Celia P. Miralles

Synaptic and nonsynaptic localization of GABAA receptors containing the α5 subunit in the rat brain

The α5 subunit of the GABAA receptors (GABAARs) has a restricted expression in the brain. Maximum expression of this subunit occurs in the hippocampus, cerebral cortex, and olfactory bulb. Hippocampal pyramidal cells show high expression of α5 subunit‐containing GABAARs (α5‐GABAARs) both in culture and in the intact brain. A large pool of α5‐GABAARs is extrasynaptic and it has been proposed to be involved in the tonic GABAergic inhibition of the hippocampus. Nevertheless, there are no studies on the localization of the α5‐GABAARs at the electron microscope (EM) level. By using both immunofluorescence of cultured hippocampal pyramidal cells and EM postembedding immunogold of the intact hippocampus we show that, in addition to the extrasynaptic pool, there is a pool of α5‐GABAARs that concentrates at the GABAergic synapses in dendrites of hippocampal pyramidal cells. The results suggest that the synaptic α5‐GABAARs might play a role in the phasic GABAergic inhibition of pyramidal neurons in hippocampus and cerebral cortex. J. Comp. Neurol. 499:458–470, 2006.

Gephyrin clustering is required for the stability of GABAergic synapses

Although gephyrin is an important postsynaptic scaffolding protein at GABAergic synapses, the role of gephyrin for GABAergic synapse formation and/or maintenance is still under debate. We report here that knocking down gephyrin expression with small hairpin RNAs (shRNAs) in cultured hippocampal pyramidal cells decreased both the number of gephyrin and GABA(A) receptor clusters. Similar results were obtained by disrupting the clustering of endogenous gephyrin by overexpressing a gephyrin-EGFP fusion protein that formed aggregates with the endogenous gephyrin. Disrupting postsynaptic gephyrin clusters also had transsynaptic effects leading to a significant reduction of GABAergic presynaptic boutons contacting the transfected pyramidal cells. Consistent with the morphological decrease of GABAergic synapses, electrophysiological analysis revealed a significant reduction in both the amplitude and frequency of the spontaneous inhibitory postsynaptic currents (sIPSCs). However, no change in the whole-cell GABA currents was detected, suggesting a selective effect of gephyrin on GABA(A) receptor clustering at postsynaptic sites. It is concluded that gephyrin plays a critical role for the stability of GABAergic synapses.

The brefeldin A-inhibited GDP/GTP exchange factor 2, a protein involved in vesicular trafficking, interacts with the β subunits of the GABAA receptors

We have found that the brefeldin A‐inhibited GDP/GTP exchange factor 2 (BIG2) interacts with the β subunits of the γ‐aminobutyric acid type‐A receptor (GABAAR). BIG2 is a Sec7 domain‐containing guanine nucleotide exchange factor known to be involved in vesicular and protein trafficking. The interaction between the 110 amino acid C‐terminal fragment of BIG2 and the large intracellular loop of the GABAAR β subunits was revealed with a yeast two‐hybrid assay. The native BIG2 and GABAARs interact in the brain since both coprecipitated from detergent extracts with either anti‐GABAAR or anti‐BIG2 antibodies. In transfected human embryonic kidney cell line 293 cells, BIG2 promotes the exit of GABAARs from endoplasmic reticulum. Double label immunofluorescence of cultured hippocampal neurons and electron microscopy immunocytochemistry of rat brain tissue show that BIG2 concentrates in the trans‐Golgi network. BIG2 is also present in vesicle‐like structures in the dendritic cytoplasm, sometimes colocalizing with GABAARs. BIG2 is present in both inhibitory GABAergic synapses that contain GABAARs and in asymmetric excitatory synapses. The results are consistent with the hypotheses that the interaction of BIG2 with the GABAAR β subunits plays a role in the exocytosis and trafficking of assembled GABAAR to the cell surface.

Disruption of postsynaptic GABAA receptor clusters leads to decreased GABAergic innervation of pyramidal neurons

We have used RNA interference (RNAi) to knock down the expression of the γ2 subunit of the GABAA receptors (GABAARs) in pyramidal neurons in culture and in the intact brain. Two hairpin small interference RNAs (shRNAs) for the γ2 subunit, one targeting the coding region and the other one the 3′‐untranslated region (UTR) of the γ2 mRNA, when introduced into cultured rat hippocampal pyramidal neurons, efficiently inhibited the synthesis of the GABAA receptor γ2 subunit and the clustering of other GABAAR subunits and gephyrin in these cells. More significantly, this effect was accompanied by a reduction of the GABAergic innervation that these neurons received. In contrast, the γ2 shRNAs had no effect on the clustering of postsynaptic α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptors, postsynaptic density protein 95 (PSD‐95) or presynaptic glutamatergic innervation. A γ2‐enhanced green fluorescent protein (EGFP) subunit construct, whose mRNA did not contain the 3′‐UTR targeted by γ2 RNAi, rescued both the postsynaptic clustering of GABAARs and the GABAergic innervation. Decreased GABAAR clustering and GABAergic innervation of pyramidal neurons in the post‐natal rat cerebral cortex was also observed after in utero transfection of these neurons with the γ2 shRNAs. The results indicate that the postsynaptic clustering of GABAARs in pyramidal neurons is involved in the stabilization of the presynaptic GABAergic contacts.

GABA A receptor γ2 subunit knockdown mice have enhanced anxiety-like behavior but unaltered hypnotic response to benzodiazepines

BackgroundGamma-aminobutyric acid type A receptors (GABAA-Rs) are the major inhibitory receptors in the mammalian brain and are modulated by a number of sedative/hypnotic drugs including benzodiazepines and anesthetics. The significance of specific GABAA-Rs subunits with respect to behavior and in vivo drug responses is incompletely understood. The γ2 subunit is highly expressed throughout the brain. Global γ2 knockout mice are insensitive to the hypnotic effects of diazepam and die perinatally. Heterozygous γ2 global knockout mice are viable and have increased anxiety-like behaviors. To further investigate the role of the γ2 subunit in behavior and whole animal drug action, we used gene targeting to create a novel mouse line with attenuated γ2 expression, i.e., γ2 knockdown mice.ResultsKnockdown mice were created by inserting a neomycin resistance cassette into intron 8 of the γ2 gene. Knockdown mice, on average, showed a 65% reduction of γ2 subunit mRNA compared to controls; however γ2 gene expression was highly variable in these mice, ranging from 10–95% of normal. Immunohistochemical studies demonstrated that γ2 protein levels were also variably reduced. Pharmacological studies using autoradiography on frozen brain sections demonstrated that binding of the benzodiazepine site ligand Ro15-4513 was decreased in mutant mice compared to controls. Behaviorally, knockdown mice displayed enhanced anxiety-like behaviors on the elevated plus maze and forced novelty exploration tests. Surprisingly, mutant mice had an unaltered response to hypnotic doses of the benzodiazepine site ligands diazepam, midazolam and zolpidem as well as ethanol and pentobarbital. Lastly, we demonstrated that the γ2 knockdown mouse line can be used to create γ2 global knockout mice by crossing to a general deleter cre-expressing mouse line.ConclusionWe conclude that: 1) insertion of a neomycin resistance gene into intron 8 of the γ2 gene variably reduced the amount of γ2, and that 2) attenuated expression of γ2 increased anxiety-like behaviors but did not lead to differences in the hypnotic response to benzodiazepine site ligands. This suggests that reduced synaptic inhibition can lead to a phenotype of increased anxiety-like behavior. In contrast, normal drug effects can be maintained despite a dramatic reduction in GABAA-R targets.

GRIP1 in GABAergic synapses.

The glutamate receptor‐interacting protein GRIP1 is present in glutamatergic synapses and interacts with the GluR2/3/4c subunits of the AMPA receptors. This interaction plays important roles in trafficking, synaptic targeting, and recycling of AMPA receptors as well as in the plasticity of glutamatergic synapses. Although GRIP1 has been shown to be present at GABAergic synapses in cultured neurons, the use of EM (electron microscopy) immunocytochemistry in the intact brain has failed to convincingly reveal the presence of GRIP1 in GABAergic synapses. Therefore, most studies on GRIP1 have focused on glutamatergic synapses. By using mild tissue fixation and embedding in EM, we show that in the intact brain the 7‐PDZ domain GRIP1a/b is present not only in glutamatergic synapses but also in GABAergic synapses. In GABAergic synapses GRIP1a/b localizes both at the presynaptic terminals and postsynaptically, being frequently localized on the synaptic membranes or the synaptic junctional complex. Considerably higher density of GRIP1a/b is found in the presynaptic GABAergic terminals than in the glutamatergic terminals, while the density of GRIP1a/b in the postsynaptic complex is similar in both types of synapses. The results also show that the 7‐PDZ and the shorter 4‐PDZ domain splice forms of GRIP1 (GRIP1c 4‐7) frequently colocalize with each other in individual GABAergic and glutamatergic synapses. The results suggest that GRIP1 splice forms might play important roles in brain GABAergic synapses. J. Comp. Neurol. 488:11–27, 2005.

Two pools of Triton X‐100‐insoluble GABAA receptors are present in the brain, one associated to lipid rafts and another one to the post‐synaptic GABAergic complex

Rat forebrain synaptosomes were extracted with Triton X‐100 at 4°C and the insoluble material, which is enriched in post‐synaptic densities (PSDs), was subjected to sedimentation on a continuous sucrose gradient. Two pools of Triton X‐100‐insoluble γ‐aminobutyric acid type‐A receptors (GABAARs) were identified: (i) a higher‐density pool (ρ = 1.10–1.15 mg/mL) of GABAARs that contains the γ2 subunit (plus α and β subunits) and that is associated to gephyrin and the GABAergic post‐synaptic complex and (ii) a lower‐density pool (ρ = 1.06–1.09 mg/mL) of GABAARs associated to detergent‐resistant membranes (DRMs) that contain α and β subunits but not the γ2 subunit. Some of these GABAARs contain the δ subunit. Two pools of GABAARs insoluble in Triton X‐100 at 4°C were also identified in cultured hippocampal neurons: (i) a GABAAR pool that forms clusters that co‐localize with gephyrin and remains Triton X‐100‐insoluble after cholesterol depletion and (ii) a GABAAR pool that is diffusely distributed at the neuronal surface that can be induced to form GABAAR clusters by capping with an anti‐α1 GABAAR subunit antibody and that becomes solubilized in Triton X‐100 at 4°C after cholesterol depletion. Thus, there is a pool of GABAARs associated to lipid rafts that is non‐synaptic and that has a subunit composition different from that of the synaptic GABAARs. Some of the lipid raft‐associated GABAARs might be involved in tonic inhibition.

Clustered and non-clustered GABAA receptors in cultured hippocampal neurons

In cultured hippocampal neurons, gamma2 subunit-containing GABA(A) Rs form large postsynaptic clusters at GABAergic synapses and small clusters outside GABAergic synapses. We now show that a pool of non-clustered gamma2 subunit-containing GABA(A) Rs are also present at the cell surface. We also demonstrate that myc- or EGFP-tagged gamma2, alpha2, beta3 or alpha1 subunits expressed in these neurons assemble with endogenous subunits, forming GABA(A) Rs that target large postsynaptic clusters, small clusters outside GABAergic synapses or a pool of non-clustered surface GABA(A) Rs. In contrast, myc- or EGFP-tagged delta subunits only form non-clustered GABA(A) Rs, which can be induced to form clusters by antibody capping. A myc-tagged chimeric gamma2 subunit possessing the large intracellular loop (IL) of the delta-subunit IL (myc gamma2S/delta-IL) assembled into GABA(A) Rs, but it did not form clusters, therefore behaving like the delta subunit. Thus, the large intracellular loops of gamma2 and delta play an important role in determining the synaptic clustering/non-clustering capacity of the GABA(A) Rs.

Biochemical analysis of GABAA receptor subunits α1, α5, β1, β2 in the hippocampus of patients with Alzheimer's disease neuropathology

Abstract Alzheimers disease (AD) is characterized by selective vulnerability of specific neuronal populations within particular brain regions. For example, hippocampal glutamatergic cell populations within the CA1/subicular pyramidal cell fields have been found to be particularly vulnerable early in AD progression. In contrast, hippocampal GABA-ergic neurons and receptors appear resistant to neurodegeneration. Despite relative sparing of GABA A receptors in AD, it is possible that the specific subunit composition of these receptors may undergo alterations with disease progression. In order to address this issue, we employed quantitative Western blot analysis to examine protein levels of GABA A receptor subunits α1, α5, β1, β2 in the hippocampus of subjects displaying increasing severity of AD neuropathology. Subjects were categorized into three groups based upon Braak staging pathologic criteria: pathologically mild (stages I/II, n =9); moderate (stages III/IV, n =8); and severe (stages V/VI, n =7). Across all subject groups, levels of subunit protein were heterogeneously distributed throughout the five hippocampal subregions analyzed (subiculum, CA1–3, dentate gyrus). Statistical analyses revealed differential preservation of GABA A receptor subunits in AD. In particular, α1, β1, and β2 displayed little difference in protein levels among pathologically mild, moderate, and severe subject groups. In contrast, although relatively modest, protein levels of the α5 subunit were significantly reduced between subjects with severe neuropathology compared with pathologically mild subjects (13.5% reduction). Collectively, our data provide evidence for heterogeneous distribution and relative sparing of GABA A receptor subunits in the hippocampus of AD patients.

Synaptic and nonsynaptic localization of protocadherin-γC5 in the rat brain

It has been proposed that γ‐protocadherins (Pcdh‐γs) are involved in the establishment of specific patterns of neuronal connectivity. Contrary to the other Pcdh‐γs, which are expressed in the embryo, Pcdh‐γC5 is expressed postnatally in the brain, coinciding with the peak of synaptogenesis. We have developed an antibody specific for Pcdh‐γC5 to study the expression and localization of Pcdh‐γC5 in brain. Pcdh‐γC5 is highly expressed in the olfactory bulb, corpus striatum, dentate gyrus, CA1 region of the hippocampus, layers I and II of the cerebral cortex, and molecular layer of the cerebellum. Pcdh‐γC5 is expressed in both neurons and astrocytes. In hippocampal neuronal cultures, and in the absence of astrocytes, a significant percentage of synapses, more GABAergic than glutamatergic, have associated Pcdh‐γC5 clusters. Some GABAergic axons show Pcdh‐γC5 in the majority of their synapses. Nevertheless, many Pcdh‐γC5 clusters are not associated with synapses. In the brain, significant numbers of Pcdh‐γC5 clusters are located at contact points between neurons and astrocytes. Electron microscopic immunocytochemistry of the rat brain shows that 1) Pcdh‐γC5 is present in some GABAergic and glutamatergic synapses both pre‐ and postsynaptically; 2) Pcdh‐γC5 is also extrasynaptically localized in membranes and in cytoplasmic organelles of neurons and astrocytes; and 3) Pcdh‐γC5 is also localized in perisynaptic astrocyte processes. The results support the notions that 1) Pcdh‐γC5 plays a role in synaptic specificity and/or synaptic maturation and 2) Pcdh‐γC5 is involved in neuron–neuron synaptic interactions and in neuron–astrocyte interactions, including perisynaptic neuron–astrocyte interactions. J. Comp. Neurol. 518:3439–3463, 2010.