One of the most likely targets of ethanol (EtOH) in the central nervous system (CNS) is the GABAA receptor (GABAAR). While the effects of EtOH have been the subject of study on most common CNS GABAAR composition, many other subunit combinations have only recently been tested for their EtOH responses. The a4b2d GABAARs are very sensitive to alcohol, with a concentration of 1 mM EtOH signifcantly enhancing GABAergic currents. Moreover, a6 and a4, when combined with b3 and d-containing subunits were associated with EtOH enhancement of function. Nevertheless, other studies were not able to obtain functional effects of low (1–30 mM) concentrations of EtOH. A plethora of studies both ‘‘in vivo’’ and ‘‘in vitro’’ show that chronic EtOH and EtOH withdrawal, can modify the gene expression of the GABAAR, but in these studies large concentrations of EtOH (50–200 mM) were used. A key question is whether GABAAR gene expression can be altered also by lower concentrations (e.g., 1–50 mM) of EtOH. In order to address this question we here used rat cerebellar granule cells in culture chronically treated with EtOH (1–100 mM). We then measured the GABAAR gene expression by RNase protection assay in two experimental conditions: After chronic EtOH (5 days) or its withdrawal (3 hours). Our results demonstrated that the only subunit affected by chronic EtOH treatment was the c2, the mRNA of which resulted decreased ()20%; p < 0.01) only at concentrations of 50 mM or higher. Neither low nor high concentrations of EtOH were able to change the gene expression of the other subunits of the GABAAR (a1, a4, a6 and d). On the contrary, EtOH withdrawal at the lowest concentration (1 mM) significantly decreased the abundance of the a1, a6 and d subunits ()36; )26 and )16% respectively; p < 0.05), but did not change the a4 subunit mRNA abundance. Similar results were observed using 10 mM EtOH for a1, a6 and d subunits ()37; )38 and 22% respectively; p < 0.05). The a4 subunit was up-regulated only by withdrawal of 100 mM EtOH (+31%; p < 0.01). This is the first report showing that low concentrations of EtOH, such as 1 mM, can modify GABAAR gene expression. These effects were subunit specific and more evident after withdrawal, suggesting that even such low EtOH concentrations when removed may disrupt neuronal excitability controlled by GABAAR; nevertheless, the key question of whether specific GABAAR subunits are more selectively sensitive to low EtOH remains still unanswered.
EFFECTS OF WITHDRAWAL OF LOW ETHANOL CONCENTRATIONS ON GABA(A) RECEPTOR GENE EXPRESSION IN RAT CEREBELLAR GRANULE NEURONS IN CULTURE
BIGGIO, FRANCESCA;FOLLESA, PAOLO
2010-01-01
Abstract
One of the most likely targets of ethanol (EtOH) in the central nervous system (CNS) is the GABAA receptor (GABAAR). While the effects of EtOH have been the subject of study on most common CNS GABAAR composition, many other subunit combinations have only recently been tested for their EtOH responses. The a4b2d GABAARs are very sensitive to alcohol, with a concentration of 1 mM EtOH signifcantly enhancing GABAergic currents. Moreover, a6 and a4, when combined with b3 and d-containing subunits were associated with EtOH enhancement of function. Nevertheless, other studies were not able to obtain functional effects of low (1–30 mM) concentrations of EtOH. A plethora of studies both ‘‘in vivo’’ and ‘‘in vitro’’ show that chronic EtOH and EtOH withdrawal, can modify the gene expression of the GABAAR, but in these studies large concentrations of EtOH (50–200 mM) were used. A key question is whether GABAAR gene expression can be altered also by lower concentrations (e.g., 1–50 mM) of EtOH. In order to address this question we here used rat cerebellar granule cells in culture chronically treated with EtOH (1–100 mM). We then measured the GABAAR gene expression by RNase protection assay in two experimental conditions: After chronic EtOH (5 days) or its withdrawal (3 hours). Our results demonstrated that the only subunit affected by chronic EtOH treatment was the c2, the mRNA of which resulted decreased ()20%; p < 0.01) only at concentrations of 50 mM or higher. Neither low nor high concentrations of EtOH were able to change the gene expression of the other subunits of the GABAAR (a1, a4, a6 and d). On the contrary, EtOH withdrawal at the lowest concentration (1 mM) significantly decreased the abundance of the a1, a6 and d subunits ()36; )26 and )16% respectively; p < 0.05), but did not change the a4 subunit mRNA abundance. Similar results were observed using 10 mM EtOH for a1, a6 and d subunits ()37; )38 and 22% respectively; p < 0.05). The a4 subunit was up-regulated only by withdrawal of 100 mM EtOH (+31%; p < 0.01). This is the first report showing that low concentrations of EtOH, such as 1 mM, can modify GABAAR gene expression. These effects were subunit specific and more evident after withdrawal, suggesting that even such low EtOH concentrations when removed may disrupt neuronal excitability controlled by GABAAR; nevertheless, the key question of whether specific GABAAR subunits are more selectively sensitive to low EtOH remains still unanswered.
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