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Microvascular insulin delivery to myocytes is rate limiting for the onset of insulin-stimulated muscle glucose uptake. The structural integrity of capillaries of the microvasculature is regulated, in part, by a family of transmembrane adhesion receptors known as integrins, which are composed of an α and a β subunit. The integrin β1 (itgβ1) subunit is highly expressed in endothelial cells (ECs). EC itgβ1 is necessary for the formation of capillary networks during embryonic development, and its knockdown in adult mice blunts the reactive hyperemia that manifests during ischemia reperfusion. In this study, we investigated the contribution of EC itgβ1 in microcirculatory function and glucose uptake, with an emphasis on skeletal muscle. We hypothesized that loss of EC itgβ1 would impair microvascular hemodynamics and glucose uptake during insulin stimulation, creating "delivery"-mediated insulin resistance. An itgβ1 knockdown mouse model was developed to avoid the lethality of embryonic gene knockout and the deteriorating health resulting from early postnatal inducible gene deletion. We found that mice with (itgβ1fl/flSCLcre) and without (itgβ1fl/fl) inducible stem cell leukemia cre recombinase (SLCcre) expression at 10 days post cre induction have comparable exercise tolerance and pulmonary and cardiac functions. We quantified microcirculatory hemodynamics using intravital microscopy and the ability of mice to respond to the high metabolic demands of insulin-stimulated muscle using a hyperinsulinemic-euglycemia clamp. We show that itgβ1fl/flSCLcre mice compared with itgβ1fl/fl littermates have 1) deficits in capillary flow rate, flow heterogeneity, and capillary density; 2) impaired insulin-stimulated glucose uptake despite sufficient transcapillary insulin efflux; and 3) reduced insulin-stimulated glucose uptake due to perfusion-limited glucose delivery. Thus, EC itgβ1 is necessary for microcirculatory function and to meet the metabolic challenge of insulin stimulation. NEW & NOTEWORTHY: The microvasculature is an important site of resistance to muscle glucose uptake. We show that microvasculature integrins determine the exchange of glucose between the circulation and muscle. Specifically, a 30% reduction in the expression of endothelial integrin β1 subunit is sufficient to cause microcirculatory dysfunction and lead to insulin resistance. This emphasizes the importance of endothelial integrins in microcirculatory function and the importance of microcirculatory function for the ability of muscle to consume glucose. [ABSTRACT FROM AUTHOR]

Background: Alcohol use disorder (AUD) is a chronic, relapsing disease, highly comorbid with anxiety and depression. The bed nucleus of the stria terminalis (BNST) and Crh+ neurons in this region play a key role in chronic ethanol‐induced increases in volitional intake, hypothesized to be driven by emergent negative affective behaviors. Excitatory N‐methyl‐d‐aspartate receptors (NMDARs) are a major target of ethanol, and chronic ethanol exposure has been shown to regulate NMDAR function and expression. Specifically, GluN2D subunit‐containing NMDARs have emerged as a target of interest due to their limited distribution and potential roles in affective behavior. Methods: Male and female mice underwent a home cage chronic drinking forced abstinence model (CDFA) to assess the impact of 1 day or 2 weeks of ethanol abstinence on BNST synaptic transmission and BNST Grin gene expression. Constitutive and conditional BNST GluN2D knockout mice were used to assess the impact of GluN2D deletion on continuous access ethanol intake as well as negative affect behaviors, using the open field, elevated zero maze, and forced swim tasks. Results: We report here that excitatory transmission undergoes time‐dependent upregulation in BNST Crh+ cells. Further, knockdown of dorsal BNST (dBNST) GluN2D expression significantly decreases ethanol intake in female, but not male, mice. While BNST Grin2b expression was significantly increased in protracted abstinence following CDFA, no differences in Grin2d expression were observed in the dBNST or dBNST Crh+ neurons. Finally, we find that deletion of GluN2D fails to alter negative affect in ethanol‐naïve female mice. Conclusions: These data suggest a role for BNST GluN2D‐containing NMDARs in ethanol drinking but not ethanol abstinence, highlighting sex differences and behavioral specificity. Overall, these data further suggest roles for BNST synaptic signaling in volitional ethanol intake that are partially independent of actions on affective behavior. [ABSTRACT FROM AUTHOR]

Metabolic state must be communicated to the brain to regulate behavior, growth, nutrient partitioning and development to meet energetic demands. While it is clearly evident that reproduction and energy balance are closely intertwined, the neural circuits integrating these two processes are poorly understood. Recent functional data suggests the melanocortin 3 receptor (MC3R) mediates the exchange of information between reproductive and metabolic state. Additionally, females exhibit uniquely aberrant metabolic and reproductive phenotypes after deletion of MC3R. In this work, we aimed to characterize the role of MC3R neural circuits in linking metabolic state with reproduction and growth in females. We found that MC3R is expressed in brain regions known to have a role in the control of reproductive and metabolic state, including the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARH). In the ARH of female mice, significant differences in overall levels of Mc3r mRNA were not observed at postnatal day 16 (P16), P28 or P48, nor did the percentage of ARH Kiss1, Esr1, Sst, Pomc or Agrp neurons doubly-labeled for Mc3r appear to change during postnatal development. However, the percentage of ARH Ghrh neurons labeled for Mc3r significantly increased from P16 to P48. Thus, MC3R in the ARH may play a potential role in conveying metabolic signals critical for growth during development. In the AVPV of female mice, overall abundance of Mc3r mRNA was low at P16, but increased significantly by P28, and was further elevated in mature mice (P48). Additionally, the percentage of Kiss1-and Esr1- expressing neurons in the AVPV labeled for Mc3r increased significantly between P16 and P48, suggesting that Mc3r may have an important role in the timing of sexual maturation. In addition to the direct regulatory action of MC3R expressed in KISS1 neurons in the AVPV, the impact of MC3R signaling may also be transmitted transynaptically via direct neural projections from MC3R neurons to KISS1 neurons in the AVPV, or to GnRH neurons in the rostral preoptic area. Clearly labeled MC3R terminals were observed in close apposition to both KISS1 neurons in the AVPV, and GnRH neurons in the rostral medial preoptic area. Although both the GnRH and KISS1 neuronal populations appeared heavily innervated by MC3R neurons, the density of MC3R terminals onto KISS1 neurons appeared to be substantially greater than that onto GnRH neurons, suggesting that inputs to AVPV neurons may be particularly impactful on reproduction. Thus, MC3R may not only regulate the activity of KISS1 neurons in the AVPV directly, but may also function transynaptically as an important neurobiological substrate for coordination of metabolic state with sexual maturation and reproductive function. [ABSTRACT FROM AUTHOR]

Microvascular insulin delivery to myocytes is rate limiting for the onset of insulin-stimulated muscle glucose uptake. The structural integrity of capillaries of the microvasculature is regulated, in part, by a family of transmembrane adhesion receptors known as integrins, which are composed of an α and β subunit. The integrin β1 (itgβ1) subunit is highly expressed in endothelial cells (EC). EC itgβ1 is necessary for the formation of capillary networks during embryonic during development and its knockdown in adult mice blunts the reactive hyperemia that manifests during ischemia reperfusion. In this study we investigated the contribution of skeletal muscle EC itgβ1 in microcirculatory function and glucose uptake. We hypothesized that loss of EC itgβ1 would impair microvascular hemodynamics and glucose uptake during insulin stimulation, creating 'delivery'-mediated insulin resistance. An itgβ1 knockdown mouse model was developed to avoid lethality of embryonic gene knockout and the deteriorating health resulting from early post-natal inducible gene deletion. We found that mice with (itgβ1 fl/fl SCLcre) and without (itgβ1 fl/fl ) inducible stem cell leukemia cre recombinase (SLCcre) expression at 10 days post cre induction have comparable exercise tolerance and pulmonary and cardiac functions. We quantified microcirculatory hemodynamics using intravital microscopy and the ability of mice to respond to the high metabolic demands of insulin-stimulated muscle using a hyperinsulinemic-euglycemia clamp. We show that itgβ1 fl/fl SCLcre mice compared to itgβ1 fl/fl littermates have, i) deficits in capillary flow rate, flow heterogeneity, and capillary density; ii) impaired insulin-stimulated glucose uptake despite sufficient transcapillary insulin efflux; and iii) reduced insulin-stimulated glucose uptake due to perfusion-limited glucose delivery. Thus, EC itgβ1 is necessary for microcirculatory function and to meet the metabolic challenge of insulin stimulation.

Nutritional fluctuations that occur early in life dictate metabolic adaptations that will affect susceptibility to weight gain and obesity later in life. The postnatal period in mice represents a time of dynamic changes in hypothalamic development and maternal consumption of a high fat diet during the lactation period (MHFD) changes the composition of milk and leads to enhanced susceptibility to obesity in offspring. Agouti-related peptide (AgRP) neurons in the arcuate nucleus of the hypothalamus (ARH) react to changes in multiple metabolic signals and distribute neuroendocrine information to other brain regions, such as the paraventricular hypothalamic nucleus (PVH), which is known to integrate a variety of signals that regulate body weight. Development of neural projections from AgRP neurons to the PVH occurs during the lactation period and these projections are reduced in MHFD offspring, but underlying developmental mechanisms remain largely unknown. Microglia are the resident immune cells of the central nervous system and are involved in refinement of neural connections and modulation of synaptic transmission. Because high fat diet exposure causes activation of microglia in adults, a similar activation may occur in offspring exposed to MHFD and play a role in sculpting hypothalamic feeding circuitry. Genetically targeted axonal labeling and immunohistochemistry were used to visualize AgRP axons and microglia in postnatal mice derived from MHFD dams and morphological changes quantified. The results demonstrate regionally localized changes to microglial morphology in the PVH of MHFD offspring that suggest enhanced surveillance activity and are temporally restricted to the period when AgRP neurons innervate the PVH. In addition, axon labeling experiments confirm a significant decrease in AgRP innervation of the PVH in MHFD offspring and provide direct evidence of synaptic pruning of AgRP inputs to the PVH. Microglial depletion with the Colony-stimulating factor 1 receptor inhibitor PLX5622 determined that the decrease in AgRP innervation observed in MHFD offspring is dependent on microglia, and that microglia are required for weight gain that emerges as early as weaning in offspring of MHFD dams. However, these changes do not appear to be dependent on the degree of microglial mediated synaptic pruning. Together, these findings suggest that microglia are activated by exposure to MHFD and interact directly with AgRP axons during development to permanently alter their density, with implications for developmental programming of metabolic phenotype., Competing Interests: Competing Interest Statement: No competing interests

Alcohol use disorder (AUD) is a chronic, relapsing disease, highly comorbid with anxiety and depression. The bed nucleus of the stria terminalis (BNST), and Crh + neurons in this region are thought to play a key role in chronic ethanol-induced increases in volitional ethanol intake. This role has been hypothesized to be driven by emergent BNST-dependent negative affective behaviors. Indeed, we report here that in female mice undergoing a home cage chronic drinking forced abstinence model (CDFA), excitatory transmission undergoes time-dependent upregulation in BNST Crh + cells. Excitatory NMDA receptors (NMDARs) are a major target of ethanol, and chronic ethanol exposure has been shown to regulate NMDAR function and expression. GluN2D subunit-containing NMDARs have emerged as a target of interest due to their limited distribution and potential roles in affective behavior. We find that knockdown of dorsal BNST (dBNST) GluN2D expression significantly decreases ethanol intake in female, but not male, mice. While BNST Grin2b expression was significantly increased in protracted abstinence following CDFA, no differences in Grin2d expression were observed in dBNST or specifically in dBNST Crh + neurons. Finally, to determine the impact of GluN2D expression on negative affective behaviors, open field, elevated zero maze, and forced swim tasks were used to measure anxiety- and depressive-like behaviors in constitutive and conditional BNST GluN2D knockout mice. Surprisingly, we find that deletion of GluN2D fails to alter negative affect in ethanol-naïve female mice. Together, these data suggest a role for BNST GluN2D-containing NMDARs in ethanol drinking behaviors but not abstinence from ethanol, highlighting potential sex differences and behavioral specificity in the context of AUD behaviors. Overall, these data further suggest roles for BNST synaptic signaling in volitional ethanol intake that are partially independent of actions on affective behavior.

α 2a -adrenergic receptor (α 2a -AR) agonists are candidate substance use disorder therapeutics due to their ability to recruit noradrenergic autoreceptors to dampen stress system engagement. However, we recently found that postsynaptic α 2a -ARs are required for stress-induced reinstatement of cocaine-conditioned behavior. Understanding the ensembles recruited by these postsynaptic receptors (heteroceptors) is necessary to understand noradrenergic circuit control. We utilized a variety of approaches in FosTRAP (Targeted Recombination in Active Populations) mice to define an ensemble of cells activated by the α 2a -AR partial agonist guanfacine ("Guansembles") in the bed nucleus of the stria terminalis (BST/BNST), a region key to stress-induced reinstatement of drug seeking. We define BNST "Guansembles" and show they differ from restraint stress-activated cells. Guanfacine produced inhibition of cAMP-dependent signaling in Guansembles, while chronic restraint stress increased cAMP-dependent signaling. Guanfacine both excited and inhibited aspects of Guansemble neuronal activity. Further, while some stressors produced overall reductions in Guansemble activity, active coping events during restraint stress and exposure to unexpected shocks were both associated with Guansemble recruitment. Using viral tracing, we define a BNST Guansemble afferent network that includes regions involved in the interplay of stress and homeostatic functions. Finally, we show that activation of Guansembles produces alterations in behavior on the elevated plus maze consistent with task-specific anxiety-like behavior. Overall, we define a population of BNST neurons recruited by α 2a -AR signaling that opposes the behavioral action of canonical autoreceptor α 2a -AR populations and which are differentially recruited by distinct stressors. Moreover, we demonstrate stressor-specific physiological responses in a specific neuronal population., (© 2022. The Author(s), under exclusive licence to American College of Neuropsychopharmacology.)