Sex-specific hypnotic effects of the neuroactive steroid (3ß,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile are mediated by peripheral metabolism into an active hypnotic steroid.

BACKGROUND: The novel synthetic neuroactive steroid (3ß,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile (3ß-OH) blocks T-type calcium channels but does not directly modulate neuronal γ-aminobutyric acid type A (GABAA) currents like other anaesthetic neurosteroids. As 3ß-OH has sex-specific hypnotic effects in adult rats, we studied the mechanism contributing to sex differences in its effects. METHODS: We used a combination of behavioural loss of righting reflex, neuroendocrine, pharmacokinetic, in vitro patch-clamp electrophysiology, and in vivo electrophysiological approaches in wild-type mice and in genetic knockouts of the CaV3.1 T-type calcium channel isoform to study the mechanisms by which 3ß-OH and its metabolite produces sex-specific hypnotic effects. RESULTS: Adult male mice were less sensitive to the hypnotic effects of 3ß-OH compared with female mice, and these differences appeared during development. Adult males had higher 3ß-OH brain concentrations despite being less sensitive to its hypnotic effects. Females metabolised 3ß-OH into the active GABAA receptor positive allosteric modulator (3α,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile (3α-OH) to a greater extent than males. The 3α-OH metabolite has T-channel blocking properties with sex-specific hypnotic and pharmacokinetic effects. Sex-dependent suppression of the cortical electroencephalogram is more pronounced with 3α-OH compared with 3ß-OH. CONCLUSIONS: The sex-specific differences in the hypnotic effect of 3ß-OH in mice are attributable to differences in its peripheral metabolism into the more potent hypnotic metabolite 3α-OH.

Systemic inflammation exacerbates developmental neurotoxicity induced by sevoflurane in neonatal rats.

BACKGROUND: General anaesthesia in the neonatal period has detrimental effects on the developing mammalian brain. The impact of underlying inflammation on anaesthesia-induced developmental neurotoxicity remains largely unknown. METHODS: Postnatal day 7 (PND7) rats were randomly assigned to receive sevoflurane (3 vol% for 3 h) or carrier gas 12 h after bacterial lipopolysaccharide (LPS; 1 µg g-1) or vehicle injection. Pharmacological inhibition of caspase-1 by Vx-765 (two doses of 50 µg g-1 body weight) was used to investigate mechanistic pathways of neuronal injury. Histomorphological injury and molecular changes were quantified 2 h after the end of anaesthesia. Long-term functional deficits were tested at 5-8 weeks of age using a battery of behavioural tests in the memory and anxiety domains. RESULTS: Sevoflurane or LPS treatment increased activated caspase-3 and caspase-9 expression in the hippocampal subiculum and CA1, which was greater when sevoflurane was administered in the setting of LPS-induced inflammation. Neuronal injury induced by LPS+sevoflurane treatment resulted in sex-specific behavioural outcomes when rats were tested at 5-8 weeks of age, including learning and memory deficits in males and heightened anxiety-related behaviour in females. Hippocampal caspase-1 and NLRP1 (NLR family pyrin domain containing 1), but not NLRP3, were upregulated by LPS or LPS+sevoflurane treatment, along with related proinflammatory cytokines, interleukin (IL)-1ß, and IL-18. Pretreatment with Vx-765, a selective caspase-1 inhibitor, led to reduced IL-1ß in LPS and LPS+sevoflurane groups. Caspase-1 inhibition by Vx-765 significantly decreased activated caspase-3 and caspase-9 immunoreactivity in the subiculum. CONCLUSIONS: Systemic inflammation promotes developmental neurotoxicity by worsening anaesthesia-induced neuronal damage with sex-specific behavioural outcomes. This highlights the importance of studying anaesthesia-induced neurotoxicity in more clinically relevant settings.

RNA-sequencing and behavioral testing reveals inherited physical inactivity co-selects for anxiogenic behavior without altering depressive-like behavior in Wistar rats.

Physical inactivity is positively associated with anxiety and depression. Considering physical inactivity, anxiety, and depression each have a genetic basis for inheritance, our lab used artificial selectively bred low-voluntary running (LVR) and wild type (WT) female Wistar rats to test if physical inactivity genes selected over multiple generations would lead to an anxiety or depressive-like phenotype. We performed next generation RNA sequencing and immunoblotting on the dentate gyrus to reveal key biological functions from heritable physical inactivity. LVR rats did not display depressive-like behavior. However, LVR rats did display anxiogenic behavior with gene networks associated with reduced neuronal development, proliferation, and function compared to WT counterparts. Additionally, immunoblotting revealed LVR deficits in neuronal development and function. To our knowledge, this is the first study to show that by selectively breeding for physical inactivity genes, anxiety-like genes were co-selected. The study also reveals molecular insights to the genetic influences that physical inactivity has on anxiety-like behavior.

Sex differences in neurodevelopmental abnormalities caused by early-life anaesthesia exposure: a narrative review.

Exposure to anaesthetic drugs during the fetal or neonatal period induces widespread neuronal apoptosis in the brains of rodents and non-human primates. Hundreds of published preclinical studies and nearly 20 clinical studies have documented cognitive and behavioural deficits many months or years later, raising the spectre that early life anaesthesia exposure is a long-term, perhaps permanent, insult that might affect the quality of life of millions of humans. Although the phenomenon of anaesthesia-induced developmental neurotoxicity is well characterised, there are important and lingering questions pertaining to sex differences and neurodevelopmental sequelae that might occur differentially in females and males. We review the relevant literature on sex differences in the field of anaesthesia-induced developmental neurotoxicity, and present an emerging pattern of potential sex-dependent neurodevelopmental abnormalities in rodent models of human infant anaesthesia exposure.

Sevoflurane Exposure Results in Sex-Specific Transgenerational Upregulation of Target IEGs in the Subiculum.

Large body of animal work and emerging clinical findings have suggested that early exposure to anesthetics may result in increased risk of learning disabilities and behavioral impairments. Recent studies have begun to investigate anesthesia-induced epigenetic modifications to elucidate their role in behavioral and neurodevelopmental abnormalities. Here we examine sevoflurane-induced transgenerational modifications of subicular neuronal DNA methylation and expression of immediate early genes (IEGs), arc and junB, crucial to synaptic plasticity and normal neuronal development. We show that 6 h sevoflurane exposure in postnatal day 7 rat pups resulted in decreased neuronal 5-methycytosine, indicating reduced DNA methylation. This effect is transgenerationally expressed in offspring born to exposed mothers which is of importance considering that decreased DNA methylation in the brain has been linked with functional decline in learning and memory. We further show that sevoflurane exposure induces upregulation of Arc and JunB mRNA expression, 42.7% and 35.2%, respectively. Transgenerational changes in Arc and JunB mRNA were sexually dimorphic only occurring in males born to exposed females, expressed as upregulation of Arc and JunB mRNA, 71.6% and 74.0%, respectively. We further investigated correlation between altered arc promoter methylation and observed upregulation of Arc mRNA and observed that sevoflurane reduced methylation in the 5-upstream promoter region of females exposed to sevoflurane. Transgenerational hypomethylation and modifications to IEGs crucial to synaptic plasticity, observed following neonatal sevoflurane exposure could contribute to morphological and cognitive deficits known to occur with neonatal sevoflurane exposure.

Cognition in female rats after blocking conversion of androgens to estrogens.

Women and non-human females have surprisingly high levels of circulating testosterone, yet the effects of androgens on non-reproductive behaviors, including cognition, of females are not well characterized. The current project used an aromatase inhibitor, letrozole, to block conversion of androgens to estrogens. Adult female rats were ovariectomized and administered either vehicle only, testosterone propionate only (400µg/kg, TP only), letrozole only (1mg/kg, Letro only), or the combination of letrozole and testosterone (TP+Letro) over 4weeks. A gonadally intact group was used for comparisons. During the last 3weeks, the animals were tested for working memory in both a spatial task (radial arm maze) and a non-spatial task (object recognition). At sacrifice, uterine weights and serum testosterone and estradiol were determined. Behavioral results were the intact animals showed better working memories on the object recognition task, but that there were no differences among the ovariectomized groups. In the radial arm maze task, groups with best to worst performance were TP only>Intact=TP+Letro>vehicle=Letro only. Highest to lowest serum titers, for testosterone, were TP+Letro>TP only>Intact=Letro only>vehicle and, for estradiol, Intact>TP only>Vehicle>Letro only=TP+Letro. Our interpretation is that testosterone enhanced spatial performance when bioavailability of both TP and E2 are high, and high testosterone can rescue spatial memory when E2 bioavailability is low.

Isoflurane exposure leads to apoptosis of neurons and oligodendrocytes in 20- and 40-day old rhesus macaques.

Previously we reported that a 5-hour exposure of 6-day-old (P6) rhesus macaques to isoflurane triggers robust neuron and oligodendrocyte apoptosis. In an attempt to further describe the window of vulnerability to anesthetic neurotoxicity, we exposed P20 and P40 rhesus macaques to 5h of isoflurane anesthesia or no exposure (control animals). Brains were collected 3h later and examined immunohistochemically to analyze neuronal and glial apoptosis. Brains exposed to isoflurane displayed neuron and oligodendrocyte apoptosis distributed throughout cortex and white matter, respectively. When combining the two age groups (P20+P40), the animals exposed to isoflurane had 3.6 times as many apoptotic cells as the control animals. In the isoflurane group, approximately 66% of the apoptotic cells were oligodendrocytes and 34% were neurons. In comparison, in our previous studies on P6 rhesus macaques, approximately 52% of the dying cells were glia and 48% were neurons. In conclusion, the present data suggest that the window of vulnerability for neurons is beginning to close in the P20 and P40 rhesus macaques, but continuing for oligodendrocytes.

Zika Virus Infection during Pregnancy in Mice Causes Placental Damage and Fetal Demise.

Zika virus (ZIKV) infection in pregnant women causes intrauterine growth restriction, spontaneous abortion, and microcephaly. Here, we describe two mouse models of placental and fetal disease associated with in utero transmission of ZIKV. Female mice lacking type I interferon signaling (Ifnar1(-/-)) crossed to wild-type (WT) males produced heterozygous fetuses resembling the immune status of human fetuses. Maternal inoculation at embryonic day 6.5 (E6.5) or E7.5 resulted in fetal demise that was associated with ZIKV infection of the placenta and fetal brain. We identified ZIKV within trophoblasts of the maternal and fetal placenta, consistent with a trans-placental infection route. Antibody blockade of Ifnar1 signaling in WT pregnant mice enhanced ZIKV trans-placental infection although it did not result in fetal death. These models will facilitate the study of ZIKV pathogenesis, in utero transmission, and testing of therapies and vaccines to prevent congenital malformations.