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Aims Preoperative (chemo)radiotherapy followed by total mesorectal excision is the current standard of care for patients with locally advanced rectal cancer. The use of intensity-modulated radiotherapy (IMRT) for rectal cancer is increasing in the UK. However, the extent of IMRT implementation and current practice was not previously known. A national survey was commissioned to investigate the landscape of IMRT use for rectal cancer and to inform the development of national rectal cancer IMRT guidance. Materials and methods A web-based survey was developed by the National Rectal Cancer IMRT Guidance working group in collaboration with the Royal College of Radiologists and disseminated to all UK radiotherapy centres. The survey enquired about the implementation of IMRT with a focus on the following aspects of the workflow: dose fractionation schedules and use of a boost; pre-treatment preparation and simulation; target volume/organ at risk definition; treatment planning and treatment verification. A descriptive statistical analysis was carried out. Results In total, 44 of 63 centres (70%) responded to the survey; 30/44 (68%) and 36/44 (82%) centres currently use IMRT to treat all patients and selected patients with rectal cancer, respectively. There was general agreement concerning several aspects of the IMRT workflow, including patient positioning, use of intravenous contrast and bladder protocols. Greater variation in practice was identified regarding rectal protocols; use of a boost to primary/nodal disease; target volume delineation; organ at risk delineation and dose constraints and treatment verification. Delineation of individual small bowel loops and daily volumetric treatment verification were considered potentially feasible by most centres. Conclusion This survey identified that IMRT is already used to treat rectal cancer in many UK radiotherapy centres, but there is heterogeneity between centres in its implementation and practice. These results have been a valuable aid in framing the recommendations within the new National Rectal Cancer IMRT Guidance., Highlights • First national UK survey of rectal cancer IMRT use and practice. • Considerable variation identified in IMRT implementation and practice. • Results of survey informed recommendations within new national consensus guideline.

Hormonal contraceptives prevent ovulation with subsequent reduction in endogenous levels of estradiol, progesterone and its neuroactive metabolite allopregnanolone. These neurosteroids modulate several brain functions, including neuronal plasticity, cognition and memory. We hypothesized that hormonal contraceptives might affect synaptic plasticity, learning and memory, as a consequence of suppressed endogenous hormones levels. Female rats were orally treated with a combination of ethinyl estradiol (EE, 0.020 mg) and levonorgestrel (LNG, 0.060 mg) once daily for four weeks. Decreased hippocampal brain-derived neurotrophic factor (BDNF) levels and altered histone H3 post-translational modifications (PTMs) were observed 14 days after discontinuation from chronic EE-LNG treatment. These effects were not accompanied by alterations in long-term plasticity at glutamatergic synapses, recognition memory in the novel object and novel place location tests, or spatial learning, memory, and behavioral flexibility in the Morris water maze test. Thus, decreased BDNF content does not affect synaptic plasticity and cognitive performance; rather it might be relevant for the occurrence of certain psychiatric symptoms, reported by some women using hormonal contraceptives. These results provide the first evidence of hippocampal epigenetic changes induced by hormonal contraceptives and complement previous studies on the neurobiological actions of hormonal contraceptives; the finding that effects of chronic EE-LNG treatment on BDNF content and histone PTMs are observed 14 days after drug discontinuation warrants further investigation to better understand the implications of such long-term consequences for women's health.

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.

Steroid hormones influence different aspects of brain function, including development, neurogenesis, neuronal excitability, and plasticity, thus affecting emotional states, cognition, sociality, and reward. In women, their levels fluctuate across the lifespan and through the reproductive stages but are also altered by exogenous administration of hormonal contraceptives (HC). HC are widely used by women throughout their fertile life both for contraceptive and therapeutic benefits. However, awareness of their effects on brain function and behavior is still poorly appreciated, despite the emerging evidence of their action at the level of the central nervous system. Here, we summarize results obtained in preclinical studies, mostly conducted in intact female rodents, aimed at investigating the neurobiological effects of HC. HC can alter neuroactive hormones, neurotransmitters, neuropeptides, as well as emotional states, cognition, social and sexual behaviors. Animal studies provide insights into the neurobiological effects of HC with the aim to improve women's health and well-being.

Exposure of female rats to estradiol during the perinatal period has profound effects on GABAergic neurotransmission that are crucial to establish sexually dimorphic brain characteristics. We previously showed that neonatal β-estradiol 3-benzoate (EB) treatment decreases brain concentrations of the neurosteroid allopregnanolone, a potent positive modulator of extrasynaptic GABAA receptors (GABAAR). We thus evaluated whether neonatal EB treatment affects GABAAR expression and function in the hippocampus of adult female rats. Neonatal EB administration increased the expression of extrasynaptic α4/δ subunit-containing GABAARs and the modulatory action of THIP on tonic currents mediated by these receptors. The same treatment decreased the expression of synaptic α1/α4/γ2 subunit-containing receptors, as well as phasic currents. These effects of neonatal EB treatment are not related to ambient allopregnanolone concentrations per se, given that vehicle-treated rats in diestrus, which have opposite neurosteroid levels than EB-treated rats, show similar changes in GABAARs. Rather, these changes may represent a compensatory mechanism to counteract the long-term reduction in allopregnanolone concentrations, induced by neonatal EB. Given that both α4/δ receptors and allopregnanolone are involved in memory consolidation, we evaluated whether neonatal EB treatment alters performance in the Morris water maze test during adulthood. Neonatal EB treatment decreased the latency and the cumulative search error to reach the platform, as well as thigmotaxis, suggesting improved learning, and also enhanced memory performance during the probe trial. These enduring changes in GABAAR plasticity may be relevant for the regulation of neuronal excitability in the hippocampus and for the etiology of psychiatric disorders that originate in development and show sex differences.

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.

Hormonal contraceptives are frequently prescribed drugs among women, mainly for their reversible contraceptive purposes but also for beneficial effects in some gynecological pathologies. Despite extensive studies aimed at elucidating the physical effects of hormonal contraceptives and ameliorating some unwanted outcomes, little is known yet about the effects of these drugs on brain function and related behavior, which are known to be modulated by endogenous steroid hormones. We describe the current literature on preclinical studies in animals undertaken to investigate effects of hormonal contraceptives on brain function and behavior. These studies suggest that hormonal contraceptives influence neurohormones, neurotransmitters, neuropeptides, and emotional, cognitive, social and sexual behaviors. Animals allow examination of the basic biological mechanisms of these drugs, devoid of the psychological aspect often associated to hormonal contraceptives' use in women. Understanding the neurobiological effects of these drugs may improve women's health and may help women making informed choices on hormonal contraception.

This chapter summarizes evidence that GABAergic neuroactive steroids are modulated by alcohol in mice, rats, monkeys, and humans. Alcohol differentially affects neuroactive steroid concentrations depending on type of exposure (acute vs chronic), as well as on species and brain regions examined. Alcohol-induced elevations in neuroactive steroids influence many of the GABAergic effects of ethanol in vivo and contribute to sensitivity to behavioral effects of ethanol in rodents. Likewise, neuroactive steroids appear to contribute to subjective effects of alcohol in humans. Moreover, neuroactive steroids modulate ethanol reinforcement and consumption, and may be deficient in ethanol dependence, suggesting that neuroactive steroid responses to alcohol may play a role in vulnerability to alcohol use disorders. These findings provide a rationale for exploration of the therapeutic effects of GABAergic neuroactive steroids in alcohol use disorders.

Women are more likely than men to suffer from anxiety disorders and major depression. These disorders share hyperresponsiveness to stress as an etiological factor. Thus, sex differences in brain arousal systems and their regulation by chronic stress may account for the increased vulnerability to these disorders in women. Social isolation is a model of early life stress that results in neurobiological alterations leading to increased anxiety-like and depressive-like behaviors. Here we investigated the sex difference in the effects of post-weaning social isolation on acute stress sensitivity and behavior in rats. In both sexes, social isolation at weaning reduced basal levels of the neuroactive steroid allopregnanolone in the brain and of corticosterone in plasma. Moreover, acute stress increased plasma corticosterone levels in both group-housed and socially isolated male and female rats; however this effect was greater in male than female rats subjected to social isolation. Intriguingly, group-housed female rats showed no change in plasma and brain levels of allopregnanolone after acute foot-shock stress. The absence of stress-induced effects on allopregnanolone synthesis might be due to the physiologically higher levels of this hormone in females vs. males. Accordingly, increasing allopregnanolone levels in male rats blunted the response to foot-shock stress in these animals. Socially isolated male, but not female, rats also display depressive-like behavior and increased hippocampal brain-derived neurotrophic factor (BDNF). The ovarian steroids could "buffer" the effect of this adverse experience in females on these parameters. Finally, the dexamethasone (DEX) suppression test indicated that the chronic stress associated with social isolation impairs feedback inhibition in both sexes in which an increase in the abundance of glucocorticoid receptors (GRs) in the hippocampus was found. Altogether, these results demonstrate that social isolation affects neuroendocrine reactivity to stress, plasticity and emotionality in a sexually dimorphic manner.

GABAA receptors containing α4 subunits are widely implicated in acute ethanol sensitivity, and their spatial and temporal regulation prominently contributes to ethanol-induced neuroplasticity in hippocampus and cortex. However, it is unknown if α4-containing GABAA receptors in the thalamus, an area of high α4 expression, display similar regulatory patterns following ethanol administration, and if so, by which molecular mechanisms. In the current study, thalamic GABAA receptor α4 subunit levels were increased following a 6-week, but not a 2-week chronic ethanol diet. Following acute high-dose ethanol administration, thalamic GABAA receptor α4 subunit levels were regulated in a temporal fashion, as a decrease was observed at 2 hours followed by a delayed transient increase. PKCγ and PKCδ levels paralleled α4 temporal expression patterns following ethanol exposure. Initial decreases in α4 subunit expression were associated with reduced serine phosphorylation. Delayed increases in expression were not associated with a change in phosphorylation state, but were prevented by inhibiting neuroactive steroid production with the 5α-reductase inhibitor finasteride. Overall, these studies indicate that thalamic GABAA receptor α4 subunit expression following acute and chronic ethanol administration exhibits similar regulatory patterns as other regions and that transient expression patterns following acute exposure in vivo are likely dependent on both subunit phosphorylation state and neuroactive steroids.

Neuroactive steroids such as (3α,5α)3-hydroxypregnan-20-one (3α,5α-THP, allopregnanolone) enhance the gamma-aminobutyric acid (GABA)-ergic effects of ethanol and modulate excessive drinking in rodents. Moreover, chronic ethanol consumption reduces 3α,5α-THP levels in human plasma, rat hippocampus and mouse limbic regions. We explored the relationship between 3α,5α-THP levels in limbic brain areas and voluntary ethanol consumption in the cynomolgus monkey following daily self-administration of ethanol for 12 months and further examined the relationship to hypothalamic-pituitary-adrenal (HPA) axis function prior to ethanol exposure. Monkeys were subjected to scheduled induction of ethanol consumption followed by free access to ethanol or water for 22 h/day over 12 months. Immunohistochemistry was performed using an anti-3α,5α-THP antibody. Prolonged voluntary drinking resulted in individual differences in ethanol consumption that ranged from 1.2 to 4.2 g/kg/day over 12 months. Prolonged ethanol consumption reduced cellular 3α,5α-THP immunoreactivity by 13 ± 2 percent (P

Background Neuroactive steroids such as (3α,5α)3-hydroxypregnan-20-one (3α,5α-THP, allopregnanolone) are potent neuromodulators that enhance GABAergic neurotransmission and produce inhibitory neurobehavioral and anti-inflammatory effects. Chronic ethanol (EtOH) consumption reduces 3α,5α-THP levels in human plasma, but has brain region- and species-specific effects on central nervous system levels of 3α,5α-THP. We explored the relationship between 3α,5α-THP levels in the hippocampus and voluntary EtOH consumption in the cynomolgus monkey following daily self-administration of EtOH for 12 months and further examined the relationship with hypothalamic–pituitary–adrenal (HPA) axis function prior to EtOH exposure. We simultaneously explored hippocampus levels of monocyte chemoattractant protein 1 (MCP-1), a pro-inflammatory cytokine that plays an important role in the neuroimmune response to EtOH, following chronic self-administration. Methods Monkeys were subjected to scheduled induction of water and EtOH consumption (0 to 1.5 g/kg) over 4 months, followed by free access to EtOH or water for 22 h/d over 12 months. Immunohistochemistry was performed using an anti-3α,5α-THP or anti-MCP-1 antibody. Prolonged voluntary drinking resulted in individual differences in EtOH consumption that ranged from 1.2 to 4.2 g/kg/d over 12 months. Results Prolonged EtOH consumption increased cellular 3α,5α-THP immunoreactivity by 12 ± 2% (p

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 (naive 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 naive 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 naive 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.

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.

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. 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. 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. 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.

Acute ethanol activates the hypothalamic–pituitary–adrenal (HPA) axis, while long-term exposure results in a blunted neuroendocrine state, particularly with regards to the primary endpoint, cortisol, the primary glucocorticoid produced in the adrenal cortex. However, it is unknown if this dampened neuroendocrine status also influences other adrenocortical steroids. Plasma concentration of the mineralocorticoid and neuroactive steroid precursor deoxycorticosterone (DOC) is altered by pharmacological challenges of the HPA axis in cynomolgus monkeys. The present study investigated HPA axis regulation of circulating DOC concentration over the course of ethanol (4% w/v) induction and self-administration in non-human primates (Macaca fasciculata, n = 10). Plasma DOC, measured by radioimmunoassay, was compared at baseline (ethanol naïve), during schedule-induced polydipsia, and following 6-months of 22 h/day access to ethanol and water. The schedule induction of ethanol drinking did not alter basal DOC levels but selectively dampened the DOC response to pharmacological challenges aimed at the anterior pituitary (ovine corticotrophin-releasing hormone) and adrenal gland (post-dexamethasone adrenocorticotropin hormone), while pharmacological inhibition of central opioid receptors with naloxone greatly enhanced the DOC response during induction. Following 6 months of ethanol self-administration, basal DOC levels were increased more than twofold, while responses to each of the challenges normalized somewhat but remained significantly different than baseline. These data show that HPA axis modulation of the neuroactive steroid precursor DOC is markedly altered by the schedule induction of ethanol drinking and long-term voluntary ethanol self-administration. The consequences of chronic ethanol consumption on HPA axis regulation of DOC point toward allostatic modification of hypothalamic and adrenal function.

Allopregnanolone is a neurosteroid involved in depression, memory, social, and sexual behavior. We have previously demonstrated that treatment with a combination of ethinylestradiol (EE) and levonorgestrel (LNG), two compounds frequently used in hormonal contraception, decreased brain allopregnanolone concentrations. These changes may contribute to some of the emotional and sexual disorders observed in hormonal contraceptive users. We thus examined whether the reduction in allopregnanolone concentrations induced by long-term EE/LNG administration was associated with altered emotional, learning, social, and sexual behaviors. Rats were orally treated with a combination of EE (0.030 mg) and LNG (0.125 mg) once a day for 4 weeks and were subjected to behavioral tests 24 h after the last administration. EE/LNG treatment reduced immobility behavior in the forced swim test, without affecting sucrose preference and spatial learning and memory. In the resident–intruder test, EE/LNG-treated rats displayed a decrease in dominant behaviors associated with a reduction in social investigation. In the paced mating test, EE/LNG treated rats showed a reduction in proceptive behaviors, while the lordosis quotient was not affected. Progesterone, but not estradiol, administration to EE/LNG-treated rats increased sexual activity and cerebrocortical allopregnanolone concentrations. Prior administration of finasteride decreased allopregnanolone concentrations and abolished the increase in proceptivity induced by progesterone administration. The decrease in brain allopregnanolone concentrations induced by EE/LNG treatment is associated with a reduction in social behavior and sexual motivation in female rats. These results might be relevant to the side effects sometimes exhibited by women taking hormonal contraceptives.

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.

Allopregnanolone plays a role in the stress response and homeostasis. Alterations in the estrogen milieu during the perinatal period influence brain development in a manner that persists into adulthood. Accordingly, we showed that a single administration of estradiol benzoate (EB) on the day of birth decreases brain allopregnanolone concentrations in adult female rats. We examined whether the persistent decrease in allopregnanolone concentrations, induced by neonatal EB treatment, might affect sensitivity to stress during adulthood. Female rats were treated with 10 μg of EB or vehicle on the day of birth. During adulthood, the response to acute foot shock stress was assessed by measuring changes in brain allopregnanolone and corticosterone levels, as well as extracellular dopamine output in the medial prefrontal cortex (mPFC). Neonatal EB treatment enhanced stress-stimulated allopregnanolone levels in the hypothalamus, as well as extracellular dopamine output in the mPFC; this latest effect is reverted by subchronic progesterone treatment. By contrast, neonatal EB treatment did not alter stress-induced corticosterone levels, sensitivity to hypothalamic-pituitary-adrenal (HPA) axis negative feedback, or abundance of glucocorticoid and mineralocorticoid receptors. The persistent decrease in brain allopregnanolone concentrations, induced by neonatal EB treatment, enhances stress-stimulated allopregnanolone levels and extracellular dopamine output during adulthood. These effects are not associated to an impairment in HPA axis activity. Heightened sensitivity to stress is a risk factor for several neuropsychiatric disorders; these results suggest that exposure to estrogen during development may predispose individuals to such disorders.

Exposure of female rats to estradiol during the perinatal period has profound effects on GABAergic neurotransmission that are crucial to establish sexually dimorphic brain characteristics. We previously showed that neonatal β-estradiol 3-benzoate (EB) treatment decreases brain concentrations of the neurosteroid allopregnanolone, a potent positive modulator of extrasynaptic GABA

Neuroactive steroids are endogenous neuromodulators synthesised in the brain that rapidly alter neuronal excitability by binding to membrane receptors, in addition to the regulation of gene expression via intracellular steroid receptors. Neuroactive steroids induce potent anxiolytic, antidepressant, anticonvulsant, sedative, analgesic and amnesic effects, mainly through interaction with the GABAA receptor. They also exert neuroprotective, neurotrophic and antiapoptotic effects in several animal models of neurodegenerative diseases. Neuroactive steroids regulate many physiological functions, such as the stress response, puberty, the ovarian cycle, pregnancy and reward. Their levels are altered in several neuropsychiatric and neurological diseases and both preclinical and clinical studies emphasise a therapeutic potential of neuroactive steroids for these diseases, whereby symptomatology ameliorates upon restoration of neuroactive steroid concentrations. However, direct administration of neuroactive steroids has several challenges, including pharmacokinetics, low bioavailability, addiction potential, safety and tolerability, which limit its therapeutic use. Therefore, modulation of neurosteroidogenesis to restore the altered endogenous neuroactive steroid tone may represent a better therapeutic approach. This review summarises recent approaches that target the neuroactive steroid biosynthetic pathway at different levels aiming to promote neurosteroidogenesis. These include modulation of neurosteroidogenesis through ligands of the translocator protein 18 kDa and the pregnane xenobiotic receptor, as well as targeting of specific neurosteroidogenic enzymes such as 17β-hydroxysteroid dehydrogenase type 10 or P450 side chain cleavage. Enhanced neurosteroidogenesis through these targets may be beneficial not only for neurodegenerative diseases, such as Alzheimer's disease and age-related dementia, but also for neuropsychiatric diseases, including alcohol use disorders.

The 3α,5α- and 3α,5β-reduced metabolites of progesterone, deoxycorticosterone, and dehydroepiandrosterone (DHEA) have potent effects on neurotransmission mediated by GABAA receptors, and dysregulation of these receptors has been implicated in depression. Using gas chromatography-mass spectrometry, we compared neuroactive steroid concentrations in women with a history of depressive disorders, but who were in full remission at the time of testing (n=11) to never depressed women (n=17) both before and after a challenge with oral micronized progesterone (300 mg). Serum concentrations of the following were obtained: four progesterone-derived GABAergic neuroactive steroids, the precursor pregnenolone, androstenedione-derived neuroactive steroids, and the precursor DHEA. As an index of conversion of progesterone to neuroactive steroids, we also examined ratios of neuroactive steroids to progesterone following the oral progesterone challenge. Results indicated that both before and after oral progesterone, women with histories of depression showed lower concentrations of all GABAergic neuroactive steroids than never depressed women. Those with a history of depression also tended to have lower cortisol concentrations. Because serum neuroactive steroids are mainly synthesized in the adrenals, we hypothesize that histories of depression may be associated with persistent adrenal suppression. Following the progesterone challenge, ratios of the progesterone-derived neuroactive steroids to plasma progesterone concentrations were elevated in women with depression histories, suggesting there may be an adaptive shift in the metabolism of progesterone that compensates for lower circulating neuroactive steroid concentrations.

Purpose Advances in radiotherapy planning and delivery have made target definition increasingly important. While CT images are required for plan calculation, MR fusion is increasingly used to more accurately define tumour and normal tissue. There is often significant variation seen between diagnostic and therapeutic imaging; hence, MR carried out in the treatment position is desirable. Materials and methods A multidisciplinary team of diagnostic and therapeutic radiographers, treatment planners, medical physicists and clinicians was convened. Planning MR was integrated into the radiotherapy pathway and carried out in the days immediately following CT simulation. All men underwent identical preparation (administration of a micro-enema and drinking 300 mL of water thirty minutes prior to imaging/treatment). Patients were set up in the treatment position using MR-compatible radiotherapy immobilisation. T2SE axial and sagittal images were acquired (Siemens Aera 1.5T E11, incorporating RT software platforms/LAP Laser Bridge/Civco RT Indexing Flat couch top/coil bridges), imported into the Eclipse planning system (V13.6, Varian), and fused to the planning CT for volume delineation. Results The service opened in mid-September 2017. 26 patients were scanned to the end of November 2017. All patients tolerated preparation and imaging without difficulty. Conclusion This service has been successfully introduced and will shortly expand to include other sites (rectum, lung, head and neck, complex palliative). A study is planned to assess the impact of the addition of MR on target delineation. Additional considerations include the need for dedicated radiology input and the potential role of collaboration with industry with a view to stand-alone MR simulation.

Background: Acute ethanol administration increases plasma and brain levels of progesterone and deoxycorticosterone-derived neuroactive steroids (3a,5a)-3-hydroxypregnan-20-one (3a,5aTHP) and (3a,5a)-3,21-dihydroxypregnan-20-one (3a,5a-THDOC) in rats. However, little is known about ethanol effects on GABAergic neuroactive steroids in mice, nonhuman primates, or humans. We investigated the effects of ethanol on plasma levels of 3a,5a- and 3a,5b-reduced GABAergic neuroactive steroids derived from progesterone, deoxycorticosterone, dehydroepiandrosterone, and testosterone using gas chromatography-mass spectrometry. Methods: Serum levels of GABAergic neuroactive steroids and pregnenolone were measured in male rats, C57BL ⁄ 6J and DBA ⁄ 2J mice, cynomolgus monkeys, and humans following ethanol administration. Rats and mice were injected with ethanol (0.8 to 2.0 g ⁄ kg), cynomolgus monkeys received ethanol (1.5 g ⁄ kg) intragastrically, and healthy men consumed a beverage containing 0.8 g ⁄ kg ethanol. Steroids were measured after 60 minutes in all species and also after 120 minutes in monkeys and humans. Results: Ethanol administration to rats increased levels of 3a,5a-THP, 3a,5a-THDOC, and pregnenolone at the doses of 1.5 g ⁄ kg (+228, +134, and +860%, respectively, p < 0.001) and 2.0 g ⁄ kg (+399, +174, and +1125%, respectively, p < 0.001), but not at the dose of 0.8 g ⁄ kg. Ethanol did not alter levels of the other neuroactive steroids. In contrast, C57BL ⁄ 6J mice exhibited a 27% decrease in serum 3a,5a-THP levels (p < 0.01), while DBA ⁄ 2J mice showed no significant effect of ethanol, although both mouse strains exhibited substantial increases in precursor steroids. Ethanol did not alter any of the neuroactive steroids in cynomolgus monkeys at doses comparable to those studied in rats. Finally, no effect of ethanol (0.8 g ⁄ kg) was observed in men. Conclusions: These studies show clear species differences among rats, mice, and cynomolgus monkeys in the effects of ethanol administration on circulating neuroactive steroids. Rats are unique in their pronounced elevation of GABAergic neuroactive steroids, while this effect was not observed in mice or cynomolgus monkeys at comparable ethanol doses.

The past decade has brought many advances in our understanding of GABA(A) receptor-mediated ethanol action in the central nervous system. We now know that specific GABA(A) receptor subtypes are sensitive to ethanol at doses attained during social drinking while other subtypes respond to ethanol at doses attained by severe intoxication. Furthermore, ethanol increases GABAergic neurotransmission through indirect effects, including the elevation of endogenous GABAergic neuroactive steroids, presynaptic release of GABA, and dephosphorylation of GABA(A) receptors promoting increases in GABA sensitivity. Ethanol's effects on intracellular signaling also influence GABAergic transmission in multiple ways that vary across brain regions and cell types. The effects of chronic ethanol administration are influenced by adaptations in GABA(A) receptor function, expression, trafficking, and subcellular localization that contribute to ethanol tolerance, dependence, and withdrawal hyperexcitability. Adolescents exhibit altered sensitivity to ethanol actions, the tendency for higher drinking and longer lasting GABAergic adaptations to chronic ethanol administration. The elucidation of the mechanisms that underlie adaptations to ethanol exposure are leading to a better understanding of the regulation of inhibitory transmission and new targets for therapies to support recovery from ethanol withdrawal and alcoholism.

The 3alpha,5alpha- and 3alpha,5beta-reduced derivatives of progesterone, deoxycorticosterone, dehydroepiandrosterone and testosterone enhance GABAergic neurotransmission and produce inhibitory neurobehavioral and anti-inflammatory effects. Despite substantial information on the progesterone derivative (3alpha,5alpha)-3-hydroxypregnan-20-one (3alpha,5alpha-THP, allopregnanolone), the physiological significance of the other endogenous GABAergic neuroactive steroids has remained elusive. Here, we describe the validation of a method using gas chromatography-mass spectrometry to simultaneously identify serum levels of the eight 3alpha,5alpha- and 3alpha,5beta-reduced derivatives of progesterone, deoxycorticosterone, dehydroepiandrosterone and testosterone. The method shows specificity, sensitivity and enhanced throughput compared to other methods already available for neuroactive steroid quantification. Administration of pregnenolone to rats and progesterone to women produced selective effects on the 3alpha,5alpha- and 3alpha,5beta-reduced neuroactive steroids, indicating differential regulation of their biosynthetic pathways. Pregnenolone administration increased serum levels of 3alpha,5alpha-THP (+1488%, p

The neurosteroid pregnenolone and its sulfated derivative enhance learning and memory in rodents. Pregnenolone sulfate also positively modulates NMDA receptors and could thus ameliorate hypothesized NMDA receptor hypofunction in schizophrenia. Furthermore, clozapine increases pregnenolone in rodent hippocampus, possibly contributing to its superior efficacy. We therefore investigated adjunctive pregnenolone for cognitive and negative symptoms in patients with schizophrenia or schizoaffective disorder receiving stable doses of second-generation antipsychotics in a pilot randomized, placebo-controlled, double-blind trial. Following a 2-week single-blind placebo lead-in, patients were randomized to pregnenolone (fixed escalating doses to 500 mg/day) or placebo, for 8 weeks. Primary end points were changes in BACS and MCCB composite and total SANS scores. Of 21 patients randomized, 18 completed at least 4 weeks of treatment (n = 9/group). Pregnenolone was well tolerated. Patients receiving pregnenolone demonstrated significantly greater improvements in SANS scores (mean change = 10.38) compared with patients receiving placebo (mean change = 2.33), p = 0.048. Mean composite changes in BACS and MCCB scores were not significantly different in patients randomized to pregnenolone compared with placebo. However, serum pregnenolone increases predicted BACS composite scores at 8 weeks in the pregnenolone group (rs = 0.81, p = 0.022). Increases in allopregnanolone, a GABAergic pregnenolone metabolite, also predicted BACS composite scores (rs = 0.74, p = 0.046). In addition, baseline pregnenolone (rs = −0.76, p = 0.037), pregnenolone sulfate (rs = − 0.83, p = 0.015), and allopregnanolone levels (rs = −0.83, p = 0.015) were inversely correlated with improvements in MCCB composite scores, further supporting a possible role for neurosteroids in cognition. Mean BACS and MCCB composite scores were correlated (rs = 0.74, p

Ethanol and the neuroactive steroids have interactive neuropharmacological effects and chronic ethanol administration blunts the ethanol-induced increase in neuroactive steroid levels in rodent plasma and brain. Few studies have explored neuroactive steroid regulation in alcohol-dependent human subjects. In fact, the regulation of adrenal neuroactive steroids has not been well defined in healthy controls. We thus explored the regulation of two neuroactive steroids, pregnenolone sulfate (PREG-S) and deoxycorticosterone, by pharmacological challenges to the hypothalamic-pituitary-adrenal (HPA) axis in healthy controls and one-month abstinent alcohol-dependent patients with co-occurring nicotine dependence. Plasma levels of PREG-S and deoxycorticosterone were measured by radioimmunoassay in controls and alcohol-dependent patients after challenges of naloxone, ovine corticotrophin releasing hormone (oCRH), dexamethasone, cosyntropin, and cosyntropin following high-dose dexamethasone. In addition, basal diurnal measures of both hormones were obtained. PREG-S plasma levels in healthy controls were increased by cosyntropin challenge (with and without dexamethasone pretreatment) and decreased by dexamethasone challenge. However, PREG-S concentrations were not altered by naloxone or oCRH challenges, suggesting that PREG-S is not solely regulated by hypothalamic or pituitary stimulation. Deoxycorticosterone, in contrast, is regulated by HPA challenge stimulation in a manner similar to cortisol. Alcohol-dependent patients had a blunted PREG-S response to cosyntropin (with and without dexamethasone pretreatment). Furthermore, the time to peak deoxycorticosterone response following oCRH was delayed in alcohol-dependent patients compared to controls. These results indicate that plasma PREG-S and deoxycorticosterone levels are differentially regulated by HPA axis modulation in human plasma. Further, alcohol-dependent patients show a blunted PREG-S response to adrenal stimulation and a delayed deoxycorticosterone response to oCRH challenge.

Gamma-aminobutyric acid (GABA) is the most widely distributed inhibitory neurotransmitter in both vertebrate and invertebrate species. GABA receptors have been described in several invertebrate species of different phyla. Despite the considerable variability of results, it is becoming clear that in many invertebrate species GABA receptors present a pharmacology comparable to that of mammalian ones without fitting precisely into the classification developed for mammalian brain. We have demonstrated that GABA is present in Hydra tissues and that Hydra responds to GABA positive allosteric modulators. To approach the molecular characterization of GABA receptors, we examined the occurrence and distribution of GABAA receptor subunits in Hydra polyps. The presence and localization of GABAA receptor subunits in Hydra was examined by Western Blot in membrane preparations and immunohistochemical analysis of whole mount preparations. The presence of GABAA receptor subunits in Hydra membrane preparations, was tested using different antibodies against a1, a2, a3, b1, b2, b3, g1, g2, g3, d, r2 and e subunits, each probed with the corresponding peptide. In membranes incubated with the a3 antibody the unique band inhibited by the specific peptide appeared at 35 kDa. Protein bands blotted with the b1 antibody occurred at 60 kDa, while those blotted with g3 and d antibodies revealed bands at approximately 50 kDa and 52 kDa, respectively. None of the other subunit antibodies examined revealed protein bands in Hydra membranes. Immunohistochemical analysis by a3 and b1 antibodies revealed a diffuse immunoreactivity in tentacles, hypostome, and upper part of the gastric region. In the peduncle, double labelling clearly showed co-localization of a3 and b1 immunoreactivity on a circular structure above the foot; in the tentacles, occasional cell bodies also exhibited double staining in the form of patches. By contrast both g3 and d antibodies obtained a strong labelling in the lower gastric region and in the peduncle; occasional immunoreactive cells were observed in the hypostome and tentacles. Double labelling showed co-localization of a3/g3 immunoreactivity in granules or cells in the gastric region, and in the peduncle, where circular fibers above the foot were clearly double-stained. Finally, co-localization of a3 and d immunoreactivity was observed in cells of the tentacles, the gastric region and the peduncle. These data indicate that populations of specific subunits of the GABAA receptor are present in Hydra vulgaris, the most primitive metazoans to have developed a nervous system.

Three-choice discrimination procedures are used to characterize how similar the discriminative stimulus effects of two drugs are in relation to each other. This procedure has suggested similarities between ethanol and ligands that positively modulate the γ-aminobutyric acid type A (GABA A ) receptor complex. As an extension to these studies, male Long-Evans rats were trained to discriminate midazolam (3.0mg/kg, i.p.) from ethanol (1.0g/kg, i.g.) from water (2.3 ml, i.g.) in a three-lever, food reinforced task. Substitution tests were conducted following administration of GABA A -positive modulators, noncompetitive N-methyl-D-aspartate (NMDA) antagonists, 5-HT 1 B agonists and isopropanol. Among the GABA A -positive modulators, diazepam was the only drug that completely substituted for midazolam; both pentobarbital and the neurosteroid allopregnanolone showed partial midazolam substitution. The NMDA antagonist dizocilpine substituted for ethanol, while phencyclidine showed no substitution for either ethanol or midazolam. The serotonin agonists tested also showed no substitution for either ethanol or midazolam. Isopropanol was the only other drug that completely substituted for ethanol. These data extend previous findings from an ethanol-pentobarbital-water discrimination and further define training conditions that result in a conditional basis for the ethanol discrimination where only those drugs with pharmacological heterogeneous effects similar to ethanol produce a full ethanol-like effect.

γ-Hydroxybutyric acid (GHB), a drug proposed in the treatment of alcohol withdrawal syndrome, increases the cerebrocortical and plasma concentrations of the neuroactive steroids allopregnanolone and allotetrahydrodeoxycorticosterone (THDOC). In the present study, we examined the role of hypothalamic–pituitary–adrenal (HPA) axis in the effect of GHB by measuring the concentrations of these steroids in the brain and plasma of adrenalectomized–orchiectomized (Adx–Orx) rats. The acute administration of GHB (500 mg/kg, i.p.) induced in 30 min an increase in the concentrations of allopregnanolone, THDOC and their precursors pregnenolone and progesterone in different brain areas (cerebral cortex, hypothalamus and cerebellum) and plasma of sham-operated rats but had no effect on the concentrations of these compounds in Adx–Orx rats, suggesting that activation of the HPA axis mediates the effect of GHB on brain and plasma concentrations of neuroactive steroids. Moreover, we evaluated whether repeated exposure of GHB induces tolerance to its steroidogenic effects. Chronic administration of GHB (500 mg/kg, i.p., twice a day for 10 days) to intact animals failed to affect the levels of progesterone, allopregnanolone, or THDOC measured 3 or 48 h after the last drug administration, whereas a challenge injection of GHB or ethanol was still able to increase the concentrations of these steroids in brain and plasma. These results indicate that repeated exposure to GHB fails to induce tolerance or cross-tolerance to the steroidogenic action of GHB or ethanol, respectively.

Nicotine, one of the most widely used psychotropic substances, is able to induce both anxiolytic and anxiogenic effects. The effect of this drug on the brain and plasma concentrations of neuroactive steroids was examined in the rat. Anxiolytic doses of nicotine (0.03-0.3 mg/kg) had no significant effect, whereas administration of anxiogenic doses (0.5 to 2 mg/kg) produced a dose- and time-dependent increase in the cerebrocortical concentrations of pregnenolone, progesterone, and allopregnanolone, with the greatest observed effects (+180%, +223%, and +124%, respectively) apparent at the dose of 2 mg/kg. In contrast, nicotine (1-2 mg/kg) decrease by 31% and 38%, respectively, the concentration of 3alpha,21-dihydroxy-5alpha-pregnan-20-one (allotetrahydrodeoxycorticosterone, or THDOC) in the cerebral cortex. Nicotine also increased the plasma concentrations of pregnenolone and progesterone, whereas failed to affect significantly those of allopregnanolone or THDOC. Nicotine induced a dose- and time-dependent increase in the plasma concentration of corticosterone, indicating that this drug activates the hypothalamic-pituitary-adrenal (HPA) axis. These results suggest that the changes in emotional behavior elicited by nicotine, similar to those induced by stressful stimuli or other anxiogenic drugs, are associated with an increase in neuroactive steroids content of the brain.

The effects of oral contraceptives (OCs) on neurosteroid concentrations were evaluated in female rats and women. In rats, ethynylestradiol and levonorgestrel (0.030 and 0.125 mg, respectively, subcutaneously) administered daily for 6 weeks reduced the concentrations of pregnenolone (-41%) progesterone (-74%) and allopregnanolone (-79%) in the cerebral cortex; the plasma concentrations of these steroids were also reduced but by smaller extents. OC administration for 3 months also reduced the serum concentrations of pregnenolone, progesterone and allopregnanolone in women. Chronic administration of OCs in rats increased the abundance of gamma-aminobutyric acid type A (GABA(A)) receptor gamma 2L and gamma 2S subunit mRNAs and the relative protein in the cerebral cortex, while the amounts of various alpha and beta subunit mRNAs were unaffected. Ovariectomy did not modify the effect of OCs administration on the concentrations of neurosteroids in the rat cerebral cortex (but not in the plasma) as well as on the GABA(A) receptor gene expression, suggesting a direct effect of OCs in brain. Finally, rats treated with OCs exhibited an anxiety-like behavior in the elevated plus-maze test. These results indicate that long-term treatment with OCs induced a persistent reduction in the concentrations of pregnenolone, progesterone and its GABA(A) receptor-active metabolite, allopregnanolone, both in rats and women. In rats this effect was associated with a plastic adaptation of GABA(A) receptor gene expression in the rat cerebral cortex.

Neuroactive steroids are endogenous neuromodulators capable of altering neuronal activity and behavior. In rodents, systemic administration of endogenous or synthetic neuroactive steroids reduces ethanol self-administration. We hypothesized this effect arises from actions within mesolimbic brain regions that we targeted by viral gene delivery. Cytochrome P450 side chain cleavage (P450scc) converts cholesterol to pregnenolone, the rate-limiting enzymatic reaction in neurosteroidogenesis. Therefore, we constructed a recombinant adeno-associated serotype 2 viral vector (rAAV2), which drives P450scc expression and neuroactive steroid synthesis. The P450scc-expressing vector (rAAV2-P450scc) or control GFP-expressing vector (rAAV2-GFP) were injected bilaterally into the ventral tegmental area (VTA) or nucleus accumbens (NAc) of alcohol preferring (P) rats trained to self-administer ethanol. P450scc overexpression in the VTA significantly reduced ethanol self-administration by 20% over the 3 week test period. P450scc overexpression in the NAc, however, did not alter ethanol self-administration. Locomotor activity was unaltered by vector administration to either region. P450scc overexpression produced a 36% increase in (3α,5α)-3-hydroxypregnan-20-one (3α,5α-THP, allopregnanolone)-positive cells in the VTA, but did not increase 3α,5α-THP immunoreactivity in NAc. These results suggest that P450scc overexpression and the resultant increase of 3α,5α-THP-positive cells in the VTA reduces ethanol reinforcement. 3α,5α-THP is localized to neurons in the VTA, including tyrosine hydroxylase neurons, but not astrocytes. Overall, the results demonstrate that using gene delivery to modulate neuroactive steroids shows promise for examining the neuronal mechanisms of moderate ethanol drinking, which could be extended to other behavioral paradigms and neuropsychiatric pathology.

Neuroactive steroids are endogenous or synthetic steroids that rapidly alter neuronal excitability via membrane receptors, primarily γ-aminobutyric acid type A (GABAA) receptors. Neuroactive steroids regulate many physiological processes including hypothalamic-pituitary-adrenal (HPA) axis function, ovarian cycle, pregnancy, aging, and reward. Moreover, alterations in neuroactive steroid synthesis are implicated in several neuropsychiatric disorders.This review will summarize the pharmacological properties and physiological regulation of neuroactive steroids, with a particular focus on divergent neuroactive steroid responses to stress and ethanol in rats, mice, and humans.GABAergic neuroactive steroids exert a homeostatic regulation of the HPA axis in rats and humans, whereby the increase in neuroactive steroid levels following acute stress counteracts HPA axis hyperactivity and restores homeostasis. In contrast, in C57BL/6J mice, acute stress decreases neurosteroidogenesis and neuroactive steroids exert paradoxical excitatory effects upon the HPA axis. Rats, mice, and humans also differ in the neuroactive steroid responses to ethanol. Genetic variation in neurosteroidogenesis may explain the different neuroactive steroid responses to stress or ethanol.Rats and mouse strains show divergent effects of stress and ethanol on neuroactive steroids in both plasma and brain. The study of genetic variation in the various processes that determine neuroactive steroids levels as well as their effects on cell signaling may underlie these differences and may play a relevant role for the potential therapeutic benefits of neuroactive steroids.