GABAA receptors (GABAARs) are ligand‐gated Cl− channels that mediate most of the fast inhibitory neurotransmission in the central nervous system (CNS). Multiple GABAAR subtypes are assembled from a ...family of 19 subunit genes, raising the question of the significance of this heterogeneity. In this review, we discuss the evidence that GABAAR subtypes represent distinct receptor populations with a specific spatio‐temporal expression pattern in the developing and adult CNS, being endowed with unique functional and pharmacological properties, as well as being differentially regulated at the transcriptional, post‐transcriptional and translational levels. GABAAR subtypes are targeted to specific subcellular domains to mediate either synaptic or extrasynaptic transmission, and their action is dynamically regulated by a vast array of molecular mechanisms to adjust the strength of inhibition to the changing needs of neuronal networks. These adaptations involve not only changing the gating or kinetic properties of GABAARs, but also modifying the postsynaptic scaffold organised by gephyrin to anchor specific receptor subtypes at postsynaptic sites. The significance of GABAAR heterogeneity is particularly evident during CNS development and adult neurogenesis, with different receptor subtypes fulfilling distinct steps of neuronal differentiation and maturation. Finally, analysis of the specific roles of GABAAR subtypes reveals their involvement in the pathophysiology of major CNS disorders, and opens novel perspectives for therapeutic intervention. In conclusion, GABAAR subtypes represent the substrate of a multifaceted inhibitory neurotransmission system that is dynamically regulated and performs multiple operations, contributing globally to the proper development, function and plasticity of the CNS.
GABAA receptor heterogeneity arises through combinatorial assembly of a large family of subunits to generate multiple receptor subtypes. It is an important facet of the variety of GABAergic signaling in adult and developing CNS, and a key factor underlying GABAergic synaptic plasticity underlying excitatory/inhibitory balance in neuronal circuits. This review presents and discusses recent progress in elucidating the relevance of GABAA receptor heterogeneity for CNS function in health and disease.
The neurotransmitters GABA and glycine mediate fast synaptic inhibition by activating ligand-gated chloride channels--namely, type A GABA (GABA(A)) and glycine receptors. Both types of receptors are ...anchored postsynaptically by gephyrin, which self-assembles into a scaffold and interacts with the cytoskeleton. Current research indicates that postsynaptic gephyrin clusters are dynamic assemblies that are held together and regulated by multiple protein-protein interactions. Moreover, post-translational modifications of gephyrin regulate the formation and plasticity of GABAergic synapses by altering the clustering properties of postsynaptic scaffolds and thereby the availability and function of receptors and other signalling molecules. Here, we discuss the formation and regulation of the gephyrin scaffold, its role in GABAergic and glycinergic synaptic function and the implications for the pathophysiology of brain disorders caused by abnormal inhibitory neurotransmission.
Immunohistochemistry is a sensitive and versatile method widely used to investigate the cyto‐ and chemoarchitecture of the brain. It is based on the high affinity and selectivity of antibodies for a ...single epitope. However, it is now recognized that the specificity of antibodies needs to be tested in control experiments to avoid false‐positive results due to non‐specific binding to tissue components or recognition of epitopes shared by several molecules. This ‘Technical Spotlight’ discusses other pitfalls, which are often overlooked, although they can strongly influence the outcome of immunohistochemical experiments. It also recapitulates the minimal set of information that should be provided in scientific publications to allow proper evaluation and replication of immunohistochemical experiments. In particular, tissue fixation and processing can have a strong impact on antigenicity by producing conformational changes to the epitopes, limiting their accessibility (epitope masking) or generating high non‐specific background. These effects are illustrated for an immunoperoxidase staining experiment with three antibodies differing in susceptibility to fixation, using tissue from mice processed under identical conditions, except for slight variations in tissue fixation. In these examples, specific immunostaining can be abolished depending on fixation strength, or detected only after prolonged postfixation. As a consequence, antibody characterization in immunohistochemistry should include their susceptibility towards fixation and determination of the optimal conditions for their use.
Aberrant epileptic activity is detectable at early disease stages in Alzheimer's disease (AD) patients and in AD mouse models. Here, we investigated in young ArcticAβ mice whether AD‐like pathology ...renders neuronal networks more susceptible to the development of acquired epilepsy induced by unilateral intrahippocampal injection of kainic acid (IHK). In this temporal lobe epilepsy model, IHK induces a status epilepticus followed after two weeks by spontaneous recurrent seizures (SRS). ArcticAβ mice exhibited more severe status epilepticus and early onset of SRS. This hyperexcitable phenotype was characterized in CA1 neurons by decreased synaptic strength, increased kainic acid‐induced LTP and reduced frequency of spontaneous inhibitory currents. However, no difference in neurodegeneration, neuroinflammation, axonal reorganization or adult neurogenesis was observed in ArcticAβ mice compared to wild‐type littermates following IHK‐induced epileptogenesis. Neuropeptide Y (NPY) expression was reduced at baseline and its IHK‐induced elevation in mossy fibres and granule cells was attenuated. However, although this alteration might underlie premature seizure onset, neutralization of soluble Aβ species by intracerebroventricular Aβ‐specific antibody application mitigated the hyperexcitable phenotype of ArcticAβ mice and prevented early SRS onset. Therefore, the development of seizures at early stages of AD is mediated primarily by Aβ species causing widespread changes in synaptic function.
Arctic Aβ mice, a model of familial Alzheimer's disease are more susceptible to epileptogenic stimuli. Neutralization of soluble Aβ species normalizes their phenotype, suggesting that Aβ interferes with GABAergic transmission.
GABAA receptors mediate most of the fast inhibitory transmission in the CNS. They form heteromeric complexes assembled from a large family of subunit genes. The existence of multiple GABAA receptor ...subtypes differing in subunit composition, localization and functional properties underlies their role for fi ne-tuning of neuronal circuits and genesis of network oscillations. The differential regulation of GABAA receptor subtypes represents a major facet of homeostatic synaptic plasticity and contributes to the excitation/inhibition (E/I) balance under physiological conditions and upon pathological challenges. The purpose of this review is to discuss recent fi ndings highlighting the signifi cance of GABAA receptor heterogeneity for the concept of E/I balance and its relevance for epilepsy. Specifi cally, we address the following issues: (1) role for tonic inhibition, mediated by extrasynaptic GABAA receptors, for controlling neuronal excitability; (2) signifi cance of chloride ion transport for maintenance of the E/I balance in adult brain; and (3) molecular mechanisms underlying GABAA receptor regulation (traffi cking, posttranslational modifi cation, gene transcription) that are important for homoeostatic plasticity. Finally, the relevance of these fi ndings is discussed in light of the involvement of GABAA receptors in epileptic disorders, based on recent experimental studies of temporal lobe epilepsy (TLE) and absence seizures and on the identifi cation of mutations in GABAA receptor subunit genes underlying familial forms of epilepsy.
The striatum is the main input nucleus of the basal ganglia, mediating motor and cognitive functions. Striatal projection neurons are GABAergic medium spiny neurons (MSN), expressing either the ...dopamine receptor type 1 (D1‐R MSN) and forming the direct, movement‐promoting pathway, or dopamine receptor type 2 (D2‐R MSN), forming the indirect movement‐suppressing pathway. Locally, activity and synchronization of MSN are modulated by several subtypes of GABAergic and cholinergic interneurons. Overall, GABAergic circuits in the striatum remain poorly characterized, and little is known about the intrastriatal connectivity of interneurons and the distribution of GABAA receptor (GABAAR) subtypes, distinguished by their subunit composition, in striatal synapses. Here, by using immunofluorescence in mouse tissue, we investigated the distribution of GABAARs containing the α1, α2, or α3 subunit in perisomatic synapses of striatal MSN and interneurons, as well as the innervation pattern of D1R‐ and D2R‐MSN soma and axonal initial segment (AIS) by GABAergic and cholinergic interneurons. Our results show that perisomatic GABAergic synapses of D1R‐ and D2R‐MSN contain the GABAAR α1 and/or α2 subunits, but not the α3 subunit; D2R‐MSN have significantly more α1‐GABAARs on their soma than D1R‐MSN. Further, interneurons have few perisomatic synapses containing α2‐GABAARs, whereas α3‐GABAARs (along with the α1‐GABAARs) are abundant in perisomatic synapses of CCK+, NPY+/SOM+, and vAChT+ interneurons. Each MSN and interneuron population analyzed received a distinct pattern of GABAergic and cholinergic innervation, complementing this postsynaptic heterogeneity. In conclusion, intra‐striatal GABAergic circuits are distinguished by cell‐type specific innervation patterns, differential expression and postsynaptic targeting of GABAAR subtypes.
The distribution of GABAARs containing the α1, α2, and α3 subunit in perisomatic GABAergic synapses was investigated in D1R‐ and D2R‐MSN, GABAergic (PV+, CCK+, NPY+, and SOM+) and cholinergic interneurons in the dorsolateral striatum. In D1R‐ and D2R‐MSN the majority of synapses contained GABAAR α1 and α2 subunits, whereas the GABAAR α3 subunit is specific for certain subpopulations of striatal interneurons. In turn, perisomatic synapses originate from different sources, indicating that the expression of GABAARs is not random, but correlates with the expression of specific neurochemical markers distinguishing specific GABAergic circuits in the dorsal striatum.
Clinical and experimental evidence indicates that inflammatory processes contribute to the pathophysiology of epilepsy, but underlying mechanisms remain mostly unknown. Using immunohistochemistry for ...CD45 (common leukocyte antigen) and CD3 (T-lymphocytes), we show here microglial activation and infiltration of leukocytes in sclerotic tissue from patients with mesial temporal lobe epilepsy (TLE), as well as in a model of TLE (intrahippocampal kainic acid injection), characterized by spontaneous, nonconvulsive focal seizures. Using specific markers of lymphocytes, microglia, macrophages, and neutrophils in kainate-treated mice, we investigated with pharmacological and genetic approaches the contribution of innate and adaptive immunity to kainate-induced inflammation and neurodegeneration. Furthermore, we used EEG analysis in mutant mice lacking specific subsets of lymphocytes to explore the significance of inflammatory processes for epileptogenesis. Blood-brain barrier disruption and neurodegeneration in the kainate-lesioned hippocampus were accompanied by sustained ICAM-1 upregulation, microglial cell activation, and infiltration of CD3(+) T-cells. Moreover, macrophage infiltration was observed, selectively in the dentate gyrus where prominent granule cell dispersion was evident. Unexpectedly, depletion of peripheral macrophages by systemic clodronate liposome administration affected granule cell survival. Neurodegeneration was aggravated in kainate-lesioned mice lacking T- and B-cells (RAG1-knock-out), because of delayed invasion by Gr-1(+) neutrophils. Most strikingly, these mutant mice exhibited early onset of spontaneous recurrent seizures, suggesting a strong impact of immune-mediated responses on network excitability. Together, the concerted action of adaptive and innate immunity triggered locally by intrahippocampal kainate injection contributes seizure-suppressant and neuroprotective effects, shedding new light on neuroimmune interactions in temporal lobe epilepsy.
Lack of dopamine (DA) in the striatum and the consequential dysregulation of thalamocortical circuits are major causes of motor impairments in Parkinson's disease. The striatum receives multiple ...cortical and subcortical afferents. Its role in movement control and motor skills learning is regulated by DA from the nigrostriatal pathway. In Parkinson's disease, DA loss affects striatal network activity and induces a functional imbalance of its output pathways, impairing thalamocortical function. Striatal projection neurons are GABAergic and form two functionally antagonistic pathways: the direct pathway, originating from DA receptor type 1‐expressing medium spiny neurons (D1R‐MSN), and the indirect pathway, from D2R‐MSN. Here, we investigated whether DA depletion in mouse striatum also affects GABAergic function. We recorded GABAergic miniature IPSCs (mIPSC) and tonic inhibition from D1R‐ and D2R‐MSN and used immunohistochemical labeling to study GABAAR function and subcellular distribution in DA‐depleted and control mice. We observed slower decay kinetics and increased tonic inhibition in D1R‐MSN, while D2R‐MSN had increased mIPSC frequency after DA depletion. Perisomatic synapses containing the GABAAR subunits α1 or α2 were not affected, but there was a strong decrease in non‐synaptic GABAARs containing these subunits, suggesting altered receptor trafficking. To broaden these findings, we also investigated GABAARs in GABAergic and cholinergic interneurons and found cell type‐specific alterations in receptor distribution, likely reflecting changes in connectivity. Our results reveal that chronic DA depletion alters striatal GABAergic transmission, thereby affecting cellular and circuit activity. These alterations either result from pathological changes or represent a compensatory mechanism to counteract imbalance of output pathways.
Chronic DA depletion alters striatal GABAergic transmission either by changing perisomatic GABAARs or by inducing pathophysiological changes, thereby affecting cellular and circuit activity. We found changes in synaptic GABAARs in striatal NPY, SOM, CCK, and cholinergic interneurons, but not in PV interneurons. In D1R‐ and D2R‐MSN, altered electrophysiological properties and GABAAR composition suggest changes in extrasynaptic GABAARs, dendritic innervation, and post‐translational modifications.
In dissociated neuronal cultures the absence of spatial and temporal cues causes the emergence of mismatched synapses, where post‐synaptic proteins of GABAergic synapses are in part apposed to ...glutamatergic pre‐synaptic terminals and vice versa. This mismatch offers an opportunity to study the mechanisms that regulate correct apposition of pre‐ and post‐synaptic elements. We report here that the IQ motif and Sec7 domain‐containing protein 3 (IQSEC3; BRAG3; synArfGEF) specifically regulates the mislocalization of GABAergic post‐synaptic density (PSD) proteins. Over‐expression of IQSEC3 constructs harboring mutations that ablate Sec7 domain or IQ motif function revealed that IQSEC3 catalytic activity is involved in the control of apposition between the GABAergic PSD and glutamatergic terminals. Neurons co‐expressing eGFP‐gephyrin with IQSEC3 Sec7 mutant displayed a drastically increased fraction of mismatched eGFP‐gephyrin clusters compared to other IQSEC3 constructs. Along with eGFP‐gephyrin, endogenous GABAA receptor cluster mismatching was increased by IQSEC3 Sec7 mutant over‐expression. Conversely, GFP‐PSD‐95 clusters were unaffected by over‐expression of any IQSEC3 construct. The GABAergic PSD mismatch phenotype was recapitulated by Arf6 dominant‐negative mutant over‐expression, suggesting that Arf6 activation by IQSEC3 is an essential step in this pathway. In addition, we provide biochemical evidence to confirm gephyrin/IQSEC3 interaction near the IQSEC3 IQ motif, which in turn binds calmodulin at low Ca2+ concentrations. Taken together, our findings identify a post‐synaptic protein which specifically regulates correct apposition of the GABAergic PSD to pre‐synaptic terminals.
During synapse formation, proteins of the post‐synaptic density are properly sorted and aggregated at sites facing the pre‐synaptic terminal releasing a specific neurotransmitter, for example, GABA versus glutamate. This study reports that the guanine nucleotide exchange factor IQSEC3, activating the small GTPase of the ADP‐ribosylation factor family Arf6, regulates the proper matching of the post‐synaptic protein gephyrin in GABAergic versus glutamatergic synapses. IQSEC3 function is modulated by binding to gephyrin and calmodulin and activation of Arf6 by IQSEC3 selectively reduces post‐synaptic clustering of gephyrin in glutamatergic synapses.
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