Extra-Synaptic GABAA Receptor Potentiation and Neurosteroid-Induced Learning Deficits Are Inhibited by GR3027, a GABAA Modulating Steroid Antagonist.

Objectives In Vitro: To study the effects of GR3027 (golexanolone) on neurosteroid-induced GABA-mediated current responses under physiological GABAergic conditions with recombinant human α5ß3γ2L and α1ß2γ2L GABAA receptors expressed in human embryonic kidney cells, using the response patch clamp technique combined with the Dynaflow™ application system. With α5ß3γ2L receptors, 0.01-3 µM GR3027, in a concentration-dependent manner, reduced the current response induced by 200 nM THDOC + 0.3 µM GABA, as well as the THDOC-induced direct gated effect. GR3027 (1 µM) alone had no effect on the GABA-mediated current response or current in the absence of GABA. With α1ß2γ2L receptors, GR3027 alone had no effect on the GABA-mediated current response or did not affect the receptor by itself. Meanwhile, 1-3 µM GR3027 reduced the current response induced by 200 nM THDOC + 30 µM GABA and 3 µM GR3027 that induced by 200 nM THDOC when GABA was not present. Objectives In Vivo: GR3027 reduces allopregnanolone (AP)-induced decreased learning and anesthesia in male Wistar rats. Rats treated i.v. with AP (2.2 mg/kg) or vehicle were given GR3027 in ratios of 1:0.5 to 1:5 dissolved in 10% 2-hydroxypropyl-beta-cyclodextrin. A dose ratio of AP:GR3027 of at least 1:2.5 antagonized the AP-induced decreased learning in the Morris Water Mase (MWM) and 1:7.5 antagonized the loss of righting reflex (LoR). GR3027 treatment did not change other functions in the rat compared to the vehicle group. Conclusions: GR3027 functions in vitro as an inhibitor of GABAA receptors holding α5ß3γ2L and α1ß2γ2L, in vivo, in the rat, as a dose-dependent inhibitor toward AP's negative effects on LoR and learning in the MWM.

Isoallopregnanolone Inhibits Estrus Cycle-Dependent Aggressive Behavior.

Among female rats, some individuals show estrus cycle-dependent irritability/aggressive behaviors, and these individual rats may be used as a model for premenstrual dysphoric disorder (PMDD). We wanted to investigate if these behaviors are related to the estrus cycle phase containing moderately increased levels of positive GABA-A receptor-modulating steroids (steroid-PAM), especially allopregnanolone (ALLO), and if the adverse behavior can be antagonized. The electrophysiology studies in this paper show that isoallopregnanolone (ISO) is a GABA-A-modulating steroid antagonist (GAMSA), meaning that ISO can antagonize the agonistic effects of positive GABA-A receptor-modulating steroids in both α1ß2γ2L and α4ß3δ GABA-A receptor subtypes. In this study, we also investigated whether ISO could antagonize the estrus cycle-dependent aggressive behaviors in female Wistar rats using a resident-intruder test. Our results confirmed previous reports of estrus cycle-dependent behaviors in that 42% of the tested rats showed higher levels of irritability/aggression at diestrus compared to those at estrus. Furthermore, we found that, during the treatment with ISO, the aggressive behavior at diestrus was alleviated to a level comparable to that of estrus. We noticed an 89% reduction in the increase in aggressive behavior at diestrus compared to that at estrus. Vehicle treatment in the same animals showed a minimal effect on the diestrus-related aggressive behavior. In conclusion, we showed that ISO can antagonize Steroid-PAM both in α1ß2γ2L and α4ß3δ GABA-A receptor subtypes and inhibit estrus cycle-dependent aggressive behavior.

Medroxyprogesterone acetate positively modulates specific GABAA-receptor subtypes - affecting memory and cognition.

Medroxyprogesterone acetate (MPA) is a progestin widely used in humans as hormone replacement therapy and at other indications. Many progestin metabolites, as the progesterone metabolite allopregnanolone, have GABAA-receptor modulatory effects and are known to affect memory, learning, appetite, and mood. In women, 4 years chronic treatment with MPA doubles the frequency of dementia and in rats, MPA causes cognitive impairment related to the GABAergic system. Activation of the membrane bound GABAA receptor results in a chloride ion flux that can be studied by whole-cell patch-clamp electrophysiological recordings. The purpose of this study was to clarify the modulatory effects of MPA and specific MPA metabolites, with structures like known GABAA-receptor modulators, on different GABAA-receptor subtypes. An additional aim was to verify the results as steroid effects on GABA response in single cells taken from rat hypothalamus. HEK-293 cell-lines permanently expressing the recombinant human GABAA-receptor subtype α1ß2γ2L or α5ß3γ2L or α2ß3γ2S were created. The MPA metabolites 3α5α-MPA,3ß5α-MPA and 3ß5ß-MPA were synthesised and purified for electrophysiological patch-clamp measurements with a Dynaflow system. The effects of MPA and tetrahydrodeoxycorticosterone were also studied. None of the studied MPA metabolites affected the responses mediated by α1ß2γ2L or α5ß3γ2L GABAA receptors. Contrary, MPA clearly acted both as a positive modulator and as a direct activator of the α5ß3γ2L and α2ß3γ2S GABAA receptors. However, in concentrations up to 10 µM, MPA was inactive at the α1ß2γ2L GABAA receptor. In the patch-clamp recordings from dissociated cells of the preoptic area in rats, MPA increased the amplitude of responses to GABA. In addition, MPA alone without added GABA, evoked a current response. In conclusion, MPA acts as a positive modulator of specific GABAA receptor subtypes expressed in HEK cells and at native GABA receptors in single cells from the hypothalamic preoptic area.