Acute and chronic alcohol modulation of extended amygdala calcium dynamics.

The central amygdala (CeA) and bed nucleus of the stria terminalis (BNST) are reciprocally connected nodes of the extended amygdala thought to play an important role in alcohol consumption. Studies of immediate-early genes indicate that BNST and CeA are acutely activated following alcohol drinking and may signal alcohol reward in nondependent drinkers, while stress signaling in the extended amygdala following chronic alcohol exposure drives increased drinking via negative reinforcement. However, the temporal dynamics of neuronal activation in these regions during drinking behavior are poorly understood. In this study, we used fiber photometry and the genetically encoded calcium sensor GCaMP6s to assess acute changes in neuronal activity during alcohol consumption in BNST and CeA before and after a chronic drinking paradigm. Activity was examined in the pan-neuronal population and separately in dynorphinergic neurons. BNST and CeA showed increased pan-neuronal activity during acute consumption of alcohol and other fluid tastants of positive and negative valence, as well as highly palatable chow. Responses were greatest during initial consummatory bouts and decreased in amplitude with repeated consumption of the same tastant, suggesting modulation by stimulus novelty. Dynorphin neurons showed similar consumption-associated calcium increases in both regions. Following three weeks of continuous alcohol access (CA), calcium increases in dynorphin neurons during drinking were maintained, but pan-neuronal activity and BNST-CeA coherence were altered in a sex-specific manner. These results indicate that BNST and CeA, and dynorphin neurons specifically, are engaged during drinking behavior, and activity dynamics are influenced by stimulus novelty and chronic alcohol.

Acute and chronic alcohol modulation of extended amygdala calcium dynamics.

The central amygdala (CeA) and bed nucleus of the stria terminalis (BNST) are reciprocally connected nodes of the extended amygdala thought to play an important role in alcohol consumption. Studies of immediate-early genes indicate that BNST and CeA are acutely activated following alcohol drinking and may signal alcohol reward in nondependent drinkers, while increased stress signaling in the extended amygdala following chronic alcohol exposure drives increased drinking via negative reinforcement. However, the temporal dynamics of neuronal activation in these regions during drinking behavior are poorly understood. In this study, we used fiber photometry and the genetically encoded calcium sensor GCaMP6s to assess acute changes in neuronal activity during alcohol consumption in BNST and CeA before and after a chronic drinking paradigm. Activity was examined in the pan-neuronal population and separately in dynorphinergic neurons. BNST and CeA showed increased pan-neuronal activity during acute consumption of alcohol and other fluid tastants of positive and negative valence, as well as highly palatable chow. Responses were greatest during initial consummatory bouts and decreased in amplitude with repeated consumption of the same tastant, suggesting modulation by stimulus novelty. Dynorphin neurons showed similar consumption-associated calcium increases in both regions. Following three weeks of continuous alcohol access (CA), calcium increases in dynorphin neurons during drinking were maintained, but pan-neuronal activity and BNST-CeA coherence were altered in a sex-specific manner. These results indicate that BNST and CeA, and dynorphin neurons specifically, are engaged during drinking behavior, and activity dynamics are influenced by stimulus novelty and chronic alcohol.

Alcohol Dependence Modifies Brain Networks Activated During Withdrawal and Reaccess: A c-Fos-Based Analysis in Mice.

BACKGROUND: High-level alcohol consumption causes neuroplastic changes in the brain that promote pathological drinking behavior. Some of these changes have been characterized in defined brain circuits and cell types, but unbiased approaches are needed to explore broader patterns of adaptations. METHODS: We used whole-brain c-Fos mapping and network analysis to assess patterns of neuronal activity during alcohol withdrawal and following reaccess in a well-characterized model of alcohol dependence. Mice underwent 4 cycles of chronic intermittent ethanol to increase voluntary alcohol consumption, and a subset underwent forced swim stress to further escalate consumption. Brains were collected either 24 hours (withdrawal) or immediately following a 1-hour period of alcohol reaccess. c-fos counts were obtained for 110 brain regions using iDISCO and ClearMap. Then, we classified mice as high or low drinkers and used graph theory to identify changes in network properties associated with high-drinking behavior. RESULTS: During withdrawal, chronic intermittent ethanol mice displayed widespread increased c-Fos expression relative to air-exposed mice, independent of forced swim stress. Reaccess drinking reversed this increase. Network modularity, a measure of segregation into communities, was increased in high-drinking mice after alcohol reaccess relative to withdrawal. The cortical amygdala showed increased cross-community coactivation during withdrawal in high-drinking mice, and cortical amygdala silencing in chronic intermittent ethanol mice reduced voluntary drinking. CONCLUSIONS: Alcohol withdrawal in dependent mice causes changes in brain network organization that are attenuated by reaccess drinking. Olfactory brain regions, including the cortical amygdala, drive some of these changes and may play an important but underappreciated role in alcohol dependence.

Dorsal raphe neuroinflammation promotes dramatic behavioral stress dysregulation.

Impulsivity, risk-taking behavior, and elevated stress responsivity are prominent symptoms of mania, a behavioral state common to schizophrenia and bipolar disorder. Though inflammatory processes activated within the brain are involved in the pathophysiology of both disorders, the specific mechanisms by which neuroinflammation drives manic behavior are not well understood. Serotonin cell bodies originating within the dorsal raphe (DR) play a major role in the regulation of behavioral features characteristic of mania. Therefore, we hypothesized that the link between neuroinflammation and manic behavior may be mediated by actions on serotonergic neurocircuitry. To examine this, we induced local neuroinflammation in the DR by viral delivery of Cre recombinase into interleukin (IL)-1ß(XAT) transgenic male and female mice, resulting in overexpressing of the proinflammatory cytokine, IL-1ß. For assertion of brain-region specificity of these outcomes, the prefrontal cortex (PFC), as a downstream target of DR serotonergic projections, was also infused. Inflammation within the DR, but not the PFC, resulted in a profound display of manic-like behavior, characterized by increased stress-induced locomotion and responsivity, and reduced risk-aversion/fearfulness. Microarray analysis of the DR revealed a dramatic increase in immune-related genes, and dysregulation of genes important in GABAergic, glutamatergic, and serotonergic neurotransmission. Behavioral and physiological changes were driven by a loss of serotonergic neurons and reduced output as measured by high-performance liquid chromatography, demonstrating inflammation-induced serotonergic hypofunction. Behavioral changes were rescued by acute selective serotonin reuptake inhibitor treatment, supporting the hypothesis that serotonin dysregulation stemming from neuroinflammation in the DR underlies manic-like behaviors.

Reproductive neuroendocrine dysfunction in polycystic ovary syndrome: insight from animal models.

Polycystic ovary syndrome (PCOS) is a common endocrinopathy with elusive origins. A clinically heterogeneous disorder, PCOS is likely to have multiple etiologies comprised of both genetic and environmental factors. Reproductive neuroendocrine dysfunction involving increased frequency and amplitude of gonadotropin-releasing hormone (GnRH) release, as reflected by pulsatile luteinizing hormone (LH) secretion, is an important pathophysiologic component in PCOS. Whether this defect is primary or secondary to other changes in PCOS is unclear, but it contributes significantly to ongoing reproductive dysfunction. This review highlights recent work in animal models, with a particular emphasis on the mouse, demonstrating the ability of pre- and postnatal steroidal and metabolic factors to drive changes in GnRH/LH pulsatility and GnRH neuron function consistent with the observed abnormalities in PCOS. This work has begun to elucidate how a complex interplay of ovarian, metabolic, and neuroendocrine factors culminates in this syndrome.

Sex differences in corticotropin-releasing factor receptor-1 action within the dorsal raphe nucleus in stress responsivity.

BACKGROUND: Women are twice as likely as men to suffer from stress-related affective disorders. Corticotropin-releasing factor (CRF) is an important link between stress and mood, in part through its signaling in the serotonergic dorsal raphe (DR). Development of CRF receptor-1 (CRFr1) antagonists has been a focus of numerous clinical trials but has not yet been proven efficacious. We hypothesized that sex differences in CRFr1 modulation of DR circuits might be key determinants in predicting therapeutic responses and affective disorder vulnerability. METHODS: Male and female mice received DR infusions of the CRFr1 antagonist, NBI 35965, or CRF and were evaluated for stress responsivity. Sex differences in indices of neural activation (cFos) and colocalization of CRFr1 throughout the DR were examined. Whole-cell patch-clamp electrophysiology assessed sex differences in serotonin neuron membrane characteristics and responsivity to CRF. RESULTS: Males showed robust behavioral and hypothalamic-pituitary-adrenal axis responses to DR infusion of NBI 35965 and CRF, whereas females were minimally responsive. Sex differences were also found for both CRF-induced DR cFos and CRFr1 co-localization throughout the DR. Electrophysiologically, female serotonergic neurons showed blunted membrane excitability and divergent inhibitory postsynaptic current responses to CRF application. CONCLUSIONS: These studies demonstrate convincing sex differences in CRFr1 activity in the DR, where blunted female responses to NBI 35965 and CRF suggest unique stress modulation of the DR. These sex differences might underlie affective disorder vulnerability and differential sensitivity to pharmacologic treatments developed to target the CRF system, thereby contributing to a current lack of CRFr1 antagonist efficacy in clinical trials.

Regulation of gonadotropin-releasing hormone neurons by glucose.

Reproduction is influenced by energy balance, but the physiological pathways mediating their relationship have not been fully elucidated. As the central regulators of fertility, gonadotropin-releasing hormone (GnRH) neurons integrate numerous physiological signals, including metabolic cues. Circulating glucose levels regulate GnRH release and might in part mediate the effects of negative energy balance on fertility. Existing evidence suggests that neural pathways originating in the hindbrain, as well as in the hypothalamic feeding nuclei, transmit information concerning glucose availability to GnRH neurons. Here we review recent evidence suggesting that GnRH neurons might directly sense changes in glucose availability by a mechanism involving AMP-activated protein kinase. These findings expand our understanding of how metabolic signaling in the brain regulates reproduction.

Glucosensing by GnRH neurons: inhibition by androgens and involvement of AMP-activated protein kinase.

GnRH neurons integrate steroidal and metabolic cues to regulate fertility centrally. Central glucoprivation reduces LH secretion, which is governed by GnRH release, suggesting GnRH neuron activity is modulated by glucose availability. Here we tested whether GnRH neurons can sense changes in extracellular glucose, and whether glucosensing is altered by the steroids dihydrotestosterone (DHT) and/or estradiol (E). Extracellular recordings were made from GnRH neurons in brain slices from ovariectomized (OVX) mice ± DHT and/or E implants. Firing rate was reduced by a switch from 4.5 to 0.2 mm glucose in cells from OVX, OVX+E, and OVX+DHT+E mice, but not OVX+DHT mice. This suggests that androgens reduce the sensitivity of GnRH neurons to changes in extracellular glucose, but E mitigates this effect. Next we investigated potential mechanisms. In the presence of the ATP-sensitive potassium channel antagonist tolbutamide, glucosensing persisted. In contrast, glucosensing was attenuated in the presence of compound C, an antagonist of AMP-activated protein kinase (AMPK), suggesting a role for AMPK in glucosensing. The AMPK activator N1-(b-D-ribofuranosyl)-5-aminoimidazole-4-carboxamide (AICAR) mimicked the effect of low glucose and was less effective in cells from DHT-treated mice. The effect of DHT to diminish responses to low glucose and AICAR was abolished by blockade of fast synaptic transmission. Both AICAR and low glucose activated a current with a reversal potential near -50 mV, suggesting a nonspecific cation current. These studies indicate that glucosensing is one mechanism by which GnRH neurons sense fuel availability and point to a novel role for AMPK in the central regulation of fertility.

Prenatal androgenization of female mice programs an increase in firing activity of gonadotropin-releasing hormone (GnRH) neurons that is reversed by metformin treatment in adulthood.

Prenatal androgenization (PNA) of female mice with dihydrotestosterone programs reproductive dysfunction in adulthood, characterized by elevated luteinizing hormone levels, irregular estrous cycles, and central abnormalities. Here, we evaluated activity of GnRH neurons from PNA mice and the effects of in vivo treatment with metformin, an activator of AMP-activated protein kinase (AMPK) that is commonly used to treat the fertility disorder polycystic ovary syndrome. Estrous cycles were monitored in PNA and control mice before and after metformin administration. Before metformin, cycles were longer in PNA mice and percent time in estrus lower; metformin normalized cycles in PNA mice. Extracellular recordings were used to monitor GnRH neuron firing activity in brain slices from diestrous mice. Firing rate was higher and quiescence lower in GnRH neurons from PNA mice, demonstrating increased GnRH neuron activity. Metformin treatment of PNA mice restored firing activity and LH to control levels. To assess whether AMPK activation contributed to the metformin-induced reduction in GnRH neuron activity, the AMPK antagonist compound C was acutely applied to cells. Compound C stimulated cells from metformin-treated, but not untreated, mice, suggesting that AMPK was activated in GnRH neurons, or afferent neurons, in the former group. GnRH neurons from metformin-treated mice also showed a reduced inhibitory response to low glucose. These studies indicate that PNA causes enhanced firing activity of GnRH neurons and elevated LH that are reversible by metformin, raising the possibility that central AMPK activation by metformin may play a role in its restoration of reproductive cycles in polycystic ovary syndrome.

Prenatal androgen exposure programs metabolic dysfunction in female mice.

Polycystic ovary syndrome (PCOS) is a common fertility disorder with metabolic sequelae. Our laboratory previously characterized reproductive phenotypes in a prenatally androgenized (PNA) mouse model for PCOS. PNA mice exhibited elevated testosterone and LH levels, irregular estrous cycles, and neuroendocrine abnormalities suggesting increased central drive to the reproductive system. In this study, we examined metabolic characteristics of female PNA mice. PNA mice exhibited increased fasting glucose and impaired glucose tolerance (IGT) that were independent of age and were not associated with changes in body composition or peripheral insulin sensitivity. IGT was associated with defects in pancreatic islet function leading to an impaired response to high glucose, consistent with impaired insulin secretion. Exposure of isolated pancreatic islets to androgen in vitro demonstrated an impaired response to glucose stimulation similar to that in PNA mice, suggesting androgens may have activational in addition to organizational effects on pancreatic islet function. PNA mice also exhibited increased size of visceral adipocytes, suggesting androgen-programed differences in adipocyte differentiation and/or function. These studies demonstrate that in addition to causing reproductive axis abnormalities, in utero androgen exposure can induce long-term metabolic alterations in female mice.