Probes for INS

Estrogen receptor (ER) α-expressing neurons in the ventrolateral area of the ventromedial hypothalamus (VMHvl) are implicated in the control of many behaviors and physiological processes, some of which are sex-specific. Recently, three sex-differentiated ERα subpopulations have been discovered in the VMHvl marked by co-expression with tachikinin1 (Tac1), reprimo (Rprm), or prodynorphin (Pdyn), that may subserve specific functions. These markers show sex differences in adulthood: females have many more Tac1/Esr1 and Rprm/Esr1 co-expressing cells, while males have more Pdyn/Esr1 cells. In this study, we sought to understand the development of these sex differences and pinpoint the sex-differentiating signal. We examined developmental changes in the number of Esr1 cells co-expressing Tac1, Rprm or Pdyn using single-molecule in situ hybridization. We found that both sexes have similarly high numbers of Tac1/Esr1 and Rprm/Esr1 cells at birth, but newborn males have many more Pdyn/Esr1 cells than females. However, the number of cells with Tac1/Esr1 and Rprm/Esr1 co-expression markedly decreases by weaning in males, but not females, leading to sex differences in neurochemical expression. Female mice administered testosterone at birth have expression patterns akin to male mice. Thus, a substantial neurochemical reorganization of the VMHvl occurs in males between birth and weaning that likely underlies the previously reported sex differences in behavioral and physiological responses to estrogens in adulthood.

Stress-linked disorders are more prevalent in women than in men and differ in their clinical presentation. Thus, investigating sex differences in factors that promote susceptibility or resilience to stress outcomes, and the circuit elements that mediate their effects, is important. In male rats, instrumental control over stressors engages a corticostriatal system involving the prelimbic cortex (PL) and dorsomedial striatum (DMS) that prevent many of the sequelae of stress exposure. Interestingly, control does not buffer against stress outcomes in females, and here, we provide evidence that the instrumental controlling response in females is supported instead by the dorsolateral striatum (DLS). Additionally, we used in vivo microdialysis, fluorescent in situ hybridization, and receptor subtype pharmacology to examine the contribution of prefrontal dopamine (DA) to the differential impact of behavioral control. Although both sexes preferentially expressed D1 receptor mRNA in PL GABAergic neurons, there were robust sex differences in the dynamic properties of prefrontal DA during controllable stress. Behavioral control potently attenuated stress-induced DA efflux in males, but not females, who showed a sustained DA increase throughout the entire stress session. Importantly, PL D1 receptor blockade (SCH 23390) shifted the proportion of striatal activity from the DLS to the DMS in females and produced the protective effects of behavioral control. These findings suggest a sex-selective mechanism in which elevated DA in the PL biases instrumental responding towards prefrontal-independent striatal circuitry, thereby eliminating the protective impact of coping with stress.

Sex differences in the sensitivity to hypertension and inflammatory processes are well characterized but insufficiently understood. In male mice, tumor necrosis factor alpha (TNFα) in the hypothalamic paraventricular nucleus (PVN) contributes to hypertension following slow-pressor angiotensin II (AngII) infusion. However, the role of PVN TNFα in the response to AngII in female mice is unknown. Using a combination of in situ hybridization, high-resolution electron microscopic immunohistochemistry, spatial-temporal gene silencing, and dihydroethidium microfluorography we investigated the influence of AngII on both blood pressure and PVN TNFα signaling in female mice. We found that chronic (14-day) infusion of AngII in female mice did not impact blood pressure, TNFα levels, the expression of the TNFα type 1 receptor (TNFR1), or the subcellular distribution of TNFR1 in the PVN. However, it was shown that blockade of estrogen receptor β (ERβ), a major hypothalamic estrogen receptor, was accompanied by both elevated PVN TNFα and hypertension following AngII. Further, AngII hypertension following ERβ blockade was attenuated by inhibiting PVN TNFα signaling by local TNFR1 silencing. It was also shown that ERβ blockade in isolated PVN-spinal cord projection neurons (i.e. sympathoexcitatory) heightened TNFα-induced production of NADPH oxidase (NOX2)-mediated reactive oxygen species, molecules that may play a key role in mediating the effect of TNFα in hypertension. These results indicate that ERβ contributes to the reduced sensitivity of female mice to hypothalamic inflammatory cytokine signaling and hypertension in response to AngII.

Sex differences occur in the structure and function of the rat cerebral cortex and hippocampus, which can change from the juvenile period through old age. Although the evidence is incomplete, it appears that in at least some portions of the cortex these differences develop due to the rise of ovarian hormones at puberty and are potentially not dependent on the perinatal rise in testosterone, which is essential for sexual differentiation of the hypothalamus and sexual behavior. During aging of female rats, the presence of continued ovarian hormone secretion after cessation of the estrous cycle also influences sex differences in neuroanatomical structure and cognitive behavior, resulting in nullification or reversal of sex differences seen in younger adults. Sex differences can be altered by experience in a stimulating environment during the juvenile/adolescent period, and sex differences in performance even can be affected by the parameters of a task. Thus, broad generalizations about differences such as “spatial ability” are to be avoided. It is clear that to understand how the brain produces behavior, sex and hormones have to be taken into account.

Decision making is a complex cognitive process that recruits a distributed network of brain regions, including the basolateral amygdala (BLA) and nucleus accumbens shell (NAcSh). Recent work suggests that communication between these structures, as well as activity of cells expressing dopamine D2 receptors (D2R) in the NAcSh, are necessary for some forms of decision making; however, the contributions of this circuit and cell population during decision making under risk of punishment are unknown. The current experiments addressed this question using circuit- and cell type-specific optogenetic approaches in rats during a decision-making task involving risk of punishment. In Experiment 1, Long-Evans rats received intra-BLA injections of halorhodopsin or mCherry (control) and in Experiment 2, D2-Cre transgenic rats received intra-NAcSh injections of Cre-dependent halorhodopsin or mCherry. Optic fibers were implanted in the NAcSh in both experiments. Following training in the decision-making task, BLA→NAcSh or D2R-expressing neurons were optogenetically inhibited during different phases of the decision process. Inhibition of the BLA→NAcSh during deliberation (the time between trial initiation and choice) increased choice of the large, risky reward (increased risk taking). Similarly, inhibition during delivery of the large, punished reward increased risk taking, but only in males. Inhibition of D2R-expressing neurons in the NAcSh during deliberation increased risk taking. In contrast, inhibition of these neurons during delivery of the small, safe reward decreased risk taking. These findings extend our knowledge of the neural dynamics of risk taking, revealing sex-dependent circuit recruitment and dissociable activity of selective cell populations during decision making.

Microglia are central to pathogenesis in many neurological conditions. Drugs targeting colony-stimulating factor-1 receptor (CSF1R) to block microglial proliferation in preclinical disease models have shown mixed outcomes, thus the therapeutic potential of this approach remains unclear. Here, we show that CSF1R inhibitors given by multiple dosing paradigms in the Tg2541 tauopathy mouse model cause a sex-independent reduction in pathogenic tau and reversion of non-microglial gene expression patterns toward a normal wild type signature. Despite greater drug exposure in male mice, only female mice have functional rescue and extended survival. A dose-dependent upregulation of immediate early genes and neurotransmitter dysregulation are observed in the brains of male mice only, indicating that excitotoxicity may preclude functional benefits. Drug-resilient microglia in male mice exhibit morphological and gene expression patterns consistent with increased neuroinflammatory signaling, suggesting a mechanistic basis for sex-specific excitotoxicity. Complete microglial ablation is neither required nor desirable for neuroprotection and therapeutics targeting microglia must consider sex-dependent effects.

The gut microbiota is increasingly recognized as a key environmental factor that shapes host development and physiology, including neural circuits formation and function. Concurrently, there has been growing concern that early-life antibiotic exposure may alter brain developmental trajectories, increasing the risk for neurodevelopmental disorders such as autism spectrum disorder (ASD). Here, we assessed whether perturbation of the maternal gut microbiota in mice during a narrow critical perinatal window (last week of pregnancy and first three postnatal days), induced by exposure to a commonly used broad-spectrum oral antibiotic (ampicillin), influences offspring neurobehavioral outcomes relevant to ASD. Our results demonstrate that neonatal offspring from antibiotic-treated dams display an altered pattern of ultrasonic communication, which was more pronounced in males. Moreover, juvenile male, but not female, offspring from antibiotic-treated dams showed reduced social motivation and social interaction, as well as context-dependent anxiety-like behavior. However, no changes were observed in locomotor or exploratory activity. This behavioral phenotype of exposed juvenile males was associated with reduced gene expression of the oxytocin receptor (OXTR) and several tight-junction proteins in the prefrontal cortex, a key region involved in the regulation of social and emotional behavior, as well as a mild inflammatory response in the colon. Further, juvenile offspring from exposed dams also showed distinct alterations in several gut bacterial species, including, Lactobacillus murinus, and Parabacteroides goldsteinii. Overall, this study highlights the importance of the maternal microbiome in early-life, and how its perturbation by a widely used antibiotic could contribute to atypical social and emotional development of offspring in a sex-dependent manner.

Chronic pain is a common and often debilitating problem that affects 100 million Americans. A better understanding of pain’s molecular mechanisms is necessary for developing safe and effective therapeutics. Microglial activation has been implicated as a mediator of chronic pain in numerous preclinical studies; unfortunately, translational efforts using known glial modulators have largely failed, perhaps at least in part due to poor specificity of the compounds pursued, or an incomplete understanding of microglial reactivity. In order to achieve a more granular understanding of the role of microglia in chronic pain as a means of optimizing translational efforts, we utilized a clinically-informed mouse model of complex regional pain syndrome (CRPS), and monitored microglial activation throughout pain progression. We discovered that while both males and females exhibit spinal cord microglial activation as evidenced by increases in Iba1, activation is attenuated and delayed in females. We further evaluated the expression of the newly identified microglia-specific marker, TMEM119, and identified two distinct populations in the spinal cord parenchyma after peripheral injury: TMEM119+ microglia and TMEM119- infiltrating myeloid lineage cells, which are comprised of Ly6G + neutrophils and Ly6G- macrophages/monocytes. Neurons are sensitized by inflammatory mediators released in the CNS after injury; however, the cellular source of these cytokines remains somewhat unclear. Using multiplex in situ hybridization in combination with immunohistochemistry, we demonstrate that spinal cord TMEM119+ microglia are the cellular source of cytokines IL6 and IL1β after peripheral injury. Taken together, these data have important implications for translational studies: 1) microglia remain a viable analgesic target for males and females, so long as duration after injury is considered; 2) the analgesic properties of microglial modulators are likely at least in part related to their suppression of microglial-released cytokines, and 3) a limited number of neutrophils and macrophages/monocytes infiltrate the spinal cord after peripheral injury but have unknown impact on pain persistence or resolution. Further studies to uncover glial-targeted therapeutic interventions will need to consider sex, timing after injury, and the exact target population of interest to have the specificity necessary for translation.

Prenatal infection increases risk for neurodevelopmental disorders such as autism in offspring. In rodents, prenatal administration of the viral mimic Polyinosinic: polycytidylic acid (Poly I: C) allows for investigation of developmental consequences of gestational sickness on offspring social behavior and neural circuit function. Because maternal immune activation (MIA) disrupts cortical development and sociability, we examined approach and avoidance in a rat social affective preference (SAP) task. Following maternal Poly I:C (0.5 mg/kg) injection on gestational day 12.5, male adult offspring (PN 60-64) exhibited atypical social interactions with stressed conspecifics whereas female SAP behavior was unaffected by maternal Poly I:C. Social responses to stressed conspecifics depend upon the insular cortex where corticotropin releasing factor (CRF) modulates synaptic transmission and SAP behavior. We characterized insular field excitatory postsynaptic potentials (fEPSP) in adult offspring of Poly I:C or control treated dams. Male MIA offspring showed decreased sensitivity to CRF (300 nM) while female MIA offspring showed greater sensitivity to CRF compared to sham offspring. These sex specific effects appear to be behaviorally relevant as CRF injected into the insula of male and female rats prior to social exploration testing had no effect in MIA male offspring but increased social interaction in female MIA offspring. We examined the cellular distribution of CRF receptor mRNA but found no effect of maternal Poly I:C in the insula. Together, these experiments reveal sex specific effects of prenatal infection on offspring responses to social affective stimuli and identify insular CRF signaling as a novel neurobiological substrate for autism risk.

Female bias is highly prevalent among adrenal cortex hyperplasia and neoplasia, but the reasons behind this phenomenon are poorly understood. In this article, we show that overexpression of the secreted WNT agonist R-spondin 1 leads to ectopic activation of WNT/β-catenin signaling and causes sex-specific adrenocortical hyperplasia in mice. While female adrenals show ectopic proliferation, male adrenals display excessive immune system activation and cortical thinning. Using a combination of genetic manipulations and hormonal treatment, we show that gonadal androgens suppress ectopic proliferation in the adrenal cortex and determine the selective regulation of WNT-related genes Axin2 and Wnt4. Notably, genetic removal of androgen receptor (AR) from adrenocortical cells restores the mitogenic effect of WNT/β-catenin signaling. This is the first demonstration that AR activity in the adrenal cortex determines susceptibility to canonical WNT signaling-induced hyperplasia.

We present a comprehensive analysis of SARS-CoV-2 infection and recovery in wild type C57BL/6 mice, demonstrating that this is an ideal model of infection and recovery that accurately phenocopies acute human disease arising from the ancestral SARS-CoV-2. Disease severity and infection kinetics are age- and sex-dependent, as has been reported for humans, with older mice and males in particular exhibiting decreased viral clearance and increased mortality. We identified key parallels with human pathology, including intense virus positivity in bronchial epithelial cells, wide-spread alveolar involvement, recruitment of immune cells to the infected lungs, and acute bronchial epithelial cell death. Moreover, older animals experienced increased virus persistence, delayed dispersal of immune cells into lung parenchyma, and morphologic evidence of tissue damage and inflammation. Parallel analysis of SCID mice revealed that the adaptive immune response was not required for recovery from COVID disease symptoms nor early phase clearance of virus but was required for efficient clearance of virus at later stages of infection. Finally, transcriptional analyses indicated that induction and duration of key innate immune gene programs may explain differences in age-dependent disease severity. Importantly, these data demonstrate that SARS-CoV-2-mediated disease in C57BL/6 mice accurately phenocopies human disease across ages and establishes a platform for future therapeutic and genetic screens for not just SARS-CoV-2 but also novel coronaviruses that have yet to emerge.