GABAA receptors mediate trophic effects of GABA on embryonic brainstem monoamine neurons in vitro

Abstract

The inhibitory neurotransmitter GABA may act as a trophic signal for developing monoamine neurons in embryonic rat brain, because GABA neurons and their receptors appear in brainstem during generation of monoamine neurons. To test this hypothesis, we used dissociated cell cultures from embryonic day 14 rat brainstem, which contains developing serotonin (5-HT), noradrenaline (tyrosine hydroxylase; TH), and GABA neurons. Immunocytochemistry and reverse transcription-PCR (RT-PCR) revealed the presence of multiple alpha, beta, gamma, and delta subunits in these cultures. Competitive RT-PCR demonstrated high levels of beta3 subunit transcripts. Expression of functional GABAA receptors was demonstrated using 36Cl- flux assays. To investigate GABAergic regulation of neuronal survival and growth, cultures were treated for 1-3 d in vitro with 10 microM GABA and/or GABAA antagonist (bicuculline or the pesticide dieldrin). The effects of treatments were quantified by analysis of immunoreactive 5-HT, TH, and GABA neurons. GABAA receptor ligands differentially regulated neuronal survival and growth depending on neurotransmitter phenotype. GABA exerted positive effects on monoamine neurons, which were countered by bicuculline (and dieldrin, 5-HT neurons only). By itself, bicuculline produced inhibitory effects on both 5-HT and TH neurons, whereas dieldrin potently inhibited 5-HT neurons only. GABA neurons responded positively to both antagonists, but more strongly to bicuculline. Taken together, these results demonstrate that the activation/inhibition of GABAA receptors produces opposite effects on the development of embryonic monoamine and GABA neurons. This suggests that these neurotransmitter phenotypes may express GABAA receptors that differ in fundamental ways, and these differences determine the developmental responses of these cells to GABAergic stimuli.

Fig. 1.

Representative 5-HT (A), TH (B), and GABA (C) immunoreactive neurons from E14 brainstem cultures. Cells were cultured for 1 d in DMEM + 10% FCS and then switched to serum-free medium (DMEM + ITS + 0.1% BSA) for 48 hr. Scale bar, 50 μm.

Fig. 2.

Expression of GABA_A receptor subunit proteins in E14 brainstem cultures. Immunocytochemistry with anti-GABA_A α1 rabbit polyclonal antibody (A) and anti-GABA_A β2/3 monoclonal antibody (B). C, Background control with α1 primary antibody omitted. D, Background control with β2/3 primary antibody omitted. Cells were cultured for 1 d in DMEM + 10% FCS and then switched to serum-free medium (DMEM + ITS + 0.1% BSA) for 48 hr. Scale bar, 50 μm.

Fig. 3.

RT-PCR analysis of GABA_A receptor subunit mRNAs in E14 rat brainstem cultures. Cells were cultured for 1 d in DMEM + 10% FCS and then for 2 d in serum-free medium (DMEM + ITS + 0.1% BSA). RT-PCR revealed expression of mRNAs encoding most of the known GABA_A receptors, except α6.

Fig. 4.

A–C, Representative gels for GABA_A receptor subunit mRNAs from cultured E14 rat brainstem analyzed by competitive RT-PCR using internal standards. A series of concentrations of internal standard cRNAs were added to each tube containing 1 μg of total RNA. The PCR products from each tube are shown in triplicate for each subunit. Top bands, PCR products of target mRNA. Bottom bands,BglII-digested internal standard PCR products. Note that increasing concentrations of internal standards compete with target mRNA for amplification. The point of equivalence was determined by linear regression analysis of the ratio of counts incorporated into the target PCR product across the series of concentrations of internal standards. The point of equivalence (when the ratio is 1) is the absolute concentration of GABA_A receptor subunit mRNA/microgram of total RNA. D, Quantification of GABA_A receptor subunit mRNA levels assayed in this study. Note that β3 is the most abundant subunit compared with α1 and γ1. Cells were cultured for 1 d in DMEM + 10% fetal calf serum and then for 2 d in serum-free medium (DMEM + ITS + 0.1% BSA).

Fig. 5.

GABA_A receptor-mediated Cl⁻ uptake into cultured E14 brainstem cells. Addition of exogenous GABA enhanced Cl⁻ uptake from basal levels of 3.3 nmol/mg protein in a dose-dependent manner to 13.6 nmol/mg protein (at 10 μm GABA). Addition of 10 μmbicuculline lowered Cl⁻ uptake stimulated by 10 μm GABA to 4.3 nmol/mg protein.

Fig. 6.

Effects of GABA receptor ligands on survival of 5-HT, TH, and GABA neurons in E14 rat brainstem cultures (number of immunoreactive neurons/mm², expressed as percentage control). Cells were cultured for 1 d in DMEM + 10% FCS and then switched to serum-free medium (DMEM + ITS + 0.1% BSA) plus ligand for 48 hr. Cultures were then fixed, stained with antibodies to 5-HT, TH, or GABA, and immunoreactive neurons were counted. Individual data from three separate experiments (n = 60 cells/treatment group) were converted to percentage control by dividing individual data points by the overall mean control value. Statistical analysis was performed by ANOVA followed by Dunnet’s multiple comparison test when ANOVA was significant (p < 0.05).