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.
Benzodiazepine tranquilizers are used in the treatment of anxiety disorders. To identify the molecular and neuronal target mediating the anxiolytic action of benzodiazepines, we generated and analyzed two mouse lines in which the alpha2 or alpha3 GABAA (gamma-aminobutyric acid type A) receptors, respectively, were rendered insensitive to diazepam by a knock-in point mutation. The anxiolytic action of diazepam was absent in mice with the alpha2(H101R) point mutation but present in mice with the alpha3(H126R) point mutation. These findings indicate that the anxiolytic effect of benzodiazepine drugs is mediated by alpha2 GABAA receptors, which are largely expressed in the limbic system, but not by alpha3 GABAA receptors, which predominate in the reticular activating system.
The heterogeneity of ␥-aminobutyric acid type A (GABAA) receptors contributes to the diversity of neuronal inhibition in the regulation of information processing. Although most GABA A receptors are located synaptically, the small population of ␣5GABAA receptors is largely expressed extrasynaptically. To clarify the role of the ␣5GABAA receptors in the control of behavior, a histidineto-arginine point mutation was introduced in position 105 of the murine ␣5 subunit gene, which rendered the ␣5GABAA receptors diazepam-insensitive. Apart from an incomplete muscle relaxing effect, neither the sedative, anticonvulsant, nor anxiolytic-like activity of diazepam was impaired in ␣5(H105R) mice. However, in hippocampal pyramidal cells, the point mutation resulted in a selective reduction of ␣5GABAA receptors, which altered the drugindependent behavior. In line with the role of the hippocampus in certain forms of associative learning, trace fear conditioning, but not delay conditioning or contextual conditioning, was facilitated in the mutant mice. Trace fear conditioning differs from delay conditioning in that the conditioned and unconditioned stimulus are separated by a time interval. Thus, the largely extrasynaptic ␣5GABAA receptors in hippocampal pyramidal cells are implicated as control elements of the temporal association of threat cues in trace fear conditioning.
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.
Hippocampal pyramidal cells, receiving domain specific GABAergic inputs, express up to 10 different subunits of the y-aminobutyric acid type A (GABAA) receptor, but only 3 different subunits are needed to form a functional pentameric channel. We have tested the hypothesis that some subunits are selectively located at subsets of GABAergic synapses. The al subunit has been found in most GABAergic synapses on all postsynaptic domains of pyramidal cells. In contrast, the a2 subunit was located only in a subset of synapses on the somata and dendrites, but in most synapses on axon initial segments innervated by axo-axonic cells. The results demonstrate that molecular specialization in the composition of postsynaptic GABAA receptor subunits parallels GABAergic cell specialization in targeting synapses to a specific domain of postsynaptic cortical neurons.Inhibition in hippocampal pyramidal cells is mediated by distinct sets of GABAergic local-circuit neurons, which govern several integrative properties such as the threshold of activation (1-4), the pattern of action potential firing (5-7), and the modification of the synaptic strength (8). Different types of GABAergic interneurons innervate separate domains of pyramidal cells and at least three types have been shown to act through y-aminobutyric acid type A (GABAA) receptors (4,(9)(10)(11)(12)(13)(14). Pentameric GABAA receptors require only three to four different subunits to display the functional properties of native channels. However, hippocampal pyramidal cells express 10 subunits of the GABAA receptor (15-18), which may be coassembled into GABAA receptor channels differing in their pharmacological and kinetic properties depending on subunit composition (19)(20)(21)(22). The multiple sources of GABAreleasing terminals on the one hand and the potential of assembling a variety of functionally distinct GABAA receptors on the other raise the possibility that the segregation of inputs coincides with a specialization in postsynaptic receptors resulting from selective targeting of particular receptor subtypes to certain synapses. To test this hypothesis, a postembedding immunogold method was applied on Lowicryl-embedded ultrathin sections to compare the cell surface distribution of the al and a2 subunits, which endow GABAA receptors with distinct pharmacological properties. MATERIALS AND METHODSPreparation of Animals and Tissues. Five adult Wistar rats were anaesthetized with Sagatal (pentobarbitone sodium, 220 mg/kg i.p.) and perfused through the heart with saline followed by fixative containing 4% paraformaldehyde, 0.05-0.1% glutaraldehyde, and -0.2% picric acid for 13-25 min. After perfusion the brains were removed, and blocks from the dorsal hippocampi were cut out and washed in several changes of 0.1 M phosphate buffer.Antibodies. Rabbit polyclonal antiserum (code no. P16) was raised to a synthetic peptide corresponding to residues 1-9 of the rat al subunit. Antibody specificity has been described (23). Immunoreactions with affinity-purified P16 antibody wer...
Vigilance, anxiety, epileptic activity, and muscle tone can be modulated by drugs acting at the benzo- (Fig. la) was constructed containing a 6.4-kb genomic region including exons 7, 9, and 10 of the y2 subunit gene isolated from a 129SV mouse genomic library. A 1.2-kb genomic Pvu II-Nco I fragment including exon 8 (coding for amino acids 306-375 of the y2 polypeptide) was replaced with the phosphoglycerate kinase (PGK)-neo cassette (11), and a tk expression cassette (12) was added at the 3' end of the y2 sequence. Splicing from exon 7 to exon 9 would result in a stop of the translational reading frame and prohibit expression of sequences downstream of exon 7. Before electroporation into E14 ES cells (13), the plasmid was linearized at a polylinker site adjacent to the 5' end of the 7y2 genomic sequence. E14 ES cells were cultured on irradiated G418-resistant feeder cells obtained from CD1-M-TKneo2 mouse embryos [BRL, Fullinsdorf (Basel)] in GMEM (Glasgow modification of Eagle's medium; Flow Laboratories) containing 10% total calf serum and leukemia inhibitory factor (103 units/ml, Life Technologies). The cells were transfected and screened for homologous recombinants (14) by using PCR and the primers y2.19 (5'-CATCT CCATC GCTAA GAATG TTCGG derived from 7y2 sequences upstream of the targeting vector and Y2.20 (5'-ATGCT CCAGA CTGCC TTGGG AAAAG C-3') derived from PGK promoter sequences (11). Chimeric mice were generated (15) and mated to C57BL/6 females, and the offspring were genotyped by PtR amplification of tail DNAs. Reactions specific for the disrupted y2 allele [(0) Abbreviations: BZ, benzodiazepine; GABA, y-aminobutyric acid; DRG, dorsal root ganglion (ganglia); ES, embryonic stem; E, embryonic day; P, postnatal day.§To whom reprint requests should be addressed. 7749The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abstract:Changes in the expression of the NMDA receptor subunits (NRs) NR2A, 2B, and 2C were investigated in histo blots of the developing rat brain with subunitspecific antisera. At birth, the NR2B subunit was detected almost ubiquitously, the NR2A subunit staining was faint and restricted to the hippocampus, cerebral cortex, and striatum, and no NR2C subunit immunoreactivity was detected. During the first 3 postnatal weeks, the NR2B subunit became confined to forebrain structures, whereas the NR2A immunoreactivity became abundantly expressed throughout the brain. The NR2C immunoreactivity emerged 5 days after birth in the olfactory bulb, thalamus, and vestibular nuclei and became very intense after 10 days in cerebellar granule cells, its primary site of expression in adulthood. After 3 weeks, NR2A and NR2B immunoreactivity decreased to adult levels, whereas NR2C immunoreactivity remained unchanged. The patterns of distribution of the subunit proteins were in agreement with those of their corresponding mRNAs, as monitored by in situ hybridization histochemistry, although the mRNA translation appeared to be delayed by several days in certain areas. Our results reveal a progressive increase in the heterogeneity of NMDA receptors due to the comparably late onset of NR2A and NR2C subunit expression and by the area-specific rearrangement of NR2B subunit expression following birth. Key Words: NMDA receptor -Subunit -specific antisera -DevelopjiientHisto blots-Rat. J. Neurochem. 68, 469-478 (1997).N-Methyl-D-aspartate (NMDA) receptors are glutamate-activated cation channels, characterized by a high Ca 2~INa~permeability ratio, a voltage-dependent Mg 2+ block, a requirement for glycine as coagonist, and slow activation and deactivation kinetics (Nowak et al
Activation of cAMP-dependent protein kinase (PKA) can enhance or reduce the function of neuronal GABAA receptors, the major sites of fast synaptic inhibition in the brain. This differential regulation depends on PKA-induced phosphorylation of adjacent conserved sites in the receptor beta subunits. Phosphorylation of beta 3 subunit-containing receptors at S408 and S409 enhanced the GABA-activated response, whereas selectively mutating S408 to alanine converted the potentiation into an inhibition, comparable to that of beta 1 subunits, which are phosphorylated solely on S409. These distinct modes of regulation were interconvertible between beta 1 and beta 3 subunits and depended upon the presence of S408 in either subunit. In contrast, beta 2 subunit-containing receptors were not phosphorylated or affected by PKA. Differential regulation by PKA of postsynaptic GABAA receptors containing different beta subunits may have profound effects on neuronal excitability.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.