Kami-shoyo-san improves ASD-like behaviors caused by decreasing allopregnanolone biosynthesis in an SKF mouse model of autism.

Dysfunctions in the GABAergic system are associated with the pathogenesis of autism spectrum disorder (ASD). However, the mechanisms by which GABAergic system dysfunctions induce the pathophysiology of ASD remain unclear. We previously demonstrated that a selective type I 5α-reductase inhibitor SKF105111 (SKF) induced ASD-like behaviors, such as impaired sociability-related performance and repetitive grooming behaviors, in male mice. Moreover, the effects of SKF were caused by a decrease in the endogenous levels of allopregnanolone (ALLO), a positive allosteric modulator of the GABAA receptor. In this study, we used SKF-treated male mice as a putative animal model of ASD and examined the effects of Kami-shoyo-san (KSS) as an experimental therapeutic strategy for ASD. KSS is a traditional Kampo formula consisting of 10 different crude drugs and has been used for the treatment of neuropsychiatric symptoms. KSS dose-dependently attenuated sociability deficits and suppressed an increase in grooming behaviors in SKF-treated mice without affecting ALLO content in the prefrontal cortex. The systemic administration of the dopamine D1 receptor antagonist SCH23390 reversed the ameliorative effects of KSS. On the other hand, the dopamine D2 receptor antagonist sulpiride and GABAA receptor antagonist bicuculline only attenuated the ameliorative effect of KSS on repetitive self-grooming behaviors. The present results indicate that KSS improves SKF-induced ASD-like behaviors by facilitating dopamine receptor-mediated mechanisms and partly by neurosteroid-independent GABAA receptor-mediated neurotransmission. Therefore, KSS is a potential candidate for the treatment of ASD.

Anti-PTSD-like effects of albiflorin extracted from Radix paeoniae Alba.

ETHNOPHARMACOLOGY RELEVANCE: Post-traumatic stress disorder (PTSD) is a severe psychiatric disorder that is characterized by symptoms of re-experiencing, avoidance and hyperarousal, as well as social and professional dysfunction at least one month after the exposure to a traumatic event. Biosynthesis of allopregnanolone has been suggested as one of the important contributors to PTSD. Albiflorin (AF) extracted from Radix paeoniae Alba had been shown to be effective in the therapy of depression. However, few studies were concerned about the anti-PTSD-like effects of AF. AIM OF THE STUDY: The current study aimed to evaluate the anti-PTSD-like effects of AF in an animal model and its possible mechanism. MATERIALS AND METHODS: To evaluate this, the single prolonged stress (SPS) model was used in the present study. The SPS rats were administered by AF (at doses of 3.5, 7 and 14.0mg/kg, i.g.) after induction of SPS from days 2-13. After the exposure to SPS, behavioral assessments were conducted, including contextual fear paradigm (CFP), elevated plus-maze test (EPMT), open-field test (OFT). The rats were decapitated at the end of the behavioral tests and levels of allopregnanolone in prefrontal cortex, hippocampus and amygdala were measured by enzyme linked immunosorbent assay (ELISA). RESULTS: It had been shown that behavioral deficits of SPS rats were reversed by AF (7.0 and 14.0mg/kg, i.g.), which attenuated the PTSD-like associated contextual freezing behavior in CFP and improved PTSD-like associated anxiogenic behavior in EPMT without affecting locomotor activity in OFT. Moreover, decreased levels of allopregnanolone in prefrontal cortex, hippocampus, and amygdala were reversed by AF (7.0 and 14.0mg/kg, i.g.), respectively. CONCLUSION: In summary, the present study indicated that AF exerted the anti-PTSD-like effects, which maybe associated with allopregnanolone biosynthesis in the brain.

Free and Easy Wanderer Plus (FEWP) improves behavioral deficits in an animal model of post-traumatic stress disorder by stimulating allopregnanolone biosynthesis.

Post-traumatic stress disorder (PTSD) is a severe psychiatric condition defined as a "trauma and stress-related disorder". Dampened allopregnanolone biosynthesis has been implicated as a possible contributor to PTSD aetiology. Free and Easy Wanderer Plus (FEWP) is a traditional Chinese medicine previously shown to be effective in PTSD treatment. However, little is known about the role of allopregnanolone in the anti-PTSD effects of FEWP. To evaluate this, the single prolonged stress (SPS) model was used in the present study. SPS-induced rats were administered FEWP (at doses of 2.5, 5.0 and 10.0 mg/kg, p.o.) after induction of SPS from days 2 through 15. After exposure to SPS, behavioral assessments were determined, including the open-field test, the contextual fear paradigm, and the elevated plus-maze test. The experimental model rats were decapitated at the end of the behavioral tests and the level of allopregnanolone in the prefrontal cortex, hippocampus and amygdala was measured by enzyme linked immunosorbent assay (ELISA). The behavioral deficits of the SPS-induced rats were significantly reversed by FEWP (at doses of 5.0 and 10.0 mg/kg, p.o.). The level of allopregnanolone was increased by administration of FEWP. In summary, this study indicated that the anti-PTSD effects of FEWP were associated with allopregnanolone biosynthesis.

The role of allopregnanolone in the anxiolytic-like effect of free and easy wanderer plus (FEWP), a polyherbal preparation.

Anxiety disorders are the serious and burdensome psychiatric illnesses, which are closely correlated with allopregnanolone. The down-regulation of allopregnanolone biosynthesis has been implicated as the possible contributor to the aetiology of anxiety disorders. Free and easy wanderer plus (FEWP) is a well-known traditional Chinese medicine that had been shown to be effective in various mood disorders. The purpose of the present study was to evaluate the anxiolytic-like effect of FEWP and its association with the level of allopregnanolone in the brain. The animal behavioral tests were processed by the acute FEWP (2.5, 5 and 10mg/kg, p.o.) treatment. It had been shown that FEWP produced anxiolytic-like effects in behavioral models, including novelty suppressed feeding (5, 10mg/kg, p.o.), Vogel-type conflict test (10mg/kg, p.o.), elevated plus-maze test (5, 10mg/kg, p.o.). The animals were decapitated after the end of the behavioral tests and measured the allopregnanolone level of the prefrontal cortex and hippocampus by enzyme-linked immunosorbent assay (ELISA). The allopregnanolone level of the prefrontal cortex and hippocampus was increased by administration of FEWP (5, 10mg/kg, p.o.). Overall, these results indicated that FEWP exerts anxiolytic-like effects that were associated with the stiumlation of the allopregnanolone biosynthesis.

The non-benzodiazepine anxiolytic drug etifoxine causes a rapid, receptor-independent stimulation of neurosteroid biosynthesis.

Neurosteroids can modulate the activity of the GABAA receptors, and thus affect anxiety-like behaviors. The non-benzodiazepine anxiolytic compound etifoxine has been shown to increase neurosteroid concentrations in brain tissue but the mode of action of etifoxine on neurosteroid formation has not yet been elucidated. In the present study, we have thus investigated the effect and the mechanism of action of etifoxine on neurosteroid biosynthesis using the frog hypothalamus as an experimental model. Exposure of frog hypothalamic explants to graded concentrations of etifoxine produced a dose-dependent increase in the biosynthesis of 17-hydroxypregnenolone, dehydroepiandrosterone, progesterone and tetrahydroprogesterone, associated with a decrease in the production of dihydroprogesterone. Time-course experiments revealed that a 15-min incubation of hypothalamic explants with etifoxine was sufficient to induce a robust increase in neurosteroid synthesis, suggesting that etifoxine activates steroidogenic enzymes at a post-translational level. Etifoxine-evoked neurosteroid biosynthesis was not affected by the central-type benzodiazepine (CBR) receptor antagonist flumazenil, the translocator protein (TSPO) antagonist PK11195 or the GABAA receptor antagonist bicuculline. In addition, the stimulatory effects of etifoxine and the triakontatetraneuropeptide TTN, a TSPO agonist, were additive, indicating that these two compounds act through distinct mechanisms. Etifoxine also induced a rapid stimulation of neurosteroid biosynthesis from frog hypothalamus homogenates, a preparation in which membrane receptor signalling is disrupted. In conclusion, the present study demonstrates that etifoxine stimulates neurosteroid production through a membrane receptor-independent mechanism.

Localization of brain 5α-reductase messenger RNA in mice selectively bred for high chronic alcohol withdrawal severity.

Several lines of evidence suggest that fluctuations in endogenous levels of the γ-aminobutyric acid (GABA)ergic neurosteroid allopregnanolone (ALLO) represent one mechanism for regulation of GABAergic inhibitory tone in the brain, with an ultimate impact on behavior. Consistent with this idea, there was an inverse relationship between ALLO levels and symptoms of anxiety and depression in humans and convulsive activity in rodents during alcohol withdrawal. Our recent studies examined the activity and expression of 5α-reductase (Srd5a1), the rate-limiting enzyme in the biosynthesis of ALLO, during alcohol withdrawal in mice selectively bred for high chronic alcohol withdrawal (Withdrawal Seizure-Prone [WSP]) and found that Srd5a1 was downregulated in the cortex and hippocampus over the time course of dependence and withdrawal. The purpose of the present studies was to extend these findings and more discretely map the regions of Srd5a1 expression in mouse brain using radioactive in situ hybridization in WSP mice that were ethanol naïve, following exposure to 72h ethanol vapor (dependent) or during peak withdrawal. In naïve animals, expression of Srd5a1 was widely distributed throughout the mouse brain, with highest expression in specific regions of the cerebral cortex, hippocampus, thalamus, hypothalamus, and amygdala. In dependent animals and during withdrawal, there was no change in Srd5a1 expression in cortex or hippocampus, which differed from our recent findings in dissected tissues. These results suggest that local Srd5a1 mRNA expression in WSP brain may not change in parallel with local ALLO content or withdrawal severity.

Palmitoylethanolamide stimulation induces allopregnanolone synthesis in C6 Cells and primary astrocytes: involvement of peroxisome-proliferator activated receptor-α.

Palmitoylethanolamide (PEA) regulates many pathophysiological processes in the central nervous system, including pain perception, convulsions and neurotoxicity, and increasing evidence points to its neuroprotective action. In the present study, we report that PEA, acting as a ligand of peroxisome-proliferator activated receptor (PPAR)-α, might regulate neurosteroidogenesis in astrocytes, which, similar to other glial cells and neurones, have the enzymatic machinery for neurosteroid de novo synthesis. Accordingly, we used the C6 glioma cell line and primary murine astrocytes. In the mitochondrial fraction from cells stimulated with PEA, we demonstrated an increase in steroidogenic acute regulatory protein (StAR) and cytochrome P450 enzyme (P450scc) expression, both comprising proteins considered to be involved in crucial steps of neurosteroid formation. The effects of PEA were completely blunted by GW6471, a selective PPAR-α antagonist, or by PPAR-α silencing by RNA interference. Accordingly, allopregnanolone (ALLO) levels were increased in supernatant of PEA-treated astrocytes, as revealed by gas chromatography-mass spectrometry, and this effect was inhibited by GW6471. Moreover, PEA showed a protective effect, reducing malondialdehyde formation in cells treated with l-buthionine-(S,R)-sulfoximine, a glutathione depletor and, interestingly, the effect of PEA was partially inhibited by finasteride, a 5α-reductase inhibitor. A similar profile of activity was demonstrated by ALLO and the lack of an additive effect with PEA suggests that the reduction of oxidative stress by PEA is mediated through ALLO synthesis. The present study provides evidence indicating the involvement of the saturated acylethanolamide PEA in ALLO synthesis through PPAR-α in astrocytes and explores the antioxidative activity of this molecule, confirming its homeostatic and protective role both under physiological and pathological conditions.

In a mouse model relevant for post-traumatic stress disorder, selective brain steroidogenic stimulants (SBSS) improve behavioral deficits by normalizing allopregnanolone biosynthesis.

The pathophysiological role of the neurosteroid 3alpha-hydroxy-5alpha-pregnan-20-one (allopregnanolone) in neuropsychiatric disorders has been highlighted in several recent investigations. For instance, allopregnanolone levels are decreased in the CSF of patients with post-traumatic stress disorder (PTSD) and major unipolar depression. Neurosteroidogenic antidepressants, including fluoxetine and analogs, correct this decrease in a manner that correlates with improved depressive symptoms. PTSD-like behavioral dysfunctions, including heightened aggression, exaggerated fear, and anxiety-like behavior associated with a decrease in corticolimbic allopregnanolone content are modeled in mice by protracted social isolation stress. Allopregnanolone is not only synthesized by principal glutamatergic and gamma-aminobutyric acid (GABA)ergic neurons, but also locally, potently, positively, and allosterically modulates GABA action at postsynaptic and extrasynaptic GABAA receptors. Hence, this paper will review preclinical studies, which show that in socially isolated mice, rather than selective serotonin reuptake inhibitor mechanisms, allopregnanolone biosynthesis in glutamatergic corticolimbic neurons offers a nontraditional target for fluoxetine to decrease signs of aggression, normalize fear responses, and decrease anxiety-like behavior. At low selective serotonin reuptake inhibitor-inactive doses, fluoxetine and related congeners potently increase allopregnanolone levels by acting as potent selective brain steroidogenic stimulants (SBSS), thereby facilitating GABAA receptor neurotransmission and improving behavioral dysfunctions. Although the precise molecular mechanisms that underlie the action of these drugs are not fully understood, findings from socially isolated mice may ultimately generate insights into novel drug targets for the treatment of psychiatric disorders, such as anxiety and panic disorders, depression, and PTSD.

Characterization of brain neurons that express enzymes mediating neurosteroid biosynthesis.

Allopregnanolone (ALLO) and tetrahydrodeoxycorticosterone (THDOC) are potent positive allosteric modulators of GABA action at GABA(A) receptors. ALLO and THDOC are synthesized in the brain from progesterone or deoxycorticosterone, respectively, by the sequential action of two enzymes: 5alpha-reductase (5alpha-R) type I and 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD). This study evaluates 5alpha-R type I and 3alpha-HSD mRNA expression level in mouse brain by using in situ hybridization combined with glutamic acid decarboxylase 67/65, vesicular glutamate transporter 2, glial fibrillary acidic protein, and S100beta immunohistochemistry. We demonstrate that 5alpha-R type I and 3alpha-HSD colocalize in cortical, hippocampal, and olfactory bulb glutamatergic principal neurons and in some output neurons of the amygdala and thalamus. Neither 5alpha-R type I nor 3alpha-HSD mRNAs are expressed in S100beta- or glial fibrillary acidic protein-positive glial cells. Using glutamic acid decarboxylase 67/65 antibodies to mark GABAergic neurons, we failed to detect 5alpha-R type I and 3alpha-HSD in cortical and hippocampal GABAergic interneurons. However, 5alpha-R type I and 3alpha-HSD are significantly expressed in principal GABAergic output neurons, such as striatal medium spiny, reticular thalamic nucleus, and cerebellar Purkinje neurons. A similar distribution and cellular location of neurosteroidogenic enzymes was observed in rat brain. Taken together, these data suggest that ALLO and THDOC, which can be synthesized in principal output neurons, modulate GABA action at GABA(A) receptors, either with an autocrine or a paracrine mechanism or by reaching GABA(A) receptor intracellular sites through lateral membrane diffusion.

Influence of 17beta-estradiol on the synthesis of reduced neurosteroids in the brain (in vivo) and in glioma cells (in vitro): possible relevance to mental disorders in women.

Brain neurosteroids modulate gamma-aminobutyric acid type A (GABAA) receptor activity, thereby playing a role in mood disorders. Alterations in 17beta-estradiol (E2) and progesterone (P) are also known to play a significant role in psychopathology in women. The aim of the present study was to evaluate the synthesis of dihydroprogesterone (DHP), tetrahydroprogesterone (THP), and the activity of 5alpha-reductase (5alphaR) which regulates the reduction of P to DHP on exposure to supraphysiological levels of E2 in vitro (C6 glioma cells) and in vivo (mouse brain). The results showed that supraphysiological levels of E2 induced a decrease in the accumulation of both neurosteroids, probably by decreasing the activity of 5alphaR. We hypothesize that the high levels of E2 in pregnancy attenuate the increase in the conversion of P to THP in the brain and that the ratio of E2/P modulates the sedative effect of THP. This process may be relevant to psychopathological disorders that are ascribed to drastic alterations in estrogen levels, such as premenstrual syndrome, pregnancy-related mental disorders, and postpartum "blues".

Effects of estradiol and raloxifene analog on brain, adrenal and serum allopregnanolone content in fertile and ovariectomized female rats.

Allopregnanolone is a neuroactive steroid synthesized in rat gonads, adrenal cortex, and central nervous system. It has been suggested that sex steroid hormones might influence allopregnanolone concentrations but no clear data have ever been reported. The aim of the present study was to investigate the effects of administration of 17beta-estradiol (17beta-E2), the raloxifene analog LY-117018 or their combination on allopregnanolone levels in fertile and ovariectomized (OVX) rats. Thirteen groups of 12 Wistar female rats each received either 17beta-E2 (0.1 or 1 microg/day) or LY-117018 (25, 250, and 1,250 microg/day), or 17beta-E2 1 microg/day plus LY-117018: 25, 250, and 1,250 microg/day for 14 days. The rats were then sacrificed and allopregnanolone content was assessed in the hypothalamus, hippocampus, pituitary, adrenals, and serum. Ovariectomy determined a significant decrease in allopregnanolone content in the hypothalamus, hippocampus, pituitary, and serum, while increasing it in the adrenals (p<0.01). In OVX rats, the administration of either 17beta-E2 or LY- 117018 restored ovariectomy-induced allopregnanolone changes. The administration of LY-117018 in addition to 17beta-E2 to OVX animals suppressed the increase in allopregnanolone levels determined by 17beta-E2 in the hippocampus, hypothalamus, and pituitary, but not in the adrenals and serum. In fertile rats, the administration of LY-117018 reproduced the effects of ovariectomy. This study shows that the raloxifene analog LY-117018 has an estrogen-like action on the central nervous system of OVX rats when administered alone, while it acts as an antiestrogen in the presence of 17beta-E2, both in OVX animals treated with 17beta-E2 and in fertile rats. A different effect was observed in the adrenal glands. The mechanism of action of this compound has still to be clarified.