Cellular distribution of the GABAA receptor-modulating 3alpha-hydroxy, 5alpha-reduced pregnane steroids in the adult rat brain

The 3alpha-hydroxy,5alpha-reduced pregnane steroids, allopregnanolone and allotetrahydrodeoxycorticosterone, are the most potent endogenous positive modulators of GABA(A) receptor-mediated inhibition. This study presents the first immunohistochemical examination of the cellular distribution of 3alpha-hydroxy,5alpha-reduced pregnane steroids across the brain. We found a widespread distribution in the adult rat, with dense immunolabelling in the olfactory bulb, striatum and cerebral cortex, and lower density labelling in the brainstem reticular formation. In general terms, this distribution accords with the regional concentrations of 3alpha-hydroxy,5alpha-reduced steroids determined, in other laboratories, by brain region sampling and either gas chromatography-mass fragmentography or radioimmunoassay. However, immunohistochemistry allowed for a more detailed examination of regional distribution and cellular specificity. All immunoreactivity was confined to the cell bodies and thick dendrites of neurones; no identifiable glia were labelled. In most brain areas, the location and morphology of labelled cells identified them as excitatory neurones. In addition, cell populations known to be projecting GABAergic neurones (e.g. cerebellar Purkinje cells) were immunoreactive, whereas local inhibitory neurones generally were not. The cellular distribution of 3alpha-hydroxy,5alpha-reduced steroids suggests that sensory, motor, limbic and homeostatic systems can be influenced by neurosteroids at multiple stages of processing.     

Neuroactive steroids and fatigue severity in patients with primary biliary cirrhosis and hepatitis C

Abstract  Fatigue is one of the most common non-specific symptoms associated with several disease states including liver diseases. Recently, it was reported that levels of progesterone metabolites such as allopregnanolone (3α,5α-tetrahydroprogesterone; 3α,5α-THP) and isopregnanolone (3β,5α-THP) were increased in plasma of patients with chronic fatigue syndrome. We hypothesize that THP metabolites might be associated with fatigue commonly observed in chronic liver diseases. We evaluated fatigue scores and plasma levels of five progesterone metabolites in 16 patients with primary biliary cirrhosis (PBC), 12 patients with chronic hepatitis C (CHC) and 11 age-matched controls. The fatigue impact scale (FIS) ratio was significantly increased ( P  < 0.01) in patients with PBC and CHC compared to controls. Plasma levels of 3α,5α-THP and pregnanolone (3α,5β-THP) were significantly increased in PBC and CHC patients. The other progesterone metabolites, i.e. 3β,5α-THP, 3β,5β-THP and 3α,5α-tetrahydrodeoxycorticosterone were either undetectable or detected only in some patients. Plasma levels of 3α,5α-THP and 3α,5β-THP were found to be significantly higher in patients with fatigue ( P  < 0.05), while those of patients without fatigue were not significantly different from controls. Both 3α,5α-THP and 3α,5β-THP are positive allosteric modulators of the gamma-aminobutyric acid type A (GABA-A) receptor and readily cross the blood–brain barrier. The present preliminary findings suggest that increased inhibition through GABA-A receptors due to the accumulation of neuroinhibitory steroids may represent an important pathophysiological mechanism of fatigue in chronic liver diseases.     

Sex,stress and steroids

The hypothalamo–pituitary–adrenal (HPA) axis plays a key role in the neuroendocrine response to stress and in maintaining physiological homoeostasis. However, stress that is chronic in nature can lead to HPA axis dysfunction and increase the risk for developing affective disorders, particularly if the stress is experienced during vulnerable periods in life. Sex differences in how the HPA axis responds to stress are well established, with females typically displaying heightened responses. The underlying cause of these sex differences is important to understand, as many neuropsychiatric disorders disproportionately affect females. Much research has provided evidence for gonadal sex steroids in underpinning sex differences in HPA axis responsivity; however, we suggest that neuroactive metabolites of these steroids also play a key role in the brain in mediating sex differences in HPA axis responses to stress. The relationship between neuroactive steroids and stress is complex. Acute stress rapidly increases neuroactive steroid production, which can in turn modulate activity of the HPA axis. However, under chronic stress conditions, stress can impact the brain's capacity to generate steroids, and this in turn has corollary effects on HPA axis function that may increase the propensity for psychopathology, given both HPA axis dysfunction and deficits in neuroactive steroids are implicated in affective disorders. Hence, here we review the evidence from animal and human studies for sex differences in the interactions between neuroactive steroids and the stress axis at various stages of life, under physiological and pathophysiological stress conditions and consider the implications for health and disease.     

Evaluation and comparison of the pharmacokinetic and pharmacodynamic properties of allopregnanolone and pregnanolone at induction of anaesthesia in the male rat

We have evaluated and compared the pharmacokinetic and pharmacodynamicproperties of allopregnanolone and pregnanolone at inductionof anaesthesia in male rats. A threshold method was used, andthe first burst suppression period of 1 s or more in theEEG was selected as the end-point after fairly slow infusions.An optimal dose of 4.0 mg kg^–1 min^–1was noted for both steroids. Brain concentrations were low atlow infusion rates, indicating that acute tolerance was notoccurring. Significant positive correlations were noted betweendose rate and serum concentrations of allopregnanolone (r =0.94, P<0.001) and pregnanolone (r = 0.88, P<0.001). Suchcorrelations were also seen in striatum, cerebellum, cortexand muscle for both steroids (P<0.01). Despite changing infusionrates, the concentrations of both steroids in brainstem, hippocampusand fat remained stable. Because no correlation between infusionrate and steroid concentration was noted in the brainstem andhippocampus, these two brain areas may be regarded as primarysites of action for allopregnanolone and pregnanolone. Pregnanoloneconcentrations in the brainstem and hippocampus were significantlyhigher than those of allopregnanolone, suggesting that allopregnanolonewas more potent than pregnanolone in inducing anaesthesia. Br J Anaesth 2001; 86: 403–12     

Sex differences in the effect of finasteride on acute ethanol withdrawal severity in C57BL/6J and DBA/2J mice

The neurosteroid allopregnanolone (ALLO) is a potent positive modulator of GABAA receptors that can modulate ethanol (EtOH) withdrawal. The 5alpha-reductase inhibitor finasteride can block the formation of ALLO and other GABAergic neurosteroids and also reduce certain effects of EtOH. Treatment with finasteride during chronic EtOH exposure decreased EtOH withdrawal severity and blood EtOH concentrations (BECs), suggesting an additional effect of finasteride on EtOH pharmacokinetics. Thus, the purpose of the present study was to determine the effect of finasteride on acute EtOH withdrawal severity, to minimize the effect of finasteride on EtOH metabolism. Male and female C57BL/6J and DBA/2J mice received a pretreatment of finasteride (50 mg/kg i.p.) or vehicle 24 h prior to an injection of EtOH (4 g/kg i.p.) or saline. Handling-induced convulsions (HICs) were scored at baseline, and then over a 24 h period after EtOH or saline injection. In another experiment, plasma estradiol and corticosterone levels were assessed at selected time points (0, 2, 8, and 24 h). In a final study, retro-orbital blood samples were collected at 30, 60, 120, and 240 min post-EtOH administration to access finasteride's effects on EtOH clearance parameters. Pretreatment with finasteride increased acute EtOH withdrawal severity in female C57BL/6J and DBA/2J mice but decreased withdrawal severity in male mice of both strains. Finasteride did not alter BECs, EtOH clearance, estradiol, or corticosterone concentrations in a manner that appeared to contribute to the sex difference in finasteride's effect on acute EtOH withdrawal severity. These findings suggest that male and female C57BL/6J and DBA/2J mice differ in their sensitivity to changes in ALLO or other GABAergic neurosteroid levels during acute EtOH withdrawal. Sex differences in the modulation of GABAergic 5alpha-reduced steroids may be an important consideration in understanding and developing therapeutic interventions in alcoholics.     

Action of Progesterone and Progesterone Metabolites in Menstrual-Cycle–Related Disorders

Progesterone and progesterone metabolites are important modulators of central nervous system function through their interactions with the γ-aminobutyric acid (GABA)_A receptor. GABA, neurosteroids, and other modulators of GABA_A, such as benzodiazepines, barbiturates, and alcohol, typically inhibit neuronal excitability. The resulting anxiolytic, anticonvulsant, sedative, and anesthetic effects are involved in mood, response to stress, and cognition.
The impact of neurosteroids has been demonstrated in women with premenstrual dysphoric disorder: onset of negative mood symptoms has been correlated with peak progesterone levels, and symptoms intensified with progesterone withdrawal in the late luteal phase of the menstrual cycle. These symptoms were not present during anovulatory cycles without the corpus luteum, the primary source of progesterone and metabolites.
The focus of this article is the paradox of why high levels of progesterone and neurosteroids, which typically are associated with anxiolytic activity, instead induce irritability, anxiety, and mood fluctuations in women with premenstrual dysphoric disorder.     

Tibolone,transdermal estradiol or oral estrogen–progestin therapies: Effects on circulating allopregnanolone,cortisol and dehydroepiandrosterone levels

The aim of the present study was to evaluate, in healthy postmenopausal women, the impact of tibolone (2.5?mg), transdermal estradiol (50?μg) (TE) and different oral estrogen–progestin regimens, conjugated equine estrogens (0.625?mg) plus medroxyprogesterone acetate (5?mg) (CEE?+?MPA) and estradiol (2?mg) plus norethisterone acetate (1?mg) (E2?+?NETA) on circulating estradiol, progesterone, allopregnanolone, cortisol and dehydroepiandrosterone (DHEA) levels. Blood samples were collected before and after 1, 3, 6 and 9 months of treatment in 85 postmenopausal women. Estradiol levels increased (p?<?0.001) in the TE, CEE?+?MPA and E2?+?NETA groups after 1 month of therapy, but did not change in the tibolone group during the entire follow-up period. Both E2?+?NETA and tibolone treatments induced an increase in progesterone levels (p?<?0.05) after 1 year of therapy. Allopregnanolone levels showed an increase in all estrogen-based groups, being significant after 3 months of treatment (p?<?0.01). Patients receiving tibolone showed a significant increase in allopregnanolone levels at 3 months (p?<?0.05), but lower than in the other groups. Cortisol levels decreased significantly in the TE and CEE?+?MPA groups after 6 months and 12 months of treatment, respectively. Neither tibolone nor E2?+?NETA treatments modified circulating cortisol levels. DHEA levels significantly (p?<?0.05) decreased after 6 months of TE or estrogen–progestin therapies independently of the presence or the type of progestin used. In contrast, DHEA remained stable throughout the 12 months of treatment with tibolone. The increase of allopregnanolone, a steroid with sedative and anxiolytic properties, in response to these different treatments could underlie, at least in part, the central effects that hormone replacement therapy and tibolone have on anxiety, mood and behavior. Unlike estrogen-based therapy, tibolone treatment did not reduce the DHEA milieu in the menopause, and thus did not enhance the androgen deficiency syndrome in postmenopausal women.     

Tolerance to allopregnanolone with focus on the GABA-A receptor

Many studies have suggested a relationship between stress, sex steroids, and negative mental and mood changes in humans. The progesterone metabolite allopregnanolone is a potent endogenous ligand of the γ-amino butyric acid -A (GABA-A) receptor, and the most discussed neuroactive steroid. Variations in the levels of neuroactive steroids that influence the activity of the GABA-A receptor cause a vulnerability to mental and emotional pathology. There are physiological conditions in which allopregnanolone production increases acutely (e.g. stress) or chronically (e.g. menstrual cycle, pregnancy), thus exposing the GABA-A receptor to high and continuous allopregnanolone concentrations. In such conditions, tolerance to allopregnanolone may develop. We have shown that both acute and chronic tolerances can develop to the effects of allopregnanolone. Following the development of acute allopregnanolone tolerance, there is a decrease in the abundance of the GABA-A receptor α4 subunit and the expression of the α4 subunit mRNA in the ventral-posteriomedial nucleus of the thalamus. Little is known about the mechanism behind allopregnanolone tolerance and its effects on assembly of the GABA-A receptor composition. The exact mechanism of the allopregnanolone tolerance phenomena remains unclear. The purpose of this review is to summarize certain aspects of current knowledge concerning allopregnanolone tolerance and changes in the GABA-A receptors.     

Role of allopregnanolone biosynthesis in acute stress‐induced anxiety‐like behaviors in mice

The neurosteroid allopregnanolone (3α, 5α‐tetra‐hydroprogesterone: ALLO) elicits anxiolytic, anticonvulsant, and hypnotic anesthetic effects in vivo similar to those induced by other positive allosteric modulators of the GABA_A receptor. Endogenous ALLO has been shown to be rapidly elevated in the brain by acute stress paradigms, such as immobilization, in animal models. The present study was designed to ascertain the role of neurosteroid biosynthesis in the anxiety‐like behavior induced by immobilization stress. Mice were exposed to an immobilization stressor for 2 h. After 24 h, the mice that had been immobilized did not behave significantly differently in the elevated plus maze (EPM) test and in the elevated open platform (EOP) test than the mice that had not been immobilized. In contrast, finasteride‐pretreated immobilization stressed mice did behave significantly differently in the EPM and EOP tests. These findings suggest that ALLO biosynthesis contributes to stress resistance. Furthermore, the ALLO mimetic drug alfaxalone appeared to antagonize the effects of finasteride by significantly changing the behavior in the EPM test or in the EOP test in finasteride (10 mg kg^?1)‐pretreated immobilized mice. In addition, alfaxalone, unlike diazepam, did not affect the muscle tone of the mice, as measured by the grip strength test. These results suggest that alfaxalone is a promising anxiolytic candidate lacking benzodiazepine‐like muscle‐relaxant effects.