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

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 gamma-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 GABA(A) 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 microM 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 the 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 alpha 2-4, beta 1-3, and gamma 2S (short form). By comparison, the P0 olfactory bulb expressed all of the subunits detectable in the SVZa and additional subunit mRNAs: alpha 1, alpha 5, gamma 1, gamma 2L (long form), gamma 3, 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 alpha 2-5, beta 1-3, and gamma 2. To relate the characteristics of GABA(A) receptors in cultured SVZa precursor cells to particular combinations of subunits, the open reading frames of the dominant subunits detected by RT-PCR (alpha 2-4, beta 3, and gamma 2S) 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 precursor cells and in CHO-K1 cells expressing various combinations of recombinant GABA(A) receptor subunits suggested that the gamma 2S subunit was present and functional in the GABA(A) receptor chloride channel complex. Thus, SVZa precursor cells are GABAergic and a subset of the GABA(A) 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 ...

GABAA Receptor Signaling Induces Osmotic Swelling and Cell Cycle Activation of Neonatal Prominin+ Precursors

“…In this study, we show that purified neonatal neuroblasts have large GABAergic currents carried by Cl À with an affinity to GABA comparable with those already reported in postnatal SEZ cells [32,33] and embryonic cortical newborn neurons [34] and that these receptors show benzodiazepine sensitivity. In comparison with neuroblasts, precursors display a lower incidence of GABAergic currents with a much smaller current density, nonetheless modulated by diazepam.…”

GABA Regulates Dendritic Growth by Stabilizing Lamellipodia in Newly Generated Interneurons of the Olfactory Bulb

“…In agreement with previous reports (Stewart et al, 2002), we observed that SVZ-derived neurons already expressed different ␣-subunit immunoreactivity (␣ 2,3,4,5) of the GABA A receptors after 1 d in vitro (DIV) (supplemental Fig. 1, available at www.…”

“…The mean resting potential of cells was Ϫ25 Ϯ 3 mV (n ϭ 6 cells) (ranging from Ϫ18 to Ϫ40 mV); we detected no difference between 1 and 6 DIV cells. These results confirmed that SVZ-derived neurons expressed GABA A receptors from early immature stages (Stewart et al, 2002;Carleton et al, 2003).…”

Autocrine/Paracrine Activation of the GABAAReceptor Inhibits the Proliferation of Neurogenic Polysialylated Neural Cell Adhesion Molecule-Positive (PSA-NCAM+) Precursor Cells from Postnatal Striatum

“…Given that previous works reported the expression of GABA A R in early postnatal neuronal progenitor cells, notably in the anterior subventricular zone, we sought to investigate the presence of these receptors in striatal PSA-NCAM ϩ neuronal precursors (Stewart et al, 2002). To characterize GABA A R subunit transcripts expressed in PSA-NCAM ϩ progenitors, total RNAs extracted from 3-DIV spheres were reverse transcribed, and the subsequent cDNAs were amplified by PCR using specific sets of primers aimed at detecting transcripts for ␣ 1-5 , ␤ 1-3 , ␥ 1-3 , and ␦GABA A R subunit genes (Table 1).…”