Changes in GABA_A Receptor Gene Expression Induced by Withdrawal of, but Not by Long-Term Exposure to, Ganaxolone in Cultured Rat Cerebellar Granule Cells

Abstract

The effects of ganaxolone, a synthetic analog of the endogenous neuroactive steroid allopregnanolone, on the function and expression of GABA A receptors were determined. Electrophysiological recordings demonstrated that ganaxolone potentiated with a potency and efficacy similar to those of allopregnanolone the Cl Ϫ currents evoked by GABA at recombinant human GABA A receptors (comprising ␣1␤2␥2L or ␣2␤2␥2L subunit assemblies) expressed in Xenopus oocytes. Exposure of cultured rat cerebellar granule cells to 1 M ganaxolone for 5 days had no effect on the abundance of mRNAs encoding the ␣1, ␣2, ␣3, ␣4, ␣5, ␥2L, or ␥2S subunits of the GABA A receptor. Withdrawal of ganaxolone after such long-term treatment, however, induced an increase in the abundance of ␣2, ␣4, and ␣5 subunit mRNAs and a decrease in the amounts of ␣1, ␥2L, and ␥2S subunit mRNAs. These changes were maximal 3 to 6 h after drug withdrawal and were reversible, being no longer apparent after 24 h. These results suggest that long-term exposure of cerebellar granule cells to ganaxolone does not affect the sensitivity of the GABA A receptor to several positive modulators. Nevertheless, the reduction in the amounts of the ␣1 and ␥2 subunit mRNAs together with the increase in the abundance of the ␣4 subunit mRNA induced by abrupt discontinuation of long-term treatment with ganaxolone suggest that withdrawal of this drug might result in a reduced response to classic benzodiazepines.GABA A receptor is the main type of inhibitory receptor in the brain and is a member of the superfamily of ligand-gated ion channels that includes the strychnine-sensitive glycine receptor, the 5-hydroxytryptamine 3 subtype of the serotonin receptor, and the nicotinic acetylcholine receptor (Betz, 1990). The binding of GABA to GABA A receptors induces the opening of an intrinsic Cl Ϫ channel with consequent hyperpolarization of the cell. The subunit composition of the pentameric GABA A receptors determines their specific physiological and pharmacological properties (Rudolph et al., 2001). The GABA A receptor is a prominent target of certain neuroactive steroids, which act as potent endogenous allosteric (positive or negative) modulators of receptor activity (ParkChung et al., 1999). Such compounds are also thought to be of potential therapeutic benefit. Indeed, neuroactive steroids have been used as intravenous anesthetics and have been shown to exert anxiolytic, hypnotic, anticonvulsant, and antiepileptic effects in animals or humans (Gasior et al., 1999).Ganaxolone (3␣-hydroxy-3␤-methyl-5␣-pregnan-20-one) is a synthetic compound that is structurally related to the endogenous neurosteroid allopregnanolone (3␣-hydroxy-5␣-pregnan-20-one) (Gasior et al., 1999). Like its endogenous analog, ganaxolone is a potent anticonvulsive and antiepileptic agent; however, it is more stable than allopregnanolone as a result of its ␤-methyl group, which prevents its metabolism and oxidation of the 3␣-hydroxy moiety (Carter et al., 1997 (Carter et al., 1997). In addition, ganaxolone is mor...

Neurosteroid withdrawal regulates GABA-A receptor α4-subunit expression and seizure susceptibility by activation of progesterone receptor-independent early growth response factor-3 pathway

“…At 24 h following neurosteroid withdrawal, there was a 3-fold upregulation in α4-subunit transcription. Similar increases in α4-subunit levels have been observed previously in response to withdrawal from allopregnanolone (Concas et al, 1998; Smith et al, 1998b; Follesa et al, 2000) and synthetic neurosteroids (Mascia et al, 2002). Enhanced α4-subunit expression is associated with a compensatory reduction in α1 expression (Shen et al, 2005).…”

A Mouse Kindling Model of Perimenstrual Catamenial Epilepsy

“…Our observation that neurosteroid withdrawal increases ␣4-subunit expression in the hippocampus is consistent with the established premise that prolonged exposure to progesterone followed by withdrawal in female rats causes up-regulation of the ␣4 subunit (Smith et al, 1998a;Gulinello et al, 2001). Similar increases in ␣4-subunit levels have been observed previously in response to withdrawal from AP (Smith et al, 1998b;Follesa et al, 2000) and synthetic neurosteroids (Mascia et al, 2002). The ␣4 subunit can coassemble with ␥2 to form synaptic GABA A receptors.…”

Allopregnanolone treatment, both as a single injection or repetitively, delays demyelination and enhances survival of Niemann‐Pick C mice

“…Similarly, alterations of GABAergic interneuron localization by neurosteroids have been shown in prefrontal cortex (Grobin et al, 2003). Treatment of cultured rat cerebellar granular cells with Ganaxolone, a synthetic analog of allopregnanolone, had no immediate effects, but its withdrawal changed the levels of several GABA receptors; thus, withdrawal of allopregnanolone might also result in potentially harmful effects (Mascia et al, 2002). It is of interest that the progesterone agonist mifepristone, but not progesterone, prevents Purkinje cell death in organotypic slice cultures, an effect not ascribed to GABA receptor responses (Ghoumari et al, 2003b), as is the effect of allopregnanolone.…”