Sex differences in corticotropin releasing factor-evoked behavior and activated networks.
ABSTRACT: Hypersecretion of corticotropin releasing factor (CRF) is linked to the pathophysiology of major depression and post-traumatic stress disorder, disorders that are more common in women than men. Notably, preclinical studies have identified sex differences in CRF receptors that can increase neuronal sensitivity to CRF in female compared to male rodents. These cellular sex differences suggest that CRF may regulate brain circuits and behavior differently in males and females. To test this idea, we first evaluated whether there were sex differences in anxiety-related behaviors induced by the central infusion of CRF. High doses of CRF increased self-grooming more in female than in male rats, and the magnitude of this effect in females was greater when they were in the proestrous phase of their estrous cycle (higher ovarian hormones) compared to the diestrous phase (lower ovarian hormones), which suggests that ovarian hormones potentiate this anxiogenic effect of CRF. Brain regions associated with CRF-evoked self-grooming were identified by correlating a marker of neuronal activation, cFOS, with time spent grooming. In the infralimbic region, which is implicated in regulating anxiety, the correlation for CRF-induced neuronal activation and grooming was positive in proestrous females, but negative for males and diestrous females, indicating that ovarian hormones altered this relationship between neuronal activation and behavior. Because CRF regulates a number of regions that work together to coordinate different aspects of responding to stress, we then examined more broadly whether CRF-activated functional connectivity networks differed between males and cycling females. Interestingly, hormonal status altered correlations for CRF-induced neuronal activation between a variety of brain regions, but the most striking differences were found when comparing proestrous females to males, particularly when comparing neuronal activation between prefrontal cortical and other forebrain regions. These results suggest that ovarian hormones alter the way brain regions work together in response to CRF, which could drive different strategies for coping with stress in males versus females. These sex differences in stress responses could also help explain female vulnerability to psychiatric disorders characterized by CRF hypersecretion.
Project description: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.
Project description:Sexually dimorphic nociception and opioid antinociception is very pervasive but poorly understood. We had demonstrated that spinal morphine antinociception in females, but not males, requires the concomitant activation of spinal ?- and ?-opioid receptors (MOR and KOR, respectively). This finding suggests an interrelationship between MOR and KOR in females that is not manifest in males. Here, we show that expression of a MOR/KOR heterodimer is vastly more prevalent in the spinal cord of proestrous vs. diestrous females and vs. males. Cross-linking experiments in combination with in vivo pharmacological analyses indicate that heterodimeric MOR/KOR utilizes spinal dynorphin 1-17 as a substrate and is likely to be the molecular transducer for the female-specific KOR component of spinal morphine antinociception. The activation of KOR within the heterodimeric MOR/KOR provides a mechanism for recruiting spinal KOR-mediated antinociception without activating the concomitant pronociceptive functions that monomeric KOR also subserves. Spinal cord MOR/KOR heterodimers represent a unique pharmacological target for female-specific pain control.
Project description:During the female reproductive cycle, estradiol exerts negative and positive feedback at both the central level to alter gonadotropin-releasing hormone (GnRH) release and at the pituitary to affect response to GnRH. Many studies of the neurobiologic mechanisms underlying estradiol feedback have been done on ovariectomized, estradiol-replaced (OVX+E) mice. In this model, GnRH neuron activity depends on estradiol and time of day, increasing in estradiol-treated mice in the late afternoon, coincident with a daily luteinizing hormone (LH) surge. Amplitude of this surge appears lower than in proestrous mice, perhaps because other ovarian factors are not replaced. We hypothesized GnRH neuron activity is greater during the proestrous-preovulatory surge than the estradiol-induced surge. GnRH neuron activity was monitored by extracellular recordings from fluorescently tagged GnRH neurons in brain slices in the late afternoon from diestrous, proestrous, and OVX+E mice. Mean GnRH neuron firing rate was low on diestrus; firing rate was similarly increased in proestrous and OVX+E mice. Bursts of action potentials have been associated with hormone release in neuroendocrine systems. Examination of the patterning of action potentials revealed a shift toward longer burst duration in proestrous mice, whereas intervals between spikes were shorter in OVX+E mice. LH response to an early afternoon injection of GnRH was greater in proestrous than diestrous or OVX+E mice. These observations suggest the lower LH surge amplitude observed in the OVX+E model is likely not attributable to altered mean GnRH neuron activity, but because of reduced pituitary sensitivity, subtle shifts in action potential pattern, and/or excitation-secretion coupling in GnRH neurons.
Project description: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.
Project description:BACKGROUND:The bed nucleus of the stria terminalis (BNST) contains the highest density of corticotropin-releasing factor (CRF)-producing neurons in the brain. CRF-immunoreactive neurons show a female-biased sexual dimorphism in the dorsolateral BNST in the rat. Since CRF neurons cannot be immunostained clearly with available CRF antibodies in the mouse, we used a mouse line, in which modified yellow fluorescent protein (Venus) was inserted to the CRF gene, and the Neo cassette was removed, to examine the morphological characteristics of CRF neurons in the dorsolateral BNST. Developmental changes of CRF neurons were examined from postnatal stages to adulthood. Gonadectomy (GDX) was carried out in adult male and female mice to examine the effects of sex steroids on the number of CRF neurons in the dorsolateral BNST. METHODS:The number of Venus-expressing neurons, stained by immunofluorescence, was compared between male and female mice over the course of development. GDX was carried out in adult mice. Immunohistochemistry, in combination with Nissl staining, was carried out, and the effects of sex or gonadal steroids were examined by estimating the number of Venus-expressing neurons, as well as the total number of neurons or glial cells, in each BNST subnucleus, using a stereological method. RESULTS:Most Venus-expressing neurons co-expressed Crf mRNA in the dorsolateral BNST. They constitute a group of neurons without calbindin immunoreactivity, which makes a contrast to the principal nucleus of the BNST that is characterized by calbindin immunostaining. In the dorsolateral BNST, the number of Venus-expressing neurons increased across developmental stages until adulthood. Sexual difference in the number of Venus-expressing neurons was not evident by postnatal day 5. In adulthood, however, there was a significant female predominance in the number of Venus expressing neurons in two subnuclei of the dorsolateral BNST, i.e., the oval nucleus of the BNST (ovBNST) and the anterolateral BNST (alBNST). The number of Venus-expressing neurons was smaller significantly in ovariectomized females compared with proestrous females in either ovBNST or alBNST, and greater significantly in orchiectomized males compared with gonadally intact males in ovBNST. The total number of neurons was also greater significantly in females than in males in ovBNST and alBNST, but it was not affected by GDX. CONCLUSION:Venus-expressing CRF neurons showed female-biased sexual dimorphism in ovBNST and alBNST of the mouse. Expression of Venus in these subnuclei was controlled by gonadal steroids.
Project description:Because binge eating and emotional eating vary through the menstrual cycle in human females, we investigated cyclic changes in binge-like eating in female rats and their control by estrogens. Binge-like eating was elicited by three cycles of 4 days of food restriction and 4 days of free feeding followed by a single frustrative nonreward-stress episode (15 min visual and olfactory exposure to a familiar palatable food) immediately before presentation of the palatable food. Intact rats showed binge-like eating during the diestrous and proestrous phases of the ovarian cycle, but not during the estrous (periovulatory) phase. Ovariectomized (OVX) rats not treated with estradiol (E2) displayed binge-like eating, whereas E2-treated OVX rats did not. The procedure did not increase signs of anxiety in an open-field test. OVX rats not treated with E2 that were subjected to food restriction and sacrificed immediately after frustrative nonreward had increased numbers of cells expressing phosphorylated extracellular signal-regulated kinases (ERK) in the central nucleus of the amygdala (CeA), paraventricular nucleus of hypothalamus (PVN), and dorsal and ventral bed nuclei of the stria terminalis (BNST) compared with nonrestricted or E2-treated rats. These data suggest that this female rat model is appropriate for mechanistic studies of some aspects of menstrual-cycle effects on emotional and binge eating in human females, that anxiety is not a sufficient cause of binge-like eating, and that the PVN, CeA, and BNST may contribute to information processing underlying binge-like eating.
Project description:This study was designed to learn more about the changes in expression of rat anterior pituitary (AP) leptin during the estrous cycle. QRT-PCR assays of cycling rat AP leptin mRNA showed 2-fold increases from metestrus to diestrus followed by an 86% decrease on the morning of proestrus. Percentages of leptin cells increased in proestrus and pregnancy to 55-60% of AP cells. Dual labeling for leptin proteins and growth hormone (GH) or gonadotropins showed that the rise in leptin protein-bearing cells from diestrus to proestrus was mainly in GH cells. Only 10-20% of leptin cells in male or cycling female rats coexpress gonadotropins. In contrast, 50-73% of leptin cells from pregnant or lactating females coexpress gonadotropins and only 19% coexpress GH, indicating plasticity in the distribution of leptin. Leptin cells expressed GnRH receptors, and estrogen and GnRH together increased the coexpression of leptin mRNA and gonadotropins. GnRH increased cellular leptin proteins three to four times and mRNA 9.8 times in proestrous rats and stimulated leptin secretion in cultures from diestrous, proestrous, and pregnant rats. These regulatory influences, and the high expression of AP leptin during proestrus and pregnancy, suggest a supportive role for leptin during key events involved with reproduction.
Project description:NELL2, a protein containing epidermal growth factor-like repeat domains, is predominantly expressed in the nervous system. In the mammalian brain, NELL2 expression is mostly neuronal. Previously we found that NELL2 is involved in the onset of female puberty by regulating the release of gonadotropin-releasing hormone (GnRH), and in normal male sexual behavior by controlling the development of the sexually dimorphic nucleus of the preoptic area (POA). In this study we investigated the effect of NELL2 on the female rat estrous cycle. NELL2 expression in the POA was highest during the proestrous phase. NELL2 mRNA levels in the POA were increased by estrogen treatment in ovariectomized female rats. Blocking NELL2 synthesis in the female rat hypothalamus decreased the expression of kisspeptin 1, an important regulator of the GnRH neuronal apparatus, and resulted in disruption of the estrous cycle at the diestrous phase. These results indicate that NELL2 is involved in the maintenance of the normal female reproductive cycle in mammals.
Project description:BACKGROUND:Corticotropin-releasing factor overexpressing (CRF-OE) male mice showed an inhibited feeding response to a fast, and lower plasma acyl ghrelin and Fos expression in the arcuate nucleus compared to wild-type (WT) mice. We investigated whether hormones and hypothalamic feeding signals are impaired in CRF-OE mice and the influence of sex. METHODS:Male and female CRF-OE mice and WT littermates (4-6 months old) fed ad libitum or overnight fasted were assessed for body, adrenal glands and perigonadal fat weights, food intake, plasma hormones, blood glucose, and mRNA hypothalamic signals. RESULTS:Under fed conditions, compared to WT, CRF-OE mice have increased adrenal glands and perigonadal fat weight, plasma corticosterone, leptin and insulin, and hypothalamic leptin receptor and decreased plasma acyl ghrelin. Compared to male, female WT mice have lower body and perigonadal fat and plasma leptin but higher adrenal glands weights. CRF-OE mice lost these sex differences except for the adrenals. Male CRF-OE and WT mice did not differ in hypothalamic expression of neuropeptide Y (NPY) and proopiomelanocortin (POMC), while female CRF-OE compared to female WT and male CRF-OE had higher NPY mRNA levels. After fasting, female WT mice lost more body weight and ate more food than male WT, while CRF-OE mice had reduced body weight loss and inhibited food intake without sex difference. In male WT mice, fasting reduced plasma insulin and leptin and increased acyl ghrelin and corticosterone while female WT showed only a rise in corticosterone. In CRF-OE mice, fasting reduced insulin while leptin, acyl ghrelin and corticosterone were unchanged with no sex difference. Fasting blood glucose was higher in CRF-OE with female > male. In WT mice, fasting increased hypothalamic NPY expression in both sexes and decreased POMC only in males, while in CRF-OE mice, NPY did not change, and POMC decreased in males and increased in females. CONCLUSIONS:These data indicate that CRF-OE mice have abnormal basal and fasting circulating hormones and hypothalamic feeding-related signals. CRF-OE also abolishes the sex difference in body weight, abdominal fat, and fasting-induced feeding and changes in plasma levels of leptin and acyl ghrelin.
Project description:Stress interacts with addictive processes to increase drug use, drug seeking, and relapse. The hippocampal formation (HF) is an important site at which stress circuits and endogenous opioid systems intersect and likely plays a critical role in the interaction between stress and drug addiction. Our prior studies demonstrate that the stress-related neuropeptide corticotropin-releasing factor (CRF) and the delta-opioid receptor (DOR) colocalize in interneuron populations in the hilus of the dentate gyrus and stratum oriens of CA1 and CA3. While independent ultrastructural studies of DORs and CRF receptors suggest that each receptor is found in CA1 pyramidal cell dendrites and dendritic spines, whether DORs and CRF receptors colocalize in CA1 neuronal profiles has not been investigated. Here, hippocampal sections of adult male and proestrus female Sprague-Dawley rats were processed for dual label pre-embedding immunoelectron microscopy using well-characterized antisera directed against the DOR for immunoperoxidase and against the CRF receptor for immunogold. DOR-immunoreactivity (-ir) was found presynaptically in axons and axon terminals as well as postsynaptically in somata, dendrites and dendritic spines in stratum radiatum of CA1. In contrast, CRF receptor-ir was predominantly found postsynaptically in CA1 somata, dendrites, and dendritic spines. CRF receptor-ir frequently was observed in DOR-labeled dendritic profiles and primarily was found in the cytoplasm rather than at or near the plasma membrane. Quantitative analysis of CRF receptor-ir colocalization with DOR-ir in pyramidal cell dendrites revealed that proestrus females and males show comparable levels of CRF receptor-ir per dendrite and similar cytoplasmic density of CRF receptor-ir. In contrast, proestrus females display an increased number of dual-labeled dendritic profiles and an increased membrane density of CRF receptor-ir in comparison to males. We further examined the functional consequences of CRF receptor-ir colocalization with DOR-ir in the same neuron using the hormone responsive neuronal cell line NG108-15, which endogenously expresses DORs, and assayed intracellular cAMP production in response to CRF receptor and DOR agonists. Results demonstrated that short-term application of DOR agonist SNC80 inhibited CRF-induced cAMP accumulation in NG108-15 cells transfected with the CRF receptor. These studies provide new insights on opioid-stress system interaction in the hippocampus of both males and females and establish potential mechanisms through which DOR activation may influence CRF receptor activity.