Biomedical subjects

J M Fritschy

Publications and source records attributed to J M Fritschy.

At least 37 records · Page 2Linked to original sources

PubMed2000

Brain S100A5 is a novel calcium-, zinc-, and copper ion-binding protein of the EF-hand superfamily.

S100A5 is a novel member of the EF-hand superfamily of calcium-binding proteins that is poorly characterized at the protein level. Immunohistochemical analysis demonstrates that it is expressed in very restricted regions of the adult brain. Here we characterized the human recombinant S100A5, especially its interaction with Ca(2+), Zn(2+), and Cu(2+). Flow dialysis revealed that the homodimeric S100A5 binds four Ca(2+) ions with strong positive cooperativity and an affinity 20-100-fold higher than the other S100 proteins studied under identical conditions. S100A5 also binds two Zn(2+) ions and four Cu(2+) ions per dimer. Cu(2+) binding strongly impairs the binding of Ca(2+); however, none of these ions change the alpha-helical-rich secondary structure. After covalent labeling of an exposed thiol with 2-(4'-(iodoacetamide)anilino)-naphthalene-6-sulfonic acid, binding of Cu(2+), but not of Ca(2+) or Zn(2+), strongly decreased its fluorescence. In light of the three-dimensional structure of S100 proteins, our data suggest that in each subunit the single Zn(2+) site is located at the opposite side of the EF-hands. The two Cu(2+)-binding sites likely share ligands of the EF-hands. The potential role of S100A5 in copper homeostasis is discussed.

Allosteric Regulation↗

PubMed2000

Selective alterations in GABAA receptor subtypes in human temporal lobe epilepsy.

Temporal lobe epilepsy (TLE) is associated with impaired inhibitory neurotransmission. Studies in animal models suggest that GABA(A) receptor dysfunction contributes to epileptogenesis. To understand the mechanisms underlying TLE in humans, it is fundamental to determine whether and how GABA(A) receptor subtypes are altered. Furthermore, identifying novel receptor targets is a prerequisite for developing selective antiepileptic drugs. We have therefore analyzed subunit composition and distribution of the three major GABA(A) receptor subtypes immunohistochemically with subunit-specific antibodies (alpha1, alpha2, alpha3, beta2,3, and gamma2) in surgical specimens from TLE patients with hippocampal sclerosis (n = 16). Profound alterations in GABA(A) receptor subtype expression were observed when compared with control hippocampi (n = 10). Although decreased GABA(A) receptor subunit staining, reflecting cell loss, was observed in CA1, CA3, and hilus, the distinct neuron-specific expression pattern of the alpha-subunit variants observed in controls was markedly changed in surviving neurons. In granule cells, prominent upregulation mainly of the alpha2-subunit was seen on somata and apical dendrites with reduced labeling on basal dendrites. In CA2, differential rearrangement of all three alpha-subunits occurred. Moreover, there was layer-specific loss of alpha1-subunit-immunoreactive interneurons in hippocampus proper, whereas surviving interneurons exhibited extensive changes in dendritic morphology. Throughout, expression patterns of beta2,3- and gamma2-subunits largely followed those of alpha-subunit variants. These results demonstrate unique subtype-specific expression of GABA(A) receptors in human hippocampus. The significant reorganization of distinct receptor subtypes in surviving hippocampal neurons of TLE patients with hippocampal sclerosis underlines the potential for synaptic plasticity in the human GABA system.

Adolescent↗

PubMed2000

Differential expression of utrophin and dystrophin in CNS neurons: an in situ hybridization and immunohistochemical study.

The cellular distribution of utrophin, the autosomal homologue of dystrophin, was investigated in developing and adult rat and mouse brain by in situ hybridization and immunohistochemistry. Digoxigenin-labeled cRNA probes complementary to N-terminal, rod-domain, and C-terminal encoding sequences of utrophin were used to differentiate between full-length and short C-terminal isoforms. Largely overlapping distribution patterns were seen for the three probes in neurons of cerebral cortex, accessory olfactory bulb, and several sensory and motor brainstem nuclei as well as in blood vessels, pia mater, and choroid plexus. The C-terminal probe was detected in addition in the main olfactory bulb, striatum, thalamic reticular nucleus, and hypothalamus, suggesting a selective expression of G-utrophin in these neurons. Western blot analysis with isoform-specific antisera confirmed the expression of both full-length and G-utrophin in brain. Immunohistochemically, only full-length utrophin was detected in neurons, in close association with the plasma membrane. In addition, intense staining was seen in blood vessels, meninges, and choroid plexus, selectively localized in the basolateral membrane of immunopositive epithelial cells. The expression pattern of utrophin was already established at early postnatal stages and did not change thereafter. Double-labeling analysis revealed that utrophin and dystrophin are differentially expressed on the cellular and subcellular levels in juvenile and adult brain. Likewise, in mice lacking full-length dystrophin isoforms (mdx mice), no change in utrophin expression and distribution could be detected in brain, although utrophin was markedly up-regulated in muscle cells. These results suggest that utrophin and dystrophin are independently regulated and have distinct functional roles in CNS neurons.

Age Factors↗

PubMed2000

Differential downregulation of GABAA receptor subunits in widespread brain regions in the freeze-lesion model of focal cortical malformations.

Focal cortical malformations comprise a heterogeneous group of disturbances of brain development, commonly associated with drug-resistant epilepsy and/or neuropsychological deficits. Electrophysiological studies on rodent models of cortical malformations demonstrated intrinsic hyperexcitability in the lesion and the structurally intact surround, indicating widespread imbalances of excitation and inhibition. Here, alterations in regional expression of GABA(A) receptor subunits were investigated immunohistochemically in adult rats with focal cortical malformations attributable to neonatal freeze-lesions. These lesions are morphologically characterized by a three- to four-layered cortex with microsulcus formation. Widespread regionally differential reduction of GABA(A) receptor subunits alpha1, alpha2, alpha3, alpha5, and gamma2 was observed. Within the cortical malformation, this downregulation was most prominent for subunits alpha5 and gamma2, whereas medial to the lesion, a significant and even stronger decrease of all subunits was detected. Lateral to the dysplastic cortex, the decrease was most prominent for subunit gamma2 and moderate for subunits alpha1, alpha2, and alpha5, whereas subunit alpha3 was not consistently altered. Interestingly, the downregulation of GABA(A) receptor subunits also involved the ipsilateral hippocampal formation, as well as restricted contralateral neocortical areas, indicating widespread disturbances in the neocortical and hippocampal network. The described pattern of downregulation of GABA(A) receptor subunits allows the conclusion that there is a considerable modulation of subunit composition. Because alterations in subunit composition critically influence the electrophysiological and pharmacological properties of GABA(A) receptors, these alterations might contribute to the widespread hyperexcitability and help to explain pharmacotherapeutic characteristics in epileptic patients.

Animals↗

PubMed2000

Colocalization of multiple GABA(A) receptor subtypes with gephyrin at postsynaptic sites.

Clustering of gamma aminobutyric acid (GABA)(A) receptors to postsynaptic sites requires the presence of both the gamma2 subunit and gephyrin. Here, we analyzed by double-immunofluorescence staining the colocalization of gephyrin and major GABA(A)-receptor subtypes distinguished by the subunits alpha1, alpha2, alpha3, or gamma2 in adult rat brain. By using confocal laser scanning microscopy, GABA(A)-receptor subunit staining revealed brightly stained clusters that were colocalized with gephyrin-positive clusters of similar size and distribution in several brain regions, including cerebellum, hippocampus, thalamus, and olfactory bulb. In addition, a diffuse staining was observed for GABA(A)-receptor subunits in the neuropil, presumably representing extrasynaptic receptors. Overall, only few gephyrin-positive clusters were not colocalized with GABA(A)-receptor subunit clusters. Electron microscopic analysis in cerebellar cortex confirmed the selective postsynaptic localization of gephyrin. High-resolution images (voxel size, 50 x 50 x 150 nm) were restored with an iterative image deconvolution procedure based on a measured point-spread function to analyze the colocalization between GABA(A)-receptor subunits and gephyrin in individual clusters. This analysis revealed a considerable heterogeneity in the micro-organization of these presumptive GABAergic postsynaptic sites. For instance, whereas gephyrin- and gamma2 subunit-positive clusters largely overlapped in the cerebellar molecular layer, the colocalization was only partial in glomeruli of the granule cell layer, where small gephyrin clusters typically were "embedded" in larger GABA(A)-receptor clusters. These findings show that gephyrin is associated with a majority of GABA(A)-receptor subtypes in brain, and document the usefulness of image deconvolution for analyzing the structural organization of the postsynaptic apparatus by fluorescence microscopy.

Animals↗

PubMed2000

Synaptic localization of GABA(A) receptor subunits in the striatum of the rat.

The inhibitory amino acid gamma-aminobutyric acid (GABA) is widely distributed in the basal ganglia. It plays a critical role in the functioning of the striatum as it is the transmitter of projection neurons and sub-populations of interneurons, as well as afferents from the globus pallidus. Some of the factors controlling GABA transmission are the type(s) of GABA receptor expressed at the site of transmission, their subunit composition, and their location in relation to GABA release sites. To address these issues, we examined the sub-cellular localization of subunits of the GABA(A) receptor in the striatum of the rat. Sections of freeze-substituted, Lowicryl-embedded striatum were immunolabelled by the post-embedding immunogold technique with antibodies specific for subunits of the GABA(A) receptor. Immunolabelling for alpha1, beta2/3, and gamma2 GABA(A) receptor subunits was primarily located at symmetrical synapses on perikarya, dendrites, and spines. Quantitative analysis of the distribution of immunolabelling for the beta2/3 subunits revealed that the majority of membrane associated immunogold particles were at synapses and that, on average for the whole population, they were evenly distributed across the synapse. Double labelling for the beta2/3 subunits and for GABA itself revealed that receptor-positive synapses were formed by at least two populations of terminals. One population (59.3%) of terminals forming receptor-positive synapses was positive for GABA, whereas the other (40.7%) had low or undetectable levels of GABA. Furthermore, the post-synaptic neurons were characterised on neurochemical and morphological grounds as both medium spiny neurons and GABA interneurons. Triple immunolabelling revealed the co-localization of alpha1, beta2/3, and gamma2 subunits at some symmetrical axodendritic synapse. It is concluded that fast GABA(A)-mediated transmission occurs primarily at symmetrical synapses within the striatum, that the populations of boutons giving rise to receptor-positive synapses are heterogeneous, and that previously reported co-existence of different subunits of the GABA(A) receptor at the cellular level also occurs at the level of individual synapses.

Animals↗

PubMed2000

Developmental switch in the expression of GABA(A) receptor subunits alpha(1) and alpha(2) in the hypothalamus and limbic system of the rat.

The GABA(A) receptor is a pentameric ligand gated ion channel complex assembled from a family of at least 17 different subunits encoded by distinct genes. Two subunits, alpha(1) and alpha(2), exhibit age dependent expression throughout several areas of the brain. In general, the density of immunoreactive product for alpha(1) is greatest in the adult brain, while alpha(2) is highest in younger tissue. Since the developmental switch in alpha(1) and alpha(2) coincides with the end of the sensitive period for steroid-mediated sexual differentiation of the brain, we hypothesized that GABA(A) receptor subunit expression may be involved in this process. We have examined the age-dependent expression of alpha(1) and alpha(2) in discrete regions of the hypothalamus and limbic system of males and females. While we did not detect any dramatic sex differences in alpha(1) or alpha(2) immunoreactive density, each region exhibited a unique developmental profile. In the ventromedial nucleus of neonatal animals immunoreactivity is highest for alpha(1), while in the adult the signal for alpha(2) is greater; the opposite of that observed in the ventrolateral thalamus. There is no age dependent change for alpha(1) in the preoptic area, while alpha(2) shows a small, but significant increase. Immunoreactive densities for both subunits increase in the arcuate nucleus and the hippocampus, but decrease in the lateral amygdala. We conclude that these regional differences in subunit expression across development determine individual characteristics of brain areas and may play a role in establishing unique physiological responses to GABA.

Aging↗

PubMed2000

Identification and characterisation of transcript and protein of a new short N-terminal utrophin isoform.

Dystrophin and utrophin are known to link the intracellular cytoskeleton to the extracellular matrix via a transmembraneous glycoprotein complex. Four short C-terminal isoforms (Dp71, Dp116, Dp140, and Dp260) are described for dystrophin and three for utrophin (Up71, Up113, and Up140). We describe here for the first time the existence of a 3.7-kb transcript and a 62-kDa protein in C6 glioma cells representing a short N-terminal isoform unique for utrophin (N-utrophin). More than 20 clones covering the entire coding region of utrophin were isolated from a rat C6 glioma cell cDNA library. Two clones were found to code for a protein with 539 amino acids. Its sequence is identical to that of the full-length utrophin, except for the last residue where Cys is replaced by Val. This isoform contains the actin binding domain (consisting of two calponin homology subdomains), followed by two spectrin-like repeats. A recombinant fragment corresponding to N-utrophin binds to F-actin in vitro with an equilibrium constant (affinity) K of 4.5 x 10(5) M(-1) and a stoichiometry of one fragment per around five actin monomers. Immunocytochemical staining of C6 glioma cells with antisera specific for different utrophin regions localised full-length utrophin in the submembraneous cortical actin layer as revealed by confocal microscopy. A distinct staining pattern for the N-utrophin was not detectable, although it was expected to localise at the actin stress fibers. It is assumed that it co-localises via the two spectrin-like repeats with the full-length utrophin at the cell membrane.

Actins↗

PubMed2000

Early loss of interneurons and delayed subunit-specific changes in GABA(A)-receptor expression in a mouse model of mesial temporal lobe epilepsy.

Unilateral injection of kainic acid (KA) into the dorsal hippocampus of adult mice induces spontaneous recurrent partial seizures and replicates histopathological changes observed in human mesial temporal lobe epilepsy (MTLE) (Bouilleret V et al., Neuroscience 1999; 89:717-729). Alterations in pre- and postsynaptic components of GABAergic neurotransmission were investigated immunohistochemically at different time points (1-120 days) in this mouse model of MTLE. Markers of GABAergic interneurons (parvalbumin, calbindin-D28k, and calretinin), the type-1 GABA transporter (GAT1), and major GABA(A)-receptor subunits expressed in the hippocampal formation were analyzed. Acutely, KA injection produced a profound loss of hilar cells but only limited damage to CA1 and CA3 pyramidal cells. In addition, parvalbumin and calbindin-D28k staining of interneurons disappeared irreversibly in CA1 and dentate gyrus (DG), whereas calretinin staining was spared. The prominent GABA(A)-receptor alpha1 subunit staining of interneurons also disappeared after KA treatment, suggesting acute degeneration of these cells. Likewise, GAT1 immunoreactivity revealed degenerating terminals at 24 h post-KA in CA1 and DC and subsided almost completely thereafter. Loss of CA1 and, to a lesser extent, CA3 neurons became evident at 7-15 days post-KA. It was more accentuated after 1 month, accompanied by a corresponding reduction of GABA(A)-receptor staining. In contrast, DC granule cells were markedly enlarged and dispersed in the molecular layer and exhibited a prominent increase in GABA(A)-receptor subunit staining. After 4 months, the dorsal CA1 area was lost almost entirely, CA3 was reduced, and the DG represented most of the remaining dorsal hippocampal formation. No significant morphological alterations were detected contralaterally. These results suggest that loss of hilar cells and GABAergic neurons contributes to epileptogenesis in this model of MTLE. In contrast, long-term degeneration of pyramidal cells and granule cell dispersion may reflect distinct responses to recurrent seizures. Finally, GABA(A)-receptor upregulation in the DG may represent a compensatory response persisting for several months in epileptic mice.

Animals↗

PubMed2000

Neurodegenerative and morphogenic changes in a mouse model of temporal lobe epilepsy do not depend on the expression of the calcium-binding proteins parvalbumin, calbindin, or calretinin.

The functional role of the calcium-binding proteins parvalbumin, calretinin, and calbindin D-28k for epileptogenesis and long-term seizure-related alterations of the hippocampal formation was assessed in single- and double-knockout mice, using a kainate model of mesial temporal lobe epilepsy. The effects of a unilateral intrahippocampal injection of kainic acid were assessed at one day, 30 days, and four months post-injection, using various markers of GABAergic interneurons (GABA-transporter type 1, GABA(A)-receptor alpha1 subunit, calretinin, calbindin D-28k, somatostatin, and neuropeptide Y). Parvalbumin-deficient, parvalbumin/calbindin-deficient, and parvalbumin/calretinin-deficient mice exhibited no difference in cytoarchitecture of the hippocampal formation and in the number, distribution, or morphology of interneurons compared to wild-type mice. Likewise, mutant mice were not more vulnerable to acute kainate-induced excitotoxicity or to long-term effects of recurrent focal seizures, and exhibited the same pattern of neurochemical alterations (e.g., bilateral induction of neuropeptide Y in granule cells) and morphogenic changes (enlargement and dispersion of dentate gyrus granule cells) as wild-type animals. Quantification of interneurons revealed no significant difference in neuronal vulnerability among the genotypes.These results indicate that the calcium-binding proteins investigated here are not essential for determining the neurochemical phenotype of interneurons. Furthermore, they are not protective against kainate-induced excitotoxicity in this model, and do not appear to modulate the overall level of excitability of the hippocampus. Finally, seizure-induced changes in gene expression in granule cells, which normally express high levels of calcium-binding proteins, apparently were not affected by the gene deletions analysed.

Animals↗

PubMed2000

Mini-review: gephyrin, a major postsynaptic protein of GABAergic synapses.

gamma-aminobutyric acid type A (GABAA) receptors are located at the majority of inhibitory synapses in the mammalian brain. However, the mechanisms by which GABAA receptor subunits are targeted to, and clustered in, the postsynaptic membrane are poorly understood. Recent studies have demonstrated that gephyrin, a protein first identified as a component of the glycine receptor (GlyR) complex, is colocalized with several subtypes of GABAA receptors and is involved in the stabilization of postsynaptic GABAA receptor clusters. Thus, gephyrin functions as a clustering protein for major subtypes of inhibitory ion channel receptors.

Animals↗

PubMed2000

Rescue of gamma2 subunit-deficient mice by transgenic overexpression of the GABAA receptor gamma2S or gamma2L subunit isoforms.

The gamma2 subunit is an important functional determinant of GABAA receptors and is essential for formation of high-affinity benzodiazepine binding sites and for synaptic clustering of major GABAA receptor subtypes along with gephyrin. There are two splice variants of the gamma2 subunit, gamma2 short (gamma2S) and gamma2 long (gamma2L), the latter carrying in the cytoplasmic domain an additional eight amino acids with a putative phosphorylation site. Here, we show that transgenic mice expressing either the gamma2S or gamma2L subunit on a gamma2 subunit-deficient background are phenotypically indistinguishable from wild-type. They express nearly normal levels of gamma2 subunit protein and [3H]flumazenil binding sites. Likewise, the distribution, number and size of GABAA receptor clusters colocalized with gephyrin are similar to wild-type in both juvenile and adult mice. Our results indicate that the two gamma2 subunit splice variants can substitute for each other and fulfil the basic functions of GABAA receptors, allowing in vivo studies that address isoform-specific roles in phosphorylation-dependent regulatory mechanisms.

Age Factors↗

PubMed2000

Chronic blockade of N-methyl-D-aspartate receptors alters gamma-aminobutyric acid type A receptor peptide expression and function in the rat.

Chronic in vivo or in vitro application of GABA(A) receptor agonists alters GABA(A) receptor peptide expression and function. Furthermore, chronic in vitro application of N-methyl-D-aspartate (NMDA) agonists and antagonists alters GABA(A) receptor function and mRNA expression. However, it is unknown if chronic in vivo blockade of NMDA receptors alters GABA(A) receptor function and peptide expression in brain. Male Sprague-Dawley rats were chronically administered the noncompetitive NMDA receptor antagonist MK-801 (0.40 mg/kg, twice daily) for 14 days. Chronic blockade of NMDA receptors significantly increased hippocampal GABA(A) receptor alpha4 and gamma2 subunit expression while significantly decreasing hippocampal GABA(A) receptor alpha2 and beta2/3 subunit expression. Hippocampal GABA(A) receptor alpha1 subunit peptide expression was not altered. In contrast, no significant alterations in GABA(A) receptor subunit expression were found in cerebral cortex. Chronic MK-801 administration also significantly decreased GABA(A) receptor-mediated hippocampal Cl- uptake, whereas no change was found in GABA(A) receptor-mediated cerebral cortical Cl- uptake. Finally, chronic MK-801 administration did not alter NMDA receptor NR1, NR2A, or NR2B subunit peptide expression in either the cerebral cortex or the hippocampus. These data demonstrate heterogeneous regulation of GABA(A) receptors by glutamatergic activity in rat hippocampus but not cerebral cortex, suggesting a new mechanism of GABA(A) receptor regulation in brain.

Animals↗

PubMed2000

Chronic ethanol administration alters immunoreactivity for GABA(A) receptor subunits in rat cortex in a region-specific manner.

BACKGROUND: Chronic ethanol administration has a plethora of physiological effects. Among the most consistently observed findings is a change in the expression pattern of gamma-aminobutyric acid type A (GABA(A)) receptor subunits in the rat brain cortex. These findings led to the hypothesis of "subunit substitution" to account for changes in receptor function without changes in receptor number. METHODS: We used subunit (alpha1 and alpha4) specific antibodies and a combination of immunohistochemistry and immunoblotting to examine subregions of cortex (prefrontal, cingulate, motor, parietal, and piriform) for their response to 2 weeks of forced ethanol administration. RESULTS: Overall, cortical immunoreactivity for the alpha1 subunit was decreased and for the alpha4 subunit increased whether measured immunohistochemically or by immunoblotting. Piriform cortex exhibited a bidirectional change in GABA(A) receptor alpha1 and alpha4 immunoreactivity, similar to that previously observed in preparations of whole cortex. However, in parietal cortex, declines in alpha1 immunoreactivity (55 +/- 12% control value [CV] and 88.3 +/- 4.3% CV; immunohistochemistry and immunoblotting, respectively) were not accompanied by concomitant increases in alpha4 immunoreactivity (104 +/- 8% CV and 116 +/- 9.3% CV; immunohistochemistry and immunoblotting, respectively). Conversely, alpha4 immunoreactivity increased in cingulate cortex (210 +/- 30% CV and 134 +/- 9.5% CV; immunohistochemistry and immunoblotting, respectively) without a decline in alpha1 immunoreactivity (90 +/- 4% CV and 91.3 +/- 3.9% CV; immunohistochemistry and immunoblotting, respectively). Prefrontal and motor cortex exhibited GABA(A) receptor subunit peptide alterations, but these changes varied with the method of analysis. CONCLUSIONS: These findings demonstrate that ethanol dependence results in nonuniform changes in GABA(A) receptor subunit peptide levels across the rat brain cortex and suggest that mechanisms which subserve functional changes in receptor activity may vary in accordance with anatomic or cellular differences within the cortex.

Cerebral Cortex↗

PubMed1999

Postsynaptic clustering of gamma-aminobutyric acid type A receptors by the gamma3 subunit in vivo.

Synaptic localization of gamma-aminobutyric acid type A (GABA(A)) receptors is a prerequisite for synaptic inhibitory function, but the mechanism by which different receptor subtypes are localized to postsynaptic sites is poorly understood. The gamma2 subunit and the postsynaptic clustering protein gephyrin are required for synaptic localization and function of major GABA(A) receptor subtypes. We now show that transgenic overexpression of the gamma3 subunit in gamma2 subunit-deficient mice restores benzodiazepine binding sites, benzodiazepine-modulated whole cell currents, and postsynaptic miniature currents, suggesting the formation of functional, postsynaptic receptors. Moreover, the gamma3 subunit can substitute for gamma2 in the formation of GABA(A) receptors that are synaptically clustered and colocalized with gephyrin in vivo. These clusters were formed even in brain regions devoid of endogenous gamma3 subunit, indicating that the factors present for clustering of gamma2 subunit-containing receptors are sufficient to cluster gamma3 subunit-containing receptors. The GABA(A) receptor and gephyrin-clustering properties of the ectopic gamma3 subunit were also observed for the endogenous gamma3 subunit, but only in the absence of the gamma2 subunit, suggesting that the gamma3 subunit is at a competitive disadvantage with the gamma2 subunit for clustering of postsynaptic GABA(A) receptors in wild-type mice.

Animals↗

PubMed1999

Benzodiazepine actions mediated by specific gamma-aminobutyric acid(A) receptor subtypes.

GABA(A) (gamma-aminobutyric acid(A)) receptors are molecular substrates for the regulation of vigilance, anxiety, muscle tension, epileptogenic activity and memory functions, which is evident from the spectrum of actions elicited by clinically effective drugs acting at their modulatory benzodiazepine-binding site. Here we show, by introducing a histidine-to-arginine point mutation at position 101 of the murine alpha1-subunit gene, that alpha1-type GABA(A) receptors, which are mainly expressed in cortical areas and thalamus, are rendered insensitive to allosteric modulation by benzodiazepine-site ligands, whilst regulation by the physiological neurotransmitter gamma-aminobutyric acid is preserved. alpha1(H101R) mice failed to show the sedative, amnesic and partly the anticonvulsant action of diazepam. In contrast, the anxiolytic-like, myorelaxant, motor-impairing and ethanol-potentiating effects were fully retained, and are attributed to the nonmutated GABA(A) receptors found in the limbic system (alpha2, alpha5), in monoaminergic neurons (alpha3) and in motoneurons (alpha2, alpha5). Thus, benzodiazepine-induced behavioural responses are mediated by specific GABA(A) receptor subtypes in distinct neuronal circuits, which is of interest for drug design.

Animals↗

PubMed1999

GABAA receptor subunit composition and functional properties of Cl- channels with differential sensitivity to zolpidem in embryonic rat hippocampal cells.

Using flow cytometry in conjunction with a voltage-sensitive fluorescent indicator dye (oxonol), we have identified and separated embryonic hippocampal cells according to the sensitivity of their functionally expressed GABAA receptors to zolpidem. Immunocytochemical and RT-PCR analysis of sorted zolpidem-sensitive (ZS) and zolpidem-insensitive (ZI) subpopulations identified ZS cells as postmitotic, differentiating neurons expressing alpha2, alpha4, alpha5, beta1, beta2, beta3, gamma1, gamma2, and gamma3 GABAA receptor subunits, whereas the ZI cells were neuroepithelial cells or newly postmitotic neurons, expressing predominantly alpha4, alpha5, beta1, and gamma2 subunits. Fluctuation analyses of macroscopic Cl- currents evoked by GABA revealed three kinetic components of GABAA receptor/Cl- channel activity in both subpopulations. We focused our study on ZI cells, which exhibited a limited number of subunits and functional channels, to directly correlate subunit composition with channel properties. Biophysical analyses of GABA-activated Cl- currents in ZI cells revealed two types of receptor-coupled channel properties: one comprising short-lasting openings, high affinity for GABA, and low sensitivity to diazepam, and the other with long-lasting openings, low affinity for GABA, and high sensitivity to diazepam. Both types of channel activity were found in the same cell. Channel kinetics were well modeled by fitting dwell time distributions to biliganded activation and included two open and five closed states. We propose that short- and long-lasting openings correspond to GABAA receptor/Cl- channels containing alpha4beta1gamma2 and alpha5beta1gamma2 subunits, respectively.

Animals↗

PubMed1999

Activity-dependent regulation of GABAA receptors.

GABAA receptor heterogeneity is based on the combinatorial assembly of a large family of subunits into distinct receptor subtypes. A neuron-specific expression pattern of receptor subtypes has been demonstrated in adult rat brain, which can be reproduced in vitro in primary neuron cultures. This suggests that genetic programs established during ontogeny govern the expression of gamma-aminobutyric acid (GABAA) receptor subtypes. Activity-dependent mechanisms nevertheless modulate on a short-term basis the cell surface expression of GABAA receptors, as demonstrated in cultured hippocampal neurons upon blockade of synaptic transmission or application of brain-derived neurotrophic factor. Preliminary evidence points to changes in protein phosphorylation as a mechanism underlying short-term activity-dependent regulation of GABAA receptors. In vivo, chronic pharmacological modulation of neuronal activity during development, while having marked effects on the rate of cortical growth, failed to influence the expression of GABAA receptor subtypes, suggesting that additional factors are involved.

Animals↗