Inhibitory neurotransmission in the brain is largely mediated by GABA(A) receptors. Potentiation of GABA receptor activation through an allosteric benzodiazepine (BZ) site produces the sedative, anxiolytic, muscle relaxant, anticonvulsant and cognition-impairing effects of clinically used BZs such as diazepam. We created genetically modified mice (alpha1 H101R) with a diazepam-insensitive alpha1 subtype and a selective BZ site ligand, L-838,417, to explore GABA(A) receptor subtypes mediating specific physiological effects. These two complimentary approaches revealed that the alpha1 subtype mediated the sedative, but not the anxiolytic effects of benzodiazepines. This finding suggests ways to improve anxiolytics and to develop drugs for other neurological disorders based on their specificity for GABA(A) receptor subtypes in distinct neuronal circuits.

Cells isolated from the embryonic, neonatal, and adult rodent central nervous system divide in response to epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2), while retaining the ability to differentiate into neurons and glia. These cultures can be grown in aggregates termed neurospheres, which contain a heterogeneous mix of both multipotent stem cells and more restricted progenitor populations. Neurospheres can also be generated from the embryonic human brain and in some cases have been expanded for extended periods of time in culture. However, the mechanisms controlling the number of neurons generated from human neurospheres are poorly understood. Here we show that maintaining cell-cell contact during the differentiation stage, in combination with growth factor administration, can increase the number of neurons generated under serum-free conditions from 8% to > 60%. Neurotrophic factors 3 and 4 (NT3, NT4) and platelet-derived growth factor (PDGF) were the most potent, and acted by increasing neuronal survival rather than inducing neuronal phenotype. Following differentiation, the neurons could survive dissociation and either replating or transplantation into the adult rat brain. This experimental system provides a practically limitless supply of enriched, non-genetically transformed neurons. These should be useful for both neuroactive drug screening in vitro and possibly cell therapy for neurodegenerative diseases.

We report the isolation and characterization of a cDNA encoding a novel member of the GABA receptor gene family, ⑀. This polypeptide is 506 amino acids in length and exhibits its greatest amino acid sequence identity with the GABA A receptor ␥3 subunit (47%), although this degree of homology is not sufficient for it to be classified as a fourth ␥ subunit. The ⑀ subunit coassembles with GABA A receptor ␣ and ␤ subunits in Xenopus laevis oocytes and transfected mammalian cells to form functional GABA-gated channels. ␣1␤1⑀ GABA A receptors, like ␣1␤1␥2s receptors, are modulated by pentobarbital and the steroid 5␣-pregnan-3␣-ol-20-one but, unlike ␣1␤1␥2s receptors, are insensitive to flunitrazepam. Additionally, ␣1␤1⑀ receptors exhibit rapid desensitization kinetics, as compared with ␣1␤1 or ␣1␤1␥2s. Northern analysis demonstrates widespread expression of a large ⑀ subunit transcript in a variety of nonneuronal tissues and expression of a smaller transcript in brain and spinal cord. Sequence analysis demonstrated that the large transcript contained an unspliced intron, whereas the small transcript represents the mature mRNA, suggesting regulation of expression of the ⑀ subunit via neuronally restricted RNA splicing. In situ hybridization and immunocytochemistry reveal a pattern of expression in the brain restricted primarily to the hypothalamus, suggesting a role in neuroendocrine regulation, and also to subfields of the hippocampus, suggesting a role in the modulation of long term potentiation and memory.

GABA A receptors (GABA A Rs) are usually formed by ␣, ␤, and ␥ or ␦ subunits. Recently, ␦-containing GABA A Rs expressed in Xenopus oocytes were found to be sensitive to low concentrations of ethanol (1-3 mM). Our objective was to replicate and extend the study of the effect of ethanol on the function of ␣ 4 ␤ 3 ␦ GABA A Rs. We independently conducted three studies in two systems: rat and human GABA A Rs expressed in Xenopus oocytes, studied through two-electrode voltage clamp; and human GABA A Rs stably expressed in the fibroblast L(tk Ϫ ) cell line, studied through patch-clamp electrophysiology. In all cases, ␣ 4 ␤ 3 ␦ GABA A Rs were only sensitive to high concentrations of ethanol (100 mM in oocytes, 300 mM in the cell line). Expression of the ␦ subunit in oocytes was assessed through the magnitude of the maximal GABA currents and sensitivity to zinc. Of the three rat combinations studied, ␣ 4 ␤ 3 was the most sensitive to ethanol, isoflurane, and 5␣-pregnan-3␣,21-diol-20-one (THDOC); ␣ 4 ␤ 3 ␦ and ␣ 4 ␤ 3 ␥ 2S were very similar in most aspects, but ␣ 4 ␤ 3 ␦ was more sensitive to GABA, THDOC, and lanthanum than ␣ 4 ␤ 3 ␥ 2S GABA A Rs. Ethanol at 30 mM did not affect tonic GABA-mediated currents in dentate gyrus reported to be mediated by GABA A Rs incorporating ␣ 4 and ␦ subunits. We have not been able to replicate the sensitivity of ␣ 4 ␤ 3 ␦ GABA A Rs to low concentrations of ethanol in four different laboratories in independent studies. This suggests that as yet unidentified factors may play a critical role in the ethanol effects on ␦-containing GABA A Rs.