GABA(A) receptor subunit mRNA expression and function in hippocampal neurons developing in vitro

Gamma - amino butyric acid (GABA) is the primary inhibitory neurotransmitter in the adult mammalian brain. The GABA_A receptor is a pentameric ligand gated chloride channel and the site of action for commonly used antiepilecptic drugs (AEDs). Numerous subunits have been cloned and grouped into families based on sequence similarity: α(1–6), β(1–3), γ(1–3), δ, ϵ, π, and &thetas;. These subunits have been found to be differentially expressed developmentally and anatomically. Little is known about how developmental changes in the subunit mRNA expression affect the pharmacology of the receptor in the maturing neuron. Furthermore, impact of chronic exposure to AEDs on the GABA_A subunit expression and synaptic pharmacology in developing animals remains to be determined. Here, we present experiments examining the relationship between subunit mRNA changes and functional pharmacology in hippocampal neurons developing in culture. Additionally, we report on the effect of chronic exposure of these neurons to clinically relevant levels of clonazepam and phenobarbital. We demonstrate that over the first 3 weeks in culture, zinc and clonazepam sensitivity diminishes. The fall in zinc inhibition is consonant with the previously described increase in γ₂ mRNA levels in these cells during the same period. However, the decrease in benzodiazepine sensitivity was surprising given these mRNA changes. We postulate that a developmental increase in the benzodiazepine insensitive α₆ subunit is responsible for the fall in clonazepam responsiveness. Furthermore, we report that chronic clonazepam and phenobarbital exposure results in statistically significant changes in levels of a number GABA_A subunit transcripts. However these changes were seen only in subunits which are likely expressed at low levels (based on detected signal). Surprisingly, physiological data indicates that chronic AED treatment does not affect the coupling of phenobarbital and clonazepam to synaptic GABA_A receptors. Finally, we demonstrate that chronic exposure of clinically relevant levels of these drugs results in neuron loss in hippocampal cultures. This suggests that these cultures may be a useful model for elucidating the mechanism of AED toxicity.