Subcellular localization of GABAB receptor subunits in rat globus pallidus

The inhibitory amino acid gamma-aminobutyric acid (GABA) is the major neurotransmitter in the globus pallidus. Although electrophysiological studies have indicated that functional GABA(B) receptors exist in rat globus pallidus, the subcellular localization of GABA(B) receptor subunits and their spatial relationship to glutamatergic and GABAergic synapses are unknown. Here, we use pre-embedding immunogold labeling to study the subcellular localization of GABA(B) receptor subunits, GABA(B1) and GABA(B2), in globus pallidus neurons and identified populations of afferent terminals. Immunolabeling for GABA(B1) and GABA(B2) was observed throughout the globus pallidus, with GABA(B1) more strongly expressed in perikarya and GABA(B2) mainly expressed in the neuropil. Electron microscopic analysis revealed that the majority of GABA(B1) labeling was localized within the cytoplasm, whereas most of GABA(B2) labeling was associated with the plasma membrane. At the subcellular level, both the GABA(B1) and GABA(B2) immunogold labeling was localized at pre- and postsynaptic sites. At asymmetric, putative excitatory, synapses, GABA(B1) and GABA(B2) immunogold labeling was found at perisynaptic sites of both pre- and postsynaptic specializations. Double immunolabeling, using the vesicular glutamate transporter 2 (VGLUT2), revealed the glutamatergic nature of most immunogold-labeled asymmetric synapses. At symmetric, putative GABAergic, synapses, including those formed by anterogradely labeled striatopallidal terminals, GABA(B1) and GABA(B2) immunogold labeling was found in the main body of both pre- and postsynaptic specializations. These results demonstrate the existence of presynaptic GABA(B) auto- and heteroreceptors and postsynaptic GABA(B) receptors, which may be involved in modulating synaptic transmission in the globus pallidus.     

Immunogold characteristics of VGLUT3‐positive GABAergic nerve terminals suggest corelease of glutamate

There is compelling evidence that glutamate can act as a cotransmitter in the mammalian brain. Interestingly, the third vesicular glutamate transporter (VGLUT3) is primarily found in neurons that were anticipated to be nonglutamatergic. Whereas the function of VGLUT3 in acetylcholinergic and serotoninergic neurons has been elucidated, the role of VGLUT3 in neurons releasing gamma‐aminobutyric acid (GABA) is not settled. We have previously shown that VGLUT3 is found together with the vesicular GABA transporter (VIAAT) on synaptic vesicle membranes in the hippocampus. Now we provide novel electron microscopic data from the rat hippocampus suggesting that glutamate is enriched in inhibitory nerve terminals containing VGLUT3 compared to those lacking VGLUT3. The opposite was found for GABA; VGLUT3‐positive inhibitory terminals contained lower density of GABA labeling compared to VGLUT3‐negative inhibitory terminals. In addition, semiquantitative confocal immunofluorescence showed that N‐methyl‐D‐aspartate (NMDA)‐receptor labeling was present more frequently in VGLUT3‐positive/VIAAT‐positive synapses versus in VGLUT3‐negative/VIAAT‐positive synapses. Electron microscopic immunogold data further suggest that NMDA receptors are enriched in VGLUT3 containing inhibitory terminals. Our data reveal new chemical characteristics of a subset of GABAergic interneurons in the hippocampus. The analyses suggest that glutamate is coreleased with GABA from hippocampal basket cell‐synapses to act on NMDA receptors. J. Comp. Neurol. 523:2698–2713, 2015. © 2015 Wiley Periodicals, Inc.     

Plasticity of synaptic inhibition in mouse spinal cord lamina II neurons during early postnatal development and after inactivation of the glycine receptor α3 subunit gene

Synaptic inhibition mediated by GABA_A receptors and glycine receptors (GlyRs) in the outer laminae of the spinal cord dorsal horn efficiently filters ascending nociceptive messages, controlling pathological pain symptoms. However, although many studies have utilized transgenic models to study spinal nociceptive processing, very little is known about the development of functional inhibitory synapses onto these interneurons in mice. Here we report that most interneurons in lamina II are placed under phasic control by both GABAergic and glycinergic synapses, a number of which exhibit dual GABA/glycine co‐release. A developmental switch is also apparent: a subpopulation of lamina II interneurons controlled exclusively by either GABAergic or glycinergic synapses becomes detectable only after postnatal days 15 and 21, respectively. Using mice older than postnatal day 21, we also characterized the plastic changes in glycinergic transmission resulting from the inactivation of the GlyR α3 subunit gene, a key player in inflammatory pain pathways. This allowed us to demonstrate that synapses containing GlyR α3 contribute in large part to synaptic inhibition in lamina II. In Glra3 knockout mice, we found that synaptic currents at the remaining glycinergic synapses, containing GlyR α1, showed faster decay kinetics with unchanged amplitudes but increased frequency. These findings explain the absence of any basal nociceptive hypersensitivity in Glra3 knockout mice, as GlyR α1 is still available for mediating synaptic inhibition at lamina II synapses, but cannot be modulated by the prostaglandin–E‐prostanoid type 2 (EP2) receptor–protein kinase A signalling cascade. Our results clearly demonstrate that presynaptic GABA/glycine release properties are influenced by the nature and complexity of postsynaptic inhibitory receptor subtypes.     

Vesicular glutamate transporter‐3 in the rodent brain: Vesicular colocalization with vesicular γ‐aminobutyric acid transporter

Vesicular glutamate transporters (VGLUT1–3) carry glutamate into synaptic vesicles. VGLUT3 has been reported to be localized in nonglutamatergic neuronal populations in the brain. However, detailed subcellular localization of VGLUT3 has not been shown. In particular, the identity of synaptic vesicles expressing VGLUT3 remains to be revealed. Here we present novel electron microscopic postembedding immunogold data from mouse and rat brains showing that small, clear, and round synaptic vesicles in γ‐aminobutyric acid (GABA)‐ergic nerve terminals contain labeling for both VGLUT3 and the vesicular GABA transporter (VGAT). Immunoisolation of synaptic vesicles confirmed the immunogold data and showed vesicular colocalization of VGLUT3 and VGAT. Moreover, we show that gold particles signaling VGLUT3 are present in synaptic vesicles in acetylcholinergic nerve terminals in the striatum. Quantitative immunogold analyses reveal that the density of VGLUT3 gold particles is similar in GABAergic terminals in the hippocampus and the neocortex to that in cholinergic terminals in the striatum. In contrast to in the hippocampus and the neocortex, VGLUT3 was absent from VGAT‐positive terminals in the striatum. The labeling pattern produced by the VGLUT3 antibodies was found to be specific; there was no labeling in VGLUT3 knockout tissue, and the observed labeling throughout the rat brain corresponds to the known light‐microscopic distribution of VGLUT3. From the present results, we infer that glutamate is released with GABA from inhibitory terminals and acetylcholine from cholinergic terminals. J. Comp. Neurol. 521: 3042–3056, 2013. © 2013 Wiley Periodicals, Inc.     

Long‐term,dynamic synaptic reorganization after GABAergic precursor cell transplantation into adult mouse spinal cord

Transplanting embryonic precursors of GABAergic neurons from the medial ganglionic eminence (MGE) into adult mouse spinal cord ameliorates mechanical and thermal hypersensitivity in peripheral nerve injury models of neuropathic pain. Although Fos and transneuronal tracing studies strongly suggest that integration of MGE‐derived neurons into host spinal cord circuits underlies recovery of function, the extent to which there is synaptic integration of the transplanted cells has not been established. Here, we used electron microscopic immunocytochemistry to assess directly integration of GFP‐expressing MGE‐derived neuronal precursors into dorsal horn circuitry in intact, adult mice with short‐ (5–6 weeks) or long‐term (4–6 months) transplants. We detected GFP with pre‐embedding avidin–biotin‐peroxidase and GABA with post‐embedding immunogold labeling. At short and long times post‐transplant, we found host‐derived synapses on GFP‐immunoreactive MGE cells bodies and dendrites. The proportion of dendrites with synaptic input increased from 50% to 80% by 6 months. In all mice, MGE‐derived terminals formed synapses with GFP‐negative (host) cell bodies and dendrites and, unexpectedly, with some GFP‐positive (i.e., MGE‐derived) dendrites, possibly reflecting autoapses or cross talk among transplanted neurons. We also observed axoaxonic appositions between MGE and host terminals. Immunogold labeling for GABA confirmed that the transplanted cells were GABAergic and that some transplanted cells received an inhibitory GABAergic input. We conclude that transplanted MGE neurons retain their GABAergic phenotype and integrate dynamically into host‐transplant synaptic circuits. Taken together with our previous electrophysiological analyses, we conclude that MGE cells are not GABA pumps, but alleviate pain and itch through synaptic release of GABA.     

Differential regulation of GABA(A) receptor and gephyrin postsynaptic clustering in immature hippocampal neuronal cultures

Gephyrin is a postsynaptic scaffolding protein involved in clustering of glycine- and GABA(A) receptors at inhibitory synapses. The role of gephyrin in GABAergic synapses, the nature of its interactions with GABA(A) receptors, and the mechanisms of targeting to GABAergic synapses are largely unknown. To gain further insights into these questions, the formation of GABA(A) receptor and gephyrin clusters and their distribution relative to presynaptic terminals were investigated in immature cultures of embryonic hippocampal neurons using triple immunofluorescence staining. GABA(A) receptor clusters, labeled for the alpha2 subunit, formed independently of gephyrin clusters, and were distributed on neurites at constant densities, either extrasynaptically or, to a lesser extent, postsynaptically, apposed to synapsin-I-positive axon terminals. In contrast, gephyrin clusters were always associated with GABA(A) receptors and were preferentially localized postsynaptically. Their density increased linearly with the extent of innervation, which developed rapidly during the first week in vitro. These results suggested that GABA(A) receptor clustering is mediated by cell-autonomous mechanisms independent of synapse formation. Their association with gephyrin is dynamically regulated and may contribute to stabilization at postsynaptic sites. Labeling for vesicular glutamate transporters revealed that most synapses in these immature cultures were presumably glutamatergic, implying that postsynaptic GABA(A) receptor and gephyrin clusters initially were located in "mismatched" synapses. However, clusters appropriately localized in GABAergic synapses were distinctly larger and more intensely stained. Altogether, these results demonstrate that the targeting of GABA(A) receptor and gephyrin clusters to GABAergic synapses occurs secondarily and is regulated by presynaptic factors that are not essential for clustering.     

GABAergic and glutamatergic axons innervate the axon initial segment and organize GABA(A) receptor clusters of cultured hippocampal pyramidal cells

We have studied gamma-aminobutyric acid (GABA)(A) receptor (GABA(A)R) clustering within the axon initial segment (AIS) in low-density cultures of hippocampal pyramidal cells following GABAergic and glutamatergic innervation of the AIS. Large, intensely fluorescent, and postsynaptic GABA(A)R clusters were present in the AIS. More than 95% of these clusters colocalized with presynaptic GABAergic or glutamatergic terminals, forming matched or mismatched synapses, respectively. Less than 5% of the GABA(A)R clusters of the AIS did not colocalize with GABAergic or glutamatergic terminals, suggesting that GABA(A)Rs normally do not form clusters unless the AIS received GABAergic or glutamatergic innervation. Few or no clusters of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors or the postsynaptic density-95 protein (PSD-95) were found in the AIS, even when the AIS was innervated by glutamatergic axons. Glutamatergic innervation of the AIS that formed mismatched synapses with postsynaptic GABA(A)R clusters mainly occurred when the AIS did not receive GABAergic innervation. However, when the AIS was innervated by GABAergic axons, the formation of matched GABAergic synapses predominated and coincided with large reductions in both the density of glutamatergic terminals from the AIS and the mismatching of GABA(A)R clusters. A similar effect was observed at axo-dendritic synapses, where GABAergic innervation also led to a large decrease in mismatched GABA(A)R clusters and a smaller, but significant, decrease in glutamatergic terminal density in dendrites that received GABAergic innervation. We hypothesize that competition between GABAergic and glutamatergic innervation of the AIS in the intact hippocampus leads to the exclusive presence of GABAergic inhibitory synapses in the AIS of pyramidal cells.     

Postsynaptic gephyrin clustering controls the development of adult‐born granule cells in the olfactory bulb

In adult rodent olfactory bulb, GABAergic signaling regulates migration, differentiation, and synaptic integration of newborn granule cells (GCs), migrating from the subventricular zone. Here we show that these effects depend on the formation of a postsynaptic scaffold organized by gephyrin—the main scaffolding protein of GABAergic synapses, which anchors receptors and signaling molecules to the postsynaptic density—and are regulated by the phosphorylation status of gephyrin. Using lentiviral vectors to selectively transfect adult‐born GCs, we observed that overexpression of the phospho‐deficient gephyrin mutant eGFP‐gephyrin(S270A), which facilitates the formation of supernumerary GABAergic synapses in vitro, favors dendritic branching and the formation of transient GABAergic synapses on spines, identified by the presence of α2‐GABA_ARs. In contrast, overexpression of the dominant‐negative eGFP‐gephyrin(L2B) (a chimera that is enzymatically active but clustering defective), curtailed dendritic growth, spine formation, and long‐term survival of GCs, pointing to the essential role of gephyrin cluster formation for its function. We could exclude any gephyrin overexpression artifacts, as GCs infected with eGFP‐gephyrin were comparable to those infected with eGFP alone. The opposite effects induced by the two gephyrin mutant constructs indicate that the gephyrin scaffold at GABAergic synapses orchestrates signaling cascades acting on the cytoskeleton to regulate neuronal growth and synapse formation. Specifically, gephyrin phosphorylation emerges as a novel mechanism regulating morphological differentiation and long‐term survival of adult‐born olfactory bulb neurons. J. Comp. Neurol. 523:1998–2016, 2015 © 2015 Wiley Periodicals, Inc.     

Localization of the GABAB receptor 1a/b subunit relative to glutamatergic synapses in the dorsal cochlear nucleus of the rat

Metabotropic gamma-aminobutyric acid receptors (GABA(B)) are involved in pre- and postsynaptic inhibitory effects upon auditory neurons and have been implicated in different aspects of acoustic information processing. To understand better the mechanisms by which GABA(B) receptors mediate their inhibitory effects, we used pre-embedding immunocytochemical techniques combined with quantification of immunogold particles to reveal the precise subcellular distribution of the GABA(B1) subunit in the rat dorsal cochlear nucleus. At the light microscopic level, GABA(B1) was detected in all divisions of the cochlear complex. The most intense immunoreactivity for GABA(B1) was found in the dorsal cochlear nucleus, whereas immunoreactivity in the anteroventral and posteroventral cochlear nuclei was very low. In the dorsal cochlear nucleus, a punctate labeling was observed in the superficial (molecular and fusiform cell) layers. At the electron microscopic level, GABA(B1) was found at both post- and presynaptic locations. Postsynaptically, GABA(B1) was localized mainly in the dendritic spines of presumed fusiform cells. Quantitative immunogold immunocytochemistry revealed that the highest concentration of GABA(B1) in the plasma membrane was in dendritic spines, followed by dendritic shafts and somata. Thus, the most intense immunoreactivity for GABA(B1) was observed in dendritic spines with a high density of immunogold particles at extrasynaptic sites, peaking around 300 nm from glutamatergic synapses. This is in contrast to GABAergic synapses, in which GABA(B1) was only occasionally found. Presynaptically, receptor immunoreactivity was detected primarily in axospinous endings, probably from granule cells, in both the active zone and extrasynaptic sites. The localization of GABA(B1) relative to synaptic sites in the DCN suggests a role for the receptor in the regulation of dendritic excitability and excitatory inputs.     

Development of gamma-aminobutyric acidergic synapses in cultured hippocampal neurons

The formation and maturation of gamma-aminobutyric acid (GABA)-ergic synapses was studied in cultured hippocampal pyramidal neurons by both performing immunocytochemistry for GABAergic markers and recording miniature inhibitory postsynaptic currents (mIPSCs). Nascent GABAergic synapses appeared between 3 and 8 days in vitro (DIV), with GABAA receptor subunit clusters appearing first, followed by GAD-65 puncta, then functional synapses. The number of GABAergic synapses increased from 7 to 14 DIV, with a corresponding increase in frequency of mIPSCs. Moreover, these new GABAergic synapses formed on neuronal processes farther from the soma, contributing to decreased mIPSC amplitude and slowed mIPSC 19-90% rise time. The mIPSC decay quickened from 7 to 14 DIV, with a parallel change in the distribution of the alpha5 subunit from diffuse expression at 7 DIV to clustered expression at 14 DIV. These alpha5 clusters were mostly extrasynaptic. The alpha1 subunit was expressed as clusters in none of the neurons at 7 DIV, in 20% at 14 DIV, and in 80% at 21 DIV. Most of these alpha1 clusters were expressed at GABAergic synapses. In addition, puncta of GABA transporter 1 (GAT-1) were localized to GABAergic synapses at 14 DIV but were not expressed at 7 DIV. These studies demonstrate that mIPSCs appear after pre- and postsynaptic elements are in place. Furthermore, the process of maturation of GABAergic synapses involves increased synapse formation at distal processes, expression of new GABAA receptor subunits, and GAT-1 expression at synapses; these changes are reflected in altered frequency, kinetics, and drug sensitivity of mIPSCs.     

Pre- and postsynaptic GABA receptors at reciprocal dendrodendritic synapses in the olfactory bulb

Presynaptic ionotropic receptors are important regulators of synaptic function; however, little is known about their organization in the presynaptic membrane. We show here a different spatial organization of presynaptic and postsynaptic GABA(A) receptors at reciprocal dendrodendritic synapses between mitral and granule cells in the rat olfactory bulb. Using postembedding electron microscopy, we have found that mitral cell dendrites express GABA(A) receptors at postsynaptic specializations of symmetric (GABAergic) synapses, as well as at presynaptic sites of asymmetric (glutamatergic) synapses. Analysis of the subsynaptic distribution of gold particles revealed that in symmetric synapses GABA(A) receptors are distributed along the entire postsynaptic membrane, whereas in asymmetric synapses they are concentrated at the edge of the presynaptic specialization. To assess the specificity of immunogold labelling, we analysed the olfactory bulbs of mutant mice lacking the alpha1 subunit of GABA(A) receptors. We found that in wild-type mice alpha1 subunit immunoreactivity was similar to that observed in rats, whereas in knockout mice the immunolabelling was abolished. These results indicate that in mitral cell dendrites GABA(A) receptors are distributed in a perisynaptic domain that surrounds the presynaptic specialization. Such presynaptic receptors may be activated by spillover of GABA from adjacent inhibitory synapses and modulate glutamate release, thereby providing a novel mechanism regulating dendrodendritic inhibition in the olfactory bulb.     

Depolarizing GABAergic synaptic input triggers endocannabinoid‐mediated retrograde synaptic signaling

Endocannabinoids released by postsynaptic neurons inhibit neurotransmitter release from presynaptic axon terminals. One typical stimulus of endocannabinoid production is an increase of calcium concentration in postsynaptic neurons. The aim of the present study was to clarify whether depolarizing GABAergic synaptic input, by increasing calcium concentration in postsynaptic neurons, can trigger endocannabinoid production. Spontaneous GABAergic inhibitory postsynaptic currents (sIPSCs) were recorded in Purkinje cells in mouse cerebellar slices with patch‐clamp pipettes containing 151 mM chloride (a usual recording mode). sIPSCs were depolarizing inward currents under this condition. Combined electrophysiological and fluorometric calcium imaging experiments indicated that sIPSCs frequently triggered calcium spikes. After the calcium spikes, a short‐term suppression of sIPSCs occurred. This suppression was prevented by the CB₁ cannabinoid receptor antagonist rimonabant and the diacylglycerol lipase inhibitor orlistat, but not changed by URB597, an inhibitor of anandamide degradation. It is, therefore, likely that CB₁ receptors and 2‐arachidonoylglycerol were involved. For testing the physiological significance of the above observation, we carried out experiments on brains of 3‐ to 5‐day‐old mice. The gramicidin‐induced perforated patch‐clamp mode was used for preserving the physiological intracellular chloride concentration of the neurons. Depolarizing GABAergic sIPSCs occurred under this condition, but at a very low rate. Rimonabant did not change the frequency of these sIPSCs, arguing against the persistence of an endocannabinoid tone. The results point to a new kind of trigger of endocannabinoid production: depolarizing GABAergic synaptic input can elicit endocannabinoid production in postsynaptic neurons by activating calcium channels. The produced endocannabinoid suppresses GABA release from presynaptic axon terminals. Synapse 63:643–652, 2009. © 2009 Wiley‐Liss, Inc.     

Organization of amyloid‐β protein precursor intracellular domain‐associated protein‐1 in the rat brain

Sustained activity‐dependent synaptic modifications require protein synthesis. Although proteins can be synthesized locally in dendrites, long‐term changes also require nuclear signaling. Amyloid‐β protein precursor intracellular domain‐associated protein‐1 (AIDA‐1), an abundant component of the biochemical postsynaptic density fraction, contains a nuclear localization sequence, making it a plausible candidate for synapse‐to‐nucleus signaling. We used immunohistochemistry to study the regional, cellular, and subcellular distribution of AIDA‐1. Immunostaining was prominent in the hippocampus, cerebral cortex, and neostriatum. Along with diffuse staining of neuropil, fluorescence microscopy revealed immunostaining of excitatory synapses throughout the forebrain, and immunoreactive puncta within and directly outside the nucleus. Presynaptic staining was conspicuous in hippocampal mossy fibers. Electron microscopic analysis of material processed for postembedding immunogold revealed AIDA‐1 label within postsynaptic densities in both hippocampus and cortex. Together with previous work, these data suggest that AIDA‐1 serves as a direct signaling link between synapses and the nucleus in adult rat brain. J. Comp. Neurol. 518:3221–3236, 2010. © 2010 Wiley‐Liss, Inc.     

Synaptogenesis in the cerebellar cortex: differential regulation of gephyrin and GABAA receptors at somatic and dendritic synapses of Purkinje cells

In rodent cerebellar cortex, synaptogenesis occurs entirely postnatally, allowing study of the mechanisms of synapse formation in vivo. Here we monitored the clustering of GABA(A) receptors and the scaffolding protein gephyrin at GABAergic postsynaptic sites during rat cerebellar development. We found that GABA(A) receptors and gephyrin co-aggregate at nascent synapses in the molecular and Purkinje cell layers with a similar time course. With few exceptions, gephyrin and GABA(A) receptor subunits clustered selectively in front of presynaptic boutons expressing the vesicular inhibitory amino acid transporter VIAAT and no ectopic localization of these molecules was observed. Surprisingly, gephyrin clusters outlining the cell body of Purkinje cells were transient, and disappeared rapidly at the end of the second postnatal week. The loss of gephyrin from perisomatic synapses was coincident with a significant reduction in the size of GABA(A) receptor clusters. Furthermore, these changes were accompanied by a developmental decrease in the size of synaptic appositions, as documented by electron microscopy. These findings suggest that gephyrin takes part in the initial assembly of postsynaptic specializations and reveal an unsuspected heterogeneity in the molecular organization of the postsynaptic apparatus at somatic and dendritic synapses of mature Purkinje cells.     

Distinct subsynaptic localization of type 1 metabotropic glutamate receptors at glutamatergic and GABAergic synapses in the rodent cerebellar cortex

Type 1 metabotropic glutamate (mGlu1) receptors play a pivotal role in different forms of synaptic plasticity in the cerebellar cortex, e.g. long‐term depression at glutamatergic synapses and rebound potentiation at GABAergic synapses. These various forms of plasticity might depend on the subsynaptic arrangement of the receptor in Purkinje cells that can be regulated by protein–protein interactions. This study investigated, by means of the freeze‐fracture replica immunogold labelling method, the subcellular localization of mGlu1 receptors in the rodent cerebellum and whether Homer proteins regulate their subsynaptic distribution. We observed a widespread extrasynaptic localization of mGlu1 receptors and confirmed their peri‐synaptic enrichment at glutamatergic synapses. Conversely, we detected mGlu1 receptors within the main body of GABAergic synapses onto Purkinje cell dendrites. Although Homer proteins are known to interact with the mGlu1 receptor C‐terminus, we could not detect Homer3, the most abundant Homer protein in the cerebellar cortex, at GABAergic synapses by pre‐embedding and post‐embedding immunoelectron microscopy. We then hypothesized a critical role for Homer proteins in the peri‐junctional localization of mGlu1 receptors at glutamatergic synapses. To disrupt Homer‐associated protein complexes, mice were tail‐vein injected with the membrane‐permeable dominant‐negative TAT‐Homer1a. Freeze‐fracture replica immunogold labelling analysis showed no significant alteration in the mGlu1 receptor distribution pattern at parallel fibre–Purkinje cell synapses, suggesting that other scaffolding proteins are involved in the peri‐synaptic confinement. The identification of interactors that regulate the subsynaptic localization of the mGlu1 receptor at neurochemically distinct synapses may offer new insight into its trafficking and intracellular signalling.     

The subcellular distribution of GABARAP and its ability to interact with NSF suggest a role for this protein in the intracellular transport of GABA(A) receptors.

GABA(A) receptors the major sites of fast synaptic inhibition in the brain are composed predominately of alpha, beta, and gamma2 subunits. The receptor gamma2 subunit interacts with a 17-kDa microtubule associated protein GABARAP, but the significance of this interaction remains unknown. Here we demonstrate that GABARAP, which immunoprecipitates with GABA(A) receptors, is not found at significant levels within inhibitory synapses, but is enriched within the Golgi apparatus and postsynaptic cisternae. We also demonstrate that GABARAP binds directly to N-ethylmaleimide-sensitive factor (NSF), a protein critical for intracellular membrane trafficking events. NSF and GABARAP complexes could be detected in neurons and these two proteins also colocalize within intracellular membrane compartments. Together our observations suggest that GABARAP may play a role in intracellular GABA(A) receptor transport but not synaptic anchoring, via its ability to interact with NSF. GABARAP may therefore have an important role in the production of GABAergic synapses.     

Localization of the clustering protein gephyrin at GABAergic synapses in the main olfactory bulb of the rat

The tubulin-binding protein gephyrin is essential for the formation of postsynaptic glycine-receptor clusters in cultured spinal neurons. In addition, there is increasing evidence that gephyrin can also be present at nonglycinergic synapses. Here we analyzed immunocytochemically the subcellular localization of gephyrin in the main olfactory bulb of the rat and compared its distribution with that of γ-aminobutyric acid (GABA) and of two major GABA_A-receptor subunits. Gephyrin was selectively localized to the postsynaptic side of symmetric synaptic junctions, where the presynaptic terminals contained GABA. Moreover, gephyrin colocalized extensively with the α1 and γ2 subunits of the GABA_A receptor. In contrast, gephyrin was not detected at presumed glutamatergic synapses. These results indicate that gephyrin is not uniquely associated with glycine receptors, but can also be found at distinct GABAergic synapses. Thus, they raise the possibility that gephyrin is involved in anchoring certain GABA_A-receptor subtypes in the postsynaptic membrane. J. Comp. Neurol. 395:231–244, 1998. © 1998 Wiley-Liss, Inc.     

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.     

Enhanced dendritic availability of μ‐opioid receptors in inhibitory neurons of the extended amygdala in mice deficient in the corticotropin‐releasing factor‐1 receptor

Activation of the corticotropin‐releasing factor‐1 (CRF‐1) receptor in the anterolateral BNST (BSTal), a key subdivision of the extended amygdala, elicits opiate‐seeking behavior exacerbated by stress. However, it is unknown whether the presence of CRF‐1 affects expression of the μ‐opioid receptor (μ‐OR) in the many GABAergic BSTal neurons implicated in the stress response. We hypothesized that deletion of the CRF‐1 receptor gene would alter the density and/or subcellular distribution of μ‐ORs in GABAergic neurons of the BSTal. We used electron microscopy to quantitatively examine μ‐OR immunogold and γ‐aminobutyric acid (GABA) immunoperoxidase labeling in the BSTal of CRFr‐1 knockout (KO) compared to wild‐type (WT) mice. To assess regional specificity, we examined μ‐OR distribution in dorsal striatum. The μ‐ORs in each region were predominantly localized in dendrites, many of which were GABA‐immunoreactive. Significantly, more cytoplasmic μ‐OR gold particles per dendritic area were observed selectively in GABA‐containing dendrites of the BSTal, but not of the dorsal striatum, in KO compared to WT mice. In both regions, however, significantly fewer GABA‐immunoreactive axon terminals were present in KO compared to WT mice. Our results suggest that the absence of CRF‐1 results in enhanced expression and/or dendritic trafficking of μ‐ORs in inhibitory BSTal neurons. They also suggest that the expression of CRF‐1 is a critical determinant of the availability of GABA in functionally diverse brain regions. These findings underscore the complex interplay between CRF, opioid, and GABA systems in limbic and striatal regions and have implications for the role of CRF‐1 in influencing the pharmacological effects of opiates active at μ‐ORs. Synapse 65:8–20, 2011. © 2010 Wiley‐Liss, Inc.     

Subcellular localization of GABA(B) receptor subunits in rat visual cortex

Although studies in the visual cortex have found gamma-aminobutyric acid B (GABA(B)) receptor-mediated pre- and postsynaptic inhibitory effects on neurons, the subcellular localization of GABA(B) receptors in different types of cortical neurons and synapses has not been shown directly. To provide this information, we have used antibodies against the GABA(B) receptor (R)1a/b and GABA(B)R2 subunits and have studied the localization of immunoreactivities in rat visual cortex. Light microscopic analyses have shown that both subunits are expressed in cell bodies and dendrites of 65-92% of corticocortically projecting pyramidal neurons and in 92-100% of parvalbumin (PV)-, calretinin (CR)-, and somatostatin (SOM)-containing GABAergic neurons. Electron microscopic analyses of immunoperoxidase- and immunogold-labeled tissue revealed staining in the nucleus, cytoplasm and cell surface membranes with both antibodies. Colocalization of both subunits was observed in all of these structures. GABA(B)R1a/b and GABA(B)R2 were concentrated in excitatory and inhibitory synapses and in extrasynaptic membranes. In GABAergic synapses, GABA(B)R1a/b and GABA(B)R2 were more strongly expressed postsynaptically on pyramidal and nonpyramidal cells than presynaptically. In type 1 synapses GABA(B)R1a/b and GABA(B)R2 was found in pre- and postsynaptic membranes. The nuclear localization of GABA(B)R1 and GABA(B)R2 subunits suggests a novel role for neurotransmitter receptors in controlling gene expression. The synaptic colocalization of GABA(B)R1 and GABA(B)R2 indicates that subunits form heteromeric assemblies of the functional receptor in inhibitory and excitatory synapses. Subunit coexpression in GABAergic synapses that include PV-containing and PV-deficient terminals suggests that pre- and postsynaptic GABA(B) receptor activation is provided by several different types of interneurons. The coexpression of both subunits in excitatory synapses suggests a role for GABA(B) receptors in the regulation of glutamate release and raises the question how these receptors are activated in the absence of pre-or postsynaptic GABAergic synaptic inputs to excitatory synapses.