Developmental changes in human γ-aminobutyric acida receptor subunit composition

γ-Aminobutyric acid (GABA) is the neurotransmitter at most inhibitory synapses in the human central nervous system. The GABA_A receptor, a ligand-gated ion channel, is the site of action of benzodiazepines, the most widely prescribed neuroactive drugs. It was recently demonstrated that there are multiple subtypes of GABA_A receptors. Studies of rodents have shown that receptor subunits are developmentally controlled. The major α subunit of the adult receptor is expressed at low levels before birth. This study, using postmortem human tissue, shows that GABA_A receptors are present in significant numbers in the human cerebellum at birth, and the numbers rise threefold by adulthood. Two subtypes of benzodiazepine receptors were detected by binding studies in the neonate, whereas only a single subtype of receptor was detected in the adult cerebellum. Comparison to recombinant human GABA_A receptors shows that receptors containing α 1 constitute 50% of the receptors at birth and the percentage rises to over 95% by adulthood. In both cerebral cortex and cerebellum, a dramatic rise in α 1 messenger RNA was observed during development, suggesting that the complement of GABA_A receptors differs in infants and adults. These findings have significant implications for normal neurodevelopment as well as for the understanding and treatment of pathophysiological states such as seizures.     

Early-onset myasthenia gravis: A recurring T-cell epitope in the adult-specific acetylcholine receptor ε subunit presented by the susceptibility allele HLA-DR52a

No immunodominant T-cell epitopes have yet been reported in the human acetylcholine receptor (AChR), the target of the pathogenic autoantibodies in myasthenia gravis (MG). We have selected and characterized T cells from MG patients by restimulation in culture with recombinant human AChR α, γ, and ε subunits; the γ and ε distinguish the fetal and adult AChR isoforms, respectively. We obtained clones specific for the ε, rather than the α or γ, subunit in 3 of the first 4 early-onset MG cases tested. They all responded to peptide ε201–219 and to low concentrations of adult but not fetal AChR. Moreover, although using different T-cell receptor genes, they were all restricted to HLA-DR52a (DRB3*0101), a member of the strongly predisposing HLA-A1-B8-DR3 haplotype. This apparently immunodominant ε201–219 epitope (plus DR52a) was also recognized by clones from an elderly patient whose MG had recently been provoked by the drug D -penicillamine. In all 4 cases, however, the serum antibodies reacted better with fetal than adult AChR and may thus be end products of determinant spreading initiated by adult AChR-specific T cell responses. Furthermore, as these T cells had a pathogenic Th1 phenotype, with the potential to induce complement-activating antibodies, they should be important targets for selective immunotherapy. Ann Neurol 1999;45:224–231     

Antisense oligonucleotide to GABAA receptor γ2 subunit induces limbic status epilepticus

γ-Aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the brain. A deficiency of GABAergic inhibition mediated via the GABA_A receptor complex has for a long time been suspected to be a central factor in epileptogenesis. Status epilepticus is a condition of sustained and prolonged excitation of neuronal circuits, as detected by epileptiform discharges in the electroencephalogram (EEG). Reduction of GABA_A receptor-mediated hippocampal inhibition has been implicated in the development of status epilepticus. The present study provides direct evidence of a link between the GABA_A receptor and epilepsy. We show that selective inhibition of the expression of the GABA_A receptor γ2 subunit in the rat hippocampus by means of antisense oligonucleotides leads to spontaneous electrographic seizures that evolve into profound limbic status epilepticus, ultimately resulting in severe neurodegenerative changes. Concurrent treatment with diazepam prevents the development of status epilepticus and markedly reduces neuronal cell loss. These findings strongly support the hypothesis that the GABA_A receptor is critically involved in the pathogenesis of seizures and status epilepticus. J. Neurosci. Res. 54:863–869, 1998. © 1998 Wiley-Liss, Inc.     

Immunocytochemical localization of the α6 subunit of the γ-aminobutyric acidA receptor in the rat nervous system

The localization in the rat central nervous system and retina of the α₆ subunit peptide of the γ-aminobutyric acid (GABA_A) receptor has been studied by light microscopy immunocytochemistry with a specific anti-α₆ antibody. The α₆ subunit was present in the granule cells of the cerebellum, the granule cells of the dorsal cochlear nucleus, axons of the olfactory nerve including the glomerular endings, layer II of the dorsal horn of the spinal cord, and in the retinal synaptic layers, particularly the inner plexiform layer. Thus, contrary to the general belief, the α₆ subunit is not exclusively localized in the granule cells of the cerebellum. It is also expressed in some sensory neurons and other neurons involved in the initial processing of sensory information. © 1996 Wiley-Liss, Inc.     

Early continuous white noise exposure alters l‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid receptor subunit glutamate receptor 2 and γ‐aminobutyric acid type a receptor subunit β3 protein expression in rat auditory cortex

Auditory experience during the postnatal critical period is essential for the normal maturation of auditory function. Previous studies have shown that rearing infant rat pups under conditions of continuous moderate‐level noise delayed the emergence of adult‐like topographic representational order and the refinement of response selectivity in the primary auditory cortex (A1) beyond normal developmental benchmarks and indefinitely blocked the closure of a brief, critical‐period window. To gain insight into the molecular mechanisms of these physiological changes after noise rearing, we studied expression of the AMPA receptor subunit GluR2 and GABA_A receptor subunit β3 in the auditory cortex after noise rearing. Our results show that continuous moderate‐level noise rearing during the early stages of development decreases the expression levels of GluR2 and GABA_Aβ3. Furthermore, noise rearing also induced a significant decrease in the level of GABA_A receptors relative to AMPA receptors. However, in adult rats, noise rearing did not have significant effects on GluR2 and GABA_Aβ3 expression or the ratio between the two units. These changes could have a role in the cellular mechanisms involved in the delayed maturation of auditory receptive field structure and topographic organization of A1 after noise rearing. © 2009 Wiley‐Liss, Inc.     

Orexin a phosphorylates the γ‐Aminobutyric acid type A receptor β2 subunit on a serine residue and changes the surface expression of the receptor in SH‐SY5Y cells exposed to propofol

Propofol activates the γ‐aminobutyric acid type A receptor (GABA_AR) and causes a reversible neurite retraction, leaving a thin, thread‐like structure behind; it also reverses the transport of vesicles in rat cortical neurons. The awakening peptide orexin A (OA) inhibits this retraction via phospholipase D (PLD) and protein kinase C? (PKC?). The human SH‐SY5Y cells express both GABA_ARs and orexin 1 and 2 receptors. These cells are used to examine the interaction between OA and the GABA_AR. The effects of OA are studied with flow cytometry and immunoblotting. This study shows that OA stimulates phosphorylation on the serine residues of the GABA_AR β₂ subunit and that the phosphorylation is caused by the activation of PLD and PKC?. OA administration followed by propofol reduces the cell surface expression of the GABA_AR, whereas propofol stimulation before OA increases the surface expression. The GABA_AR β₂ subunit is important for receptor recirculation, and the effect of OA on propofol‐stimulated cells may be due to a disturbed recirculation of the GABA_AR. © 2015 Wiley Periodicals, Inc.     

PET imaging of acetylcholinesterase inhibitor‐induced effects on α4β2 nicotinic acetylcholine receptor binding

Acetylcholinesterase inhibitors (AChEIs) are drugs that increase synaptic acetylcholine (ACh) concentrations and are under investigation as treatments for symptoms accompanying Alzheimer's disease. The goal of this work was to use PET imaging to evaluate alterations of in vivo α4β2 nicotinic acetylcholine receptor (nAChR) binding induced by the AChEIs physostigmine (PHY) and galanthamine (GAL). The α4β2 nAChR‐specific radioligand [¹⁸F]nifene was used to examine the effects of 0.1–0.2 mg/kg PHY, 5 mg/kg GAL, and saline in three separate experiments all performed on each of two rat subjects. A 60‐min bolus‐infusion protocol was used with drug administered after 30 min. Data from the thalamus and cortex were analyzed with a graphical model accounting for neurotransmitter activation using the cerebellum as a reference region to test for transient competition with bound [¹⁸F]nifene. Significant [¹⁸F]nifene displacement was detected in both regions during one PHY and both GAL studies, while no significant competition was observed in both saline studies. This preliminary work indicates the viability of [¹⁸F]nifene in detecting increases in synaptic ACh induced by AChEIs. Synapse 67:882–886, 2013. © 2013 Wiley Periodicals, Inc.     

A β-subunit mutation in the acetylcholine receptor channel gate causes severe slow-channel syndrome

Point mutations in the genes encoding the acetylcholine receptor (AChR) subunits have been recognized in some patients with slow-channel congenital myasthenic syndromes (CMS). Clinical, electrophysiological, and pathological differences between these patients may be due to the distinct effects of individual mutations. We report that a spontaneous mutation of the β subunit that interrupts the leucine ring of the AChR channel gate causes an eightfold increase in channel open time and a severe CMS characterized by severe endplate myopathy and extensive remodeling of the postsynaptic membrane. The pronounced abnormalities in neuromuscular synaptic architecture and function, muscle fiber damage and weakness, resulting from a single point mutation are a dramatic example of a mutation having a dominant gain of function and of hereditary excitotoxicity.     

Nicotinic acetylcholine receptors in rat cochlear nucleus: [125I]-α-bungarotoxin receptor autoradiography and in situ hybridization of α7 nAChR subunit mRNA

The cochlear nucleus (CN) is the first site in the central nervous system (CNS) for processing auditory information. Acetylcholine in the CN is primarily extrinsic and is an important neurotransmitter in efferent pathways thought to provide CNS modulation of afferent signal processing. Although muscarinic acetylcholine receptors have been studied in the CN, the role of nicotinic receptors has not. We examined the distribution of one nicotinic acetylcholine receptor subtype, the α-bungarotoxin receptor (αBgt), in the CN. Quantitative autoradiography was used to localize receptors and in situ hybridization was used to localize α7 mRNA in CN neurons that express the αBgt receptor. Binding sites for αBgt are abundant in the anterior ventral, posterior ventral, and dorsal divisions of the CN, and receptor density is low in the granule cell layer and interstitial nucleus. Heterogeneity in CN subregions is described. Four distinct patterns of αBgt binding were observed: (1) binding over and around neuronal cell bodies, (2) receptors locally surrounding neurons, (3) dense punctate binding in the dorsal CN (DCN) not associated with neuronal cell bodies, and (4) diffuse fields of αBgt receptors prominent in the DCN molecular layer, a field underlying the granule cell layer and in the medial sheet. The perikaryial receptors are abundant in the ventral CN (VCN) and are always associated with neurons expressing mRNA for the receptor. Other neurons in the VCN also express α7 mRNA, but without αBgt receptor expression associated with the cell body. In general, αBgt receptor distribution parallels cholinergic terminal distribution, except in granule cell regions rich in cholinergic markers but low in αBgt receptors. The findings indicate that αBgt receptors are widespread in the CN but are selectively localized on somata, proximal dendrites, or distal dendrites depending on the specific CN subregion. The data are consistent with the hypothesis that descending cholinergic fibers modulate afferent auditory signals by regulating intracellular Ca²⁺ through αBgt receptors. J. Comp. Neurol. 397:163–180, 1998. © 1998 Wiley-Liss, Inc.