Postsynaptic colocalization of gephyrin and GABAA receptors.
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Biomedical subjects
J M Fritschy
Publications and source records attributed to J M Fritschy.
Neuronal activity influences the growth of barrels in developing rat primary somatosensory cortex without affecting the expression pattern of four major GABAA receptor alpha subunits.
Thalamic innervation plays a major role in parcellation of neocortex and maturation of cortical circuits. While the underlying mechanisms are unknown, lesion studies have identified GABAA receptors in neocortex as molecular targets of thalamic regulation [J. Paysan, A. Kossel, J. Bolz, J.M. Fritschy, Area-specific regulation of gamma-aminobutyric acid A receptor subtypes by thalamic afferents in developing rat neocortex, Proc. Natl. Acad. Sci. USA 94 (1997) 6995-7000]. To determine the factors regulating the expression of GABAA receptors, the overall level of neuronal activity was chronically modulated in neonatal rat cortex. Slices of Elvax polymer loaded with the N-methyl-D-asparate (NMDA) receptor antagonist MK-801 or with brain derived neurotrophic factor (BDNF) were placed unilaterally over the left parietal cortex in newborn animals. Unlike thalamic lesions (Paysan et al., 1997), these chronic drug treatments did not alter the laminar distribution or the expression level of the four major GABAA receptor alpha subunit isoforms (alpha 1, alpha 2, alpha 3, alpha 5) in primary somatosensory cortex (S1), as assessed immunohistochemically after one week. In particular, the staining of the barrel field in layers III-IV, which is very prominent with the alpha 1-subunit, was preserved in the drug-treated hemisphere. Even systemic administration of MK-801 at birth, which resulted in pronounced retardation of cortical development, had no effect on the laminar distribution and staining intensity of the four GABAA receptor alpha subunit variants. However, the size of barrels in S1, as measured in tangential sections stained for the GABAA receptor alpha 1 subunit, was enlarged upon chronic, topical blockade of NMDA receptors with MK-801 and was reduced to the same extent upon chronic exposure to BDNF. Thus, these pharmacological treatments modulated cortical growth, possibly by exerting opposite effects on neuronal activity in S1. The results suggest that the parcellation of somatosensory cortex and the laminar distribution of GABAA receptor subtypes are governed primarily by factors independent of thalamocortical activity.
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GABAB receptors make it to the top--as dimers.
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GABAergic neurons and GABA(A)-receptors in temporal lobe epilepsy.
Mesial temporal lobe epilepsy (MTLE) is the most prevalent form of epilepsy, characterized by recurrent complex partial seizures and hippocampal sclerosis. The pathophysiology underlying this disorder remains unidentified. While a loss of benzodiazepine binding sites is a key diagnostic feature of MTLE, experimental studies have shown enhanced inhibitory transmission and increased expression of GABA(A)-receptors, suggesting that compensatory mechanisms are operative in epileptic hippocampus. In the present study, changes in the expression and cellular distribution of major GABA(A)-receptor subunits were investigated in the hippocampus of pilocarpine-treated rats during the phase of spontaneous recurrent seizures. A uniform decrease in GABA(A)-receptor subunit-immunoreactivity was observed in regions of extensive neuronal death (i.e. CA1, CA3, hilus). whereas a prominent increase occurred in the dentate gyrus (DG). Most strikingly, the increase was largest for the alpha3- and alpha5-subunits, which are expressed at very low levels in the DG of control rats, suggesting the formation of novel GABA(A)-receptor subtypes in epileptic tissue. Furthermore, an extensive loss of interneurons expressing the alpha1-subunit, representing presumptive basket cells, was seen in the DG. These changes were very similar to those reported in a novel mouse model of MTLE, based on the unilateral injection of kainic acid into the dorsal hippocampus (Bouilleret et al., 1999). This indicates that the regulation of GABA(A)-receptor expression is related to chronic recurrent seizures, and is not due to the extrahippocampal neuronal damage affecting pilocarpine-treated rats. These results allow causal relationships in the induction and maintenance of chronic recurrent seizures to be distinguished. The loss of a critical number of interneurons in the DG is a possible cause of seizure initiation, whereas the long-lasting upregulation of GABA(A)-receptors in granule cells represents a compensatory response to seizure activity.
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Decreased GABAA-receptor clustering results in enhanced anxiety and a bias for threat cues.
Patients with panic disorders show a deficit of GABAA receptors in the hippocampus, parahippocampus and orbitofrontal cortex. Synaptic clustering of GABAA receptors in mice heterozygous for the gamma2 subunit was reduced, mainly in hippocampus and cerebral cortex. The gamma2 +/- mice showed enhanced behavioral inhibition toward natural aversive stimuli and heightened responsiveness in trace fear conditioning and ambiguous cue discrimination learning. Implicit and spatial memory as well as long-term potentiation in hippocampus were unchanged. Thus gamma2 +/- mice represent a model of anxiety characterized by harm avoidance behavior and an explicit memory bias for threat cues, resulting in heightened sensitivity to negative associations. This model implicates GABAA-receptor dysfunction in patients as a causal predisposition to anxiety disorders.
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GABAB-receptor splice variants GB1a and GB1b in rat brain: developmental regulation, cellular distribution and extrasynaptic localization.
GABAB (gamma-aminobutyric acid)-receptors have been implicated in central nervous system (CNS) functions, e.g. cognition and pain perception, and dysfunctions including spasticity and absence epilepsy. To permit an analysis of the two known GABAB-receptor splice variants GABAB-R1a (GB1a) and GABAB-R1b (GB1b), their distribution pattern has been differentiated in the rat brain, using Western blotting and immunohistochemistry with isoform-specific antisera. During postnatal maturation, the expression of the two splice variants was differentially regulated with GB1a being preponderant at birth. In adult brain, GB1b-immunoreactivity (-IR) was predominant, and the two isoforms largely accounted for the pattern of GABAB-receptor binding sites in the brain. Receptor heterogeneity was pronounced in the hippocampus, where both isoforms occurred in CA1, but only GB1b in CA3. Similarly, in the cerebellum, GB1b was exclusively found in Purkinje cells in a zebrin-like pattern. The staining was most pronounced in Purkinje cell dendrites and spines. Using electron microscopy, over 80% of the spine profiles in which a synaptic contact with a parallel fibre was visible contained GB1b-IR at extrasynaptic sites. This subcellular localization is unrelated to GABAergic inputs, indicating that the role of GABAB-receptors in vivo extends beyond synaptic GABAergic neurotransmission and may, in the cerebellum, involve taurine as a ligand.
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Short communication: altered synaptic clustering of GABAA receptors in mice lacking dystrophin (mdx mice).
Dystrophin is selectively localized in the postsynaptic density of neurons in cerebral cortex, hippocampus and cerebellum. Here, we show by double-immunofluorescence staining that dystrophin is extensively colocalized with GABAA receptor subunit clusters in these brain regions. To determine the relevance of this observation, we investigated in mdx mice, which provide a model of Duchenne muscular dystrophy, whether the absence of dystrophin affects the synaptic clustering of GABAA receptors. A marked reduction in the number of clusters immunoreactive for the alpha1 and alpha2 subunits was observed in, respectively, cerebellum and hippocampus of mdx mice, but not in striatum, which is normally devoid of dystrophin. Furthermore, these alterations were not accompanied by a change in gephyrin staining, although gephyrin is colocalized with the majority of GABAA receptor clusters in these regions. These results indicate that dystrophin may play an important role in the clustering or stabilization of GABAA receptors in a subset of central inhibitory synapses. These deficits may underlie the cognitive impairment seen in Duchenne patients.
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Localization of the organic anion transporting polypeptide 2 (Oatp2) in capillary endothelium and choroid plexus epithelium of rat brain.
In this study we investigated the distribution of a recently cloned polyspecific organic anion transporting polypeptide (Oatp2) in rat brain by nonradioactive in situ hybridization histochemistry and immunofluorescence microscopy. The results demonstrate that Oatp2 is expressed in brain capillary and in plexus epithelial cells. At the blood-brain barrier (BBB), Oatp2 expression could be co-localized with the endothelial marker vWF (von Willebrand factor) but not with the astrocyte marker GFAP (glial fibrillary acidic protein). In choroid plexus epithelial cells, Oatp2 could be localized to the basolateral cell pole, whereas the first member of the Oatp gene family of membrane transporters to be cloned (Oatp1) co-localized with the alpha(1)-subunit of Na,K-ATPase at the apical plasma membrane domain. Because Oatp1 and Oatp2 have been previously shown to mediate transmembrane transport of a wide variety of amphipathic organic compounds, including many drugs and other xenobiotics, the histochemical localization of Oatp2 at the BBB and of Oatp1 and Oatp2 in the choroid plexus imply a role for these transporters in the active exchange of amphipathic solutes between the blood, brain, and cerebrospinal fluid compartments. (J Histochem Cytochem 47:1255-1263, 1999)
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GABA, GABA transporters, GABA(A) receptor subunits, and GAD mRNAs in the rat parabrachial and Kölliker-Fuse nuclei.
In the present study, we investigated the key molecules that determine gamma-aminobutyric acid (GABA)ergic signal transduction in the parabrachial/Kölliker-Fuse complex (PB/KF) by means of immunocytochemistry and in situ hybridization. Our data demonstrate a dense plexus of GABA-immunoreactive (-ir) varicosities throughout the nuclei of the PB and the KF. The number of neurons expressing GAD65 or GAD67 mRNA was fairly low in the PB, whereas caudally in the KF an accumulation of GAD-expressing neurons was observed. The GABA transporter-3 (GAT-3) was detected in all parts of the PB/KF, whereas immunolabeling for GAT1 was not observed. All nuclei of the PB and the KF exhibited immunoreactivity for the gamma2-, alpha2-, and alpha3-subunits of the GABA(A) receptor. Gamma2-ir was strong and similar in all PB/KF nuclei. In contrast, alpha2-labeling was particularly intense in the superior lateral PB, and alpha3-labeling was most prominent in the external lateral and external medial PB, compared with the remaining nuclei. With respect to the subcellular localization, we found gamma2-ir in cell bodies and higher order dendrites, whereas alpha2- and alpha3-ir was predominantly found in cell bodies. Immunolabeling for the beta2/3- and the alpha1-subunit was seen in cell bodies and presumed dendritic profiles. The staining intensity was strongest in the dorsal lateral PB. Most importantly, the external lateral PB and the waist area were totally devoid of beta2/3- and alpha1-ir. Our data suggest that neural processing in the PB/KF is under a strong GABAergic inhibition that is apparently mediated by different types of GABA(A) receptors in functionally different pathways through the PB/KF.
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GABA(A) receptors in the primate basal ganglia: an autoradiographic and a light and electron microscopic immunohistochemical study of the alpha1 and beta2,3 subunits in the baboon brain.
The distribution of gamma-aminobutyric acid(A) (GABA(A)) receptors was investigated in the basal ganglia in the baboon brain by using receptor autoradiography and the immunohistochemical localisation of the alpha1 and beta2,3 subunits of the GABA(A) receptor by light and electron microscopy. In the caudate-putamen, the alpha1 subunit was distributed in high densities in the matrix compartment, and the beta2,3 subunits were more homogeneously distributed; the globus pallidus showed lower levels of the alpha1 and beta2,3 subunits. Four types of alpha1 subunit immunoreactive neurons were identified in the baboon striatum: the most numerous (75%) were type 1 medium-sized aspiny neurons; type 2 (2%) were large aspiny neurons with an indented nuclear membrane located in the ventral striatum; type 3 neurons were the least numerous (1%) and were comprised of large neurons in the ventromedial regions of the striatum; and type 4 (22%) neurons were medium to large aspiny neurons located in striosomes. At the ultrastructural level, alpha1 and beta2,3 subunit immunoreactivity was localised in the neuropil of the striatum in both symmetrical and asymmetrical synaptic contacts. In the globus pallidus, alpha1 and beta2,3 subunits were localised on large neurons and were found in three types of synaptic terminals: type 1 terminals were small and established symmetrical synapses; type 2 terminals were large; and type 3 terminals formed small synaptic terminals with subjunctional dense bodies. These results show that the subunit composition of GABA(A) receptors varies between the striosome and the matrix compartments in the striatum and that there is receptor subunit homogeneity in the globus pallidus.
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Independent assembly and subcellular targeting of GABA(A)-receptor subtypes demonstrated in mouse hippocampal and olfactory neurons in vivo.
The ability of neurons to display more than a single GABA(A)-receptor subtype per cell requires intricate targeting mechanisms. Analysis by confocal laser scanning microscopy revealed that the alpha2- and alpha5-subunits differed strikingly in their subcellular distribution in hippocampal pyramidal cells and olfactory bulb granule cells, while the distribution of the gamma2-subunit was rather uniform. In mutant mice lacking the alpha5-subunit gene due to a chromosomal deletion, the absence of the alpha5-subunit was accompanied by a corresponding decrease of the gamma2-subunit immunoreactivity. In striking contrast, the subcellular distribution of the alpha2-subunit was unchanged in these mutant mice. These findings indicate that the assembly of distinct GABA(A)-receptor subtypes in the same neuron is regulated independently. Furthermore, the alpha-subunit is a prime candidate for providing domains which direct subcellular targeting.
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Influence of gender on chronic ethanol-induced alterations in GABAA receptors in rats.
Ethanol dependence, arising from chronic ethanol exposure, is associated with neuroadaptations of GABAA receptors, evidenced by alterations in various behaviors, receptor responsiveness and subunit gene expression. The present studies explored the effects of ethanol dependence in female rats for comparison with previous studies in our laboratory using male rats. We found that ethanol dependence resulted in differential effects on GABAA receptor gene expression in female rat cerebral cortex compared to ethanol dependent male rats. Notably, chronic ethanol consumption did not change GABAA receptor alpha 1 subunit peptide levels in ethanol dependent female rat cortex, in contrast to previously observed decreases in alpha 1 subunit expression in ethanol dependent male rat cortex. The effects of ethanol dependence on additional GABAA receptor subunit peptide levels (alpha 4, beta 2/3 and gamma 2) were similar, but not identical, between female and male rat cortex. When directly compared within the experiment, male and female rats had similar baseline bicuculline seizure thresholds and displayed a similar increase in seizure susceptibility during ethanol withdrawal. Ethanol withdrawn female rats were cross tolerant to the anticonvulsant effects of diazepam, similar to the findings in ethanol withdrawn male rats. Ethanol withdrawn female rats showed a dose-dependent enhancement of the anticonvulsant effect of the neuroactive steroid, THDOC (3 alpha,21-dihydroxy-5 alpha-pregnan-20-one) compared to control animals. This finding is similar to previous observations of increased sensitivity to the anticonvulsant effect of 3 alpha,5 alpha-THP (3 alpha-hydroxy-5 alpha-pregnan-20-one) in ethanol withdrawn male and female rats. In addition, low dose administration of THDOC elevated seizure thresholds in ethanol withdrawn female but not male rats, suggesting that ethanol withdrawn female rats were more responsive to the anticonvulsant effects of this neurosteroid than were ethanol withdrawn male rats. These findings show that gender impacts on adaptations in GABAA receptors elicited by ethanol dependence. However, the physiological outcomes of the differential alterations are not clear. Taken together, these studies suggest that additional mechanisms, beyond effects on GABAA receptor gene expression are involved in the mediation of ethanol dependence and withdrawal.
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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 gamma-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 alpha1 and gamma2 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.
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Several GABAA receptor subunits are expressed in LHRH neurons of juvenile female rats.
Gamma aminobutyric acid (GABA), the dominant inhibitory neurotransmitter in brain, is involved in the developmental regulation of LHRH secretion. Morphological studies in rodents have demonstrated that LHRH neurons are innervated by GABA-containing processes, suggesting that LHRH secretion is under direct transsynaptic GABAergic control. While GABA acts through two different receptors, GABAA and GABAB, to exert its effects, it appears that GABAA receptors are able to mediate both inhibitory and stimulatory effects of GABA on LHRH neurons. GABAA receptors are heterooligomeric ligand-gated anion channels that exhibit a diverse array of functional and pharmacological properties. This diversity is determined by the structural heterogeneity of the receptors, which are assembled from the combination of different classes of subunits with multiple isoforms. Although several studies have described the effect of GABAA receptor stimulation on LHRH and/or gonadotropin release in prepubertal animals, nothing is known about the receptor subunits that may be expressed in LHRH neurons at this phase in development. Double immunohistofluorescence followed by confocal laser microscopy revealed that subsets of prepubertal LHRH neurons are endowed with alpha 1, alpha 2, beta 2/3, and gamma 2 GABAA receptor subunits. Combined immunohistochemistry for LHRH neurons and in situ hybridization for GABAA subunit mRNAs confirmed that the genes encoding the alpha 1, alpha 2, beta 3 and gamma 2 subunits, but not the gamma 1 subunit, are expressed in LHRH neurons. Notwithstanding the relative insensitivity of these methods, both the immunohistochemical and hybridization histochemical approaches employed indicate that only a fraction of LHRH neurons are endowed with GABAA receptors. This arrangement suggests that those LHRH neurons bearing the appropriate GABAA receptors are responsible for either the entire secretory response to direct GABAergic inputs or for its initiation.
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Synapse-specific localization of NMDA and GABA(A) receptor subunits revealed by antigen-retrieval immunohistochemistry.
Conventional immunohistochemistry provides little evidence for the synaptic localization of ionotropic neurotransmitter receptors, suggesting that their epitopes are not readily accessible in situ. Here, we have adapted antigen retrieval procedures based on microwave irradiation to enhance the immunohistochemical staining of gamma-aminobutyric acid type A (GABA[A]) and N-methyl-D-aspartate (NMDA) receptor subunits in rat brain tissue. Microwave irradiation of fixed tissue produced a marked reduction of nonspecific staining, allowing an improved detection of GABA(A) receptor subunits. However, staining of NMDA receptor subunits remained suboptimal. In contrast, microwave irradiation of cryostat sections prepared from fresh tissue resulted in a major enhancement of both NMDA and GABAA receptor subunit staining. The diffuse, partially intracellular signals were largely replaced by numerous, intensely immunoreactive puncta outlining neuronal somata and dendrites, highly suggestive ofsynaptic receptors. In hippocampus CA1-CA3 fields, the NR2Aand NR2B subunit positive puncta exhibited an extensive colocalization in the stratum oriens and radiatum, whereas pyramidal cell bodies, which receive no excitatory synapses, were unstained. In addition, the NR2A subunit, but not the NR2B subunit, was selectively detected on pyramidal cell dendrites in the stratum lucidum of CA3, suggesting a selective targeting to sites of mossy fiber input. For the GABAA receptor subunits, the most striking change induced by this protocol was the selective staining of the axon initial segment of cortical and hippocampal pyramidal cells. The alpha2 subunit immunoreactivity was particularly prominent in these synapses. In control experiments, the staining of cytoskeletal proteins (neurofilaments, glial fibrillary acid protein) was not influenced by prior microwave irradiation. The enhancement of cell-surface-associated staining is therefore strongly suggestive of an 'unmasking' of subunit epitopes by the microwave treatment. These results reveal a remarkable specificity in the synaptic targeting of NMDA and GABAA receptor subunits in hippocampal and neocortical neurons, suggesting that individual neurons can express multiple receptor subtypes in functionally distinct synapses.
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Comparison of the rat dorsal and ventral striatopallidal system. A study using the GABA(A)-receptor alpha1-subunit and parvalbumin immunolabeling.
The ventral striatum is more closely related to limbic brain regions than the dorsal striatum in spite of the remarkable similarities in the structural organization between these two brain regions. The present study is focused on the comparison of ventral striatopallidal territories and the dorsal striatopallidal system regarding the GABA(A)-receptor alpha1-subunit and parvalbumin immunoreactivity, as these markers showed specific distribution patterns and coexpression sites in the more intensely studied dorsal regions. Our investigations revealed that: (1) Parvalbumin single-labeled cells and a moderate number of neurons single-labeled with the GABA(A)-receptor alpha1-subunit exist not only in the dorsal but also in the ventral striatum, including the striatal cell bridges. In addition, morphologically similar neurons positive for the alpha1-subunit were also found in the corpus callosum and anterior commissure. (2) A small number of double-labeled neurons was seen not only in dorsal but also in ventral striatal regions. Such cells were mainly located near the border with the globus pallidus and ventral pallidum. They are likely to represent a further type of striatal neuron. (3) The vast majority of neurons in the entopeduncular nucleus, the homologue of the primate internal globus pallidus segment, coexpressed alpha1-subunit and parvalbumin immunoreactivity, as reported previously for the other pallidal compartments. (4) The islands of Calleja adjoining the ventral pallidal extensions in the olfactory tubercle exhibited a strong alpha1-subunit immunoreactivity in the neuropil as well as somata single- or double-labeled for both markers. Our findings indicate that the dorsal and ventral striatopallidal compartments are similarly organized in general with respect to the occurrence and distribution of single- and double-labeled parvalbumin-immunoreactive and GABA(A)-receptor alpha1-subunit-immunoreactive neurons.
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GABA(A)-receptor assembly in vivo: lessons from subunit mutant mice.
The rules governing the assembly of GABA(A) receptors in vivo were assessed in subunit mutant mice. The transcription of individual subunit genes was regulated independently. The lack of a particular subunit did not result in a molecular rescue by an enhanced transcription of other subunits. In addition, the availability of an alpha- and beta-subunit was essential for receptor formation. Finally, highly selective recognition processes directed the subcellular targeting of receptors. The loss of a particular receptor subtype (alpha5) did not lead to a subcellular redistribution of the remaining subtype (alpha2) present in the same cell.
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Immunohistochemical evidence for dysregulation of the GABAergic system ipsilateral to photochemically induced cortical infarcts in rats.
Deficits of GABAergic transmission have been reported to occur in tissue surrounding ischemic cortical lesions between a few days and several weeks after the insult. In the present experiments, we used immunohistochemistry with antibodies against parvalbumin and two major subunits of the GABA(A) receptor (alpha1, alpha2) to characterize the events that underlie these changes at different levels of circuit organization. Neocortical infarcts (2 mm diameter) consistently affecting medial parts of the primary somatosensory cortex were induced photochemically in adult male Wistar rats; animals were allowed to recover for one week before perfusion-fixation. When compared to controls the pattern of immunoreactivity had changed for the al subunit of the GABA(A) receptor seven days after the insult. Ipsilateral to the ischemic lesions, we found a decrease in staining intensity reaching up to 4 mm laterally, resulting in a partial or complete absence of the normal laminar staining pattern. No consistent changes were observed for the alpha2 subunit. Parvalbumin staining revealed pathological alterations in a rim of tissue surrounding the infarct, measuring up to 1 mm from the border of the infarcts. Parvalbumin-positive interneurons in this region showed signs of degeneration; both a reduction of the number of dendrites and, to a lesser extent and only immediately adjacent to the ischemic lesions, a reduction of the number of parvalbumin-positive neurons was readily apparent. The results provide evidence for both a differential regulation of two GABA(A) receptor subunits and degenerative changes of parvalbumin-containing interneurons ipsilateral to cortical infarcts. The relevance of these findings for mechanisms underlying long-term recovery, transient functional deficits and postinfarct seizures warrants further investigation.
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