Neuroanatomical distribution of fluorophores within adult RXFP3 Cre-tdTomato/YFP mouse brain.

Relaxin-family peptide 3 receptor (RXFP3) is activated by relaxin-3 in the brain to influence arousal and related functions, such as feeding and stress responses. Two transgenic mouse lines have recently been developed that co-express different fluorophores within RXFP3-expressing neurons: either yellow fluorescent protein (YFP; RXFP3-Cre/YFP mice) or tdTomato (RXFP3-Cre/tdTomato mice). To date, the characteristics of neurons that express RXFP3-associated fluorophores in these mice have only been investigated in the bed nucleus of the stria terminalis and the hypothalamic arcuate nucleus. To better determine the utility of these fluorophore-expressing mice for further research, we characterised the neuroanatomical distribution of fluorophores throughout the brain of these mice and compared this to the published distribution of Rxfp3 mRNA (detected by in situ hybridisation) in wildtype mice. Coronal sections of RXFP3-Cre/YFP (n = 8) and RXFP3-Cre/tdTomato (n = 8) mouse brains were imaged, and the density of fluorophore-expressing cells within various brain regions/nuclei was qualitatively assessed. Comparisons with our previously reported RXFP3 mRNA distribution revealed that of 212 brain regions that contained either fluorophore or RXFP3 mRNA, approximately half recorded densities that were within two qualitative measurements of each other (on a 9-point scale), including hippocampal dentate gyrus and amygdala subregions. However, many brain areas with likely non-authentic, false-positive, or false-negative fluorophore expression were also detected, including the cerebellum. Therefore, this study provides a guide to which brain regions should be prioritized for future study of RXFP3 in these mice, to better understand the neuroanatomy and function of this intriguing, neuronal peptide receptor.

Cocaine and amphetamine regulated transcript (CART) mediates sex differences in binge drinking through central taste circuits.

The neuropeptide cocaine- and amphetamine-regulated transcript (CART) has been implicated in alcohol consumption and reward behaviours, yet mechanisms mediating these effects have yet to be identified. Using a transgenic CART knockout (KO) mouse line we uncovered a sexually dimorphic effect of CART in binge drinking, with male CART KO mice increasing intake, whilst female CART KO mice decreased their alcohol intake compared to controls. Female CART KO mice show greater sensitivity to bitter solutions that can be overshadowed through addition of a sweetener, implicating taste as a factor. Further we identify that this is not driven through peripherally circulating sex hormones, but the central nucleus of the amygdala (CeA) is a locus where CART contributes to the regulation of alcohol consumption, with CeA CART neutralisation specifically reducing plain alcohol, but not sweetened alcohol consumption in female mice. These findings may have implications for the development of sex-specific treatment options for alcohol use disorders through targeting the CART system.

A paraventricular thalamus to insular cortex glutamatergic projection gates "emotional" stress-induced binge eating in females.

It is well-established that stress and negative affect trigger eating disorder symptoms and that the brains of men and women respond to stress in different ways. Indeed, women suffer disproportionately from emotional or stress-related eating, as well as associated eating disorders such as binge eating disorder. Nevertheless, our understanding of the precise neural circuits driving this maladaptive eating behavior, particularly in women, remains limited. We recently established a clinically relevant model of 'emotional' stress-induced binge eating whereby only female mice display binge eating in response to an acute "emotional" stressor. Here, we combined neuroanatomic, transgenic, immunohistochemical and pathway-specific chemogenetic approaches to investigate whole brain functional architecture associated with stress-induced binge eating in females, focusing on the role of Vglut2 projections from the paraventricular thalamus (PVTVglut2+) to the medial insular cortex in this behavior. Whole brain activation mapping and hierarchical clustering of Euclidean distances revealed distinct patterns of coactivation unique to stress-induced binge eating. At a pathway-specific level, PVTVglut2+ cells projecting to the medial insular cortex were specifically activated in response to stress-induced binge eating. Subsequent chemogenetic inhibition of this pathway suppressed stress-induced binge eating. We have identified a distinct PVTVglut2+ to insular cortex projection as a key driver of "emotional" stress-induced binge eating in female mice, highlighting a novel circuit underpinning this sex-specific behavior.

Organisation of enkephalin inputs and outputs of the central nucleus of the amygdala in mice.

The central nucleus of the amygdala (CeA) is a key hub integrating sensory inputs and modulating behavioural outputs. The CeA is a complex structure with discrete subdivisions, high peptidergic heterogeneity and broad CNS afferent and efferent projections. While several neuropeptide systems within the CeA have been examined in detail, less is known about CeA preproenkephalin (ppENK) cells. Here, we used a recently developed transgenic Penk-Cre mouse line to advance our understanding of the efferent and afferent connectivity of ppENK in the CeA. First, to determine the fidelity of Cre expression in Penk-Cre transgenic mice, we conducted RNAscope in the CeA of Penk-Cre mice. Our analysis revealed that 96.6 % of CeA Cre+ neurons co-expressed pENK mRNA, and 99.7 % of CeA pENK+ neurons co-expressed Cre mRNA, indicating faithful recapitulation of Cre expression in CeA ppENK-expressing cells, supporting the fidelity of the Penk-Cre reporter mouse. Anterograde tracing of CeAPenk cells showed strong efferent projections to the extended amygdala, midbrain and hindbrain PBN and NTS. Retrograde tracing of Penk afferents to the CeA were more restricted, with primary innervation originating within the amygdala complex and bed nucleus of the stria terminalis, and minor innervation from the parabrachial nucleus and nucleus of the solitary tract. Together, our data provide a comprehensive map of ENKergic efferent and afferent connectivity of the CeA in Penk-Cre mice. Further, we highlight both the utility and limitations of the Penk-Cre mice to study the function of CeA, PBN and NTS ppENK cells.

The Impact of Removal of Ovarian Hormones on Cholinergic Muscarinic Receptors: Examining Prepulse Inhibition and Receptor Binding.

Ovarian hormones, such as estrogens and progesterone, are known to exert beneficial effects on cognition and some psychiatric disorders. The basis of these effects is not fully understood, but may involve altered cholinergic neurotransmission. This study aimed to investigate how a lack of ovarian hormones would impact muscarinic receptor-induced deficits in prepulse inhibition (PPI) and muscarinic receptor density in several brain regions. Adult female rats were either ovariectomized, to remove the source of ovarian hormones, or left intact (sham-operated). PPI is a measure of sensorimotor gating that is typically impaired in schizophrenia patients, and similar deficits can be induced in rats by administering scopolamine, a muscarinic receptor antagonist. Our results revealed no significant effects of ovariectomy on PPI after saline or scopolamine treatment. Autoradiography was performed to measure cholinergic muscarinic receptor binding density using [3H]-pirenzepine, [3H]-AF-DX, and [3H]-4-DAMP, to label M1, M2/M4, and M3 receptors, respectively. We examined the amygdala, caudate putamen, dorsal hippocampus, motor cortex, retrosplenial cortex, and ventromedial hypothalamus. There were no significant group differences in any region for any muscarinic receptor type. These results suggest that removing peripheral ovarian hormones does not influence the cholinergic muscarinic receptor system in the context of PPI or receptor binding density.

Differential Level of RXFP3 Expression in Dopaminergic Neurons Within the Arcuate Nucleus, Dorsomedial Hypothalamus and Ventral Tegmental Area of RXFP3-Cre/tdTomato Mice.

RXFP3 (relaxin-family peptide 3 receptor) is the cognate G-protein-coupled receptor for the neuropeptide, relaxin-3. RXFP3 is expressed widely throughout the brain, including the hypothalamus, where it has been shown to modulate feeding behavior and neuroendocrine activity in rodents. In order to better characterize its potential mechanisms of action, this study determined whether RXFP3 is expressed by dopaminergic neurons within the arcuate nucleus (ARC) and dorsomedial hypothalamus (DMH), in addition to the ventral tegmental area (VTA). Neurons that express RXFP3 were visualized in coronal brain sections from RXFP3-Cre/tdTomato mice, which express the tdTomato fluorophore within RXFP3-positive cells, and dopaminergic neurons in these areas were visualized by simultaneous immunohistochemical detection of tyrosine hydroxylase-immunoreactivity (TH-IR). Approximately 20% of ARC neurons containing TH-IR coexpressed tdTomato fluorescence, suggesting that RXFP3 can influence the dopamine pathway from the ARC to the pituitary gland that controls prolactin release. The ability of prolactin to reduce leptin sensitivity and increase food consumption therefore represents a potential mechanism by which RXFP3 activation influences feeding. A similar proportion of DMH neurons containing TH-IR expressed RXFP3-related tdTomato fluorescence, consistent with a possible RXFP3-mediated regulation of stress and neuroendocrine circuits. In contrast, RXFP3 was barely detected within the VTA. TdTomato signal was absent from the ARC and DMH in sections from Rosa26-tdTomato mice, suggesting that the cells identified in RXFP3-Cre/tdTomato mice expressed authentic RXFP3-related tdTomato fluorescence. Together, these findings identify potential hypothalamic mechanisms through which RXFP3 influences neuroendocrine control of metabolism, and further highlight the therapeutic potential of targeting RXFP3 in feeding-related disorders.

A model of emotional stress-induced binge eating in female mice with no history of food restriction.

Overeating is a major contributing factor to obesity and related health complications. For women, in particular, negative emotions such as stress strongly influence eating behavior and bingeing episodes. Modeling this type of binge eating in rodents presents challenges: firstly, stress-induced anorexia is commonly observed in rodents therefore a mild stressor is required in order to observe an orexigenic effect. Second, many studies report using calorie restriction to observe the required behavior; yet this does not necessarily reflect the human condition. Thus, the aim of this study was to develop a model of emotional stress-induced bingeing independent of caloric restriction. Female and male C57BL/6J mice were divided into ad libitum (n = 20 per sex) and food-restricted (n = 20 per sex) groups which were both further split into a control group and a group exposed to frustration stress (n = 10 per group). All mice were provided intermittent access to a highly palatable food in 2 cycles. At the end of each cycle the stress group was subjected to a 15-minute frustration episode where highly palatable food was within the home cage but inaccessible. Both groups were then given free access for 15 minutes. Frustrated female mice from the ad libitum displayed binge-like behavior compared with controls (P = .0001). Notably, this behavior was absent in males. Ovariectomy had no impact on binge-like behavior. Collectively, these data validate a novel model of emotional stress-induced binge eating specific to female mice which does not require caloric restriction and is not driven by ovarian hormones.

Phenotyping neurons activated in the mouse brain during restoration of salt debt.

Salt overconsumption contributes to hypertension, which is a major risk factor for stroke, heart and kidney disease. Characterising neuronal pathways that may control salt consumption is therefore important for developing novel approaches for reducing salt overconsumption. Here, we identify neurons within the mouse central amygdala (CeA), lateral parabrachial nucleus (LPBN), intermediate nucleus of the solitary tract (iNTS), and caudal NTS (cNTS) that are activated and display Fos immunoreactivity in mice that have consumed salt in order to restore a salt debt, relative to salt replete and salt depleted controls. Double-label immunohistochemical studies revealed that salt restoring mice had significantly greater densities of activated enkephalin neurons within the CeA and iNTS, while statistically significant changes within the LPBN and cNTS were not observed. Furthermore, within the CeA, restoration of salt debt conferred a significant increase in the density of activated calretinin neurons, while there was no change relative to control groups in the density of activated neurons that co-expressed protein kinase C delta (PKC-δ). Taken together, these studies highlight the importance of opioid systems within the CeA and iNTS in neuronal processes associated with salt restoration, and may aid the development of future pharmacological and other strategies for reducing salt overconsumption.

Characterization of the relaxin family peptide receptor 3 system in the mouse bed nucleus of the stria terminalis.

The bed nucleus of the stria terminalis (BNST) is a critical node involved in stress and reward-related behaviors. Relaxin family peptide receptor 3 (RXFP3) signaling in the BNST has been implicated in stress-induced alcohol seeking behavior. However, the neurochemical phenotype and connectivity of BNST RXFP3-expressing (RXFP3+) cells have yet to be elucidated. We interrogated the molecular signature and electrophysiological properties of BNST RXFP3+ neurons using a RXFP3-Cre reporter mouse line. BNST RXFP3+ cells are circumscribed to the dorsal BNST (dBNST) and are neurochemically heterogeneous, comprising a mix of inhibitory and excitatory neurons. Immunohistochemistry revealed that ~48% of BNST RXFP3+ neurons are GABAergic, and a quarter of these co-express the calcium-binding protein, calbindin. A subset of BNST RXFP3+ cells (~41%) co-express CaMKIIα, suggesting this subpopulation of BNST RXFP3+ neurons are excitatory. Corroborating this, RNAscope® revealed that ~35% of BNST RXFP3+ cells express vVGluT2 mRNA, indicating a subpopulation of RXFP3+ neurons are glutamatergic. RXFP3+ neurons show direct hyperpolarization to bath application of a selective RXFP3 agonist, RXFP3-A2, while around 50% of cells were depolarised by exogenous corticotrophin releasing factor. In behaviorally naive mice the majority of RXFP3+ neurons were Type II cells exhibiting Ih and T type calcium mediated currents. However, chronic swim stress caused persistent plasticity, decreasing the proportion of neurons that express these channels. These studies are the first to characterize the BNST RXFP3 system in mouse and lay the foundation for future functional studies appraising the role of the murine BNST RXFP3 system in more complex behaviors.

The subfornical organ in sodium appetite: Recent insights.

To maintain sodium homeostasis, animals will readily seek and ingest salt when salt-depleted, even at concentrations that they typically find aversive when sodium replete. This innate behaviour is known as sodium (or salt) appetite. Salt appetite is subserved by a conserved brain network that senses sodium need and promotes the ingestion of salty substances when sodium-deficient. The subfornical organ (SFO) is a circumventricular organ that has diverse roles encompassing cardiovascular regulation, energy balance, immune responses, reproduction, and hydromineral balance. The SFO acts as a central sensor of sodium need and is essential for the generation of salt appetite. In this review, we discuss recent findings on the neurochemical and circuit-level organisation of the SFO in the context of sodium appetite. This article is part of the Special Issue entitled 'Hypothalamic Control of Homeostasis'.

Glucose Availability Predicts the Feeding Response to Ghrelin in Male Mice, an Effect Dependent on AMPK in AgRP Neurons.

Metabolic feedback from the periphery to the brain results from a dynamic physiologic fluctuation of nutrients and hormones, including glucose and fatty acids, ghrelin, leptin, and insulin. The specific interactions between humoral factors and how they influence feeding is largely unknown. We hypothesized that acute glucose availability may alter how the brain responds to ghrelin, a hormonal signal of energy availability. Acute glucose administration suppressed a range of ghrelin-induced behaviors as well as gene expression changes in hypothalamic neuropeptide Y (NPY) and agouti-related peptide (AgRP) neurons after ghrelin administration. Knockdown of the energy-sensing molecule AMP-activated protein kinase (AMPK) in AgRP neurons resulted in loss of the glucose effect, and mice responded as though pretreated with saline. Conversely, 2-deoxyglucose (2-DG), which decreases glucose availability, potentiated ghrelin-induced feeding and increased hypothalamic NPY mRNA levels. AMPK knockdown did not alter the additive effect of 2-DG and ghrelin on feeding. Our findings support the idea that computation of energy status is dynamic, is informed by multiple signals, and responds to acute fluctuations in metabolic state. These observations are broadly relevant to the investigation of neuroendocrine control of feeding and highlight the underappreciated complexity of control within these systems.

Investigational drugs for alcohol use disorders: a review of preclinical data.

INTRODUCTION: Alcohol use disorders (AUDs) are one of the leading causes of preventable death in the developed world. In the U.S., only three FDA-approved pharmacotherapies for AUDs currently exist, but at a population level they display poor efficacy, low compliance rates, and adverse side effects. Therefore, identifying novel neurobiological targets for pharmacological treatment of AUDs is of urgent concern. AREAS COVERED: We discuss recent preclinical data on investigational drugs that have been assessed for their therapeutic potential in AUDs. We focus on three neurobiological domains underlying AUDs: neuropeptide systems, neuroinflammatory/neuroimmune mediators, and epigenetic modifications. We iterate the therapeutic potential of ghrelin receptor antagonists, oxytocin, neurokinin 1 receptor antagonists, and glucagon-like peptide-1 receptor agonists. In the context of neuroinflammatory/neuroimmune modulators, we draw attention to P2X4 receptor positive allosteric modulators and phosphodiesterase inhibitors. Finally, we highlight the prospects of histone deacetylase inhibitors and DNA methyltransferases that modulate the dysregulated epigenetic landscape in alcohol dependence. EXPERT OPINION: We propose that several of the compounds discussed may be suitable to be repurposed for AUD treatment. We allude to the possibility of combined pharmacotherapy for AUDs and anticipate the efforts that must be enacted to advance the field of personalised medicine for the treatment of this devastating condition.

The intersection of stress and reward: BNST modulation of aversive and appetitive states.

The bed nucleus of the stria terminalis (BNST) is widely acknowledged as a brain structure that regulates stress and anxiety states, as well as aversive and appetitive behaviours. The diverse roles of the BNST are afforded by its highly modular organisation, neurochemical heterogeneity, and complex intrinsic and extrinsic circuitry. There has been growing interest in the BNST in relation to psychopathologies such as anxiety and addiction. Although research on the human BNST is still in its infancy, there have been extensive preclinical studies examining the molecular signature and hodology of the BNST and their involvement in stress and reward seeking behaviour. This review examines the neurochemical phenotype and connectivity of the BNST, as well as electrophysiological correlates of plasticity in the BNST mediated by stress and/or drugs of abuse.

Distribution of the orexin-1 receptor (OX1R) in the mouse forebrain and rostral brainstem: A characterisation of OX1R-eGFP mice.

We have utilised a transgenic reporter mouse in which green fluorescent protein (GFP) expression is driven by the orexin-1 receptor (OX1R) promoter to systematically map the distribution of OX1R-expressing neurons throughout the mouse forebrain and rostral brainstem. GFP labelling was observed in perikarya and fibres in an extensive range of brain loci encompassing the olfactory and cerebral cortices, dorsal and ventral pallidum, hippocampus, amygdaloid regions, septal areas, thalamic nuclei, hypothalamic nuclei and several brainstem regions, consistent with previous studies of OX1R mRNA expression. This is the first study to systematically characterise the neuroanatomical distribution of OX1R in the OX1R-eGFP mouse, confirming its veracity as a faithful reporter of OX1R expression and utility for future studies assessing the role of OX1R in more complex behaviours.