Neurosteroid Allopregnanolone Research Articles

Age and sex effects of a validated LC-MS/MS method for the simultaneous quantification of testosterone, allopregnanolone, and its isomers in human serum

Despite the great relevance of the neurosteroid allopregnanolone and related isomers to various health conditions, quantification typically involves immunoassay, which suffers from serious issues with cross-reactivity of closely related molecules. This article describes the development and partial validation of a liquid chromatography coupled with tandem mass spectrometry assay for the simultaneous quantification of allopregnanolone, pregnanolone, isopregnanolone, epi-allopregnanolone, and testosterone in the human serum of healthy males and females aged 5–85 years. 1-amino-4-methylpiperazine (AMP) was used as a derivatisation reagent to enhance the ionisation signal. Linearity was calculated at 0.99 with a lower limit of quantification of 10.08 pg/mL for allopregnanolone, along with a linearity of 0.98 and a lower limit of quantification of 42.32 pg/mL for testosterone. Application of the method showed sex and age effects across the lifespan for both allopregnanolone and testosterone, whereas a comparative immunoassay for allopregnanolone was not able to detect differences in the same samples. Our partial validation of this method should provide a new tool for researchers to discover the role of allopregnanolone and its isomers in human health, and how it compares to testosterone and sex hormones.

Differently increased volumes of multiple brain areas in Npc1 mutant mice following various drug treatments.

Niemann-Pick disease type C1 (NPC1, MIM 257220) is a heritable lysosomal storage disease characterized by a progressive neurological degeneration that causes disability and premature death. A murine model of Npc1-/- displays a rapidly progressing form of Npc1 disease, which is characterized by weight loss, ataxia, and increased cholesterol storage. Npc1-/- mice receiving a combined therapy (COMBI) of miglustat (MIGLU), the neurosteroid allopregnanolone (ALLO) and the cyclic oligosaccharide 2-hydroxypropyl-β-cyclodextrin (HPßCD) showed prevention of Purkinje cell loss, improved motor function and reduced intracellular lipid storage. Although therapy of Npc1-/- mice with COMBI, MIGLU or HPßCD resulted in the prevention of body weight loss, reduced total brain weight was not positively influenced. In order to evaluate alterations of different brain areas caused by pharmacotherapy, fresh volumes (volumes calculated from the volumes determined from paraffin embedded brain slices) of various brain structures in sham- and drug-treated wild type and mutant mice were measured using stereological methods. In the wild type mice, the volumes of investigated brain areas were not significantly altered by either therapy. Compared with the respective wild types, fresh volumes of specific brain areas, which were significantly reduced in sham-treated Npc1-/- mice, partly increased after the pharmacotherapies in all treatment strategies; most pronounced differences were found in the CA1 area of the hippocampus and in olfactory structures. Volumes of brain areas of Npc1-/- mice were not specifically changed in terms of functionality after administering COMBI, MIGLU, or HPßCD. Measurements of fresh volumes of brain areas in Npc1-/- mice could monitor region-specific changes and response to drug treatment that correlated, in part, with behavioral improvements in this mouse model.

51. A First-In-Human Phase I Study of LYT-300, a First-In-Class Orally Bioavailable Prodrug of the Neurosteroid Allopregnanolone That is Absorbed via the Lymphatic System

51. A First-In-Human Phase I Study of LYT-300, a First-In-Class Orally Bioavailable Prodrug of the Neurosteroid Allopregnanolone That is Absorbed via the Lymphatic System

Neurosteroid Modulation of Synaptic and Extrasynaptic GABAA Receptors of the Mouse Nucleus Accumbens.

The recent approval of formulations of the endogenous neurosteroid allopregnanolone (brexanolone) and the synthetic neuroactive steroid SAGE-217 (zuranolone) to treat postpartum depression (PPD) has encouraged further research to elucidate why these potent enhancers of GABAAR function are clinically effective in this condition. Dopaminergic projections from the ventral tegmental area (VTA) to the nucleus accumbens are associated with reward/motivation and brain imaging studies report that individuals with PPD show reduced activity of this pathway in response to reward and infant engagement. However, the influence of neurosteroids on GABA-ergic transmission in the nucleus accumbens has received limited attention. Here, we investigate, in the medium spiny neurons (MSNs) of the mouse nucleus accumbens core, the effect of allopregnanolone, SAGE-217 and other endogenous and synthetic steroids of interest on fast phasic and tonic inhibition mediated by synaptic (α1/2βγ2) and extrasynaptic (α4βδ) GABAARs, respectively. We present evidence suggesting the resident tonic current results from the spontaneous opening of δ-GABAARs, where the steroid-enhanced tonic current is GABA-dependent. Furthermore, we demonstrate local neurosteroid synthesis in the accumbal slice preparation and reveal that GABA-ergic neurotransmission of MSNs is influenced by an endogenous neurosteroid tone. Given the dramatic fluctuations in allopregnanolone levels during pregnancy and postpartum, this neurosteroid-mediated local fine-tuning of GABAergic transmission in the MSNs will probably be perturbed.

Evaluation of Midazolam-Ketamine-Allopregnanolone Combination Therapy against Cholinergic-Induced Status Epilepticus in Rats.

Status epilepticus (SE) is a life-threatening development of self-sustaining seizures that becomes resistant to benzodiazepines when treatment is delayed. Benzodiazepine pharmacoresistance is thought in part to result from internalization of synaptic GABAA receptors, which are the main target of the drug. The naturally occurring neurosteroid allopregnanolone is a therapy of interest against SE for its ability to modulate all isoforms of GABAA receptors. Ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, has been partially effective in combination with benzodiazepines in mitigating SE-associated neurotoxicity. In this study, allopregnanolone as an adjunct to midazolam or midazolam-ketamine combination therapy was evaluated for efficacy against cholinergic-induced SE. Adult male rats implanted with electroencephalographic (EEG) telemetry devices were exposed to the organophosphorus chemical (OP) soman (GD) and treated with an admix of atropine sulfate and HI-6 at 1 minute after exposure followed by midazolam, midazolam-allopregnanolone, or midazolam-ketamine-allopregnanolone 40 minutes after seizure onset. Neurodegeneration, neuronal loss, and neuroinflammation were assessed 2 weeks after GD exposure. Seizure activity, EEG power integral, and epileptogenesis were also compared among groups. Overall, midazolam-ketamine-allopregnanolone combination therapy was effective in reducing cholinergic-induced toxic signs and neuropathology, particularly in the thalamus and hippocampus. Higher dosage of allopregnanolone administered in combination with midazolam and ketamine was also effective in reducing EEG power integral and epileptogenesis. The current study reports that there is a promising potential of neurosteroids in combination with benzodiazepine and ketamine treatments in a GD model of SE. SIGNIFICANCE STATEMENT: Allopregnanolone, a naturally occurring neurosteroid, reduced pathologies associated with soman (GD) exposure such as epileptogenesis, neurodegeneration, and neuroinflammation, and suppressed GD-induced toxic signs when used as an adjunct to midazolam and ketamine in a delayed treatment model of soman-induced status epilepticus (SE) in rats. However, protection was incomplete, suggesting that further studies are needed to identify optimal combinations of antiseizure medications and routes of administration for maximal efficacy against cholinergic-induced SE.

Cryo-EM structures reveal native GABAA receptor assemblies and pharmacology.

Type A γ-aminobutyric acid receptors (GABAARs) are the principal inhibitory receptors in the brain and the target of a wide range of clinical agents, including anaesthetics, sedatives, hypnotics and antidepressants1-3. However, our understanding of GABAAR pharmacology has been hindered by the vast number of pentameric assemblies that can be derived from 19 different subunits4 and the lack of structural knowledge of clinically relevant receptors. Here, we isolate native murine GABAAR assemblies containing the widely expressed α1 subunit and elucidate their structures in complex with drugs used to treat insomnia (zolpidem(ZOL) and flurazepam) and postpartum depression (the neurosteroid allopregnanolone(APG)). Using cryo-electron microscopy (cryo-EM)analysis and single-molecule photobleaching experiments, we uncover three major structural populations in the brain: the canonical α1β2γ2 receptor containing two α1 subunits, and two assemblies containing one α1 and either an α2 or α3 subunit, in which the single α1-containing receptors feature a more compact arrangement between the transmembrane and extracellular domains. Interestingly, APG is bound at the transmembrane α/β subunit interface, even when not added to the sample, revealing an important role for endogenous neurosteroids in modulating native GABAARs. Together with structurally engaged lipids, neurosteroids produce global conformational changes throughout the receptor that modify the ion channel pore and the binding sites for GABA and insomnia medications. Our data reveal the major α1-containing GABAAR assemblies, bound with endogenous neurosteroid, thus defining a structural landscape from which subtype-specific drugs can be developed.

Divergent Transcriptomic Effects of Allopregnanolone in Postpartum Depression.

Brexanolone, a formulation of the neurosteroid allopregnanolone (ALLO), is approved for treating postpartum depression (PPD) and is being investigated for therapeutic efficacy across numerous neuropsychiatric disorders. Given ALLO's beneficial effects on mood in women with PPD compared to healthy control women, we sought to characterize and compare the cellular response to ALLO in women with (n = 9) or without (n = 10, i.e., Controls) past PPD, utilizing our previously established patient-derived lymphoblastoid cell lines (LCLs). To mimic in vivo PPD ALLO-treatment, LCLs were exposed to ALLO or DMSO vehicle for 60 h and RNA-sequenced to detect differentially expressed genes (DEGs, pnominal < 0.05). Between ALLO-treated Control and PPD LCLs, 269 DEGs were identified, including Glutamate Decarboxylase 1 (GAD1), which was decreased 2-fold in PPD. Network analysis of PPD:ALLO DEGs revealed enriched terms related to synaptic activity and cholesterol biosynthesis. Within-diagnosis analyses (i.e., DMSO vs. ALLO) detected 265 ALLO-induced DEGs in Control LCLs compared to only 98 within PPD LCLs, with just 11 DEGs overlapping. Likewise, the gene ontologies underlying ALLO-induced DEGs in PPD and Control LCLs were divergent. These data suggest that ALLO may activate unique and opposing molecular pathways in women with PPD, which may be tied to its antidepressant mechanism.

Prefrontal allopregnanolone mediates the adverse effects of acute stress in a mouse model of tic pathophysiology.

Ample evidence suggests that acute stress can worsen symptom severity in Tourette syndrome (TS); however, the neurobiological underpinnings of this phenomenon remain poorly understood. We previously showed that acute stress exacerbates tic-like and other TS-associated responses via the neurosteroid allopregnanolone (AP) in an animal model of repetitive behavioral pathology. To verify the relevance of this mechanism to tic pathophysiology, here we tested the effects of AP in a mouse model recapitulating the partial depletion of dorsolateral cholinergic interneurons (CINs) seen in post-mortem studies of TS. Mice underwent targeted depletion of striatal CINs during adolescence and were tested in young adulthood. Compared with controls, partially CIN-depleted male mice exhibited several TS-relevant abnormalities, including deficient prepulse inhibition (PPI) and increased grooming stereotypies after a 30-min session of spatial confinement - a mild acute stressor that increases AP levels in the prefrontal cortex (PFC). These effects were not seen in females. Systemic and intra-PFC AP administration dose-dependently worsened grooming stereotypies and PPI deficits in partially CIN-depleted males. Conversely, both AP synthesis inhibition and pharmacological antagonism reduced the effects of stress. These results further suggest that AP in the PFC mediates the adverse effects of stress on the severity of tics and other TS-related manifestations. Future studies will be necessary to confirm these mechanisms in patients and define the circuitry responsible for the effects of AP on tics.

Prefrontal allopregnanolone synergizes with D1 receptor activation to disrupt sensorimotor gating in male Sprague-Dawley rats.

The prepulse inhibition (PPI) of the startle reflex is the best-established index of sensorimotor gating. We documented that the neurosteroid allopregnanolone (AP) is necessary to reduce PPI in response to D1 dopamine receptor agonists. Since Sprague-Dawley (SD) rats are poorly sensitive to the PPI-disrupting effects of these drugs, we hypothesized that AP might increase this susceptibility. We tested whether AP is sufficient to increase the vulnerability of SD rats to PPI deficits in response to the D1 receptor full agonist SKF82958. SD rats were tested for PPI after treatment with SKF82958 (0.05-0.3 mg/kg, SC) in combination with either intraperitoneal (1-10 mg/kg) or intracerebral (0.5 μg/μl/side) AP administration into the medial prefrontal cortex (mPFC) or nucleus accumbens shell. To rule out potential confounds, we measured whether SKF82958 affected the endogenous mPFC levels of AP. SD rats exhibited marked PPI deficits in response to the combination of systemic and intra-mPFC AP with SKF82958 but not with the D2 receptor agonist quinpirole (0.3-0.6 mg/kg, SC). SKF82958 did not elevate mPFC levels of AP but enhanced the content of its precursor progesterone. The PPI deficits caused by SKF82958 in combination with AP were opposed by the AP antagonist isoallopregnanolone (10 mg/kg, IP) and the glutamate NMDA receptor positive modulator CIQ (5 mg/kg, IP). These results suggest that AP enables the detrimental effects of D1 receptor activation on sensorimotor gating. AP antagonism or glutamatergic modulation counters these effects and may have therapeutic potential for neuropsychiatric disorders characterized by gating deficits.

Tolerability and pharmacokinetics of intravenous allopregnanolone with and without midazolam pretreatment in two healthy dogs.

The neurosteroid allopregnanolone (ALLO) is under investigation as a treatment for benzodiazepine-refractory status epilepticus (SE). Here we assess the cardiopulmonary safety of intravenous ALLO by itself and after a clinically recommended dose of midazolam (MDZ) in two healthy adult beagles. Each dog received ALLO (1 mg/kg, IV), and after a washout period of 2 weeks, the dog was dosed with MDZ (0.2 mg/kg, IV) followed 10 min later by ALLO. Behavioral state, vital signs, arterial blood gasses, blood chemistries, and plasma ALLO concentrations were monitored for up to 6 h after dosing. The dogs appeared sleepy but were fully responsive after both treatments. No depression of mean arterial pressure or respiratory rate was noted. Blood gas measurements failed to show evidence of drug-induced acute respiratory acidosis. Estimated maximum plasma ALLO concentrations were in the range of 1500 to 3000 ng/mL. The results indicate that intravenous ALLO can be used safely to treat benzodiazepine-refractory SE, even when administered shortly after a benzodiazepine.

Emotion-induced brain activation across the menstrual cycle in individuals with premenstrual dysphoric disorder and associations to serum levels of progesterone-derived neurosteroids

Premenstrual dysphoric disorder (PMDD) is a debilitating disorder characterized by severe mood symptoms in the luteal phase of the menstrual cycle. PMDD symptoms are hypothesized to be linked to an altered sensitivity to normal luteal phase levels of allopregnanolone (ALLO), a GABAA-modulating progesterone metabolite. Moreover, the endogenous 3β-epimer of ALLO, isoallopregnanolone (ISO), has been shown to alleviate PMDD symptoms through its selective and dose-dependent antagonism of the ALLO effect. There is preliminary evidence showing altered recruitment of brain regions during emotion processing in PMDD, but whether this is associated to serum levels of ALLO, ISO or their relative concentration is unknown. In the present study, subjects with PMDD and asymptomatic controls underwent functional magnetic resonance imaging (fMRI) in the mid-follicular and the late-luteal phase of the menstrual cycle. Brain responses to emotional stimuli were investigated and related to serum levels of ovarian steroids, the neurosteroids ALLO, ISO, and their ratio ISO/ALLO. Participants with PMDD exhibited greater activity in brain regions which are part of emotion-processing networks during the late-luteal phase of the menstrual cycle. Furthermore, activity in key regions of emotion processing networks - the parahippocampal gyrus and amygdala - was differentially associated to the ratio of ISO/ALLO levels in PMDD subjects and controls. Specifically, a positive relationship between ISO/ALLO levels and brain activity was found in PMDD subjects, while the opposite was observed in controls. In conclusion, individuals with PMDD show altered emotion-induced brain responses in the late-luteal phase of the menstrual cycle which may be related to an abnormal response to physiological levels of GABAA-active neurosteroids.

Antiepileptogenic effects of trilostane in the kainic acid model of temporal lobe epilepsy.

Epileptogenesis after status epilepticus (SE) has a faster onset in rats treated to reduce brain levels of the anticonvulsant neurosteroid allopregnanolone with the 5α-reductase inhibitor finasteride; however, it still has to be evaluated whether treatments aimed at increasing allopregnanolone levels could result in the opposite effect of delaying epileptogenesis. This possibility could be tested using the peripherally active inhibitor of 3β-hydroxysteroid dehydrogenase/Δ5-4 isomerase trilostane, which has been shown repeatedly to increase allopregnanolone levels in the brain. Trilostane (50 mg/kg) was administered subcutaneously once daily for up to six consecutive days, starting 10min after intraperitoneal administration of kainic acid (15 mg/kg). Seizures were evaluated by video-electrocorticographic recordings for 70 days maximum, and endogenous neurosteroid levels were assessed by liquid chromatography-electrospray tandem mass spectrometry. Immunohistochemical staining was performed to evaluate the presence of brain lesions. Trilostane did not alter the latency of kainic acid-induced SE onset or its overall duration. When compared to the vehicle-treated group, rats receiving six daily trilostane injections presented a remarkable delay of the first spontaneous electrocorticographic seizure and subsequent tonic-clonic spontaneous recurrent seizures (SRSs). Conversely, rats treated with only the first trilostane injection during SE did not differ from vehicle-treated rats in developing the SRSs. Notably, trilostane did not modify neuronal cell densities or the overall damage in the hippocampus. In comparison to the vehicle group, repeated administration of trilostane significantly decreased the activated microglia morphology in the subiculum. As expected, allopregnanolone and other neurosteroid levels were remarkably increased in the hippocampus and neocortex of rats treated for 6 days with trilostane, but pregnanolone was barely detectable. Neurosteroids returned to basal levels after a week of trilostane washout. Overall, these results suggest that trilostane led to a remarkable increase in allopregnanolone brain levels, which was associated with protracted effects on epileptogenesis.

The Mechanism of Enantioselective Neurosteroid Actions on GABAA Receptors.

The neurosteroid allopregnanolone (ALLO) and pregnanolone (PREG), are equally effective positive allosteric modulators (PAMs) of GABAA receptors. Interestingly, the PAM effects of ALLO are strongly enantioselective, whereas those of PREG are not. This study was aimed at determining the basis for this difference in enantioselectivity. The oocyte electrophysiology studies showed that ent-ALLO potentiates GABA-elicited currents in α1β3 GABAA receptors with lower potency and efficacy than ALLO, PREG or ent-PREG. The small PAM effect of ent-ALLO was prevented by the α1(Q242L) mutation in the intersubunit neurosteroid binding site between the β3 and α1 subunits. Consistent with this result, neurosteroid analogue photolabeling with mass spectrometric readout, showed that ent-ALLO binds weakly to the β3-α1 intersubunit binding site in comparison to ALLO, PREG and ent-PREG. Rigid body docking predicted that ent-ALLO binds in the intersubunit site with a preferred orientation 180° different than ALLO, PREG or ent-PREG, potentially explaining its weak binding and effect. Photolabeling studies did not identify differences between ALLO and ent-ALLO binding to the α1 or β3 intrasubunit binding sites that also mediate neurosteroid modulation of GABAA receptors. The results demonstrate that differential binding of ent-ALLO and ent-PREG to the β3-α1 intersubunit site accounts for the difference in enantioselectivity between ALLO and PREG.

Behavioral Despair Is Blocked by the Flavonoid Chrysin (5,7-Dihydroxyflavone) in a Rat Model of Surgical Menopause.

Women have a high susceptibility to the negative effects of stress. Hormonal changes experienced throughout their reproductive life partially contribute to a higher incidence of anxiety and depression symptoms, particularly, during natural or surgical menopause. In preclinical research, the flavonoid chrysin (5,7-dihydroxyflavone) exerts anxiolytic- and anti-despair-like effects; however, it is unknown whether chrysin exerts a protective effect against the behavioral changes produced by acute stress on locomotor activity and behavioral despair in rats at 12-weeks post-ovariectomy. Ovariectomized female Wistar rats were assigned to eight groups: vehicle group (10% DMSO), three groups with chrysin and three groups with the same dose of allopregnanolone (0.5, 1, and 2 mg/kg), and one group with diazepam (2 mg/kg). The treatments were administered for seven consecutive days and the effects were evaluated in the locomotor activity and swimming tests. Chrysin (2 mg/kg) increased the latency to first immobility and decreased the total immobility time in the swimming test as the reference drugs allopregnanolone and diazepam (2 mg/kg); while locomotor activity prevented the behavioral changes produced by swimming. In conclusion, chrysin exerts a protective effect against the behavioral changes induced by acute stress, similarly to the neurosteroid allopregnanolone and the benzodiazepine diazepam in rats subjected to a surgical menopause model.

Clinical Implications of the Neurosteroid Allopregnanolone in Reproductive Depression.

Certain women develop depression with fluctuations in hormone levels whereas other women do not; this hormonally driven depression has been termed reproductive depression. The pathophysiology of reproductive depression differs from that of major depressive disorder, and this distinction has important clinical-including treatment-implications. Recent advances have revealed that the neurosteroid, allopregnanolone, plays a central role in reproductive depression. Appreciation of allopregnanolone's role in reproductive depression aids in selecting targeted treatments and in predicting symptom worsening during subsequent reproductive stages, and it can be used to reduce risk of relapse. This knowledge is also guiding the development of new pharmacologic treatments for reproductive depression.

Alfaxalone anaesthesia increases brain derived neurotrophic factor levels and preserves postoperative cognition by activating pregnane-X receptors: an in vitro study and a double blind randomised controlled trial

BackgroundAlfaxalone is a fast acting intravenous anaesthetic with high therapeutic index. It is an analogue of the naturally-occurring neurosteroid allopregnanolone responsible for maintenance of cognition and neuroprotection by activation of brain pregnane X receptors and consequent increased production of mature brain-derived neurotrophic factor (m-BDNF). Two studies are reported here: an in vitro study investigated whether alfaxalone activates human pregnane X receptors (h-PXR) as effectively as allopregnanolone; and a clinical study that measured postoperative changes in serum m-BDNF and cognition in patients after alfaxalone anaesthesia compared with propofol and sevoflurane.MethodsIn vitro Activation of h-PXR by allopregnanolone and alfaxalone solutions (206 - 50,000 nM) was measured using human embryonic kidney cells expressing h-PXR hybridised and linked to the firefly luciferase gene. Light emission by luciferase stimulated by each ligand binding with h-PXR was measured.Clinical A double blind prospective randomised study of patients undergoing hip arthroplasty anaesthetised with alfaxalone TIVA (n = 8) or propofol TIVA (n = 3) or propofol plus sevoflurane inhalational anaesthesia (n = 4). The doses of anaesthetics were titrated to the same depth of anaesthesia (BIS 40-60). Subjects’ cognitive performance was assessed using the Grooved Pegboard Test, Digit Symbol Substitution Test (DSST) and Mini Mental State examination (MMSE) for 7 days postoperatively. Serum m-BDNF concentrations were measured for 7 postoperative days.ResultsIn vitro Allopregnanolone and alfaxalone both activated h-PXR, alfaxalone being more efficacious than allopregnanolone: 50,000 nM, p = 0.0019; 16,700 nM, p = 0.0472; 5600 nM, p = 0.0031.Clinical Alfaxalone treated subjects scored better than propofol and sevoflurane anaesthetised patients in the cognition tests: (MMSE p = 0.0251; Grooved Pegboard test dominant hand pre v post anaesthesia scores p = 0.8438 for alfaxalone and p = 0.0156 for propofol and propofol/sevoflurane combined). The higher cognition scores were accompanied by higher serum m-BDNF levels in the alfaxalone anaesthetised patients (p < 0.0001).ConclusionsThese results suggest that sedation and anaesthesia induced by the synthetic neuroactive steroid alfaxalone may be accompanied by effects normally caused by physiological actions of allopregnanolone at PXR, namely, increased secretion of m-BDNF and consequent neuroprotection and preservation of cognition.Trial registrationThe clinical trial was registered on 17/01/2018 with the Australian New Zealand Clinical Trials Registry: registration number ACTRN12618000064202 [Universal Trial Number U1111-1198-0412].

Ganaxolone for management of seizures associated with CDKL5 deficiency disorder

STRUCTURE: Ganaxolone is a 3β-methylated synthetic analog of the natural neurosteroid allopregnanolone, a metabolite of progesterone. It is also called 3α-hydroxy-3β-methyl-5α-pregnan-20-one and has a molecular formula C22H36O2, with a molecular weight of 332.5 g/mol. The 3β-methyl substitution prevents rapid metabolism and thus increases its stability (terminal half-life: 34 h). Ganaxolone is a white crystalline powder that is insoluble in water but soluble in organic solvents.

Effect of progesterone administration in male and female smokers on nicotine withdrawal and neural response to smoking cues: role of progesterone conversion to allopregnanolone

BackgroundProgesterone administration has therapeutic effects in tobacco use disorder (TUD), with females benefiting more than males. Conversion of progesterone to the neurosteroid allopregnanolone is hypothesized to partly underlie the therapeutic effects of progesterone; however, this has not been investigated clinically.MethodsSmokers (n = 18 males, n = 21 females) participated in a randomized, double-blind, placebo-controlled crossover study of 200 mg progesterone daily across 4 days of abstinence. The ratio of allopregnanolone:progesterone was analyzed in relationship to nicotine withdrawal, smoking urges, mood states, subjective nicotine effects, and neural response to smoking cues.ResultsAllopregnanolone:progesterone ratio interacted with sex to predict withdrawal symptoms (p = 0.047), such that females with higher allopregnanolone:progesterone ratios reported lower withdrawal severity (b = − 0.98 [− 1.95, − 0.01]; p = 0.048). In addition, allopregnanolone:progesterone ratio interacted with sex to predict confusion (p = 0.014) and fatigue (p = 0.034), such that females with higher allopregnanolone:progesterone ratios reported less confusion (b = − 0.45 [− 0.78, − 0.12]; p = 0.008) and marginally lower fatigue (b = − 0.50 [− 1.03, 0.02]; p = 0.062. Irrespective of sex, higher ratios of allopregnanolone:progesterone were associated with stronger “good effects” of nicotine (b = 8.39 [2.58, 14.20]); p = 0.005) and weaker “bad effects” of nicotine (b = − 7.13 [− 13.53, − 0.73]; p = 0.029).ConclusionsConversion of progesterone to allopregnanolone correlated with smoking-related outcomes in both sex-dependent and sex-independent ways. Sex-dependent effects suggest that conversion of progesterone to allopregnanolone may contribute to greater therapeutic benefits in females but not males with TUD.Trial registration Clinicaltrials.gov registration, retrospectively registered: NCT01954966; https://clinicaltrials.gov/ct2/show/NCT01954966\\

Acute stress impairs sensorimotor gating via the neurosteroid allopregnanolone in the prefrontal cortex

Ample evidence indicates that environmental stress impairs information processing, yet the underlying mechanisms remain partially elusive. We showed that, in several rodent models of psychopathology, the neurosteroid allopregnanolone (AP) reduces the prepulse inhibition (PPI) of the startle, a well-validated index of sensorimotor gating. Since this GABAA receptor activator is synthesized in response to acute stress, we hypothesized its participation in stress-induced PPI deficits. Systemic AP administration reduced PPI in C57BL/6J mice and Long-Evans, but not Sprague-Dawley rats. These effects were reversed by isoallopregnanolone (isoAP), an endogenous AP antagonist, and the GABAA receptor antagonist bicuculline and mimicked by AP infusions in the medial prefrontal cortex (mPFC). Building on these findings, we tested AP's implication in the PPI deficits produced by several complementary regimens of acute and short-term stress (footshock, restraint, predator exposure, and sleep deprivation). PPI was reduced by acute footshock, sleep deprivation as well as the combination of restraint and predator exposure in a time- and intensity-dependent fashion. Acute stress increased AP concentrations in the mPFC, and its detrimental effects on PPI were countered by systemic and intra-mPFC administration of isoAP. These results collectively indicate that acute stress impairs PPI by increasing AP content in the mPFC. The confirmation of these mechanisms across distinct animal models and several acute stressors strongly supports the translational value of these findings and warrants future research on the role of AP in information processing.

Positive allosteric modulators of GABAA receptor restore chloride current from blockade by competitive antagonists in a ligand-dependent manner

γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter. GABA receptor type A (GABAAR) possesses binding sites for a large group of pharmacological agents which are supposed to interact allosterically with each other. The aim of this work was to study the interaction between the positive allosteric modulators (PAMs) and the competitive antagonists of GABAARs. The GABA-induced chloride current (IGABA) was measured in isolated Purkinje cells of rat cerebellum using the patch-clamp technique. PAMs, neurosteroid allopregnanolone (Allo) and zolpidem (Zolp), a drug that positively modulates the GABAAR through interaction with the benzodiazepine (BDZ) site, doubled the IGABA amplitude in the control solution. Competitive antagonist of GABAARs, bicuculline (Bic, 5 µM) blocked the IGABA by 90%. The addition of 1 μM Allo or 0.5 µM Zolp to the Bic solution caused an unblocking effect, so that the IGABA amplitude increased 10 and 4 times from control value, correspondingly. This unblocking effect developed slowly, as evidenced by a threefold increase in the current rise time. Competitive antagonist of GABAARs, gabazine (GBZ, 0.5 µM) blocked the IGABA by 87%. The addition of 1 μM Allo to the GBZ solution caused an unblocking effect, so that the IGABA amplitude increased 7-fold. However, the addition of 0.5 µM Zolp to the GBZ solution did not cause an unblocking effect. So, Allo appeared to have a stronger unblocking potential than Zolp, and Bic binding site showed a higher sensitivity to the action of unblocking PAMs than GBZ binding site. The results indicate for the first time the existence of an allosteric relationship between the sites binding PAMs and the competitive antagonists of GABAAR.