Targeted blockade of aberrant sodium current in a stem cell-derived neuron model of SCN3A encephalopathy.

Missense variants in SCN3A encoding the voltage-gated sodium (Na+) channel α subunit Nav1.3 are associated with SCN3A-related neurodevelopmental disorder (SCN3A-NDD), a spectrum of disease that includes epilepsy and malformation of cortical development. How genetic variation in SCN3A leads to pathology remains unclear, as prior electrophysiological work on disease-associated variants has been performed exclusively in heterologous cell systems. To further investigate the mechanisms of SCN3A-NDD pathogenesis, we used CRISPR/Cas9 gene editing to modify a control human induced pluripotent stem cell (iPSC) line to express the recurrent de novo missense variant SCN3A c.2624T>C (p.Ile875Thr). With the established Ngn2 rapid induction protocol, we generated glutamatergic forebrain-like neurons (iNeurons), which we showed to express SCN3A mRNA and Nav1.3-mediated Na+ currents. We performed detailed whole-cell patch clamp recordings to determine the effect of the SCN3A-p.Ile875Thr variant on endogenous Na+ currents in, and intrinsic excitability of, human neurons. Compared to control iNeurons, variant-expressing iNeurons exhibit markedly increased slowly-inactivating/persistent Na+ current, abnormal firing patterns with paroxysmal bursting and plateau-like potentials with action potential failure, and a hyperpolarized voltage threshold for action potential generation. We then validated these findings using a separate iPSC line generated from a patient harbouring the SCN3A-p.Ile875Thr variant compared to a corresponding CRISPR-corrected isogenic control line. Finally, we found that application of the Nav1.3-selective blocker ICA-121431 normalizes action potential threshold and aberrant firing patterns in SCN3A-p.Ile1875Thr iNeurons; in contrast, consistent with action as a Na+ channel blocker, ICA-121431 decreases excitability of control iNeurons. Our findings demonstrate that iNeurons can model the effects of genetic variation in SCN3A yet reveal a complex relationship between gain-of-function at the level of the ion channel versus impact on neuronal excitability. Given the transient expression of SCN3A in the developing human nervous system, selective blockade or suppression of Nav1.3-containing Na+ channels could represent a therapeutic approach towards SCN3A-NDD.

Corticotropin releasing factor in the nucleus basalis of Meynert impairs attentional performance and reduces levels of glutamate and taurine in male and female rats.

Psychiatric disorders that are characterized by impairments in sustained attention, including attention deficit hyperactivity disorder (ADHD), post-traumatic stress disorder (PTSD), and major depression are also sensitive to exacerbation by stress. Sustained attention relies on cholinergic and non-cholinergic projections from the nucleus basalis of Meynert (NBM) in the basal forebrain to the medial prefrontal cortex (mPFC). We have previously shown that central administration of the stress neuropeptide corticotropin releasing factor (CRF) impairs performance on the sustained attention task (SAT) in adult male and female rats. The present study investigated whether this effect was mediated by CRF's action in the NBM. Rats were administered CRF in the NBM and subsequent SAT performance was measured. A high dose of CRF (100 ng) significantly impaired performance on non-signaled events across sex. Because performance on non-signaled events is believed to depend on non-cholinergic (i.e., GABA and glutamate) signaling, high performance liquid chromatography was used to quantify amino acid levels in the NBM and mPFC. We found females have higher levels of glutamate, glutamine, GABA glycine, and alanine in the NBM than males. Importantly, CRF in the NBM led to a local decrease of taurine and several amino acids involved in glutamate synthesis in males and females, changes which may mediate the CRF-induced SAT performance deficit. Together these studies suggest that CRF regulation of amino acids in the NMB contributes to stress-induced attention deficits.

The effects of early life adversity on growth, maturation, and steroid hormones in male and female rats.

Early life adversity is a risk factor for psychiatric disorders, yet the mechanisms by which adversity increases this risk are still being delineated. Here, we used a limited bedding and nesting (LBN) manipulation in rats that models a low resource environment to examine effects on growth, developmental milestones, and endocrine endpoints. In LBN, dams and pups, from pups' postnatal days 2-9, are exposed to an environment where dams lack proper materials to build a nest. This manipulation is compared to control housing conditions, where rat dams have access to ample nesting materials and enrichment throughout pups' development. We found that the LBN condition altered maternal care, increasing pup-directed behaviors while reducing self-care. This, perhaps compensatory, increase in nursing and attention to pups did not mitigate against changes in metabolism, as LBN reduced weight gain in both sexes and this effect persisted into adulthood. Although adult stress hormone levels in both sexes and vaginal opening and estrous cycle length in females were not disrupted, there was other evidence of endocrine dysregulation. Compared to controls, LBN rats of both sexes had shortened anogenital distances, indicating reduced androgen exposure. LBN males also had higher plasma estradiol levels in adulthood. This combination of results suggests that LBN causes a demasculinizing effect in males that could contribute to lasting changes in the brain and behavior. Importantly, alterations in metabolic and endocrine systems due to early life adversity could be one mechanism by which stress early in life increases risk for later disease.

Sex differences in corticotropin releasing factor regulation of medial septum-mediated memory formation.

Stress can disrupt memory and contribute to cognitive impairments in psychiatric disorders, including schizophrenia and attention deficit hyperactivity disorder. These diseases are more common in men than in women, with men showing greater cognitive impairments. Mnemonic deficits induced by stress are mediated, in part, by corticotropin releasing factor (CRF). However, where CRF is acting to regulate memory, and sex differences therein, is understudied. Here we assessed whether CRF in the medial septum (MS), which projects to the hippocampus, affected memory formation in male and female rats. CRF in the MS did not alter hippocampal-independent object recognition memory, but impaired hippocampal-dependent object location memory in both sexes. Interestingly, males were more sensitive than females to the disruptive effect of a low dose of CRF in the MS. Female resistance was not due to circulating ovarian hormones. However, compared to males, females had higher MS expression of CRF binding protein, which reduces CRF bioavailability and thus may mitigate the effect of the low dose of CRF in females. In contrast, there was no sex difference in CRF1 expression in the MS. Consistent with this finding, CRF1 antagonism blocked the memory impairment caused by the high dose of CRF in the MS in both sexes. Collectively, these results suggest that males are more vulnerable than females to the memory impairments caused by CRF in the MS. In both sexes, CRF1 antagonists prevented MS-mediated memory deficits caused by high levels of CRF, and such levels can result from very stressful events. Thus, CRF1 antagonists may be a viable option for treating cognitive deficits in stressed individuals with psychiatric disorders.

Sex differences in circuits activated by corticotropin releasing factor in rats.

Women are more likely than men to suffer from psychiatric disorders characterized by corticotropin releasing factor (CRF) hypersecretion, suggesting sex differences in CRF sensitivity. In rodents, sex differences in the sensitivity of specific brain regions to CRF have been identified. However, regions do not work in isolation, but rather form circuits to coordinate distinct responses to stressful events. Here we examined whether CRF activates different circuits in male and female rats. Following central administration of CRF or artificial cerebrospinal fluid (aCSF), neuronal activation in stress-related areas was assessed using cFOS. Functional connectivity was gauged by correlating the number of cFOS-positive cells between regions and then identifying differences within each sex in correlations for aCSF-treated and CRF-treated groups. This analysis revealed that CRF altered different circuits in males and females. As an example, CRF altered correlations involving the dorsal raphe in males and the bed nucleus of the stria terminalis in females, suggesting sex differences in stress-activated circuits controlling mood and anxiety. Next, plasma estradiol and progesterone levels were correlated with cFOS counts in females. Negative correlations between estradiol and neuronal activation in the regions within the extended amygdala were found in CRF-treated, but not aCSF-treated females. This result suggests that estrogens and CRF together modulate the fear and anxiety responses mediated by these regions. Collectively, these studies reveal sex differences in the way brain regions work together in response to CRF. These differences could drive different stress coping strategies in males and females, perhaps contributing to sex biases in psychopathology.

Sex differences in stress regulation of arousal and cognition.

There are sex differences in the prevalence and presentation of many psychiatric disorders. For example, posttraumatic stress disorder (PTSD) and major depression are more common in women than men, and women with these disorders present with more hyperarousal symptoms than men. In contrast, attention deficit hyperactivity disorder (ADHD) and schizophrenia are more common in men than women, and men with these disorders have increased cognitive deficits compared to women. A shared feature of the aforementioned psychiatric disorders is the contribution of stressful events to their onset and/or severity. Here we propose that sex differences in stress responses bias females towards hyperarousal and males towards cognitive deficits. Evidence from clinical and preclinical studies is detailed. We also describe underlying neurobiological mechanisms. For example, sex differences in stress receptor signaling and trafficking in the locus coeruleus-arousal center are detailed. In learning circuits, evidence for sex differences in dendritic morphology is provided. Finally, we describe how evaluating sex-specific mechanisms for responding to stress in female and male rodents can lead to better treatments for stress-related psychiatric disorders.