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Introduction: The mechanism underlying endometriosis‐related pain remains poorly understood. Previous studies have indicated that γ‐aminobutyric acid (GABA) type A (GABAA) receptors and GABAergic substances (eg endogenous neurosteroids) play important mechanistic roles in various pain conditions. Our primary objective was to compare GABAA receptor function between women with endometriosis and healthy controls by performing a challenge test with diazepam, a GABAA receptor agonist, using the saccadic eye velocity as the main outcome. The secondary objective was to investigate the relation between GABAA receptor function and serum levels of allopregnanolone, an endogenous positive modulator of the GABAA receptor, in the participating women. Material and methods: 15 women with pelvic pain and laparoscopically confirmed endometriosis and 10 healthy, symptom‐free, control women, aged 18–40 years, underwent the diazepam challenge test during the follicular phase of the menstrual cycle. Basal serum allopregnanolone levels were measured prior to diazepam injection. Results: Compared with healthy controls, women with pelvic pain and confirmed endometriosis had a significantly smaller change in saccadic eye velocity after GABAA receptor stimulation with diazepam, indicating lower sensitivity to diazepam. The saccadic eye velocity response was not correlated with the serum allopregnanolone levels. Conclusions: Women with painful endometriosis show altered GABAA receptor function, depicted as a muted response to an exogenous GABAA receptor agonist. [ABSTRACT FROM AUTHOR]

The prevalence of cognitive dysfunction, dementia, and neurodegenerative disorders such as Alzheimer's disease (AD) is increasing in parallel with an aging population. Distinct types of chronic stress are thought to be instrumental in the development of cognitive impairment in central nervous system (CNS) disorders where cognitive impairment is a major unmet medical need. Increased GABAergic tone is a mediator of stress effects but is also a result of other factors in CNS disorders. Positive GABA‐A receptor modulating stress and sex steroids (steroid‐PAMs) such as allopregnanolone (ALLO) and medroxyprogesterone acetate can provoke impaired cognition. As such, ALLO impairs memory and learning in both animals and humans. In transgenic AD animal studies, continuous exposure to ALLO at physiological levels impairs cognition and increases degenerative AD pathology, whereas intermittent ALLO injections enhance cognition, indicating pleiotropic functions of ALLO. We have shown that GABA‐A receptor modulating steroid antagonists (GAMSAs) can block the acute negative cognitive impairment of ALLO on memory in animal studies and in patients with cognitive impairment due to hepatic encephalopathy. Here we describe disorders affected by steroid‐PAMs and opportunities to treat these adverse effects of steroid‐PAMs with novel GAMSAs. [ABSTRACT FROM AUTHOR]

Hepatic encephalopathy (HE) is one of the primary complications of liver cirrhosis. Current treatments for HE, mainly directed to reduction of ammonia levels, are not effective enough because they cannot completely eliminate hyperammonemia and inflammation, which induce the neurological alterations. Studies in animal models show that overactivation of GABAA receptors is involved in cognitive and motor impairment in HE and that reducing this activation restores these functions. We have developed a new compound, GR3027, that selectively antagonizes the enhanced activation of GABAA receptors by neurosteroids such as allopregnanolone and 3α,21-dihydroxy-5α-pregnan-20-one (THDOC). This work aimed to assess whether GR3027 improves motor incoordination, spatial learning, and circadian rhythms of activity in rats with HE. GR3027 was administered subcutaneously to two main models of HE: rats with chronic hyperammonemia due to ammonia feeding and rats with portacaval shunts (PCS). Motor coordination was assessed in beam walking and spatial learning and memory in the Morris water maze and the radial maze. Circadian rhythms of ambulatory and vertical activity were also assessed. In both hyperammonemic and PCS rats, GR3027 restores motor coordination, spatial memory in the Morris water maze, and spatial learning in the radial maze. GR3027 also partially restores circadian rhythms of ambulatory and vertical activity in PCS rats. GR3027 is a novel approach to treatment of HE that would normalize neurological functions altered because of enhanced GABAergic tone, affording more complete normalization of cognitive and motor function than current treatments for HE. [ABSTRACT FROM AUTHOR]

Animal studies suggest regulatory effects on the hypothalamic-pituitary-gonad axis by allopregnanolone, an endogenous gamma-aminobutyric acid A (GABAA) receptor agonist. Elevated levels of allopregnanolone in women with hypothalamic amenorrhea have been seen. Isoallopregnanolone is an isomer to allopregnanolone, but without GABAA receptor effects. The purpose of this study was to investigate effects of allopregnanolone and isoallopregnanolone on gonadotropin levels in healthy women of fertile age. Ten women were given allopregnanolone and five women isoallopregnanolone intravenously in follicular phase. Repeated blood samples were drawn during the test day. Main outcomes were changes in serum levels of follicle-stimulating hormone (FSH), luteinising hormone (LH), oestradiol, and progesterone. Serum-FSH decreased between 5 and 105 min after the allopregnanolone injection ( F(16,144)==2.18, p==0.008). Serum-LH was reduced between 5 and 35 min following the allopregnanolone injection ( F(16,144)==2.63, p==0.001). Serum-oestradiol and -progesterone were not significantly changed after allopregnanolone injections. No effect on gonadotropin levels were seen after administration of isoallopregnanolone. Allopregnanolone reduces FSH and LH levels in women and the effect might be mediated via a specific GABAA receptor activation since isoallopregnanolone lacked this effect. Although the number of women was small, the results suggest a regulatory mechanism on the hypothalamic-pituitary-gonadal axis by allopregnanolon. [ABSTRACT FROM AUTHOR]

1. It is known that the α4-subunit is likely to occur in the brain predominantly in α4β3δ receptors at extrasynaptic sites. Recent studies have revealed that the α1-, α4-, γ2- and δ-subunits may colocalize extrasynaptically in dentate granule cells of the hippocampus. In the present study, we characterized a series of recombinant GABAA receptors containing human (H) and rat (R) α1/α4-, β2/β3- and γ2S/δ-subunits in Xenopus oocytes using the two-electrode voltage-clamp technique. 2. Both Hα1β3δ and Hα4β3γ2S receptors were sensitive to activation by GABA and pentobarbital. Contrary to earlier findings that the α4β3δ combination was more sensitive to agonist action than the α4β3γ2S receptor, we observed extremely small GABA- and pentobarbital-activated currents at the wild-type Hα4β3δ receptor. However, GABA and pentobarbital activated the wild-type Rα4β3δ receptor with high potency (EC50 = 0.5 ± 0.7 and 294 ± 5 μmol/L, respectively). 3. Substituting the Hα4 subunit with Rα4 conferred a significant increase in activation on the GABA and pentobarbital site in terms of reduced EC50 and increased Imax. When the Hα4 subunit was combined with the Rβ3 and Rδ subunit in a heteropentameric form, the amplitude of GABA- and pentobarbital-activated currents increased significantly compared with the wild-type Hα4β3δ receptor. 4. Thus, the results indicate that the Rα4β3δ, Hα1β3δ and Hα4β3γ2S combinations may contribute to functions of extrasynaptic GABAA receptors. The presence of the Rα4 subunit at recombinant GABAA receptors containing the δ-subunit is a strong determinant of agonist action. The recombinant Hα4β3δ receptor is a less sensitive subunit composition in terms of agonist activation. [ABSTRACT FROM AUTHOR]

Allopregnanolone is an endogenous neuroactive steroid that, through its binding to the γ-aminobutyric acid (GABA) A receptor, has GABA-active properties. Animal studies indicate that allopregnanolone administration results in diminished learning and memory impairment. The aim of the current study was to investigate the effect of intravenously administered allopregnanolone on episodic memory, semantic memory, and working memory in healthy women. Twenty-eight healthy women were included in the study. The participants were scheduled for the memory tests twice in the follicular phase. During the test sessions, an intravenous allopregnanolone and placebo infusion were administered in a double-blinded, randomized order at intervals of 48 h. Before and 10 min after the allopregnanolone/placebo injections, memory tasks were performed. The study demonstrated that allopregnanolone impaired episodic memory in healthy women. There was a significant difference between pre- and postallopregnanolone injection episodic memory scores ( p < 0.05), whereas there was no change in episodic memory performance following the placebo injections. There was also a significant difference between allopregnanolone and placebo postinjection episodic memory scores ( p < 0.05). There were no effects of allopregnanolone on the semantic memory task or working memory task. Intravenous allopregnanolone impairs episodic memory in healthy women, but there is a high degree of individual variability. [ABSTRACT FROM AUTHOR]

Abstract: The two neurosteroids 3α-hydroxy-5α-pregnane-20-one (allopregnanolone; AlloP) and pregnenolone sulfate (PregS) affect neuronal GABAA receptors differently. While AlloP mainly potentiates the currents through GABAA receptors, PregS reduces such currents. The present study aimed at clarifying the interaction of AlloP and PregS at GABAA receptors in neurons from the medial preoptic nucleus of male rat. AlloP has previously been shown to dramatically prolong GABA-mediated spontaneous inhibitory postsynaptic currents (sIPSCs) in these neurons. Here, by recording sIPSCs under voltage-clamp conditions with the perforated-patch technique, it was shown that PregS by itself did not significantly affect the amplitude or time course of such currents. However, PregS, in a concentration-dependent manner, reduced the AlloP-evoked prolongation of sIPSC decay when the two neurosteroids were applied together. In contrast to sIPSC amplitude and time course, sIPSC frequency was significantly reduced by 10 μM PregS alone. Further, although 1.0 μM AlloP alone induced a clear increase in sIPSC frequency, the frequency was not significantly different from control when 1.0 μM AlloP was applied in combination with 10 μM PregS. In addition to the effects on sIPSC parameters, PregS reduced the baseline current evoked by 1.0 μM AlloP in the absence of GABA application or synaptic activity. PregS by itself did not significantly affect the baseline current. The main effects of AlloP and PregS on the sIPSC time course were mimicked by a simplified model with AlloP assumed to reduce the rate of GABA unbinding from the receptor and PregS assumed to increase the rate of desensitization. [Copyright &y& Elsevier]

That 3alpha-hydroxy-5alpha/beta-pregnane steroids (GABA steroids) have modulatory effects on the GABA-A receptor is well known. In behavioral studies in animals high exogenous dosages give concentrations not usually reached in the brain under physiological conditions. Animal and human studies show that GABA-A receptor-positive modulators like barbiturates, benzodiazepines, alcohol, and allopregnanolone have a bimodal effect. In pharmacological concentrations they are CNS depressants, anesthetic, antiepileptic, and anxiolytic. In low dosages and concentrations, reached endogenously, they can induce adverse emotional reactions in up to 20% of individuals. GABA steroids can also induce tolerance to themselves and similar substances, and rebound occurs at withdrawal. Menstrual cycle-linked disorders can be understood by the concept that they are caused by the action of endogenously produced GABA-steroids through three mechanisms: (a) direct action, (b) tolerance induction, and (c) withdrawal effect. Examples of symptoms and disorders caused by the direct action of GABA steroids are sedation, memory and learning disturbance, clumsiness, increased appetite, worsening of petit mal epilepsy, negative mood as tension, irritability and depression during hormone treatments, and the premenstrual dysphoric disorder (PMDD). A continuous exposure to GABA steroids causes tolerance, and women with PMDD are less sensitive to GABA-A modulators. A malfunctioning GABA-A receptor system is related to stress sensitivity, concentration difficulties, loss of impulse control, irritability, anxiety, and depression. An example of withdrawal effect is "catamenial epilepsy," when seizures increase during menstruation after the withdrawal of GABA steroids. Similar phenomena occur at stress since the adrenals produce GABA steroids during stress. [ABSTRACT FROM AUTHOR]

Chronic stress in various forms increases the risk for cognitive dysfunction, dementia and Alzheimer's disease. While the pathogenesis behind these findings is unknown, growing evidence suggests that chronic increase in neurosteroid levels, such as allopregnanolone, is part of the mechanism. We treated wild-type C57BL/6J mice with allopregnanolone for 5 months, using osmotic pumps. This treatment led to moderately increased levels of allopregnanolone, equivalent to that of mild chronic stress. After an interval of no treatment for 1 month, female mice showed impaired learning and memory function in the Morris water maze (MWM) in combination with diminished hippocampus weight and increased cerebellum weight, both correlating to MWM performance. Male mice showed a minor reduction in memory function and no differences in brain structure. We conclude that chronic allopregnanolone elevation can lead to cognitive dysfunction and negative brain alterations. We suggest that allopregnanolone could play a key role in the pathogenesis of stress-induced cognitive disturbances and perhaps dementia. [ABSTRACT FROM AUTHOR]

Abstract: Both acute and chronic tolerance can develop to allopregnanolone—a gamma-aminobutyric acid (GABA)-modulatory progesterone metabolite. Here we investigated if acute tolerance to allopregnanolone persisted for 1 or 2 days after the induction and thus could be the initial part of chronic tolerance. Male rats were anaesthetised with allopregnanolone (i.v) to the deep anaesthesia level of the silent second (SS), which is an EEG burst suppression of 1 s or more. They were divided into four groups: SS1—anaesthesia to the first silent second; LAn (long anaesthesia)—90 min anaesthesia at the SS level; SS2;D1—90 min anaesthesia and SS induction 1 day later; SS2;D2—90 min anaesthesia and SS induction 2 days later. Allopregnanolone concentrations in tissue and serum were analysed. Levels of the GABAA receptor α2, α4, γ2(S+L) and δ subunits mRNAs were analysed by in situ hybridisation. Acute tolerance was induced during the 90 min anaesthesia. Tolerance persisted for 1 day, since the dose of allopregnanolone needed to induce a new SS anaesthesia was increased after 1 day. The level of α4 subunit mRNA expression in the ventral posteriomedial nucleus of thalamus was negatively related to the tolerance parameters, the SS dose of allopregnanolone and ΔSS (SS dose difference between days). Allopregnanolone threshold anaesthesia lasting 90 min induces acute tolerance that persisted for at least 1 day, which could be used as the start of a chronic tolerance. The α4 subunit may be involved in allopregnanolone caused effects in the brain. [Copyright &y& Elsevier]

Altered gamma-aminobutyric acid (GABA)-ergic function is associated with neurological and psychiatric disorders. Certain progesterone metabolite 3α-hydroxy-5α-pregnan-20-one, or allopregnanolone (ALLO), increases the GABA-mediated chloride ion (Cl−) flux through GABAA receptors in a similar fashion as benzodiazepines and barbiturates. We have studied the effect of its 3β diastereomer, 3β-hydroxy-5α-pregnan-20-one, or isoallopregnanolone (ISO), on the Cl− flux and investigated the interaction between ISO and ALLO on GABA-mediated Cl− uptake in cortical homogenates from adult male Wistar rats. We found that ISO from 1 μM to 1 mM does not affect baseline Cl− uptake in rat cortical homogenates. Neither does ISO at dose range of 100 nM to 100 μM interact with 10 μM GABA in the Cl− uptake assay. In addition, ISO at the dose range of 1–30 μM does not affect flunitrazepam and pentobarbital-induced increase of Cl− uptake. We conclude that ISO selectively inhibits the ALLO-induced Cl− uptake with respect to ALLO concentrations. The IC50 of ISO inhibition on 1 μM ALLO-induced Cl− uptake was calculated to be 12.25 μM. On the other hand, we have studied the effect of 30 μM ISO on ALLO (0.01 nM to 1 μM) induced displacement of tert-butylbicyclophosphorothionate (TBPS) binding. We did not note any interaction between ALLO and ISO on TBPS binding assay. These results indicate that ISO may be useful functional blocker of GABAA receptor potentiating steroid ALLO when used at concentrations that do not affect baseline GABAergic neurotransmission. [Copyright &y& Elsevier]

Context Allopregnanolone is a metabolite from progesterone and a positive modulator of the GABA A receptor. This endogenous steroid may induce negative mood in sensitive women when present in serum levels comparable to the premenstrual phase. Its endogenous isomer, isoallopregnanolone, has been shown to antagonize allopregnanolone effects in experimental animal and human models. Objective The objective was to test whether inhibition of allopregnanolone by treatment with the GABA A modulating steroid antagonist (GAMSA) Sepranolone (UC1010) during the premenstrual phase could reduce symptoms of the premenstrual dysphoric disorder (PMDD). The pharmacokinetic parameters of UC1010 when given as a subcutaneous injection were measured in healthy women prior to the study in women with PMDD. Design This was an explorative randomized, double-blind, placebo-controlled study. Setting Swedish multicentre study with 10 centers. Participants Participants were 26 healthy women in a pharmacokinetic phase I study part, and 126 women with PMDD in a phase II study part. Diagnosis followed the criteria for PMDD in DSM-5 using Daily Record of Severity of Problems (DRSP) and Endicott’s algorithm. Intervention Subjects were randomized to treatment with UC1010 (10 or 16 mg) subcutaneously every second day during the luteal phase or placebo during one menstrual cycle. Outcome measures The primary outcome measure was the sum of all 21 items in DRSP (Total DRSP score). Secondary outcomes were Negative mood score i.e. the ratings of the 4 key symptoms in PMDD (anger/irritability, depression, anxiety and lability) and impairment (impact on daily life). Results 26 healthy women completed the pharmacokinetic phase I study and the dosing in the following trial was adjusted according to the results. 106 of the 126 women completed the phase II study. Within this group, a significant treatment effect with UC1010 compared to placebo was obtained for the Total DRSP score (p = 0.041) and borderline significance (p = 0.051) for the sum of Negative mood score. Nineteen participants however showed symptoms during the follicular phase that might be signs of an underlying other conditions, and 27 participants had not received the medication as intended during the symptomatic phase. Hence, to secure that the significant result described above was not due to chance, a post hoc sub-group analysis was performed, including only women with pure PMDD who completed the trial as intended (n = 60). In this group UC1010 reduced Total DRSP scores by 75% compared with 47% following placebo; the effect size 0.7 (p = 0.006), and for sum of Negative mood score (p = 0.003) and impairment (p = 0.010) with the effect size 0.6. No severe adverse events were reported during the treatment and safety parameters (vital signs and blood chemistry) remained normal during the study. Conclusions This explorative study indicates promising results for UC1010 as a potential treatment for PMDD. The effect size was comparable to that of SSRIs and drospirenone containing oral contraceptives. UC1010 was well tolerated and deemed safe. [ABSTRACT FROM AUTHOR]

Summary: Certain women experience negative mood symptoms as a result of progesterone during the luteal phase of the menstrual cycle, progestagens in hormonal contraceptives, or the addition of progesterone or progestagens in sequential hormone therapy (HT). This phenomenon is believed to be mediated via the action of the progesterone metabolites on the GABAA system, which is the major inhibitory system in the mammalian CNS. The positive modulators of the GABAA receptor include allopregnanolone and pregnanolone, both neuroactive metabolites of progesterone, as well as benzodiazepines, barbiturates, and alcohol. Studies on the effect of GABAA receptor modulators have shown contradictory results; although human and animal studies have revealed beneficial properties such as anaesthesia, sedation, anticonvulsant effects, and anxiolytic effects, recent reports have also indicated adverse effects such as anxiety, irritability, and aggression. It has actually been suggested that several GABAA receptor modulators, including allopregnanolone, have biphasic effects, in that low concentrations increase an adverse, anxiogenic effect whereas higher concentrations decrease this effect and show beneficial, calming properties. The allopregnanolone increase during the luteal phase in fertile women, as well as during the addition of progesterone in HT, has been shown to induce adverse mood in women. The severity of these mood symptoms is related to the allopregnanolone serum concentrations in a manner similar to an inverted U-shaped curve. Negative mood symptoms occur when the serum concentration of allopregnanolone is similar to endogenous luteal phase levels, while low and high concentrations have less effect on mood. It has also been shown that progesterone/allopregnanolone treatment in women increases the activity in the amygdala (as measured with functional magnetic resonance imaging) in a similar way to the changes seen during anxiety reactions. However, it is evident that only certain women experience adverse mood during progesterone or GABAA receptor modulator treatments. Women with premenstrual dysphoric disorder (PMDD) have severe luteal phase related symptoms; in this phase, they show changes in GABAA receptor sensitivity and GABA concentrations that are related to the severity of the condition. These findings suggest that negative mood symptoms in women with PMDD are caused by the paradoxical effect of allopregnanolone mediated via the GABAA receptor. Conclusion: Progesterone and progestagens induce negative mood, most probably via their GABAA receptor active metabolites. In postmenopausal women treated with progesterone and animals treated with allopregnanolone, there is a bimodal association between serum allopregnanolone concentration and adverse mood, resembling an inverted U-shaped curve. In humans, the maximal effective concentration of allopregnanolone for producing negative mood is within the range of physiological luteal phase serum concentrations. [Copyright &y& Elsevier]

Abstract: The endogenous progesterone metabolite allopregnanolone has a number of properties including anesthetic, sedative, antiepileptic, anxiolytic, impaired memory function and negative mood symptoms. Allopregnanolone is a potent positive GABAA receptor function modulators. In contrast, 3β-hydroxy-steroids (3β-steroids) usually modulate the GABAA receptor negatively. They have attracted some interest for their possible use as therapeutic agents that could counteract the negative symptoms induced by allopregnanolone. Two hypotheses for the action of 3β-steroids have been proposed: 1) 3β-steroids act in a similar way to pregnenolone sulphate, which non-competitively reduces GABAA receptor activity. 2) 3β-steroids specifically antagonize the effect of allopregnanolone. We have therefore tried to clarify this issue by comparing the effect of pregnenolone sulphate and 5β-pregnane-3β, 20(R)-diol on the GABA-evoked currents by the patch clamp technique on neurons from the medial preoptic nucleus. Both pregnenolone sulphate and 5β-pregnane-3β, 20(R)-diol increase the desensitization rate of the current response evoked by a 2 s GABA application. However, their effects on other parameters of the GABA evoked currents differed in degree and sometimes even in direction. The actions of pregnenolone sulphate and 5β-pregnane-3β, 20(R)-diol were not altered in the presence of allopregnanolone, which indicates that they do not directly interact with allopregnanolone. In addition, when 5β-pregnane-3β, 20(R)-diol was tested on spontaneous inhibitory postsynaptic currents (sIPSCs), it dramatically reduced the allopregnanolone-induced prolongation of the decay time constant but it had no effect on the decay under control conditions. In conclusion, the effect of 5β-pregnane-3β, 20(R)-diol on GABA-evoked currents is different to that of pregnenolone sulphate in medial preoptic nucleus neurons. [Copyright &y& Elsevier]

Abstract: Neurosteroids directly modulate ligand gated ion channels such as GABAA receptors. Two such molecules, 3β-OH A-ring reduced pregnane steroids and pregnenolone sulfate (PS), inhibit recombinant GABAA receptor. Using a two-electrode voltage-clamp technique, we compared the effect of 5α-pregnan-3β,20(S)-diol (UC1019), 5β-pregnan-3β, 20(R)-diol (UC1020) and PS on the activation onset and offset times of the recombinant GABAA receptor (rat α1β2γ2L) in Xenopus oocytes. Rapid solution changes allowed the kinetic analysis of GABA-evoked currents. Steroids were co-applied with 30 µM GABA for 10 s, followed by a 80 s washout period. PS (≥0.3 µM) moderately increased the slow onset rate (k on-S) of GABA-response. PS had no significant effects on the fast onset rate (k on-F). UC1019 and UC1020 decreased the k on-S of the GABA-response in a concentration-dependent manner with no significant effects on the k on-F. Like PS, UC1019 and UC1020 decreased the slow offset rates (k off-S). In addition, PS increased the fast offset rate (k off-F) in a concentration-dependent manner, while UC1019 and UC1020 decreased k off-F. The EC50 of PS to increase k off-F was calculated as 0.47±0.1 µM. The corresponding IC50 values of UC1019 and UC1020 to decrease k off-F were 5.0±0.5 µM and 8.4±0.9 µM, respectively. These results suggest differential actions of PS and 3β, 20(R/S)-pregnandiols on the offset time course of GABA-site activation. [Copyright &y& Elsevier]

Abstract: 3β-hydroxysteroids are pregnenolone sulfate-like GABAA receptor antagonists. The aim of the current study was to compare the functional differences between 3β-hydroxysteroids and pregnenolone sulfate to inhibit GABAA receptors expressed in Xenopus oocytes. Recombinant rat GABAA receptors encoding wild type α1β2γ2L receptor, mutant α1V256Sβ2γ2L and α1β2A252Sγ2L receptors were examined using a two-electrode voltage-clamp technique. A homologous mutation of the residue at 2′position closest to the cytoplasmic end of the M2 helix to serine on both α1 and β2 subunit, α1V256S and β2A252S, reduced the slow desensitization components of GABA-activated currents at saturating doses. Compared to the wild type receptor, the potency of GABA increased significantly in the α1V256Sβ2γ2L receptor (P <0.05), whereas it decreased moderately in the α1β2A252Sγ2L receptor. We found that 5α-pregnan-3β, 20(S)-diol (UC1019) and 5β-pregnan-3β, 20(R)-diol (UC1020) were the most effective blockers of maximal GABA responses among a panel of 3β-hydroxysteroids. Pregnenolone sulfate, UC1019 and UC1020 were potent antagonists in the wild type receptor with calculated IC50s of 0.20±0.07 μM; 1.88±0.32 μM and 2.58±0.58 μM, respectively. The inhibitory effect of pregnenolone sulfate was significantly reduced in both mutants α1V256Sβ2γ2L and α1β2A252Sγ2L receptors (P <0.05), whereas the inhibitory effects of UC1019 and UC1020 were reduced only in the mutant α1V256Sβ2γ2L receptor. Pregnenolone sulfate promoted slow desensitization with prolonged GABA application in a dose-dependent manner in the wild type receptor, but not mutant receptors. On the contrary, UC1019 and UC1020 (≤20 μM) did not promote desensitization in both wild type and mutant receptors. In conclusion, the GABAA receptor inhibition by pregnenolone sulfate, but not 3β-hydroxysteroids, was dependent on desensitization kinetics of the Cl- channels. A point mutation at M2 helix of the β2-subunit (β2A252S) can dramatically reduce the inhibitory effect of pregnenolone sulfate on the GABAA receptors without affecting the inhibitory properties of 3β-hydroxysteroids. These results are consistent with the hypothesis that pregnenolone sulfate-inhibition does not share with 3β-hydroxysteroids the coincident channel property at the GABAA receptor. [Copyright &y& Elsevier]

Abstract: GABAA receptors containing α5-subunit have an important role in cognitive function. As the agonistic effect of 3α-hydroxy ring-A reduced steroids depends on subunit combinations of the GABAA receptor, the antagonistic effect of pregnenolone sulfate and 3β-hydroxypregnane steroids may vary between α5-subunit and α1-subunit containing receptors. We investigated the effect of agonist and antagonist steroids in the recombinant rat α1β2γ2L and α5β2γ2L receptors expressed in Xenopus oocytes using a two electrodes voltage-clamp technique. We did not find any significant difference in potency and efficacy of GABA response between α1β2γ2L and α5β2γ2L receptors. Compared to the α1β2γ2L receptor, a significantly lower degree of desensitization was observed in the α5β2γ2L receptor. In addition, the potencies of 3α-OH-5α-pregnan-20-one (3α5αP), 5α-pregnan-3α,21-diol-20-one (3α5αTHDOC) and 5α-androstane-3α,17β-diol (3α5αADL) to enhance GABA response were significantly higher in the α5β2γ2L receptor, whereas their efficacies remained unchanged between two receptors. In either receptor, the efficacy of 3α5αTHDOC was significantly higher than 3α5αP and 3α5αADL. The efficacies of 5β-pregnan-3β,21-diol-20-one(UC1015) and 5α-pregnan-3β,20α-diol(UC1019) to inhibit 30 μM GABA response, and the efficacies of 3β-OH-5β-pregnan-20-one (UC1014) and 5β-pregnan-3β, 20β-diol (UC1020) to inhibit 3 μM 3α5αTHDOC+3 μM GABA response were higher in the α5β2γ2L receptor compared to the α1β2γ2L receptor. The potencies of pregnenolone sulfate and 3β-hydroxypregnane steroids to inhibit the GABA response and the 3α5αTHDOC+GABA response did not vary between two receptors. Interestingly, the potencies and efficacies of pregnenolone sulfate and 3β-hydroxypregnane steroids to inhibit the GABA response were positively correlated to their potencies and efficacies to inhibit the 3α5αTHDOC+GABA response. Results from the current study revealed a different modulation pattern by neurosteroids between the α1β2γ2L and α5β2γ2L receptor. [Copyright &y& Elsevier]

Abstract: To study acute tolerance, rats were anesthetized with interrupted i.v. allopregnanolone infusions where the “silent second” in the electroencephalogram (EEG) was the target. Animals were killed either directly at the first silent second or at the silent second level after 30 or 90min of anaesthesia. Acute tolerance was demonstrated at 90min of anaesthesia as earlier shown. In situ hybridization showed a decreased expression of the γ-aminobutyric acidA (GABAA) receptor subunit α4 mRNA amount in the thalamus ventral–posteriomedial nucleus of the tolerant rats. A parallel change in the abundance of the α4 subunit was detected with immunohistochemistry. The increase in maintenance dose rate (MDR) was significantly negatively correlated with the α4 mRNA in the thalamus ventral–posteriomedial nucleus, and positively correlated with α2 mRNA in different hippocampal subregions. There was also a positive relationship between the α1 mRNA amounts in the different hippocampal subregions, with significant differences between groups. These changes in GABAA receptor subunits mRNA expression and protein (α4) might be of importance for the development of acute tolerance to allopregnanolone. [Copyright &y& Elsevier]