Ethanol exposure and withdrawal differentially affect the GABA - A receptor plasticity and function in two different neuronal populations

Gamma-Aminobutyric acid type A (GABA-A) receptors have been implicated as major target sites for the acute and chronic ac- tions of ethanol (EtOH). Prolonged exposure to and withdrawal from EtOH are associated with alterations in GABA-A receptor subunit gene expression as well as in receptor function and phar- macological sensitivity in different in vivo and in vitro experi- mental models. Here, we focus on the effects of chronic EtOH ex- posure and withdrawal on the expression of the delta subunit of the GABA-A receptor in cultured rat hippocampal neurons (HP) and cerebellar granule (CG) neurons. GABA-A receptors contain- ing the delta subunit are preferentially extrasynaptic, are respon- sible for the tonic inhibition, and possess an enhanced sensitivity to the agonist THIP, to the neurosteroid allopregnanolone as well as to low concentrations of EtOH. Immunocytochemical and con- focal microscopy studies showed that delta subunit is preferen- tially expressed on the soma of CG cells, whereas it is mainly den- dritic in HP neurons. Long-term EtOH exposure increased delta subunit mRNA and peptide levels in HP neurons, while it did not significantly modify its expression in CG cells. EtOH withdrawal was associated to a rapid (6 h) marked reduction of delta subunit expression in CG cells, and to a persistent enhancement in HP neurons during the first 6h from withdrawal compared to un- treated control. Patch clamp recordings revealed that these EtOH induced changes in delta subunit expression are associated, in HP and CG cells, to an opposite effect, increase and decrease, respec- tively, of the efficacy of THIP and allopregnanolone to potentiate the chloride currents. Our data demonstrate that the changes in gene expression and function of GABA-A receptor containing the delta subunit induced by long-term EtOH exposure and with- drawal are opposite in HP and CG cells, suggesting a putative dif- ferent role of these extrasynaptic receptors in controlling tonic in- hibition in these two different cell types. The results indicate also a strict association existing between the GABA-A receptors sub- unit diversity and their differential sensitivity to drugs and en- dogenous modulators. This evidence together with the different cellular localization of GABA-A receptors may explain the differ- ent threshold of excitability of selective neuronal populations in specific brain areas. It is well established that ethanol increases plasma and brain levels of GABA-A receptor active neurosteroids by activating the HPA axis. We now show that, in isolated rat hip- pocampal slices, EtOH dose dependently increased the concentra- tion of allopregnanolone as well as the amplitude of GABA-A re- ceptor-mediated inhibitory postsynaptic currents recorded from CA1 pyramidal neurons. This latter effect appears biphasic, con- sisting of a rapid, direct modulatory effect, insensitive to the 5 alfa reductase blocker finasteride, and a delayed, allopregnanolone- mediated action reversed by finasteride. These observations sug- gest that EtOH may modulate GABA-A receptor function through an increase in de novo neurosteroid synthesis in brain that is in- dependent from the HPA axis. Given that neurosteroids play a major role in the physiological modulation of GABA-A receptor plasticity and function this novel mechanism may be important in mediating some effects of ethanol in physiological and pathologi- cal conditions (menstrual cycle, pregnancy, menopause, premen- strual symdrome) and a variety of psychiatric and neurological disorders in which the steroidogenesis undergoes dramatic func- tional changes.