Neurons In Female Rats Research Articles

Activating transcription factor 3 (ATF3) and calcitonin gene-related peptide (CGRP) increase in trigeminal ganglion neurons in female rats after photorefractive keratectomy (PRK)-like corneal abrasion

Photorefractive keratectomy (PRK) is a type of eye surgery that involves removal of the corneal epithelium and its associated nerves, which causes intense acute pain in most people. We used a rat model of corneal epithelium removal (corneal abrasion) to examine underlying cellular and molecular mechanisms. In this study, we used immunohistochemistry of trigeminal ganglion (TG) to assess neuronal content of CGRP and ATF3, as well as orbital tightening (OT) to assess spontaneous pain behaviors. CGRP is an important neuropeptide in pain modulation and ATF3 is often used as a nerve injury marker. We found dynamic changes in CGRP and ATF3 in TG; both increased significantly at 24 h following corneal abrasion and females had a more pronounced increase at 24 h compared to males. Interestingly, there was no sex difference in OT behaviors. Additionally, the number of cells containing either CGRP or ATF3 in each animal correlate significantly with their OT behavior at the assessed timepoint. Since CGRP increased most in females, we tested the effectiveness of Olcegepant, a CGRP antagonist, at reducing OT behaviors following corneal abrasion in female rats. Olcegepant (1 mg/kg) was given prior to and again at 24 h after abrasion but did not change OT behaviors at any time over a 1-week period. Examination of CGRP and ATF3 together in TG showed that they rarely colocalized, indicating that the cells with upregulated CGRP are distinct from those responding to epithelial nerve injury. The studies also show that underlying molecular responses may be sex specific.

Raphe glucose-sensing serotonergic neurons stimulate KNDy neurons to enhance LH pulses via 5HT2CR: rat and goat studies

Dysfunction of central serotonergic neurons is known to cause depressive disorders in humans, who often show reproductive and/or glucose metabolism disorders. This study examined whether dorsal raphe (DR) serotonergic neurons sense high glucose availability to upregulate reproductive function via activating hypothalamic arcuate (ARC) kisspeptin neurons (= KNDy neurons), a dominant stimulator of gonadotropin-releasing hormone (GnRH)/gonadotropin pulses, using female rats and goats. RNA-seq and histological analysis revealed that stimulatory serotonin-2C receptor (5HT2CR) was mainly expressed in the KNDy neurons in female rats. The serotonergic reuptake inhibitor administration into the mediobasal hypothalamus (MBH), including the ARC, significantly blocked glucoprivic suppression of luteinizing hormone (LH) pulses and hyperglycemia induced by intravenous 2-deoxy-D-glucose (2DG) administration in female rats. A local infusion of glucose into the DR significantly increased in vivo serotonin release in the MBH and partly restored LH pulses and hyperglycemia in the 2DG-treated female rats. Furthermore, central administration of serotonin or a 5HT2CR agonist immediately evoked GnRH pulse generator activity, and central 5HT2CR antagonism blocked the serotonin-induced facilitation of GnRH pulse generator activity in ovariectomized goats. These results suggest that DR serotonergic neurons sense high glucose availability to reduce gluconeogenesis and upregulate reproductive function by activating GnRH/LH pulse generator activity in mammals.

Exercise leads to sex-specific recovery of behavior and pathological AD markers following adolescent ethanol exposure in the TgF344-AD model.

Human epidemiological studies suggest that heavy alcohol consumption may lead to earlier onset of Alzheimer's Disease (AD), especially in individuals with a genetic predisposition for AD. Alcohol-related brain damage (ARBD) during a critical developmental timepoint, such as adolescence, interacts with AD-related pathologies to accelerate disease progression later in life. The current study investigates if voluntary exercise in mid-adulthood can recover memory deficits caused by the interactions between adolescence ethanol exposure and AD-transgenes. Male and female TgF344-AD and wildtype F344 rats were exposed to an intragastric gavage of water (control) or 5 g/kg of 20% ethanol (adolescent intermittent ethanol; AIE) for a 2 day on/off schedule throughout adolescence (PD27-57). At 6 months old, rats either remained in their home cage (stationary) or were placed in a voluntary wheel running apparatus for 4 weeks and then underwent several behavioral tests. The number of cholinergic neurons in the basal forebrain and measure of neurogenesis in the hippocampus were assessed. Voluntary wheel running recovers spatial working memory deficits selectively in female TgF344-AD rats exposed to AIE and improves pattern separation impairment seen in control TgF344-AD female rats. There were sex-dependent effects on brain pathology: Exercise improves the integration of recently born neurons in AIE-exposed TgF344-AD female rats. Exercise led to a decrease in amyloid burden in the hippocampus and entorhinal cortex, but only in male AIE-exposed TgF344-AD rats. Although the number of basal forebrain cholinergic neurons was not affected by AD-transgenes in either sex, AIE did reduce the number of basal forebrain cholinergic neurons in female rats. These data provide support that even after symptom onset, AIE and AD related cognitive decline and associated neuropathologies can be rescued with exercise in unique sex-specific ways.

Neonatal olfactory bulbectomy causes dendritic spine retraction in dorsal hippocampal CA3 neurons in female rats and spatial learning deficits in male rats.

Olfactory bulbectomy (OBX) is an experimental strategy that is widely employed because it produces changes at different levels (from behavioral to molecular) that can be related to symptoms of depression in humans. This procedure has been widely studied in adult rats, but little information has been obtained of its effect in neonatal rats. The objective of the present study was to evaluate learning and memory capacity and dendritic spine density in dorsal hippocampal CA3 neurons. Seven-day-old male and female Wistar rats were subjected to nOBX by suction, we included an intact group as a control (CON) and a sham-operated group (SHAM), too. Spatial learning and memory were measured at 56days of age using a Morris water maze. A different cohort of experimental groups was used to measure dendritic spine density by Golgi-Cox impregnation. Male rats with nOBX showed a pronounced spatial learning deficit than female rats. Also, there was a significant decrease in basilar dendritic spine density in female rats with nOBX compared to the CON group. No changes were observed in this variable in male rats with nOBX. Our results allow us to suggest that there is sexual dimorphism in the effect of nOBX on the dorsal hippocampus and its relationship with spatial learning and memory processes.

Decreased PPARgamma in the trigeminal spinal subnucleus caudalis due to neonatal injury contributes to incision-induced mechanical allodynia in female rats

Whisker pad skin incision in infancy causes the prolongation of mechanical allodynia after re-incision in adulthood. A recent study also proposed the importance of sex differences in pain signaling in the spinal cord. However, the sex difference in re-incision-induced mechanical allodynia in the orofacial region is not fully understood. In the rats that experienced neonatal injury in the whisker pad skin, the mechanical allodynia in the whisker pad was significantly prolonged after re-incision in adulthood compared to sham injury in infancy. No significant sex differences were observed in the duration of mechanical allodynia. The duration of mechanical allodynia in male rats was shortened by intracisternal administration of minocycline. However, minocycline had no effects on the duration of mechanical allodynia in female rats. In contrast, intracisternal administration of pioglitazone markedly suppressed mechanical allodynia in female rats after re-incision. Following re-incision, the number of peroxisome proliferator-activated receptor gamma (PPARgamma)-positive cells were reduced in the trigeminal spinal subnucleus caudalis (Vc) in female rats that experienced neonatal injury. Immunohistochemical analyses revealed that PPARgamma was predominantly expressed in Vc neurons. Pioglitazone increased the number of PPARgamma-positive Vc neurons in female rats whose whisker pad skin was incised in both infancy and adulthood stages. Pioglitazone also upregulated heme oxygenase 1 and downregulated NR1 subunit in the Vc in female rats after re-incision. Together, PPARgamma signaling in Vc neurons is a female-specific pathway for whisker pad skin incision-induced mechanical allodynia.

Partial loss of arcuate kisspeptin neurons in female rats stimulates luteinizing hormone and decreases prolactin secretion induced by estradiol.

Kisspeptin, neurokinin, and dynorphin (KNDy) neurons in the arcuate nucleus (ARC) control luteinizing hormone (LH) and prolactin (PRL) release, although their role in conveying the effects of estradiol (E2 ) to these hormones is not well understood. We performed a longitudinal evaluation of female rats in which KNDy neurons were ablated using a neurokinin-3 receptor agonist conjugated with saporin (NK3-SAP) to investigate the impact of the reduction of KNDy neurons on the E2 regulation of gonadal and PRL axes. NK3-SAP rats, bearing a moderate loss of ARC kisspeptin-immunoreactive (-IR) neurons (50%-90%), displayed irregular estrous cycles but essentially unaltered follicular development and a normal number of corpora lutea. Rats were then ovariectomized (OVX) and treated with a positive-feedback dose of E2 (OVX + E2 ). LH and PRL were measured in the tail blood by an enzyme-linked immunosorbent assay. The E2 -induced LH surge was amplified, whereas the PRL rise was decreased in NK3-SAP rats compared to Blank-SAP control. After 10 days of no hormonal treatment, basal LH levels were equally elevated in NK3-SAP and controls. Tyrosine hydroxylase (TH) phosphorylation in the median eminence, in turn, was increased in NK3-SAP rats, with no change in the number of ARC TH-IR neurons. Thus, KNDy neurons exert concurrent and opposite roles in the E2 -induced surges of LH and PRL. The partial loss of KNDy neurons disrupts ovarian cyclicity but does not preclude ovulation, consistent with the disinhibition of the LH preovulatory surge. Conversely, KNDy neurons tonically inhibit the enzymatic activity of tuberoinfundibular dopaminergic neurons, which appears to facilitate PRL release in response to E2 .

Estrogens influence female itch sensitivity via the spinal gastrin-releasing peptide receptor neurons

There are sex differences in somatosensory sensitivity. Circulating estrogens appear to have a pronociceptive effect that explains why females are reported to be more sensitive to pain than males. Although itch symptoms develop during pregnancy in many women, the underlying mechanism of female-specific pruritus is unknown. Here, we demonstrate that estradiol, but not progesterone, enhances histamine-evoked scratching behavior indicative of itch in female rats. Estradiol increased the expression of the spinal itch mediator, gastrin-releasing peptide (GRP), and increased the histamine-evoked activity of itch-processing neurons that express the GRP receptor (GRPR) in the spinal dorsal horn. The enhancement of itch behavior by estradiol was suppressed by intrathecal administration of a GRPR blocker. In vivo electrophysiological analysis showed that estradiol increased the histamine-evoked firing frequency and prolonged the response of spinal GRP-sensitive neurons in female rats. On the other hand, estradiol did not affect the threshold of noxious thermal pain and decreased touch sensitivity, indicating that estradiol separately affects itch, pain, and touch modalities. Thus, estrogens selectively enhance histamine-evoked itch in females via the spinal GRP/GRPR system. This may explain why itch sensation varies with estrogen levels and provides a basis for treating itch in females by targeting GRPR.

Sexually dimorphic response of colorectal motility to noxious stimuli in the colorectum in rats.

This study showed a remarkable sex difference in responses of colorectal motility to noxious stimuli in the colorectum in rats: colorectal motility was enhanced in response to intracolonic administration of a noxious stimulant, capsaicin, in male rats but not in female rats. The difference in descending neurons from the brain to spinal cord operating after noxious stimulation could be responsible for the sex difference. In male rats, serotoninergic and dopaminergic neurons are dominantly activated, both of which activate the spinal defaecation centre. In female rats, GABAergic neurons in addition to serotoninergic neurons are activated. GABA may compete for facilitative action of 5-HT in the spinal defaecation centre, and thereby colorectal motility is not enhanced in response to intracolonic administration of capsaicin. The findings provide a novel insight into pathophysiological mechanisms of sex differences in functional defaecation disorders such as irritable bowel syndrome. We previously demonstrated that noxious stimuli in the colorectum enhance colorectal motility through activation of descending pain inhibitory pathways in male rats. It can be expected that the regulatory mechanisms of colorectal motility differ in males and females owing to remarkable sex differences in descending pain inhibitory pathways. Thus, we aimed to clarify sex differences in responses of colorectal motility to noxious stimuli in rats. Colorectal motility was measured in vivo in anaesthetized rats. Administration of a noxious stimulant, capsaicin, into the colorectal lumen enhanced colorectal motility in male rats but not in female rats. Quantitative PCR and immunohistochemistry showed that TRPV1 expression levels in the dorsal root ganglia and in the colorectal mucosa were comparable in male and female rats. When a GABAA receptor inhibitor was intrathecally administered to the L6-S1 level of the spinal cord, colorectal motility was facilitated in response to intracolonic capsaicin even in female rats. The capsaicin-induced response in the presence of the GABA blocker in female rats was inhibited by intrathecal administration of 5-HT2 and -3 receptor antagonists but not by a D2-like dopamine receptor antagonist. Our findings demonstrate that intracolonic noxious stimulation activates GABAergic and serotoninergic descending neurons in female rats, whereas serotoninergic and dopaminergic neurons are dominantly activated in male rats. Thus, the difference in the descending neurons operating after noxious stimulation would be responsible for the sexually dimorphic responses of colorectal motility. Our findings provide a novel insight into pathophysiological mechanisms of sex differences in functional defaecation disorders such as irritable bowel syndrome.

Paraventricular Dynorphin A Neurons Mediate LH Pulse Suppression Induced by Hindbrain Glucoprivation in Female Rats.

Malnutrition suppresses reproductive functions in mammals, which is considered to be mostly due to the inhibition of pulsatile gonadotropin-releasing hormone (GnRH)/gonadotropin secretion. Accumulating evidence suggests that kisspeptin neurons in the arcuate nucleus (ARC) play a critical role in the regulation of pulsatile GnRH/gonadotropin release. The present study aimed to examine if the hypothalamic dynorphin A (Dyn) neurons mediate the suppression of GnRH/luteinizing hormone (LH) pulses during malnutrition. Ovariectomized rats treated with a negative feedback level of estradiol-17β-treated (OVX+E2) were administered with intravenous (iv) or fourth cerebroventricle (4V) 2-deoxy-D-glucose (2DG), an inhibitor of glucose utilization, to serve as a malnutrition model. Central administration of a Dyn receptor antagonist blocked the iv- or 4V-2DG-induced suppression of LH pulses in OVX+E2 rats. The 4V 2DG administration significantly increased the number of Pdyn (Dyn gene)-positive cells co-expressing fos in the paraventricular nucleus (PVN), but not in the ARC and supraoptic nucleus (SON), and the iv 2DG treatment significantly increased the number of fos and Pdyn-co-expressing cells in the PVN and SON, but decreased it in the ARC. The E2 treatment significantly increased Pdyn expression in the PVN, but not in the ARC and SON. Double in situ hybridization for Kiss1 (kisspeptin gene) and Oprk1 (Dyn receptor gene) revealed that around 60% of ARC Kiss1-expressing cells co-expressed Oprk1. These results suggest that the PVN Dyn neurons, at least in part, mediate LH pulse suppression induced by the hindbrain or peripheral glucoprivation, and Dyn neurons may directly suppress the ARC kisspeptin neurons in female rats.

Early life stress-induced alterations in the activity and morphology of ventral tegmental area neurons in female rats

Childhood maltreatment, which can take the form of physical or psychological abuse, is experienced by more than a quarter of all children. Early life stress has substantial and long-term consequences, including an increased risk of drug abuse and psychiatric disorders in adolescence and adulthood, and this risk is higher in women than in men. The neuronal mechanisms underlying the influence of early life adversities on brain functioning remain poorly understood; therefore, in the current study, we used maternal separation (MS), a rodent model of early-life neglect, to verify its influence on the properties of neurons in the ventral tegmental area (VTA), a brain area critically involved in reward and motivation processing. Using whole-cell patch-clamp recordings in brain slices from adolescent female Sprague-Dawley rats, we found an MS-induced increase in the excitability of putative dopaminergic (DAergic) neurons selectively in the medial part of the VTA. We also showed an enhancement of excitatory synaptic transmission in VTA putative DAergic neurons. MS-induced alterations in electrophysiology were accompanied by an increase in the diameter of dendritic spine heads on lateral VTA DAergic neurons, although the overall dendritic spine density remained unchanged. Finally, we reported MS-related increases in basal plasma ACTH and corticosterone levels. These results show the long-term consequences of early life stress and indicate the possible neuronal mechanisms of behavioral disturbances in individuals who experience early life neglect.

Chemogenetic inhibition of dopaminergic projections to the nucleus accumbens has sexually dimorphic effects in the rat gambling task.

Women and men can differ in their propensity to take risks and develop impulse control and addiction disorders. Sexual dimorphisms in behavioral control by the mesolimbic dopaminergic reward system may underlie these phenomena, given its sensitivity to gonadal hormones. However, this is hard to test experimentally using humans. Using the rat gambling task (rGT), we investigated what impact acute inhibition of accumbal dopamine had on decision-making and impulsivity in animals of both sexes. We expressed an inhibitory designer receptor exclusively activated by designer drugs (hM4D[Gi]) in the accumbal dopaminergic efferents of female and male transgenic (Tg) rats, engineered to selectively express cre recombinase in neurons synthesizing tyrosine hydroxylase. We then trained the rats to perform the rGT and assessed the effect of an acute clozapine-n-oxide (0-3 mg/kg) challenge. Chemogenetic inhibition of dopaminergic projections to the accumbens did not affect choice in females, perhaps due to low levels of risky choice in Tg+ animals at baseline, but induced a switch from risky to optimal decision-making in males performing the cued rGT. This manipulation also decreased motor impulsivity but only in females. These data support the hypothesis that cue-driven risky choice is mediated, at least in males, by activity of accumbal dopaminergic neurons. However, motor impulsivity is more sensitive to inhibition of accumbal dopamine neurons in female rats. These data may help explain differences in the manifestation of addictions across gender and reinforce the importance of examining both sexes when seeking to attribute control of behavior to specific monoaminergic pathways. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

Sex Differences in Demand for Highly Palatable Foods: Role of the Orexin System.

BackgroundThe prevalence of eating disorders, including binge eating disorder, is significantly higher in women. These findings are mirrored by preclinical studies, which indicate that female rats have a higher preference for palatable food and show greater binge-like eating compared with male rats.MethodsHere, we describe a novel within-session behavioral-economic paradigm that allows for the simultaneous measurement of the intake at null cost (Q0) and normalized demand elasticity (α) of 3 types of palatable food (low fat, high fat, and chocolate sucrose pellets) via demand curve analysis. In light of evidence that the orexin (hypocretin) system is critically involved in reward and feeding behaviors, we also examined the role of orexin function in sex differences of economic demand for palatable foods.ResultsThe novel within-session behavioral-economic approach revealed that female rats have higher intake (demand) than males for all palatable foods at low cost (normalized to body weight) but no difference in intake at higher prices, indicating sex-dependent differences in the hedonic, but not motivational, aspects of palatable food. Immediately following behavioral-economic testing, we observed more orexin-expressing neurons and Fos expression (measure of recent neural activation) in these neurons in female rats compared with male rats. Moreover, the orexin-1 receptor antagonist SB334867 reduced both low- and high-cost intake for palatable food in both male and female rats.ConclusionsThese findings provide evidence of higher demand at low prices for palatable food in females and indicate that these behavioral differences may be associated with sexual dimorphism in orexin system function.

Social and olfactory experiences modify neuronal morphology of orbital frontal cortex.

Structural modifications in the dendritic morphology of neurons occur following many forms of experience, including exposure to drugs, complex housing, and training in specific behavioral tasks. The present study examined morphological changes in orbitofrontal (OFC) and medial prefrontal cortex (mPFC) neurons of female rats following experience with a variety of social partners or nonsocial olfactory stimuli. We reasoned that experience with various social partners or olfactory stimuli, and the associated behavioral adaptations, would drive structural modifications in prefrontal cortex neurons engaged by these stimuli. Social experience was manipulated by providing rats with a novel cage-mate or housing the animal with the same cage-mate throughout the study. Similarly, olfactory experience was manipulated by introducing novel, nonsocial odors in the home cage or exposing the animals to the same home-cage odor throughout the study. Both forms of experience resulted in altered dendritic morphology in OFC neurons, whereas morphological changes in mPFC were comparatively small and limited to changes in spine density. These observations indicate that OFC and mPFC neurons respond differently to social and nonsocial olfactory stimulation in adulthood and join the growing body of data illustrating differential effects of experience on structural plasticity in OFC and mPFC. (PsycINFO Database Record (c) 2020 APA, all rights reserved).

GnRH(1-5), a metabolite of gonadotropin-releasing hormone, enhances luteinizing hormone release via activation of kisspeptin neurons in female rats.

Accumulating evidence suggests that kisspeptin neurons in the arcuate nucleus (ARC), which coexpress neurokinin B and dynorphin, are involved in gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) pulse generation, while the anteroventral periventricular nucleus (AVPV) kisspeptin neurons are responsible for GnRH/LH surge generation. The present study aims to examine whether GnRH(1-5), a GnRH metabolite, regulates LH release via kisspeptin neurons. GnRH(1-5) was intracerebroventricularly injected to ovariectomized and estrogen-treated Wistar-Imamichi female rats. Immediately after the central GnRH(1-5) administration at 2 nmol, plasma LH concentration increased, resulting in significantly higher levels of the area under the curve and baseline of plasma LH concentrations compared to vehicle-injected controls. On the other hand, in Kiss1 knockout rats, GnRH(1-5) administration failed to affect LH secretion, suggesting that the facilitatory effect of GnRH(1-5) on LH release is mediated by kisspeptin neurons. Double in situ hybridization (ISH) for Kiss1 and Gpr101, a GnRH(1-5) receptor gene, revealed that few Kiss1-expressing cells coexpress Gpr101 in both ARC and AVPV. On the other hand, double ISH for Gpr101 and Slc17a6, a glutamatergic marker gene, revealed that 29.2% of ARC Gpr101-expressing cells coexpress Slc17a6. Further, most of the AVPV and ARC Kiss1-expressing cells coexpress Grin1, a gene encoding a subunit of NMDA receptor. Taken together, these results suggest that the GnRH(1-5)-GPR101 signaling facilitates LH release via indirect activation of kisspeptin neurons and that glutamatergic neurons may mediate the signaling. This provides a new aspect of kisspeptin- and GnRH-neuronal communication with the presence of stimulation from GnRH to kisspeptin neurons in female rats.

Hypothalamic Kisspeptin Expression in Hyperandrogenic Female Rats and Aging Rats.

Hypothalamic kisspeptin neurons stimulate gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) release. Kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) of rats induce an LH surge for ovulation, and those in the arcuate nucleus (ARC) regulate pulsatile LH secretion for follicle development and spermatogenesis. Dysfunction of kisspeptin neurons thus reduces the reproductive function. This review focuses on the effect of androgen or aging on kisspeptin expression in rats. Although androgen directly suppresses ARC kisspeptin neurons in female rats, the AVPV kisspeptin neurons are hardly affected. In rats, plasma LH concentrations decrease in both sexes with aging, and ARC kisspeptin expression also decreases in old rats compared with young rats. In addition, kisspeptin neurons may be associated with hyperprolactinemia in old female rats because they are known to release prolactin through hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons. Hypothalamic kisspeptin neurons are thus the main regulator to secrete LH, and inhibition of kisspeptin expression leads to various kinds of reproductive dysfunction.

Sex- and Stress-Dependent Effects on Dendritic Morphology and Spine Densities in Putative Orexin Neurons

We recently found that non-stressed female rats have higher basal prepro-orexin expression and activation of orexinergic neurons compared to non-stressed males, which lead to impaired habituation to repeated restraint stress at the behavioral, neural, and endocrine level. Here, we extended our study of sex differences in the orexin system by examining spine densities and dendritic morphology in putative orexin neurons in adult male and female rats that were exposed to 5 consecutive days of 30-min restraint. Analysis of spine distribution and density indicated that putative orexinergic neurons in control non-stressed females had significantly more dendritic spines than those in control males, and the majority of these were mushroom spines. This morphological finding may suggest more excitatory input onto orexin neurons in female rats. As orexin neurons are known to promote the hypothalamic–pituitary–adrenal response, this morphological change in orexin neurons could underlie the impaired habituation to repeated stress in female rats. Dendritic complexity did not differ between non-stressed males and females, however repeated restraint stress decreased total dendritic length, nodes, and branching primarily in males. Thus, reduced dendritic complexity of putative orexinergic neurons is observed in males but not in females after 5days of repeated restraint stress. This morphological change might be reflective of decreased orexin system function, which may allow males to habituate more fully to repeated restraint than females. These results extend our understanding of the role of orexin neurons in regulating habituation and demonstrate changes in putative orexin cell morphology and spines that may underlie sex differences in habituation.

The Role of the Basolateral Amygdala Projections to the Bed Nucleus of the Stria Terminalis in Anxiety‐like Behaviors in Male and Female Rats

Men and women display different symptomology in anxiety disorders, yet our understanding of the sex differences in the neurobiology related to these disorders is limited. The basolateral amygdala (BLA) is a critical component of the neurocircuitry involved in anxiety, is hyperactive in patients with anxiety disorders and displays a sexual dimorphism with respect to neural activity. Differences in BLA activity may be a contributing factor to sex differences in the pathophysiology of anxiety disorders. Prior studies have demonstrated that sex differences in BLA neuronal activity exist; however, it is not known whether specific subsets of BLA neurons exhibit this disparity. BLA outputs to the lateral bed nucleus stria terminalis (BSTL) play a critical role in the expression of prolonged cued fear and general anxiety‐like behavior with female rats expressing less general anxiety‐like behaviors compared to males. The purpose of this study was to determine if BLA‐BSTL neurons in female rats are less active than in males. We utilized in vivo single‐unit extracellular electrophysiological recordings to record anti‐dromically identified BLA‐BSTL neurons in anesthetized male and cycling female rats. We found that BLA‐BSTL neurons in females have lower firing frequencies compared to males, despite females having greater spontaneous BLA neuronal activity overall. We also examined whether prolonged cued fear conditioning, which is dependent, in part, on BLA‐BSTL connections, is less robust in female rats compared to males. Rats were conditioned to an 8 min tone, during which footshocks (0.3mA) were delivered at random. Four days later (when females were in the same estrous stage as training), rats were tested in a novel context and time spent freezing during the presentation of the tone was measured. No sex differences were observed during training, but proestrous females froze less during training compared to males and metestrous females. However, during extinction, females extinguished freezing behavior more quickly than males, and proestrous females froze less than metestrous females. To determine if our electrophysiological data suggesting decreased BLA‐BSTL activity in females leads to a decreased freezing behavior during prolonged cued fear, we utilized a chemogenic approach to selectively inhibit BLA‐BSTL neurons. We performed bilateral Cre‐dependent designer receptor exclusively activated by designer drug (DREADD) injections then assessed anxiety‐like behavior and freezing responses during prolonged cued fear. Preliminary results suggest that inhibition of BLA‐BSTL neurons during the open field task has opposite effects in male and female rats. Specifically, we found that BLA‐BSTL inhibition reduced latency to enter the center in males, but increased latency to enter center in females. Together this suggests that in addition to BLA‐BSTL neurons displaying sex differences in activity, these same neurons may also mediate different behaviors in male and female rats. These results may help explain the apparent disconnect between overall sex differences in BLA neuronal firing and its relation to particular anxiety behaviors.Support or Funding InformationR01MH100536This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Recurrent binge ethanol is associated with significant loss of dentate gyrus granule neurons in female rats despite concomitant increase in neurogenesis

Binge drinking is becoming increasingly common among American women and girls. We have previously shown significant cell loss, downregulation of neurotrophins and microgliosis in female rats after a single 4-day ethanol exposure. To determine whether recurrent binge exposure would produce similar effects, we administered ethanol (5 g/kg) or iso-caloric control diet once-weekly for 11 weeks to adult female rats. As we have previously shown exercise neuroprotection against binge-induced damage, half the rats were given access to exercise wheels. Blood ethanol concentration (BEC) did not differ between sedentary and exercised groups, nor did it change across time. Using stereology, we quantified the number and/or size of neurons in the medial prefrontal cortex (mPFC) and hippocampal dentate gyrus (DG), as well as the number and activation state of microglia. Binged sedentary rats had significant cell loss in the dentate gyrus, but exercise eliminated this effect. Compared to sedentary controls, sedentary binged rats and all exercised rats showed increased neurogenesis in the DG. Number and nuclear volume of neurons in the mPFC were not changed. In the hippocampus and mPFC, the number of microglia with morphology indicative of partial activation was increased by recurrent binge ethanol and decreased by exercise. In summary, we show significant binge-induced loss of DG granule neurons despite increased neurogenesis, suggesting an unsuccessful compensatory response. Although exercise eliminated cell loss, our results indicate that infrequent, but recurrent exposure to clinically relevant BEC is neurotoxic.

Activity-based anorexia activates CRF immunoreactive neurons in female rats

Activity-based anorexia (ABA) is a well-established animal model mimicking the eating disorder anorexia nervosa (AN). Since the pathophysiology of AN is yet poorly understood and specific drug treatments are lacking so far, animal models might be useful to further understand this disease. ABA consists of time-restricted access to food for 1.5 h/day and the possibility to exercise in a running wheel for 24 h/day. This combination leads to robust body weight loss as observed in AN. Here, we investigated the activation of brain corticotropin-releasing factor (CRF) neurons, a transmitter involved in the response to stress, emotional processes and also food intake. After development of ABA, rat brains were processed for c-Fos and CRF double immunohistochemistry. ABA increased the number of c-Fos/CRF double labeled neurons in the paraventricular nucleus (PVN) and the dorsomedial hypothalamic nucleus (DMH) compared to the ad libitum (AL, ad libitum fed, no running wheel) and activity (AC, ad libitum fed, running wheel, p < 0.05) but not to the restricted feeding (RF, food for 1.5 h/day, no running wheel, p > 0.05) group. Also the number of CRF neurons was increased in the DMH of ABA rats compared to AL and AC (p < 0.05). In the Edinger-Westphal nucleus (EW) the number of c-Fos positive neurons was increased in ABA and RF compared to AC (p < 0.05), while the number of double labeled neurons was not different (p > 0.05). Taken together, brain CRF activated under conditions of ABA might play a role in the development and maintenance of this animal model and possibly also in human AN.

Co-expression of the calcitonin receptor gene in the hypothalamic kisspeptin neurons in female rats.

PurposeHypothalamic kisspeptin neurons are considered to play a critical role in regulating mammalian reproduction and integrating humoral and neuronal inputs that control gonadotropin‐releasing hormone (GnRH)/gonadotropin release. The present study aimed to investigate the upstream regulator candidates for kisspeptin neurons.MethodsVisualized kisspeptin neurons that were taken from the arcuate nucleus (ARC) of Kiss1‐tdTomato rats were subjected to next‐generation sequencing (NGS) analysis. In situ hybridization (ISH) for the calcitonin receptor gene (Calcr) was performed throughout the whole forebrain of ovariectomized wild‐type female rats that had been implanted with a negative feedback level of estrogen, because the Calcr expression was evident in the ARC kisspeptin neurons from the NGS analysis. Then, a double ISH was performed for the Calcr and kisspeptin gene (Kiss1) in the brain regions, containing either the anteroventral periventricular nucleus (AVPV) or ARC of the female rats.ResultsThe NGS analysis revealed that the Calcr was highly expressed in the ARC kisspeptin neurons. It was found that the Calcr was co‐expressed in 12% and 22% of the Kiss1‐expressing cells in the ARC and AVPV, respectively.ConclusionThe present study suggests that calcitonin receptor signaling could be involved in the regulation of reproductive function through the direct control of the ARC and/or AVPV kisspeptin neurons, and then GnRH/gonadotropin release.