Changes in gamma-aminobutyrate type A receptor subunit mRNAs, translation product expression, and receptor function during neuronal maturation in vitro.

Abstract

The amounts of mRNAs encoding al, a'6, 132, 133, y2, and 8 subunits of y-aminobutyrate type A (GABAA) receptors and the gold immunolabeling density of their translation products were monitored during the growth of neonatal rat granule cells in primary culture. We investigated possible correlations (i) between temporal changes in mRNA content and expression density of their respective translation products and (ii) between the quantitative changes of receptor subunit expression, the GABA EC5. for Cl-channel activation, and diazepam efficacy in modulating GABA action on the Clchannels. At 3 days in vitro, the amount of GABAA receptor subunit mRNAs and the expression of their respective translation products were very low. During the next 2 weeks both parameters for every subunit studied increased asynchronously; moreover, at 14 days in vitro the sum of V2 and 8 subunit expression was smaller than the expression of the al or a6 or 182/133 subunits. This suggests that during in vitro maturation each subunit may be regulated independently and invites speculation as to possible changes in specific GABAA receptor subtype abundance during development in vitro. The maximal current intensity elicited by GABA failed to increase from 5 to 14 days in vitro, though the amount of mRNA encoding various subunits and the expression density of their respective translation products increased. Thus, qualitative changes in the GABAA receptor subtypes expressed and/or abnormalities in the subunit assembly very likely account for the uniformity of the maximal current intensity elicited by GABA during in vitro development. Also, during maturation of neuronal cultures from 5 to 20 days in vitro the extent of the positive modulation of GABA action by diazepam decreased dramatically. This finding might be related to an increase in the abundance of GABAA receptors including the a6 subunit and/or to the expression, during granule cell maturation in vitro, of GABAA receptors devoid of y2 subunits. To study whether changes in GABAA receptor subunit mRNA and protein expression occur synchronously during neuronal maturation in vitro (13) and whether eventual maturational variations in subunit expression could be associated with changes in GABAA receptor function, we used a competitive reverse transcription-polymerase chain reaction (RT-PCR) technique (14-16) to quantify specific GABAA subunit mRNAs, the label-fracture immunogold technique to monitor the expression of GABAA receptor subunits in the plasma membrane of cerebellar granule neurons (4, 5), and whole-cell recordings to assess GABA action and its modulation by diazepam (8). MATERIALS AND METHODSCell Culture. Primary cultures of cerebellar granule cells were prepared from 8-day-old Sprague-Dawley rat pups (13).Cells were dispersed with trypsin and plated at 2 x 10W cells per cm2 on 100-or 35-mm Nunc dishes coated with poly(Dlysine). Cultures were maintained in a humidified 6% CO2 atmosphere at 370C in basal Eagle's medium containing 10%6heat-inactivated fetal bovine serum, 2 mM ...

Effects of Ethanol on Tonic GABA Currents in Cerebellar Granule Cells and Mammalian Cells Recombinantly Expressing GABAAReceptors

“…These results indicate that the apparent affinity of the receptor for GABA increase as granule cells become more matured. Our data are in accord with those reported previously using the cerebellar granule cells (Zheng et al, 1994).…”

“…2). These findings might be related to an increase in the amount of GABA A receptors containing the ␣6 subunit during granule cell maturation in vitro (Zheng et al, 1994).…”

“…The sensitivity to GABA in cerebellar granule cells in primary culture is known to increase during development in vitro (Zheng et al, 1994). To characterize the GABA A receptors in cerebellar granule cells, whole-cell currents were recorded at a holding potential of Ϫ80 mV.…”

Plasticity in GABAAReceptor Subunit mRNA Expression by Hypothalamic Magnocellular Neurons in the Adult Rat

“…Although the present study has not evaluated whether the fluctuations in ␣ 1 subunit mRNA expression observed here during pregnancy are translated into ␣ 1 subunit protein, previous studies have indicated a positive correlation between ␣ 1 subunit mRNA expression and ␣ 1 subunit protein in vitro (Zheng et al, 1994) and in vivo (Huntsman et al, 1994). It is not unreasonable, therefore, to suggest that the 30 -80% variations in mRNA expression observed here may result in changes in functional subunit protein.…”

Lanthanum‐mediated modification of GABAA receptor deactivation, desensitization and inhibitory synaptic currents in rat cerebellar neurons

“…with development in vivo and in vitro (Laurie et al 1992;Zheng et al 1993;Mathews et al 1994;Zheng et al 1995). The distinct subunit expression of GABAA receptors in the cerebellum possibly results in the observed heterogeneity of receptor function and pharmacological modulation (Puia et al 1994;Tia et al 1996a;Zhu et al 1996;Mellor & Randall, 1997).…”