, we determined the role of presynaptic GABA(B) receptors in regulation of glutamatergic and GABAergic inputs to spinally projecting PVN neurons. The GABA(B) receptor agonist baclofen (1-50 microM) dose-dependently decreased the frequency but not the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) and inhibitory postsynaptic currents (sIPSCs). The effect of baclofen on sEPSCs and sIPSCs was completely blocked by 10 microM CGP52432, a selective GABA(B) receptor antagonist. Baclofen also significantly reduced the frequency of both miniature excitatory and miniature inhibitory postsynaptic currents (mEPSCs and mIPSCs). Furthermore, uncoupling pertussis toxin-sensitive G(i/o) proteins with N-ethylmaleimide abolished baclofen-induced inhibition of mEPSCs and mIPSCs. However, the inhibitory effect of baclofen on the frequency of mIPSCs and mEPSCs persisted in the presence of either Cd2+, a voltage-gated Ca2+ channel blocker, or 4-aminopyridine, a blocker of voltage-gated K+ channels. Our results suggest that activation of presynaptic GABA(B) receptors inhibits synaptic GABA and glutamate release to PVN presympathetic neurons. This presynaptic action of GABA(B) receptors is mediated by the N-ethylmaleimide-sensitive G(i/o) proteins, but independent of voltage-gated Ca2+ and K+ channels.     

Drug interactions at GABA(A) receptors

Neurotransmitter receptor systems have been the focus of intensive pharmacological research for more than 20 years for basic and applied scientific reasons, but only recently has there been a better understanding of their key features. One of these systems includes the type A receptor for the gamma-aminobutyric acid (GABA), which forms an integral anion channel from a pentameric subunit assembly and mediates most of the fast inhibitory neurotransmission in the adult vertebrate central nervous system. Up to now, depending on the definition, 16-19 mammalian subunits have been cloned and localized on different genes. Their assembly into proteins in a poorly defined stoichiometry forms the basis of functional and pharmacological GABA(A) receptor diversity, i.e. the receptor subtypes. The latter has been well documented in autoradiographic studies using ligands that label some of the receptors' various binding sites, corroborated by recombinant expression studies using the same tools. Significantly less heterogeneity has been found at the physiological level in native receptors, where the subunit combinations have been difficult to dissect.This review focuses on the characteristics, use and usefulness of various ligands and their binding sites to probe GABA(A) receptor properties and to gain insight into the biological function from fish to man and into evolutionary conserved GABA(A) receptor heterogeneity. We also summarize the properties of the novel mouse models created for the study of various brain functions and review the state-of-the-art imaging of brain GABA(A) receptors in various human neuropsychiatric conditions.The data indicate that the present ligands are only partly satisfactory tools and further ligands with subtype-selective properties are needed for imaging purposes and for confirming the behavioral and functional results of the studies presently carried out in gene-targeted mice with other species, including man.     

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.     

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.     

Presynaptic GABA(B) receptors modulate organum vasculosum lamina terminalis-evoked postsynaptic currents in rat hypothalamic supraoptic neurons

Whole-cell patch-clamp recordings obtained from 36 hypothalamic supraoptic nucleus neurons in explant preparations evaluated a role for GABA(B) receptors in modulating postsynaptic inhibitory and excitatory currents evoked by electrical stimulation in the organum vasculosum of the lamina terminalis. At a holding current of -65 mV, application of baclofen (1-10 microM) induced a dose-dependent reduction in the amplitude of pharmacologically isolated inhibitory and excitatory postsynaptic currents, converted paired-pulse depression in inhibitory postsynaptic currents to paired-pulse facilitation, and enhanced paired-pulse ratios for excitatory postsynaptic currents. In media containing 2-hydroxysaclofen (200-400 microM), baclofen-associated events were blocked and paired-pulse depression in evoked inhibitory postsynaptic currents was abolished. In addition, a progressive increase in the amplitude of inhibitory postsynaptic currents implied that GABA was endogenously active at presynaptic GABA(B) receptors. In contrast, no paired-pulse depression was observed for inhibitory postsynaptic currents evoked in six non-magnocellular neurons. Neither baclofen nor 2-hydroxysaclofen altered holding currents or input resistances in supraoptic neurons, or altered the kinetics of the evoked responses.These observations imply that the terminals of both inhibitory (GABAergic) and excitatory (glutamatergic) afferents to supraoptic nucleus neurons from organum vasculosum lamina terminalis neurons are subject to modulation by presynaptic GABA(B) receptors, and that this modulation is preferentially directed to the inhibitory inputs.     

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.     

Functional GABA(B) receptors are expressed at the cone photoreceptor terminals in bullfrog retina

GABA(B) receptors at the cone terminals in bullfrog retina were characterized by immunocytochemical and whole-cell patch clamp techniques in retinal slice preparations. Somata, axons and synaptic terminals (pedicles) of cones were both GABA(B) receptor (GABA(B)R) 1 and GABA(B)R2 immunoreactive. Physiologically, barium/calcium currents of cones to voltage steps were significantly reduced in size when GABA was puffed to cone terminals in the presence of picrotoxin that is supposed to block both GABA(A) and GABA(C) receptors. Similar reduction in barium currents was obtained with puff application of baclofen to cone terminals. These results suggest the presence of functional GABA(B) receptors at the bullfrog cone terminals. Suppression of barium currents of cones by baclofen was dose-dependent. Moreover, barium currents of cones were potentiated by background illumination, as compared with those recorded in the dark. 6,7-Dinitroquinoxaline-2,3-dione, an antagonist of non-NMDA receptors that hyperpolarizes horizontal cells and reduces GABA release from these cells, and saclofen, a GABA(B) receptor antagonist, both potentiated barium currents of cones in the dark, thereby mimicking the effects of background illumination. It is suggested that changes in calcium influx into the cone synaptic terminals due to activation of GABA(B) receptors may provide a negative feedback mechanism for regulating signal transmission between cones and second-order neurons in the retina by modifying the amount of glutamate released from the cones.     

Endogenous zinc inhibits GABA(A) receptors in a hippocampal pathway

Depending on their subunit composition, GABA(A) receptors can be highly sensitive to Zn(2+). Although a pathological role for Zn(2+)-mediated inhibition of GABA(A) receptors has been postulated, no direct evidence exists that endogenous Zn(2+) can modulate GABAergic signaling in the brain. A possible explanation is that Zn(2+) is mainly localized to a subset of glutamatergic synapses. Hippocampal mossy fibers are unusual in that they are glutamatergic but have also been reported to contain GABA and Zn(2+). Here, we show, using combined Timm's method and post-embedding immunogold, that the same mossy fiber varicosities can contain both GABA and Zn(2+). Chelating Zn(2+) with either calcium-saturated EDTA or N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine had no effect on stratum-radiatum-evoked inhibitory postsynaptic currents (IPSCs), but enhanced IPSCs evoked by stimuli designed to recruit dentate granule cells. We also show that IPSCs recorded in CA3 pyramidal neurons in acute hippocampal slices are depressed by exogenous Zn(2+). This depression was of similar amplitude whether the IPSCs were evoked by stimulation in s. radiatum (to recruit local interneurons) or in the s. granulosum of the dentate gyrus (to recruit mossy fibers). These results show for the first time that GABAergic IPSCs can be modulated by endogenous Zn(2+) and are consistent with GABA release at Zn(2+)-containing mossy fiber synapses.     

Differential subcellular and subsynaptic distribution of GABA(A) and GABA(B) receptors in the monkey subthalamic nucleus

The activation of GABA receptor subtype A (GABA(A)) and GABA receptor subtype B (GABA(B)) receptors mediates differential effects on GABAergic and non-GABAergic transmission in the basal ganglia. To further characterize the anatomical substrate that underlies these functions, we used immunogold labeling to compare the subcellular and subsynaptic localization of GABA(A) and GABA(B) receptors in the subthalamic nucleus (STN). Our findings demonstrate major differences and some similarities in the distribution of GABA(A) and GABA(B) receptors in the monkey STN. The immunoreactivity for GABA(A) receptor alpha1 subunits is mostly bound to the plasma membrane, whereas GABA(B) R1 subunit alpha1 immunoreactivity is largely expressed intracellularly. Plasma membrane-bound GABA(A) alpha1 subunit aggregate in the main body of putative GABAergic synapses, while GABA(B) R1 receptors are found at the edges of putative glutamatergic or GABAergic synapses. A large pool of plasma membrane-bound GABA(A) and GABA(B) receptors is extrasynaptic. In conclusion, these findings demonstrate a significant degree of heterogeneity between the distributions of the two major GABA receptor subtypes in the monkey STN. Their pattern of synaptic localization puts forward interesting questions regarding their mechanisms of activation and functions at GABAergic and non-GABAergic synapses.     

Subcellular localization and complements of GABA(A) and GABA(C) receptors on bullfrog retinal bipolar cells

gamma-Aminobutyric acid (GABA) receptors on retinal bipolar cells (BCs) are highly relevant to spatial and temporal integration of visual signals in the outer and inner retina. In the present work, subcellular localization and complements of GABA(A) and GABA(C) receptors on BCs were investigated by whole cell recordings and local drug application via multi-barreled puff pipettes in the bullfrog retinal slice preparation. Four types of the BCs (types 1-4) were identified morphologically by injection of Lucifer yellow. According to the ramification levels of the axon terminals and the responses of these cells to glutamate (or kainate) applied at their dendrites, types 1 and 2 of BCs were supposed to be OFF type, whereas types 3 and 4 of BCs might be ON type. Bicuculline (BIC), a GABA(A) receptor antagonist, and imidazole-4-acetic acid (I4AA), a GABA(C) receptor antagonist, were used to distinguish GABA receptor-mediated responses. In all BCs tested, not only the axon terminals but also the dendrites showed high GABA sensitivity mediated by both GABA(A) and GABA(C) receptors. Subcellular localization and complements of GABA(A) and GABA(C) receptors at the dendrites and axon terminals were highly related to the dichotomy of OFF and ON BCs. In the case of OFF BCs, GABA(A) receptors were rather evenly distributed at the dendrites and axon terminals, but GABA(C) receptors were predominantly expressed at the axon terminals. Moreover, the relative contribution of GABA(C) receptors to the axon terminals was prevalent over that of GABA(A) receptors, while the situation was reversed at the dendrites. In the case of ON BCs, GABA(A) and GABA(C) receptors both preferred to be expressed at the axon terminals; relative contributions of these two GABA receptor subtypes to both the sites were comparable, while GABA(C) receptors were much less expressed than GABA(A) receptors. GABA(A), but not GABA(C) receptors, were expressed clusteringly at axons of a population of BCs. In a minority of BCs, I4AA suppressed the GABA(C) responses at the dendrites, but not at the axon terminal, implying that the GABA(C) receptors at these two sites may be heterogeneous. Taken together, these results suggest that GABA(A) and GABA(C) receptors may play different roles in the outer and inner retina and the differential complements of the two receptors on OFF and ON BCs may be closely related to physiological functions of these cells.     

Effects of learned helplessness on brain GABA receptors

GABA is involved in both clinical depression and in animal models of depression; however, the roles of GABA(A) and GABA(B) receptors in specific brain regions are not clear. Changes in densities of both GABA(A) and GABA(B) receptors have been reported with the learned helplessness animal model of depression and with chronic antidepressant drug treatment. However, some of these findings are discrepant. Thus, we used quantitative autoradiography to study the GABA(A) and GABA(B) receptors in learned helplessness and we used an experimental paradigm that allows non-specific effects of stress to be differentiated from learned helplessness. Densities of GABA binding were measured in prefrontal cortex, septum, hippocampus, hypothalamus and amygdala. In the septum, learned helpless rats had increased densities of GABA(A) receptors and rats that did not become helpless after inescapable stress had decreased GABA(B) receptor densities. No significant group differences of GABA(A) or GABA(B) receptor densities were observed in any other brain region studied. These results suggest a unique role for the septum in modulating GABA in the learned helplessness animal model of depression.     

Activation of cerebellar granule cells GABA(A) receptors by guanidinoacetate

The extracellular concentration of guanidinoacetate (GAA) in the brain increases in guanidino acetate methyl transferase (GAMT) deficiency, an inherited disorder. We tested whether the levels which this substance can reach in the brain in GAMT deficiency are able to activate GABA(A) receptors in key cerebellar neurons such as the cerebellar granules. GAA in fact activates these receptors in rat cerebellar granules in culture although at quite high concentrations, in the millimolar range. However, these millimolar GAA levels are not reached extracellularly in the brain in GAMT deficiency. In addition, GAA does not act as a partial agonist on granules' GABA(A) receptors. This appears to deny an effect by this molecule on cerebellar function in the disease via interference with granule cells' GABA(A) receptors. Study of partial blockage by furosemide of chloride currents activated by GABA and GAA in granule cells allowed us to distinguish two populations of GABA(A) receptors presumably involved in granule cells' tonic inhibition. One is devoid of alpha6 subunit and another one contains it. The latter when activated by GABA has a decay kinetics much slower than the former. GAA does not distinguish between these two populations. In any case, the very high extracellular GAA concentrations able to activate them are not likely to be reached in GAMT deficiency.     

Feed-forward facilitation of glutamate release by presynaptic GABA(A) receptors

Disynaptic GABAergic inputs from Schaffer collateral (SC) afferents on to the soma of glutamatergic CA1 pyramidal neurons are involved in feed-forward inhibition in the hippocampal neural circuits. Here we report the functional roles of presynaptic GABA(A) receptors on SC afferents projecting to CA1 pyramidal neurons. Muscimol (0.5 microM), a selective GABA(A) receptor agonist, increased SC-evoked EPSC amplitude and decreased paired-pulse ratio in the slice preparation, in addition, it facilitated spontaneous glutamate release on to mechanically dissociated CA1 pyramidal neurons in an external Ca2+-dependent manner. In field recordings, muscimol at low concentrations (< or = 0.5 microM) increased not only the excitability of SC afferents but glutamate release, however, it at high concentrations (> or = 1 microM) changed bidirectionally. These results suggest that the moderate activation of presynaptic GABA(A) receptors depolarizes SC afferents and enhances SC-mediated glutamatergic transmission. When endogenous GABA was disynaptically released by brief trains of stimulation of SC afferents, the axonal excitability in addition to glutamate release was increased. The effects of endogenous GABA on the excitability of SC afferents were blocked by either SR95531 or AMPA receptor blockers, which would be expected to block disynaptic feed-forward neural circuits. The present results provide a novel form of presynaptic modulation (feed-forward facilitation) of glutamatergic transmission by presynaptic GABA(A) receptors within the intrinsic hippocampal neural circuits.     

Investigation of Gamma-aminobutyric acid (GABA) A receptors genes and migraine susceptibility

Background  

Migraine is a neurological disorder characterized by recurrent attacks of severe headache, affecting around 12% of Caucasian populations. It is well known that migraine has a strong genetic component, although the number and type of genes involved is still unclear. Prior linkage studies have reported mapping of a migraine gene to chromosome Xq 24–28, a region containing a cluster of genes for GABA A receptors (GABRE, GABRA3, GABRQ), which are potential candidate genes for migraine. The GABA neurotransmitter has been implicated in migraine pathophysiology previously; however its exact role has not yet been established, although GABA receptors agonists have been the target of therapeutic developments. The aim of the present research is to investigate the role of the potential candidate genes reported on chromosome Xq 24–28 region in migraine susceptibility. In this study, we have focused on the subunit GABA A receptors type ε (GABRE) and type θ (GABRQ) genes and their involvement in migraine.     

Effects of etifoxine on ligand binding to GABA(A) receptors in rodents

The GABA(A) receptor/chloride ionophore is allosterically modulated by several classes of anxiolytic and anticonvulsant agents, including benzodiazepines, barbiturates and neurosteroids. Etifoxine, an anxiolytic and anticonvulsant compound competitively inhibited the binding of [(35)S]t-butylbicyclophosphoro-thionate (TBPS), a specific ligand of the GABA(A) receptor chloride channel site. To investigate the etifoxine modulatory effects on the different binding sites of the GABA(A) receptor complex, we have examined the effects of etifoxine on binding of the receptor agonist [(3)H]muscimol and the benzodiazepine modulator [(3)H]flunitrazepam in rat brain membrane preparations. The anticonvulsant properties of etifoxine combined with muscimol and flunitrazepam were performed in mice with picrotoxin-induced clonic seizures. Etifoxine modestly enhanced binding of [(3)H]muscimol and of [(3)H]flunitrazepam by increasing the number of binding sites without changing the binding affinity of [(3)H]flunitrazepam. In contrast, the compound decreased the affinity of muscimol for its binding site. In vivo, the combination of subactive doses of etifoxine with muscimol or flunitrazepam produced an anticonvulsant additive effect against the picrotoxin-induced clonic seizures in mice. These results suggest that the interaction of etifoxine on the GABA(A) receptor complex would allosterically modify different binding sites due to conformational changes. Functionally, the resulting facilitation of GABA transmission underlies the pharmacological properties of etifoxine.