Developmental Expression of GABAA Receptor Subunit mRNAs in Individual Hippocampal Neurons In Vitro and In Vivo

Abstract: The GABA_A receptor is a heterooligomeric protein complex composed of multiple receptor subunits. Developmental changes in the pattern of expression of 11 GABA_A receptor subunits in individual rat embryonic hippocampal neurons on days 1–21 in culture and acutely dissociated hippocampal neurons from postnatal day (PND) 5 rat pups were investigated using the technique of single-cell mRNA amplification. We demonstrate that multiple GABA_A receptor subunits are expressed within individual hippocampal neurons, with most cells simultaneously expressing α1, α2, α5, β1, and γ2 mRNAs. Further, relative expression of several GABA_A receptor subunit mRNAs changes significantly in embryonic hippocampal neurons during in vitro development, with the relative abundance (compared with β-actin) of α1, α5, and γ2 mRNAs increasing 2.3-, 2.7-, and 3.8-fold, respectively, from days 1 to 14, and β1 increasing 5-fold from days 1 to 21. In situ hybridization with antisense digoxigenin-labeled α1, β1, and γ2 RNA probes demonstrates a similar increase in expression of subunit mRNAs as embryonic hippocampal neurons mature in vitro. Relative abundances of α1, β1, and γ2 subunit mRNAs in acutely dissociated PND 5 hippocampal neurons are also significantly greater than in embryonic day 17 neurons on day 1 in vitro and exceed the peak values seen in cultured neurons on days 14–21, suggesting that GABA_A receptor subunit mRNA expression within individual hippocampal neurons follows a similar, if somewhat delayed, developmental pattern in vitro compared with in vivo. These findings suggest that embryonic hippocampal neuronal culture provides a useful model in which to study the developmental regulation of GABA_A receptor expression and that developmental changes in GABA_A receptor subunit expression may underlie some of the differences in functional properties of GABA_A receptors in neonatal and mature hippocampal neurons.     

Distinct Subunit-specific α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Trafficking Mechanisms in Cultured Cortical and Hippocampal Neurons in Response to Oxygen and Glucose Deprivation

Brain ischemia occurs when the blood supply to the brain is interrupted, leading to oxygen and glucose deprivation (OGD). This triggers a cascade of events causing a synaptic accumulation of glutamate. Excessive activation of glutamate receptors results in excitotoxicity and delayed cell death in vulnerable neurons. Following global cerebral ischemia, hippocampal CA1 pyramidal neurons are more vulnerable to injury than their cortical counterparts. The mechanisms that underlie this difference are unclear. Cultured hippocampal neurons respond to OGD with a rapid internalization of AMPA receptor (AMPAR) subunit GluA2, resulting in a switch from GluA2-containing Ca²⁺-impermeable receptors to GluA2-lacking Ca²⁺-permeable subtypes (CP-AMPARs). GluA2 internalization is a critical component of OGD-induced cell death in hippocampal neurons. It is unknown how AMPAR trafficking is affected in cortical neurons following OGD. Here, we show that cultured cortical neurons are resistant to an OGD insult that causes cell death in hippocampal neurons. GluA1 is inserted at the plasma membrane in both cortical and hippocampal neurons in response to OGD. In contrast, OGD causes a rapid endocytosis of GluA2 in hippocampal neurons, which is absent in cortical neurons. These data demonstrate that populations of neurons with different vulnerabilities to OGD recruit distinct cell biological mechanisms in response to insult, and that a crucial aspect of the mechanism leading to OGD-induced cell death is absent in cortical neurons. This strongly suggests that the absence of OGD-induced GluA2 trafficking contributes to the relatively low vulnerability of cortical neurons to ischemia.     

Topiramate attenuates cerebral ischemia/reperfusion injury in gerbils via activating GABAergic signaling and inhibiting astrogliosis

Impaired GABAergic inhibitory synaptic transmission plays an essential role in the pathogenesis of selective neuronal cell death following transient global ischemia. GABA_A receptor (GABA_AR), K⁺-Cl⁻ co-transporter 2 (KCC2), Na⁺-K⁺-Cl⁻ co-transporter 1 (NKCC1) and astrocytes are of particular importance to GABAergic transmission. The present study was designed to explore whether the neuroprotective effect of topiramate (TPM) was linked with the alterations of GABAergic signaling and astrocytes. The bilateral carotid arteries were occluded, and TPM (80 mg/kg/day (divided twice daily), i.p.) was injected into gerbils. At day 1, 3 and 7 post-ischemia, neurological deficit was scored and changes in hippocampal neuronal cell death were evaluated by Nissl staining. The apoptosis-related regulatory proteins (procaspase-3, caspase-3, Bax and Bcl-2) and GABAergic signal molecules (GABA_AR α1, GABA_AR γ2, KCC2 and NKCC1) were also detected using western blot assay. In addition, the fluorescent intensity and protein level of glial fibrillary acidic protein (GFAP), a major component of astrocyte, were examined by confocal and immunoblot analysis. Our results showed that TPM treatment significantly decreased neurological deficit scores, attenuated the ischemia-induced neuronal loss and remarkably decreased the expression levels of procaspase-3, caspase-3 as well as the ratio of Bax/Bcl-2. Besides, treatment with TPM also resulted in the increased protein expressions of GABA_AR α1, GABA_AR γ2 and KCC2 together with the decreased protein level of NKCC1 in gerbils hippocampus. Furthermore, fluorescent intensity and protein level of GFAP were evidently reduced in TPM-treated gerbils. These findings suggest that the therapeutic effect of TPM on global ischemia/reperfusion injury appears to be associated with the enhancement of GABAergic signaling and the inhibition of astrogliosis in gerbils.     

Ring Finger Protein 34 (RNF34) Interacts with and Promotes γ-Aminobutyric Acid Type-A Receptor Degradation via Ubiquitination of the γ2 Subunit

We have found that the large intracellular loop of the γ2 GABA_A receptor (R) subunit (γ2IL) interacts with RNF34 (an E3 ubiquitin ligase), as shown by yeast two-hybrid and in vitro pulldown assays. In brain extracts, RNF34 co-immunoprecipitates with assembled GABA_ARs. In co-transfected HEK293 cells, RNF34 reduces the expression of the γ2 GABA_AR subunit by increasing the ratio of ubiquitinated/nonubiquitinated γ2. Mutating several lysines of the γ2IL into arginines makes the γ2 subunit resistant to RNF34-induced degradation. RNF34 also reduces the expression of the γ2 subunit when α1 and β3 subunits are co-assembled with γ2. This effect is partially reversed by leupeptin or MG132, indicating that both the lysosomal and proteasomal degradation pathways are involved. Immunofluorescence of cultured hippocampal neurons shows that RNF34 forms clusters and that a subset of these clusters is associated with GABAergic synapses. This association is also observed in the intact rat brain by electron microscopy immunocytochemistry. RNF34 is not expressed until the 2nd postnatal week of rat brain development, being highly expressed in some interneurons. Overexpression of RNF34 in hippocampal neurons decreases the density of γ2 GABA_AR clusters and the number of GABAergic contacts that these neurons receive. Knocking down endogenous RNF34 with shRNA leads to increased γ2 GABA_AR cluster density and GABAergic innervation. The results indicate that RNF34 regulates postsynaptic γ2-GABA_AR clustering and GABAergic synaptic innervation by interacting with and ubiquitinating the γ2-GABA_AR subunit promoting GABA_AR degradation.     

GABAA receptor plasticity in Jurkat T cells

GABA_A receptors (GABA_AR) mediate inhibitory neurotransmission in the human brain. Neurons modify subunit expression, cellular distribution and function of GABA_AR in response to different stimuli, a process named plasticity. Human lymphocytes have a functional neuronal-like GABAergic system with GABA_AR acting as inhibitors of proliferation. We here explore if receptor plasticity occurs in lymphocytes. To this end, we analyzed human T lymphocyte Jurkat cells exposed to different physiological stimuli shown to mediate plasticity in neurons: GABA, progesterone and insulin. The exposure to 100 μM GABA differently affected the expression of GABA_AR subunits measured at both the mRNA and protein level, showing an increase of α1, β3, and γ2 subunits but no changes in δ subunit. Exposure of Jurkat cells to different stimuli produced different changes in subunit expression: 0.1 μM progesterone decreased δ and 0.5 μM insulin increased β3 subunits. To identify the mechanisms underlying plasticity, we evaluated the Akt pathway, which is involved in the phosphorylation of β subunits and receptor translocation to the membrane. A significant increase of phosphorylated Akt and on the expression of β3 subunit in membrane occurred in cells exposed 15 h to GABA. To determine if plastic changes are translated into functional changes, we performed whole cell recordings. After 15 h GABA-exposure, a significantly higher percentage of cells responded to GABA application when compared to 0 and 40 h exposure, thus indicating that the detected plastic changes may have a role in GABA-modulated lymphocyte function.     

Differential Regulation of GABAB Receptor Trafficking by Different Modes of N-methyl-d-aspartate (NMDA) Receptor Signaling

Inhibitory GABA_B receptors (GABA_BRs) can down-regulate most excitatory synapses in the CNS by reducing postsynaptic excitability. Functional GABA_BRs are heterodimers of GABA_B1 and GABA_B2 subunits and here we show that the trafficking and surface expression of GABA_BRs is differentially regulated by synaptic or pathophysiological activation of NMDA receptors (NMDARs). Activation of synaptic NMDARs using a chemLTP protocol increases GABA_BR recycling and surface expression. In contrast, excitotoxic global activation of synaptic and extrasynaptic NMDARs by bath application of NMDA causes the loss of surface GABA_BRs. Intriguingly, exposing neurons to extreme metabolic stress using oxygen/glucose deprivation (OGD) increases GABA_B1 but decreases GABA_B2 surface expression. The increase in surface GABA_B1 involves enhanced recycling and is blocked by the NMDAR antagonist AP5. The decrease in surface GABA_B2 is also blocked by AP5 and by inhibiting degradation pathways. These results indicate that NMDAR activity is critical in GABA_BR trafficking and function and that the individual subunits can be separately controlled to regulate neuronal responsiveness and survival.     

Cell Type-Specific Properties of Subicular GABAergic Currents Shape Hippocampal Output Firing Mode

GABAergic function of the subiculum is central to the regulation of hippocampal output activity. Subicular neuronal networks are indeed under potent control by local inhibition. However, information about the properties of GABAergic currents generated by neurons of this parahippocampal area in normal tissue is still missing. Here, we describe GABA_A receptor (GABA_AR)-mediated phasic and tonic currents generated by principal cells (PCs) and interneurons (INs) of the rat subiculum. We show that in spite of similar synaptic current densities, INs generate spontaneous IPSCs (sIPSCs) that occur less frequently and exhibit smaller charge transfer, thus receiving less synaptic total current than PCs. Further distinction of PCs between intrinsically bursting (IB) and regular-spiking (RS) neurons suggested that sIPSCs generated by the two PC sub-types are likely to be similar. PCs and INs are also controlled by a similar tonic inhibition. However, whereas a comparable tonic current density is found in RS cells and INs, IB neurons are constrained by a greater inhibitory tone. Finally, pharmacological blockade of GABA_AR did not promote functional switch of RS neurons to IB mode, but influenced the bursting propensity of IB cells and released fast spiking activity in INs. Our findings reveal differences in GABAergic currents between PCs and INs as well as within PC sub-types. We propose that GABAergic inhibition may shape hippocampal output activity by providing cell type-specific fine-tuning of subicular excitatory and inhibitory drives.     

Pharmacological characterization of GABA<Subscript>A</Subscript> receptors in taurine-fed mice

Background

Taurine is one of the most abundant free amino acids especially in excitable tissues, with wide physiological actions. Chronic supplementation of taurine in drinking water to mice increases brain excitability mainly through alterations in the inhibitory GABAergic system. These changes include elevated expression level of glutamic acid decarboxylase (GAD) and increased levels of GABA. Additionally we reported that GABA_A receptors were down regulated with chronic administration of taurine. Here, we investigated pharmacologically the functional significance of decreased / or change in subunit composition of the GABA_A receptors by determining the threshold for picrotoxin-induced seizures. Picrotoxin, an antagonist of GABA_A receptors that blocks the channels while in the open state, binds within the pore of the channel between the β2 and β3 subunits. These are the same subunits to which GABA and presumably taurine binds.

Methods

Two-month-old male FVB/NJ mice were subcutaneously injected with picrotoxin (5 mg kg^-1) and observed for a) latency until seizures began, b) duration of seizures, and c) frequency of seizures. For taurine treatment, mice were either fed taurine in drinking water (0.05%) or injected (43 mg/kg) 15 min prior to picrotoxin injection.

Results

We found that taurine-fed mice are resistant to picrotoxin-induced seizures when compared to age-matched controls, as measured by increased latency to seizure, decreased occurrence of seizures and reduced mortality rate. In the picrotoxin-treated animals, latency and duration were significantly shorter than in taurine-treated animas. Injection of taurine 15 min before picrotoxin significantly delayed seizure onset, as did chronic administration of taurine in the diet. Further, taurine treatment significantly increased survival rates compared to the picrotoxin-treated mice.

Conclusions

We suggest that the elevated threshold for picrotoxin-induced seizures in taurine-fed mice is due to the reduced binding sites available for picrotoxin binding due to the reduced expression of the beta subunits of the GABA_A receptor. The delayed effects of picrotoxin after acute taurine injection may indicate that the two molecules are competing for the same binding site on the GABA_A receptor. Thus, taurine-fed mice have a functional alteration in the GABAergic system. These include: increased GAD expression, increased GABA levels, and changes in subunit composition of the GABA_A receptors. Such a finding is relevant in conditions where agonists of GABA_A receptors, such as anesthetics, are administered.

     

Regulation of GABAergic inhibition by serotonin signaling in prefrontal cortex: molecular mechanisms and functional implications

Serotonergic neurotransmission in prefrontal cortex (PFC) plays a key role in regulating emotion and cognition under normal and pathological conditios. Increasing evidence suggests that serotonin receptors are involved in the complex regulation of GABAergic inhibitory transmission in PFC. Activation of postsynaptic 5-HT₂ receptors in PFC pyramidal neurons inhibits GABA_A-receptor currents via phosphorylation of GABA_A receptor γ2 subunits by RACK1-anchored PKC. In contrast, activation of postsynaptic 5-HT₄ receptors produces an activity-dependent bi-directional regulation of GABA-evoked currents in PFC pyramidal neurons, which is mediated through phosphorylation of GABA_A-receptor β subunits by anchored PKA. On the presynaptic side, GABAergic inhibition is regulated by 5-HT through the activation of 5-HT₂, 5-HT₁, and 5-HT₃ receptors on GABAergic intereneurons. These data provide a molecular and cellular mechanism for serotonin to dynamically regulate synaptic transmission and neuronal excitability in the PFC network, which may underlie the actions of many antidepressant and antipsychotic drugs.     

The cell adhesion molecule neuroplastin-65 is a novel interaction partner of γ-aminobutyric acid type A receptors

γ-Aminobutyric acid type A (GABA_A) receptors are pentameric ligand-gated ion channels that mediate fast inhibition in the central nervous system. Depending on their subunit composition, these receptors exhibit distinct pharmacological properties and differ in their ability to interact with proteins involved in receptor anchoring at synaptic or extra-synaptic sites. Whereas GABA_A receptors containing α1, α2, or α3 subunits are mainly located synaptically where they interact with the submembranous scaffolding protein gephyrin, receptors containing α5 subunits are predominantly found extra-synaptically and seem to interact with radixin for anchorage. Neuroplastin is a cell adhesion molecule of the immunoglobulin superfamily that is involved in hippocampal synaptic plasticity. Our results reveal that neuroplastin and GABA_A receptors can be co-purified from rat brain and exhibit a direct physical interaction as demonstrated by co-precipitation and Förster resonance energy transfer (FRET) analysis in a heterologous expression system. The brain-specific isoform neuroplastin-65 co-localizes with GABA_A receptors as shown in brain sections as well as in neuronal cultures, and such complexes can either contain gephyrin or be devoid of gephyrin. Neuroplastin-65 specifically co-localizes with α1 or α2 but not with α3 subunits at GABAergic synapses. In addition, neuroplastin-65 also co-localizes with GABA_A receptor α5 subunits at extra-synaptic sites. Down-regulation of neuroplastin-65 by shRNA causes a loss of GABA_A receptor α2 subunits at GABAergic synapses. These results suggest that neuroplastin-65 can co-localize with a subset of GABA_A receptor subtypes and might contribute to anchoring and/or confining GABA_A receptors to particular synaptic or extra-synaptic sites, thus affecting receptor mobility and synaptic strength.     

Stimulation of TM3 Leydig cell proliferation via GABA<Subscript>A</Subscript> receptors: A new role for testicular GABA

The neurotransmitter gamma-aminobutyric acid (GABA) and subtypes of GABA receptors were recently identified in adult testes. Since adult Leydig cells possess both the GABA biosynthetic enzyme glutamate decarboxylase (GAD), as well as GABA_A and GABA_B receptors, it is possible that GABA may act as auto-/paracrine molecule to regulate Leydig cell function. The present study was aimed to examine effects of GABA, which may include trophic action. This assumption is based on reports pinpointing GABA as regulator of proliferation and differentiation of developing neurons via GABA_A receptors. Assuming such a role for the developing testis, we studied whether GABA synthesis and GABA receptors are already present in the postnatal testis, where fetal Leydig cells and, to a much greater extend, cells of the adult Leydig cell lineage proliferate. Immunohistochemistry, RT-PCR, Western blotting and a radioactive enzymatic GAD assay evidenced that fetal Leydig cells of five-six days old rats possess active GAD protein, and that both fetal Leydig cells and cells of the adult Leydig cell lineage possess GABA_A receptor subunits. TM3 cells, a proliferating mouse Leydig cell line, which we showed to possess GABA_A receptor subunits by RT-PCR, served to study effects of GABA on proliferation. Using a colorimetric proliferation assay and Western Blotting for proliferating cell nuclear antigen (PCNA) we demonstrated that GABA or the GABA_A agonist isoguvacine significantly increased TM3 cell number and PCNA content in TM3 cells. These effects were blocked by the GABA_A antagonist bicuculline, implying a role for GABA_A receptors. In conclusion, GABA increases proliferation of TM3 Leydig cells via GABA_A receptor activation and proliferating Leydig cells in the postnatal rodent testis bear a GABAergic system. Thus testicular GABA may play an as yet unrecognized role in the development of Leydig cells during the differentiation of the testicular interstitial compartment.     

Blocking L-type Voltage-gated Ca2+ Channels with Dihydropyridines Reduces ��-Aminobutyric Acid Type A Receptor Expression and Synaptic Inhibition

γ-Aminobutyric acid type A receptors (GABA_ARs) are the major sites of fast inhibitory neurotransmission in the brain, and the numbers of these receptors at the cell surface can determine the strength of GABAergic neurotransmission. Chronic changes in neuronal activity lead to an adaptive modulation in the efficacy of GABAergic synaptic inhibition, brought about in part by changes in the number of synaptic GABA_ARs, a mechanism known as homeostatic synaptic plasticity. Reduction in the number of GABA_ARs in response to prolonged neuronal activity blockade is dependent on the ubiquitin-proteasome system. The underlying biochemical pathways linking chronic activity blockade to proteasome-dependent degradation of GABA_ARs are unknown. Here, we show that chronic blockade of L-type voltage-gated calcium channels (VGCCs) with nifedipine decreases the number of GABA_ARs at synaptic sites but not the overall number of inhibitory synapses. In parallel, blockade of L-type VGCCs decreases the amplitude but not the frequency of miniature inhibitory postsynaptic currents or expression of the glutamic acid decarboxylase GAD65. We further reveal that the activation of L-type VGCCs regulates the turnover of newly translated GABA_AR subunits in a mechanism dependent upon the activity of the proteasome and thus regulates GABA_AR insertion into the plasma membrane. Together, these observations suggest that activation of L-type VGCCs can regulate the abundance of synaptic GABA_ARs and the efficacy of synaptic inhibition, revealing a potential mechanism underlying the homeostatic adaptation of fast GABAergic inhibition to prolonged changes in activity.     

Synaptic localization of α5 GABA (A) receptors via gephyrin interaction regulates dendritic outgrowth and spine maturation

GABA_A receptor subunit composition is a critical determinant of receptor localization and physiology, with synaptic receptors generating phasic inhibition and extrasynaptic receptors producing tonic inhibition. Extrasynaptically localized α5 GABA_A receptors are largely responsible for tonic inhibition in hippocampal neurons. However, we show here that inhibitory synapses also contain a constant level of α5 GABA_A receptors throughout neuronal development, as measured by its colocalization with gephyrin, the inhibitory postsynaptic scaffolding protein. Immunoprecipitation of the α5 subunit from both cultured neurons and adult rat brain coimmunoprecipitated gephyrin, confirming this interaction in vivo. Furthermore, the α5 subunit can interact with gephyrin independent of other synaptically localized alpha subunits, as shown by immunoprecipitation experiments in HEK cells. By replacing the α5 predicted gephyrin binding domain (Residues 370–385) with either the high affinity gephyrin binding domain of the α2 subunit or homologous residues from the extrasynaptic α4 subunit that does not interact with gephyrin, α5 GABA_A receptor localization shifted into or out of the synapse, respectively. These shifts in the ratio of synaptic/extrasynaptic α5 localization disrupted dendritic outgrowth and spine maturation. In contrast to the predominant view of α5 GABA_A receptors being extrasynaptic and modulating tonic inhibition, we identify an intimate association of the α5 subunit with gephyrin, resulting in constant synaptic levels of α5 GABA_AR throughout circuit formation that regulates neuronal development. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1241–1251, 2015     

Homeostatic Competition between Phasic and Tonic Inhibition

The GABA_A receptors are the major inhibitory receptors in the brain and are localized at both synaptic and extrasynaptic membranes. Synaptic GABA_A receptors mediate phasic inhibition, whereas extrasynaptic GABA_A receptors mediate tonic inhibition. Both phasic and tonic inhibitions regulate neuronal activity, but whether they regulate each other is not very clear. Here, we investigated the functional interaction between synaptic and extrasynaptic GABA_A receptors through various molecular manipulations. Overexpression of extrasynaptic α6β3δ-GABA_A receptors in mouse hippocampal pyramidal neurons significantly increased tonic currents. Surprisingly, the increase of tonic inhibition was accompanied by a dramatic reduction of the phasic inhibition, suggesting a possible homeostatic regulation of the total inhibition. Overexpressing the α6 subunit alone induced an up-regulation of δ subunit expression and suppressed phasic inhibition similar to overexpressing the α6β3δ subunits. Interestingly, blocking all GABA_A receptors after overexpressing α6β3δ receptors could not restore the synaptic GABAergic transmission, suggesting that receptor activation is not required for the homeostatic interplay. Furthermore, insertion of a gephyrin-binding-site (GBS) into the α6 and δ subunits recruited α6_GBSβ3δ_GBS receptors to postsynaptic sites but failed to rescue synaptic GABAergic transmission. Thus, it is not the positional effect of extrasynaptic α6β3δ receptors that causes the down-regulation of phasic inhibition. Overexpressing α5β3γ2 subunits similarly reduced synaptic GABAergic transmission. We propose a working model that both synaptic and extrasynaptic GABA_A receptors may compete for limited receptor slots on the plasma membrane to maintain a homeostatic range of the total inhibition.     

Response of the GABAergic System to Axotomy of the Rat Facial Nerve

The responses of inhibitory neurons/synapses to motoneuron injury in the cranial nervous system remain to be elucidated. In this study, we analyzed GABA_A receptor (GABA_AR) and GABAergic neurons at the protein level in the transected rat facial nucleus. Immunoblotting revealed that the GABA_ARα1 protein levels in the axotomized facial nucleus decreased significantly 5–14 days post-insult, and these levels remained low for 5 weeks. Immunohistochemical analysis indicated that the GABA_ARα1-expressing cells were motoneurons. We next examined the specific components of GABAergic neurons, including glutamate decarboxylase (GAD), vesicular GABA transporter (VGAT) and GABA transporter-1 (GAT-1). Immunoblotting indicated that the protein levels of GAD, VGAT and GAT-1 decreased transiently in the transected facial nucleus from 5 to 14 days post-insult, but returned to the control levels at 5 weeks post-insult. Although GABA_ARα1 protein levels in the transected nucleus did not return to their control levels for 5 weeks post-insult, the administration of glial cell line—derived neurotrophic factor at the cut site significantly ameliorated the reductions. Through these findings, we verified that the injured facial motoneurons suppressed the levels of GABA_ARα1 protein over the 5 weeks post-insult, presumably due to the deprivation of neurotrophic factor. On the other hand, the levels of the GAD, VGAT and GAT-1 proteins in GABAergic neurons were transiently reduced in the axotomized facial nucleus at 5–14 days post-insult, but recovered at 4–5 weeks post-insult.     

Antibodies to the Human γ2 Subunit of the γ-Aminobutyric AcidA/Benzodiazepine Receptor

Abstract: A γ-aminobutyric acid_A (GABA_A) receptor (GABA_AR) γ₂ subunit (short form) was cloned from an adult human cerebral cortex cDNA library in bacteriophage λgt11. The 261-bp intracellular loop (IL) located between M3 and M4 was amplified using the polymerase chain reaction and inserted into the expression vectors λgt11 and pGEX-3X. Both γ-galactosidase (LacZ) and glutathione-S-transferase (GST) fusion proteins containing the γ₂IL were purified, and a rabbit antibody to the LacZ–γ₂IL was made. The antibody reacted with the γ₂IL of both LacZ and GST fusion proteins and immunoprecipitated the GABA_AR/ benzodiazepine receptor (GABA_AR/BZDR) from bovine and rat brain. The antibody reacted in affinity-purified GABA_AR/BZDR immunoblots with a wide peptide band of 44,000–49,000 M_r. Immunoprecipitation studies with the anti-γ₂IL antibody suggest that in the cerebral cortex, 87% of the GABA_ARs with high affinity for benzodiazepines and 70% of the GABA_ARs with high affinity for muscimol contain at least a γ subunit, probably a γ₂. These results indicate that there are [³H]muscimol binding GABA_ARs that do not bind [³H]flunitrazepam with high affinity. Immunoprecipitations with this and other anti-GABA_AR/BZDR antibodies indicate that the most abundant combination of GABA_AR subunits in the cerebral cortex involves α₁, γ₂ (or other γ), and β₂ and/or β₃ subunits. These subunits coexist in >60% of the GABA_AR/BZDRs in the cerebral cortex. The results also show that a considerable proportion (20–25%) of the cerebellar GABA_AR/BZDRs is clonazepam insensitive. At least 74% of these cerebellar receptors, which likely contain α₆, also contain γ₂ (or other γ) subunit(s). The α₁ and β₂ or β₃ subunits are also frequently associated with γ₂ (or other γ) and α₆ in these cerebellar receptors.     

GABA<Subscript>A</Subscript> Receptor and Glycine Receptor Activation by Paracrine/Autocrine Release of Endogenous Agonists: More Than a Simple Communication Pathway

It is a common and widely accepted assumption that glycine and GABA are the main inhibitory transmitters in the central nervous system (CNS). But, in the past 20 years, several studies have clearly demonstrated that these amino acids can also be excitatory in the immature central nervous system. In addition, it is now established that both GABA receptors (GABARs) and glycine receptors (GlyRs) can be located extrasynaptically and can be activated by paracrine release of endogenous agonists, such as GABA, glycine, and taurine. Recently, non-synaptic release of GABA, glycine, and taurine gained further attention with increasing evidence suggesting a developmental role of these neurotransmitters in neuronal network formation before and during synaptogenesis. This review summarizes recent knowledge about the non-synaptic activation of GABA_ARs and GlyRs, both in developing and adult CNS. We first present studies that reveal the functional specialization of both non-synaptic GABA_ARs and GlyRs and we discuss the neuronal versus non-neuronal origin of the paracrine release of GABA_AR and GlyR agonists. We then discuss the proposed non-synaptic release mechanisms and/or pathways for GABA, glycine, and taurine. Finally, we summarize recent data about the various roles of non-synaptic GABAergic and glycinergic systems during the development of neuronal networks and in the adult.     

Diversity matters: combinatorial information coding by GABAA receptor subunits during spatial learning and its allosteric modulation

In the hippocampus, GABA inhibition tunes network oscillations and shapes synchronous activity during spatial learning and memory coding. Once released from the presynapse, GABA primarily binds to ionotropic GABA_A receptors (GABA_ARs), which are heteropentamers combinatorially assembled from nineteen known subunits to induce Cl^- currents postsynaptically. Dissecting GABA_AR subtype specificities in neurobiology is daunting because of differences in their developmental dynamics, regional distribution and subcellular compartmentalization. Here, we review recent data to show that the combination of single-cell mRNA-seq and neuroanatomy can reveal unprecedented cell-type and network-specificity of GABA_AR subunits and limit the permutation in subunit configurations, thus rationalizing GABA_AR physiology and pharmacology. By comparing RNA-seq data on principal cells and interneurons we discuss a tight match between GABA_AR subunit allocation, diversity in the origins of GABA inputs and operational rules at synaptic and extrasynaptic sites. We propose that coincident analysis of all GABA_AR subunits, particularly in relation to specific behaviors, could overcome existing pitfalls of the genetic and pharmacological manipulation of single subunits. By using α1 and α5 GABA_AR subunits, we single out hippocampal spatial learning as a process in which, despite the many studies available to date, neither consensus nor causality exists with regards to GABA_AR subtype requirements, curtailing a unifying concept on postsynaptic coding of GABA signals. Finally, we address the modulation of GABA_AR subunits by dopamine and endocannabinoids through receptor heteromerization, cross-modulation of signal transduction and allostery. In sum, data in this review infer that multiparametric computation gains momentum to improve knowledge on GABA_ARs function in cognition and neuropsychiatric illnesses.     

GABAA receptor diversity and pharmacology

Because of its control of spike-timing and oscillatory network activity, γ-aminobutyric acid (GABA)-ergic inhibition is a key element in the central regulation of somatic and mental functions. The recognition of GABA_A receptor diversity has provided molecular tags for the analysis of distinct neuronal networks in the control of specific pharmacological and physiological brain functions. Neurons expressing α₁GABA_A receptors have been found to mediate sedation, whereas those expressing α₂GABA_A receptors mediate anxiolysis. Furthermore, associative temporal and spatial memory can be regulated by modulating the activity of hippocampal pyramidal cells via extrasynaptic α₅GABA_A receptors. In addition, neurons expressing α₃GABA_A receptors are instrumental in the processing of sensory motor information related to a schizophrenia endophenotype. Finally, during the postnatal development of the brain, the maturation of GABAergic interneurons seems to provide the trigger for the experience-dependent plasticity of neurons in the visual cortex, with α₁GABA_A receptors setting the time of onset of a critical period of plasticity. Thus, particular neuronal networks defined by respective GABA_A receptor subtypes can now be linked to the regulation of various clearly defined behavioural patterns. These achievements are of obvious relevance for the pharmacotherapy of certain brain disorders, in particular sleep dysfunctions, anxiety disorders, schizophrenia and diseases associated with memory deficits.