Acute, but not repeated, cocaine exposure alters allopregnanolone levels in the midbrain of male and female rats.

RATIONALE: Multiple psychiatric disorders are associated with altered brain and serum levels of neuroactive steroids, including the endogenous GABAergic steroid, allopregnanolone. Clinically, chronic cocaine use was correlated with decreased levels of pregnenolone. Preclinically, the effect of acute cocaine on allopregnanolone levels in rodents has had mixed results, showing an increase or no change in allopregnanolone levels in some brain regions. OBJECTIVE: We hypothesized that cocaine acutely increases allopregnanolone levels, but repeated cocaine exposure decreases allopregnanolone levels compared to controls. METHODS: We performed two separate studies to determine how systemic administration of 15 mg/kg cocaine (1) acutely or (2) chronically alters brain (olfactory bulb, frontal cortex, dorsal striatum, and midbrain) and serum allopregnanolone levels in adult male and female Sprague-Dawley rats. RESULTS: Cocaine acutely increased allopregnanolone levels in the midbrain, but not in olfactory bulb, frontal cortex, or dorsal striatum. Repeated cocaine did not persistently (24 h later) alter allopregnanolone levels in any region in either sex. However, allopregnanolone levels varied by sex across brain regions. In the acute study, we found that females had significantly higher allopregnanolone levels in serum and olfactory bulb relative to males. In the repeated cocaine study, females had significantly higher allopregnanolone levels in olfactory bulb, frontal cortex, and serum. Finally, acute cocaine increased allopregnanolone levels in the frontal cortex of females in proestrus, relative to non-proestrus stages. CONCLUSION: Collectively these results suggest that allopregnanolone levels vary across brain regions and by sex, which may play a part in differential responses to cocaine by sex.

Emerging evidence for endogenous neurosteroid modulation of pro-inflammatory and anti-inflammatory pathways that impact neuropsychiatric disease.

This mini-review presents emerging evidence that endogenous neurosteroids modulate both pro- and anti-inflammatory signaling by immune cells and brain cells that contribute to depression, alcohol use disorders, and other inflammatory conditions. We first review the literature on pregnenolone and allopregnanolone inhibition of proinflammatory neuroimmune pathways in the periphery and the brain - effects that are independent of GABAergic mechanisms. We follow with evidence for neurosteroid enhancement of anti-inflammatory and protective pathways in brain and immune cells. These studies draw clinical relevance from a large body of evidence that pro-inflammatory immune signaling is dysregulated in many brain disorders and the fact that neurosteroids inhibit the same inflammatory pathways that are activated in depression, alcohol use disorders and other inflammatory conditions. Thus, we describe evidence that neurosteroid levels are decreased and neurosteroid supplementation has therapeutic efficacy in these neuropsychiatric conditions. We conclude with a perspective that endogenous regulation of immune balance between pro- and anti-inflammatory pathways by neurosteroid signaling is essential to prevent the onset of disease. Deficits in neurosteroids may unleash excessive pro-inflammatory activation which progresses in a feed-forward manner to disrupt brain networks that regulate stress, emotion and motivation. Neurosteroids can block various inflammatory pathways in mouse and human macrophages, rat brain and human blood and therefore provide new hope for treatment of intractable conditions that involve excessive inflammatory signaling.

Deleterious Interaction between the Neurosteroid (3α,5α)3-Hydroxypregnan-20-One (3α,5α-THP) and the Mu-Opioid System Activation during Forced Swim Stress in Rats.

The neurosteroid 3α,5α-THP is a potent GABAA receptor-positive modulator and its regulatory action on the HPA axis stress response has been reported in numerous preclinical and clinical studies. We previously demonstrated that 3α,5α-THP down-regulation of HPA axis activity during stress is sex-, brain region- and stressor-dependent. In this study, we observed a deleterious submersion behavior in response to 3α,5α-THP (15 mg/kg) during forced swim stress (FSS) that led us to investigate how 3α,5α-THP might affect behavioral coping strategies engaged in by the animal. Given the well-established involvement of the opioid system in HPA axis activation and its interaction with GABAergic neurosteroids, we explored the synergic effects of 3α,5α-THP/opiate system activation in this behavior. Serum ß-endorphin (ß-EP) was elevated by FSS and enhanced by 3α,5α-THP + FSS. Hypothalamic Mu-opiate receptors (MOP) were increased in female rats by 3α,5α-THP + FSS. Pretreatment with the MOP antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP; 2 mg/kg, IP) reversed submersion behavior in males. Moreover, in both males and females, CTAP pretreatment decreased immobility episodes while increasing immobility duration but did not alter swimming duration. This interaction between 3α,5α-THP and the opioid system in the context of FSS might be important in the development of treatment for neuropsychiatric disorders involving HPA axis activation.

Brexanolone therapeutics in post-partum depression involves inhibition of systemic inflammatory pathways.

BACKGROUND: Brexanolone has rapid, long-lasting, and remarkable efficacy in the treatment of post-partum depression (PPD). We test the hypothesis that brexanolone inhibits proinflammatory modulators and macrophage activation in PPD patients, which may promote clinical recovery. METHODS: PPD patients (N = 18) provided blood samples before and after brexanolone infusion according to the FDA-approved protocol. Patients were unresponsive to prior treatment before brexanolone therapy. Serum was collected to determine neurosteroid levels and whole blood cell lysates were examined for inflammatory markers and in vitro responses to the inflammatory activators lipopolysaccharide (LPS) and imiquimod (IMQ). FINDINGS: Brexanolone infusion altered multiple neuroactive steroid levels (N = 15-18), reduced levels of inflammatory mediators (N = 11) and inhibited their response to inflammatory immune activators (N = 9-11). Specifically, brexanolone infusion reduced whole blood cell tumor necrosis factor-α (TNF-α, p = 0.003), and interleukin-6 (IL-6, p = 0.04) and these effects were correlated with HAM-D score improvement (TNF-α, p = 0.049; IL-6, p = 0.02). Furthermore, brexanolone infusion prevented LPS and IMQ-induced elevation of TNF-α (LPS: p = 0.02; IMQ: p = 0.01), IL-1ß (LPS: p = 0.006; IMQ: p = 0.02) and IL-6 (LPS: p = 0.009; IMQ: p = 0.01), indicating inhibition of toll-like receptor (TLR)4 and TLR7 responses. Finally, inhibition of TNF-α, IL-1ß and IL-6 responses to both LPS and IMQ were correlated with HAM-D score improvements (p < 0.05). INTERPRETATION: Brexanolone actions involve inhibition of inflammatory mediator production and inhibition of inflammatory responses to TLR4 and TLR7 activators. The data suggest that inflammation plays a role in post-partum depression and that inhibition of inflammatory pathways contributes to the therapeutic efficacy of brexanolone. FUNDING: The Foundation of Hope, Raleigh, NC and UNC School of Medicine, Chapel Hill.

Pharmacological administration of 3α,5α-THP into the nucleus accumbens core increases 3α,5α-THP expression and reduces alcohol self-administration.

BACKGROUND: Alcohol affects multiple circuits in the brain, mainly disrupting the delicate balance between inhibitory γ-aminobutyric acid (GABA) transmission and excitatory glutamate signaling in brain areas involved in reward circuits. These include the amygdala, nucleus accumbens (Acb), and ventral tegmental area (VTA). This action impairs circuits that regulate behavioral control of craving and alcohol seeking and intake. Studies in both rodent models and postmortem human brain of patients with alcohol use disorder (AUD) have highlighted the association between the loss of GABAergic inhibition and the development of addiction. The neurosteroid (3α,5α)-3-hydroxypregnan-20-one (3α,5α-THP) is a potent positive modulator of GABAA receptors. Chronic alcohol consumption reduces 3α,5α-THP levels, resulting in decreased GABA inhibition. We previously demonstrated that enhancing neurosteroid biosynthesis by overexpression of the cholesterol side-chain cleavage enzyme P450scc decreased alcohol intake in male alcohol-preferring rats (P-rats). While most of the evidence of alcohol-induced alterations comes from studies in male subjects, some data show that females are more vulnerable to alcohol's effects than males. METHODS: In this study, we investigated the ability of 3α,5α-THP direct infusions in two brain regions that contribute to alcohol reinforcement, the VTA and Acb core (AcbC), to regulate alcohol self-administration in female P-rats. RESULTS: Administration of 3α,5α-THP into the AcbC increased 3α,5α-THP-positive cell expression in this area and reduced alcohol self-administration. By contrast, 3α,5α-THP infusion into the VTA did not significantly affect alcohol self-administration, though trends for a reduction were found. CONCLUSIONS: Our results show that local increases in 3α,5α-THP in the AcbC may alter mesolimbic activity that drives a reduction in alcohol self-administration.

(3α,5α)3-Hydroxypregnan-20-one (3α,5α-THP) Regulation of the HPA Axis in the Context of Different Stressors and Sex.

Corticotropin-releasing factor (CRF) regulates the stress response in the hypothalamus and modulates neurotransmission across the brain through CRF receptors. Acute stress increases hypothalamic CRF and the GABAergic neurosteroid (3α,5α)3-hydroxypregnan-20-one (3α,5α-THP). We previously showed that 3α,5α-THP regulation of CRF is sex and brain region dependent. In this study, we investigated 3α,5α-THP regulation of stress-induced hypothalamic CRF, CRF receptor type 1 (CRFR1), CRF binding protein (CRFBP), pro-opiomelanocortin (POMC), and glucocorticoid receptor (GR) by western blot and circulating corticosterone (CORT) by enzyme-linked immunosorbent assay (ELISA) in male and female Sprague Dawley rats. Tissue was collected after rats were injected with 3α,5α-THP (15 mg/kg, IP) or vehicle 15 min prior to 30 min of restraint stress (RS), or 10 min of forced swim stress (FSS) and 20 min recovery. The initial exposure to a stress stimulus increased circulating CORT levels in both males and females, but 3α,5α-THP attenuated the CORT response only in females after RS. 3α,5α-THP reduced GR levels in male and females, but differently between stressors. 3α,5α-THP decreased the CRF stress response after FSS in males and females, but after RS, only in female rats. 3α,5α-THP reduced the CRFR1, CRFBP, and POMC increases after RS and FSS in males, but in females only after FSS. Our results showed different stress responses following different types of stressors: 3α,5α-THP regulated the HPA axis at different levels, depending on sex.

Sex- and subtype-specific adaptations in excitatory signaling onto deep-layer prelimbic cortical pyramidal neurons after chronic alcohol exposure.

Long-term alcohol use results in behavioral deficits including impaired working memory, elevated anxiety, and blunted inhibitory control that is associated with prefrontal cortical (PFC) dysfunction. Preclinical observations demonstrate multiple impairments in GABAergic neurotransmission onto deep-layer principal cells (PCs) in the prelimbic cortex that suggest dependence-related cortical dysfunction is the product of elevated excitability in these cells. Despite accumulating evidence showing alcohol-induced changes in interneuron signaling onto PCs differ between sexes, there is limited data explicitly evaluating sex-specific ethanol effects on excitatory signaling onto deep-layer PCs that may further contribute to deficits in PFC-dependent behaviors. To address this, we conducted electrophysiological and behavioral tests in both male and female Sprague-Dawley rats to evaluate the effects of chronic ethanol exposure. Among our observations, we report a marked enhancement in glutamatergic signaling onto deep-layer PCs in male, but not female, rats after alcohol exposure. This phenomenon was furthermore specific to a sub-class of PC, sub-cortically projecting Type-A cells, and coincided with enhanced anxiety-like behavior, but no observable deficit in working memory. In contrast, female rats displayed alcohol-induced facilitation in working memory performance with no change in expression of anxiety-like behavior. Together, these results suggest fundamental differences in alcohol effects on cell activity, cortical sub-circuits, and PFC-dependent behaviors across male and female rats.

Neurosteroid allopregnanolone (3α,5α-THP) inhibits inflammatory signals induced by activated MyD88-dependent toll-like receptors.

We have shown that endogenous neurosteroids, including pregnenolone and 3α,5α-THP inhibit toll-like receptor 4 (TLR4) signal activation in mouse macrophages and the brain of alcohol-preferring (P) rat, which exhibits innate TLR4 signal activation. The current studies were designed to examine whether other activated TLR signals are similarly inhibited by 3α,5α-THP. We report that 3α,5α-THP inhibits selective agonist-mediated activation of TLR2 and TLR7, but not TLR3 signaling in the RAW246.7 macrophage cell line. The TLR4 and TLR7 signals are innately activated in the amygdala and NAc from P rat brains and inhibited by 3α,5α-THP. The TLR2 and TLR3 signals are not activated in P rat brain and they are not affected by 3α,5α-THP. Co-immunoprecipitation studies indicate that 3α,5α-THP inhibits the binding of MyD88 with TLR4 or TLR7 in P rat brain, but the levels of TLR4 co-precipitating with TRIF are not altered by 3α,5α-THP treatment. Collectively, the data indicate that 3α,5α-THP inhibits MyD88- but not TRIF-dependent TLR signal activation and the production of pro-inflammatory mediators through its ability to block TLR-MyD88 binding. These results have applicability to many conditions involving pro-inflammatory TLR activation of cytokines, chemokines, and interferons and support the use of 3α,5α-THP as a therapeutic for inflammatory disease.

(3α,5α)3-hydroxypregnan-20-one (3α,5α-THP) regulation of hypothalamic and extrahypothalamic corticotropin releasing factor (CRF): Sexual dimorphism and brain region specificity in Sprague Dawley rats.

CRF is the main activator of the hypothalamic-pituitary-adrenal (HPA) axis in response to stress. CRF neurons are found mainly in the hypothalamus, but CRF positive cells and CRF1 receptors are also found in extrahypothalamic structures, including amygdala (CeA), hippocampus, NAc and VTA. CRF release in the hypothalamus is regulated by inhibitory GABAergic interneurons and extrahypothalamic glutamatergic inputs, and disruption of this balance is found in stress-related disorders and addiction. (3α,5α)3-hydroxypregnan-20-one (3α,5α-THP), the most potent positive modulator of GABAA receptors, attenuates the stress response reducing hypothalamic CRF mRNA expression and ACTH and corticosterone serum levels. In this study, we explored 3α,5α-THP regulation of hypothalamic and extrahypothalamic CRF mRNA and peptide expression, in male and female Sprague Dawley rats, following vehicle or 3α,5α-THP administration (15 mg/kg). In the hypothalamus, we found sex differences in CRF mRNA expression (females +74%, p < 0.01) and CRF peptide levels (females -71%, p < 0.001). 3α,5α-THP administration reduced hypothalamic CRF mRNA expression only in males (-50%, p < 0.05) and did not alter CRF peptide expression in either sex. In hippocampus and CeA, 3α,5α-THP administration reduced CRF peptide concentrations only in the male (hippocampus -29%, p < 0.05; CeA -62%, p < 0.01). In contrast, 3α,5α-THP injection increased CRF peptide concentration in the VTA of both males (+32%, p < 0.01) and females (+26%, p < 0.01). The results show sex and region-specific regulation of CRF signals and the response to 3α,5α-THP administration. This data may be key to successful development of therapeutic approaches for stress-related disorders and addiction.

A Rationale for Allopregnanolone Treatment of Alcohol Use Disorders: Basic and Clinical Studies.

For many years, research from around the world has suggested that the neuroactive steroid (3α,5α)-3-hydroxypregnan-20-one (allopregnanolone or 3α,5α-THP) may have therapeutic potential for treatment of various symptoms of alcohol use disorders (AUDs). In this critical review, we systematically address all the evidence that supports such a suggestion, delineate the etiologies of AUDs that are addressed by treatment with allopregnanolone or its precursor pregnenolone, and the rationale for treatment of various components of the disease based on basic science and clinical evidence. This review presents a theoretical framework for understanding how endogenous steroids that regulate the effects of stress, alcohol, and the innate immune system could play a key role in both the prevention and the treatment of AUDs. We further discuss cautions and limitations of allopregnanolone or pregnenolone therapy with suggestions regarding the management of risk and the potential for helping millions who suffer from AUDs.

Pleiotropic actions of allopregnanolone underlie therapeutic benefits in stress-related disease.

For several years, research from around the world has suggested that the neuroactive steroid (3α,5α)-3-hydroxypregnan-20-one (allopregnanolone) may have therapeutic potential for treatment of various stress-related diseases including post-traumatic stress disorder (PTSD), depression, alcohol use disorders (AUDs), as well as neurological and psychiatric conditions that are worsened in the presence of stress, such as multiple sclerosis, schizophrenia, and seizure disorders. In this review, we make the argument that the pleiotropic actions of allopregnanolone account for its ability to promote recovery in such a wide variety of illnesses. Likewise, the allopregnanolone precursors, pregnenolone and progesterone, share many actions of allopregnanolone. Of course, pregnenolone and progesterone lack direct effects on GABAA receptors, but these compounds are converted to allopregnanolone in vivo. This review presents a theoretical framework for understanding how endogenous neurosteroids that regulate 1) γ-aminobutyric acid (GABA)A receptors, 2) corticotropin releasing factor (CRF) and 3) pro-inflammatory signaling in the innate immune system and brain could play a key role in both the prevention and treatment of stress-related disease. We further discuss cautions and limitations of allopregnanolone or precursor therapy as well as the need for more clinical studies.

Are preconceptional stressful experiences crucial elements for the aetiology of autism spectrum disorder? Insights from an animal model.

Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders characterized by changes in social interactions, impaired language and communication, fear responses and presence of repetitive behaviours. Although the genetic bases of ASD are well documented, the recent increase in clinical cases of idiopathic ASD indicates that several environmental risk factors could play a role in ASD aetiology. Among these, maternal exposure to psychosocial stressors during pregnancy has been hypothesized to affect the risk for ASD in offspring. Here, we tested the hypothesis that preconceptional stressful experiences might also represent crucial elements in the aetiology of ASD. We previously showed that social isolation stress during adolescence results in a marked decrease in the brain and plasma concentrations of progesterone and in the quality of maternal care that these female rats later provide to their young. Here we report that male offspring of socially isolated parents showed decreased agonistic behaviour and social transmission of flavour preference, impairment in reversal learning, increased seizure susceptibility, reduced plasma oxytocin levels, and increased plasma and brain levels of BDNF, all features resembling an ASD-like phenotype. These alterations came with no change in spatial learning, aggression, anxiety and testosterone plasma levels, and were sex-dependent. Altogether, the results suggest that preconceptional stressful experiences should be considered as crucial elements for the aetiology of ASD, and indicate that male offspring of socially isolated parents may be a useful animal model to further study the neurobiological bases of ASD, avoiding the adaptations that may occur in other genetic or pharmacologic experimental models of these disorders.

Impaired glucocorticoid-mediated HPA axis negative feedback induced by juvenile social isolation in male rats.

We previously demonstrated that socially isolated rats at weaning showed a significant decrease in corticosterone and adrenocorticotropic hormone (ACTH) levels, associated with an enhanced response to acute stressful stimuli. Here we shown that social isolation decreased levels of total corticosterone and of its carrier corticosteroid-binding globulin, but did not influence the availability of the free active fraction of corticosterone, both under basal conditions and after acute stress exposure. Under basal conditions, social isolation increased the abundance of glucocorticoid receptors, while it decreased that of mineralocorticoid receptors. After acute stress exposure, socially isolated rats showed long-lasting corticosterone, ACTH and corticotrophin releasing hormone responses. Moreover, while in the hippocampus and hypothalamus of group-housed rats glucocorticoid receptors expression increased with time and reached a peak when corticosterone levels returned to basal values, in socially isolated rats expression of glucocorticoid receptors did not change. Finally, social isolation also affected the hypothalamic endocannabinoid system: compared to group-housed rats, basal levels of anandamide and cannabinoid receptor type 1 were increased, while basal levels of 2-arachidonoylglycerol were decreased in socially isolated rats and did not change after acute stress exposure. The present results show that social isolation in male rats alters basal HPA axis activity and impairs glucocorticoid-mediated negative feedback after acute stress. Given that social isolation is considered an animal model of several neuropsychiatric disorders, such as generalized anxiety disorder, depression, post-traumatic stress disorder and schizophrenia, these data could contribute to better understand the alterations in HPA axis activity observed in these disorders.

Combined effect of gestational stress and postpartum stress on maternal care in rats.

Variations in maternal care in the rat influence the development of individual differences in behavioral and endocrine responses to stress. This study aimed to examine the interaction between intragastric intubation during late gestation and postpartum stress, induced by pup separation, on maternal behavior and on dams' emotional state and HPA axis function. Rats received intragastric intubation of water on days 12-20 of gestation or remained untreated in their home cage (naïve dams). Pup separation was used as a model of postpartum stress. The procedure consisted of a daily separation of the dam from its litter for 3h from PND 3 until PND 15. Pup separation was carried out in both naïve and intubated dams. The behavioral results indicate that the association of these two stressors significantly decreased arched-back nursing (ABN) and licking and grooming (LG), behaviors considered important parameters to discriminate the high quality of maternal care. Moreover, dams that received both stressors displayed less nest building and blanket nursing behaviors; no effect on the frequency of passive and total nursing was recorded. The analysis of single effects on ABN and LG, revealed that dams that underwent gestational stress induced by intragastric intubation displayed less LG, but ABN was overall unchanged. On the contrary, pup separation stress significantly increased ABN and LG upon reunion of naïve dams with their pups. Treatments per se or the association of both induced modest changes in plasma levels of allopregnanolone and corticosterone that likely did not influence maternal care. These data show that the association of a mild stress during gestation with an unfavorable experience after parturition had a significant impact on maternal care. This effect seems independent from HPA axis activation or from changes in emotional state; further studies would be necessary to ascertain the neural changes that could contribute to altered maternal behavior in stressed mothers. Moreover, these results suggest that the use of intragastric intubation during gestation would interfere with measures of drug-induced changes in maternal behavior and likely their consequences on the offspring.

Juvenile social isolation affects maternal care in rats: involvement of allopregnanolone.

RATIONALE: Social isolation of rats immediately after weaning is thought to represent an animal model of anxiety-like disorders. Socially isolated virgin females showed a significant decrease in allopregnanolone levels, associated with increased anxiety-related behavior compared with group-housed rats. OBJECTIVES: The present study investigates whether post-weaning social isolation affects maternal behavior and assesses neuroactive steroid levels in adult female rats during pregnancy and postpartum. RESULTS: Socially isolated dams displayed a reduction in the frequency of arched back nursing (ABN) behavior compared to group-housed dams. In addition, both total and active nursing were lower in socially isolated dams compared to group-housed dams. Compared to virgin females, pregnancy increases allopregnanolone levels in group-housed as well as isolated dams and such increase was greater in the latter group. Compared to pregnancy levels, allopregnanolone levels decreased after delivery and this decrease was more pronounced in isolated than group-housed dams. Moreover, the fluctuations in plasma corticosterone levels that occur in late pregnancy and during lactation follow a different pattern in socially isolated vs. group-housed rats. CONCLUSIONS: The present results show that social isolation in female rats decreases maternal behavior; this effect is associated with lower allopregnanolone concentrations at postpartum, which may account, at least in part, for the poor maternal care observed in socially isolated dams. In support of this conclusion is the finding that finasteride-treated dams, which display a decrease in plasma allopregnanolone levels, also showed a marked reduction in maternal care, suggesting that allopregnanolone may contribute to the quality of maternal care.