Neural progenitor cells of the neonatal rat anterior subventricular zone express functional GABAA receptors

Authors

  • R.R. Stewart,

    Corresponding author
    1. Laboratory of Molecular and Cellular Neurobiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, 12420 Parklawn Drive, Bethesda, Maryland 20892-8115
    Current affiliation:
    1. NIH/NINDS, Neuroscience Building, Room 2135, 6001 Executive Blvd., MSC 9523, Bethesda, MD 20892-9523
    • Laboratory of Molecular and Cellular Neurobiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, 12420 Parklawn Drive, Bethesda, Maryland 20892-8115
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  • G.J. Hoge,

    1. Laboratory of Molecular and Cellular Neurobiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, 12420 Parklawn Drive, Bethesda, Maryland 20892-8115
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  • T. Zigova,

    1. Department of Neurosurgery, College of Medicine, University of South Florida, 12901 Bruce B. Downs Boulevard, Tampa, Florida 33612
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  • M.B. Luskin

    1. Department of Cell Biology, Emory University School of Medicine, 1648 Pierce Drive, Atlanta, Georgia 30322-3030
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Abstract

The interneurons of the olfactory bulb arise from precursor cells in the anterior part of the neonatal subventricular zone, the SVZa, and 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 complement of ionotropic neurotransmitter receptors that they express in vitro. For this analysis, we tested the sensitivity of SVZa progenitor cells to γ-amino-n-butyric 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 GABAA receptor-specific agonist, but were insensitive to ATP, kainate, NMDA, and ACh. In addition, GABA- or muscimol-activated chloride currents were blocked nearly completely by 30 μM bicuculline, the GABAA receptor-specific antagonist, suggesting that GABAB and GABAC receptors are absent. Measurements of the chloride reversal potential by gramicidin-perforated patch clamp revealed that currents generated by activation of GABAA 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 the messenger RNA (mRNA) coding for glutamic acid decarboxylase 67 (GAD67), used in the synthesis of GABA and for GABAA receptor subunits. Both postnatal day (P0) SVZa and olfactory bulb possessed detectable mRNA coding for GAD67. In P0 SVZa, the GABAA receptor subunits detected with RT-PCR included α2-4, β1-3, and γ2S (short form). By comparison, the P0 olfactory bulb expressed all of the subunits detectable in the SVZa and additional subunit mRNAs: α1, α5, γ1, γ2L (long form), γ3, and δ subunit mRNAs. Antibodies recognizing GABA, GAD, and various GABAA 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 GABAA receptor subunits α2-5, β1-3, and γ2. To relate the characteristics of GABAA receptors in cultured SVZa precursor cells to particular combinations of subunits, the open reading frames of the dominant subunits detected by RT-PCR (α2-4, β3, and γ2S) 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 GABAA receptors in SVZa precursor cells and in CHO-K1 cells expressing various combinations of recombinant GABAA receptor subunits suggested that the γ2S subunit was present and functional in the GABAA receptor chloride channel complex. Thus, SVZa precursor cells are GABAergic and a subset of the GABAA receptor subunits detected in the olfactory bulb was found in the SVZa, as might be expected because SVZa progenitor cells migrate to the bulb as they differentiate. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 305–322, 2002; DOI 10.1002/neu.10038

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