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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 ...

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