Synaptic Mechanisms And Alcohol Actions

Alcohol Abuse And Alcoholism

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Abstract

The mammalian nervous system generates new neurons well into adulthood from a population of precursor cells in the anterior region of the subventricular zone (SVZa) of the forebrain. The progeny of SVZa progenitor cells migrate long distances from the SVZa to the olfactory bulb, within which they differentiate into two types of cells: periglomerular and granule interneurons. The progenitor cells are distinctive in that they possess a neuronal phenotype and yet undergo cell division. To characterize the differentiation of neonatal SVZa progenitor cells, we analyzed the compliment of ionotropic neurotransmitter receptors that they express in vitro. For this analysis, we tested the sensitivity of SVZa progenitor cells to gamma-aminobutyric acid (GABA), adenosine triphosphate (ATP), kainate, N-methyl-D-aspartate (NMDA), and acetylcholine (ACh) after 1 day in vitro. SVZa progenitor cells had chloride currents activated by GABA and muscimol (the GABA-A receptor-specific agonist), but were insensitive to ATP, kainate, NMDA and ACh. In addition, GABA- or muscimol-activated chloride currents were blocked by 30 uM bicuculline (the GABA-A receptor-specific antagonist), suggesting that GABA-B and GABA-C receptors are absent. Measurements of the chloride reversal potential by gramicidin- perforated patch-clamp revealed that currents generated by activation of GABA-A receptors were inward, and thus depolarizing. A set of complementary experiments was undertaken to determine by reverse transcription and polymerase chain reaction (RT-PCR) whether SVZa progenitor cells express messenger RNA (mRNA) coding for glutamic acid decarboxylase 67 (GAD67), used in the synthesis of GABA and for GABA-A receptor subunits. Both postnatal day (P0) SVZa and olfactory bulb possessed detectable mRNA coding for GAD67. In P0 SVZa, the GABA-A receptor subunits detected with RT-PCR included alpha2-4, beta1-3 and gamma2S (short form). By comparison, the P0 olfactory bulb expressed all of the subunits detectable in the SVZa and additional subunit mRNAs: alpha1, alpha5, gamma2L (long form), gamma3 and delta subunit mRNAs. Antibodies recognizing GABA, GAD and various GABA-A receptor subunits were used to label SVZa cells harvested from P0-1 rats and cultured for 1 day. The cells were immunoreactive for GABA, GAD and the GABA-A receptor subunits alpha2-5, beta1-3 and gamma2. To relate the characteristics of GABA-A receptors in cultured SVZa progenitor cells to particular combinations of subunits, the open reading frames of the dominant subunits detected by PCR (alpha2-4, beta3 and gamma2S) were cloned into a mammalian cell expression vector and different combinations were transfected into Chinese hamster ovary-K1 (CHO-K1) cells. A comparison of the sensitivity to inhibition by zinc of GABA-A receptors in SVZa progenitor cells and in CHO-K1 cells expressing various combinations of recombinant GABA-A receptor subunits suggested that the gamma2S subunit was present and functional. Thus, SVZa progenitor cells are GABAergic and a subset of the GABA-A receptor subunits detected in the olfactory bulb was found in the SVZa progenitor cells, as might be expected because the SVZa progenitor cells migrate to the olfactory bulb, where they differentiate. In addition, activation of GABA-A receptors was found to have an excitatory action in the SVZa progenitor cells. Experiments are also in progress to elucidate the cellular mechanisms of alcohol and neuroactive substance action on neurotransmitter receptors such as NMDA receptors, GABA-A receptors, serotonin type 3 (5-HT3) receptors, nicotinic acetylcholine (nACh) receptors and ATP receptors. These studies hold the promise that such physiological approaches will advance our knowledge of the cellular mechanisms of alcohol and neuroactive substance action in the nervous system and provide a foundation for understanding the cellular basis of alcohol abuse and alcoholism.