Neurosteroid regulation of GABA(A) receptors: Focus on the alpha4 and delta subunits

Abstract

Neurosteroids, such as the progesterone metabolite 3alpha-OH-5alpha[beta]-pregnan-20-one (THP or [allo]pregnanolone), function as potent positive modulators of the GABA(A) receptor (GABAR) when acutely administered. However, fluctuations in the circulating levels of this steroid at puberty, across endogenous ovarian cycles, during pregnancy or following chronic stress produce periods of prolonged exposure and withdrawal, where changes in GABAR subunit composition may occur as compensatory responses to sustained levels of inhibition. A number of laboratories have demonstrated that both chronic administration of THP as well as its withdrawal transiently increase expression of the alpha4 subunit of the GABAR in several areas of the central nervous system (CNS) as well as in in vitro neuronal systems. Receptors containing this subunit are insensitive to benzodiazepine (BDZ) modulation and display faster deactivation kinetics, which studies suggest underlie hyperexcitability states. Similar increases in alpha4 expression are triggered by withdrawal from other GABA-modulatory compounds, such as ethanol and BDZ, suggesting a common mechanism. Other studies have reported puberty or estrous cycle-associated increases in delta-GABAR, the most sensitive target of these steroids which underlies a tonic inhibitory current. In the studies reported here, the effect of steroids on inhibition, which influence anxiety state and seizure susceptibility, depend not only on the subunit composition of the receptor but also on the direction of Cl(-) current generated by these target receptors. The effect of neurosteroids on GABAR function thus results in behavioral outcomes relevant for pubertal mood swings, premenstrual dysphoric disorder and catamenial epilepsy, which are due to fluctuations in endogenous steroids.

Fig. 1

Time course of progesterone effects on GABAR subunit composition and pharmacology. Levels of α4 expression were detected with Western blot techniques on a daily or weekly basis during sustained exposure to progesterone (and thus THP) via subcutaneous capsule for 3 weeks. Levels of α4 expression increased significantly by 2-3 days of progesterone exposure compared to vehicle; then levels recovered to control values across the remainder of the exposure period until progesterone withdrawal (24 hr after capsule removal), when significant increases in α4 expression were again observed (P<0.001). Concomitant pharmacological evaluation of BDZ LZM potentiation of GABA-gated current evaluated in pyramidal neurons acutely isolated from CA1 hippocampus revealed a LZM insensitivity closely correlated with α4 upregulation. *P<0.05 (from Smith, 2003, reprinted with permission).

Fig. 2

Suppression of α4 expression alters the kinetics of mIPSCs after chronic neurosteroid exposure. For this study, female rats were administered THP for 48 hr (10 mg/kg, i.p.) in conjunction with α4 antisense (or missense) oligonucleotide administered intraventricularly to suppress α4 expression. mIPSCs were recorded from the soma with whole cell patch clamp techniques from CA1 hippocampal pyramidal cells in the slice at near physiological temperature (33-35 °C). Representative traces (left) and averaged currents (right) reveal that THP treatment results in a faster τ-fast of decay, an effect prevented by antisense suppression of α4 expression (n = 30-34 rats; from Hsu et al., 2003, reprinted with permission).

Fig. 3

α4-containing GABAR exhibit faster deactivation kinetics than other receptor subtypes. Representative current traces illustrate the different kinetics exhibited by α1β2γ2, α4β2γ2, α4β2δ, and α5β2γ2 recorded from HEK-293 cells using outside-out patch recording techniques and rapid application of saturating concentrations of agonist. Both α4-containing GABAR deactivate with an accelerated τ-fast compared with the other subtypes. These results are representative of those from 6 to 8 cells/group (from Smith & Gong, 2005).

Fig. 4

Withdrawal from THP increases hippocampal α4 expression and results in a BDZ insensitivity. (A) 24 hr withdrawal from chronic administration of THP (10 mg/kg, i.p. for 3 weeks) to adult, female rats increased expression of α4 protein (left) and α4 mRNA (right) in hippocampal membranes compared to vehicle-injected controls. (B) This THP withdrawal state produced a relative insensitivity to modulation of GABA (10 μM)-gated current by the BDZ LZM in pyramidal neurons acutely isolated from CA1 hippocampus and recorded with whole-cell patch clamp techniques. In contrast, LZM was effective in potentiating GABA-gated current up to 2-fold in neurons from control rats (n = 6-8 cells/group).

Fig. 5

THP withdrawal reduces paired-pulse inhibition. Following 24 hr withdrawal from chronically administered THP (10 mg/kg, i.p. for 3 weeks) to adult, female rats hippocampal slices were prepared, and population EPSPs recorded from the pyramidal cell layer of CA 1 hippocampus in response to 2 stimulating pulses (4× threshold, 10 ms interpulse interval) delivered to the Schaffer collaterals. In contrast to the vehicle-injected controls, which exhibited a highly significant reduction in population spike amplitude in response to the second (test) pulse compared to the first (conditioning) pulse, reduction in PS amplitude in response to the second stimulus was not significantly different from the response to the first stimulus after THP withdrawal. This demonstrates a decrease in feedback inhibition after THP withdrawal. (A) Representative traces. (B) Population data (n = 10-12 slices/group).

Fig. 6

THP increases anxiety in a shock-paired plus maze paradigm following THP withdrawal in mice. For this study, mice were subjected to withdrawal from their naturally high levels of endogenous THP with a 5α-reductase blocker (finasteride, 50 mg/kg, i.p., 3 days). When tested in the elevated plus maze following a brief shock (left panel), THP decreased open arm time following THP Wd, in contrast to its effect in control mice, where it increased this parameter. (*P<0.05 vs. vehicle, n = 17-26). The total number of arm entries (lower panel) was not altered by any experimental treatment. This anxiogenic effect of THP (right panel) was not observed after THP Wd in δ knock-out mice, implicating δ-GABAR in this paradoxical effect of the steroid.