Glucoregulation and coping behavior after chronic stress in rats: Sex differences across the lifespan.

The purpose of the current study was to determine how biological sex shapes behavioral coping and metabolic health across the lifespan after chronic stress. We hypothesized that examining chronic stress-induced behavioral and endocrine outcomes would reveal sex differences in the biological basis of susceptibility. During late adolescence, male and female Sprague-Dawley rats experienced chronic variable stress (CVS). Following completion of CVS, all rats experienced a forced swim test (FST) followed 3 days later by a fasted glucose tolerance test (GTT). The FST was used to determine coping in response to a stressor. Endocrine metabolic function was evaluated in the GTT by measuring glucose and corticosterone, the primary rodent glucocorticoid. Rats then aged to 15 months when the FST and GTT were repeated. In young rats, chronically stressed females exhibited more passive coping and corticosterone release in the FST. Additionally, chronically stressed females had elevated corticosterone and impaired glucose clearance in the GTT. Aging affected all measurements as behavioral and endocrine outcomes were sex specific. Furthermore, regression analysis between hormonal and behavioral responses identified associations depending on sex and stress. Collectively, these data indicate increased female susceptibility to the effects of chronic stress during adolescence. Further, translational investigation of coping style and glucose homeostasis may identify biomarkers for stress-related disorders.

Chemogenetic Inhibition of Infralimbic Prefrontal Cortex GABAergic Parvalbumin Interneurons Attenuates the Impact of Chronic Stress in Male Mice.

Hypofunction of the prefrontal cortex (PFC) contributes to stress-related neuropsychiatric illnesses. Mechanisms leading to prefrontal hypoactivity remain to be determined. Prior evidence suggests that chronic stress leads to an increase in activity of parvalbumin (PV) expressing GABAergic interneurons (INs) in the PFC. The purpose of the study was to determine whether reducing PV IN activity in the Infralimbic (IL) PFC would prevent stress-related phenotypes. We used a chemogenetic approach to inhibit IL PFC PV INs during stress. Mice were first tested in the tail suspension test (TST) to determine the impact of PV IN inhibition on behavioral responses to acute stress. The long-term impact of PV IN inhibition during a modified chronic variable stress (CVS) was tested in the forced swim test (FST). Acute PV IN inhibition reduced active (struggling) and increased passive coping behaviors (immobility) in the TST. In contrast, inhibition of PV INs during CVS increased active and reduced passive coping behaviors in the FST. Moreover, chronic inhibition of PV INs attenuated CVS-induced changes in Fos expression in the prelimbic cortex (PrL), basolateral amygdala (BLA), and ventrolateral periaqueductal gray (vlPAG) and also attenuated adrenal hypertrophy and body weight loss associated with chronic stress. Our results suggest differential roles of PV INs in acute versus chronic stress, indicative of distinct biological mechanisms underlying acute versus chronic stress responses. Our results also indicate a role for PV INs in driving chronic stress adaptation and support literature evidence suggesting cortical GABAergic INs as a therapeutic target in stress-related illnesses.

Conditional deletion of glucocorticoid receptors in rat brain results in sex-specific deficits in fear and coping behaviors.

Glucocorticoid receptors (GR) have diverse functions relevant to maintenance of homeostasis and adaptation to environmental challenges. Understanding the importance of tissue-specific GR function in physiology and behavior has been hampered by near-ubiquitous localization in brain and body. Here we use CRISPR/Cas9 gene editing to create a conditional GR knockdown in Sprague Dawley rats. To test the impact of cell- and region-specific GR knockdown on physiology and behavior, we targeted GR knockdown to output neurons of the prelimbic cortex. Prelimbic knockdown of GR in females caused deficits in acquisition and extinction of fear memory during auditory fear conditioning, whereas males exhibited enhanced active-coping behavior during forced swim. Our data support the utility of this conditional knockdown rat to afford high-precision knockdown of GR across a variety of contexts, ranging from neuronal depletion to circuit-wide manipulations, leveraging the behavioral tractability and enhanced brain size of the rat as a model organism.

Enhanced Glucose Control Following Vertical Sleeve Gastrectomy Does Not Require a ß-Cell Glucagon-Like Peptide 1 Receptor.

Bariatric surgeries, including vertical sleeve gastrectomy (VSG), resolve diabetes in 40-50% of patients. Studies examining the molecular mechanisms underlying this effect have centered on the role of the insulinotropic glucagon-like peptide 1 (GLP-1), in great part because of the ∼10-fold rise in its circulating levels after surgery. However, there is currently debate over the role of direct ß-cell signaling by GLP-1 to mediate improved glucose tolerance following surgery. In order to assess the importance of ß-cell GLP-1 receptor (GLP-1R) for improving glucose control after VSG, a mouse model of this procedure was developed and combined with a genetically modified mouse line allowing an inducible, ß-cell-specific Glp1r knockdown (Glp1rß-cell-ko). Mice with VSG lost ∼20% of body weight over 30 days compared with sham-operated controls and had a ∼60% improvement in glucose tolerance. Isolated islets from VSG mice had significantly greater insulin responses to glucose than controls. Glp1r knockdown in ß-cells caused glucose intolerance in diet-induced obese mice compared with obese controls, but VSG improved glycemic profiles to similar levels during oral and intraperitoneal glucose challenges in Glp1rß-cell-ko and Glp1rWT mice. Therefore, even though the ß-cell GLP-1R seems to be important for maintaining glucose tolerance in obese mice, in these experiments it is dispensable for the improvement in glucose tolerance after VSG. Moreover, the metabolic physiology activated by VSG can overcome the deficits in glucose regulation caused by lack of ß-cell GLP-1 signaling in obesity.

Deletion of Glucocorticoid Receptors in Forebrain GABAergic Neurons Alters Acute Stress Responding and Passive Avoidance Behavior in Female Mice.

The glucocorticoid receptor (GR) is critically involved in regulation of stress responses [inhibition of the hypothalamic-pituitary-adrenal (HPA) axis], emotional behavior and cognition via interactions with forebrain corticolimbic circuity. Work to date has largely focused on GR actions in forebrain excitatory neurons; however, recent studies suggest a potential role mediated by interneurons. Here, we targeted GR deletion in forebrain GABAergic neurons, including the cortical interneurons, using a Dlx5/6-Cre driver line to test the role of forebrain interneuronal GR in HPA axis regulation and behavior. Our data indicate that GR deletion in GABAergic neurons causes elevated corticosterone stress responsiveness and decreased cross-over latencies in a passive avoidance task in females, but not males. Dlx5/6-Cre driven gene deletion caused loss of GR in interneurons in the prefrontal cortex (PFC) and hippocampus, but also in select diencephalic GABAergic neurons (including the reticular thalamic nucleus and dorsomedial hypothalamus). Our data suggest that GR signaling in interneurons is differentially important in females, which may have implications for GR-directed therapies for stress-related affective disease states.

Desacyl Ghrelin Decreases Anxiety-like Behavior in Male Mice.

Ghrelin is a 28-amino acid polypeptide that regulates feeding, glucose metabolism, and emotionality (stress, anxiety, and depression). Plasma ghrelin circulates as desacyl ghrelin (DAG) or, in an acylated form, acyl ghrelin (AG), through the actions of ghrelin O-acyltransferase (GOAT), exhibiting low or high affinity, respectively, for the growth hormone secretagogue receptor (GHSR) 1a. We investigated the role of endogenous AG, DAG, and GHSR1a signaling on anxiety and stress responses using ghrelin knockout (Ghr KO), GOAT KO, and Ghsr stop-floxed (Ghsr null) mice. Behavioral and hormonal responses were tested in the elevated plus maze and light/dark (LD) box. Mice lacking both AG and DAG (Ghr KO) increased anxiety-like behaviors across tests, whereas anxiety reactions were attenuated in DAG-treated Ghr KO mice and in mice lacking AG (GOAT KO). Notably, loss of GHSR1a (Ghsr null) did not affect anxiety-like behavior in any test. Administration of AG and DAG to Ghr KO mice with lifelong ghrelin deficiency reduced anxiety-like behavior and decreased phospho-extracellular signal-regulated kinase phosphorylation in the Edinger-Westphal nucleus in wild-type mice, a site normally expressing GHSR1a and involved in stress- and anxiety-related behavior. Collectively, our data demonstrate distinct roles for endogenous AG and DAG in regulation of anxiety responses and suggest that the behavioral impact of ghrelin may be context dependent.

Disruption of Glucagon-Like Peptide 1 Signaling in Sim1 Neurons Reduces Physiological and Behavioral Reactivity to Acute and Chronic Stress.

Organismal stress initiates a tightly orchestrated set of responses involving complex physiological and neurocognitive systems. Here, we present evidence for glucagon-like peptide 1 (GLP-1)-mediated paraventricular hypothalamic circuit coordinating the global stress response. The GLP-1 receptor (Glp1r) in mice was knocked down in neurons expressing single-minded 1, a transcription factor abundantly expressed in the paraventricular nucleus (PVN) of the hypothalamus. Mice with single-minded 1-mediated Glp1r knockdown had reduced hypothalamic-pituitary-adrenal axis responses to both acute and chronic stress and were protected against weight loss associated with chronic stress. In addition, regional Glp1r knockdown attenuated stress-induced cardiovascular responses accompanied by decreased sympathetic drive to the heart. Finally, Glp1r knockdown reduced anxiety-like behavior, implicating PVN GLP-1 signaling in behavioral stress reactivity. Collectively, these findings support a circuit whereby brainstem GLP-1 activates PVN signaling to mount an appropriate whole-organism response to stress. These results raise the possibility that dysfunction of this system may contribute to stress-related pathologies, and thereby provide a novel target for intervention. SIGNIFICANCE STATEMENT: Dysfunctional stress responses are linked to a number of somatic and psychiatric diseases, emphasizing the importance of precise neuronal control of effector pathways. Pharmacological evidence suggests a role for glucagon-like peptide-1 (GLP-1) in modulating stress responses. Using a targeted knockdown of the GLP-1 receptor in the single-minded 1 neurons, we show dependence of paraventricular nucleus GLP-1 signaling in the coordination of neuroendocrine, autonomic, and behavioral responses to acute and chronic stress. To our knowledge, this is the first direct demonstration of an obligate brainstem-to-hypothalamus circuit orchestrating general stress excitation across multiple effector systems. These findings provide novel information regarding signaling pathways coordinating central control of whole-body stress reactivity.

Chronic Stress Increases Prefrontal Inhibition: A Mechanism for Stress-Induced Prefrontal Dysfunction.

BACKGROUND: Multiple neuropsychiatric disorders, e.g., depression, are linked to imbalances in excitatory and inhibitory neurotransmission and prefrontal cortical dysfunction, and are concomitant with chronic stress. METHODS: We used electrophysiologic (n = 5-6 animals, 21-25 cells/group), neuroanatomic (n = 6-8/group), and behavioral (n = 12/group) techniques to test the hypothesis that chronic stress increases inhibition of medial prefrontal cortex (mPFC) glutamatergic output neurons. RESULTS: Using patch clamp recordings from infralimbic mPFC pyramidal neurons, we found that chronic stress selectively increases the frequency of miniature inhibitory postsynaptic currents with no effect on amplitude, which suggests that chronic stress increases presynaptic gamma-aminobutyric acid release. Elevated gamma-aminobutyric acid release under chronic stress is accompanied by increased inhibitory appositions and terminals onto glutamatergic cells, as assessed by both immunohistochemistry and electron microscopy. Furthermore, chronic stress decreases glucocorticoid receptor immunoreactivity specifically in a subset of inhibitory neurons, which suggests that increased inhibitory tone in the mPFC after chronic stress may be caused by loss of a glucocorticoid receptor-mediated brake on interneuron activity. These neuroanatomic and functional changes are associated with impairment of a prefrontal-mediated behavior. During chronic stress, rats initially make significantly more errors in the delayed spatial win-shift task, an mPFC-mediated behavior, which suggests a diminished impact of the mPFC on decision making. CONCLUSIONS: Taken together, the data suggest that chronic stress increases synaptic inhibition onto prefrontal glutamatergic output neurons, limiting the influence of the prefrontal cortex in control of stress reactivity and behavior. Thus, these data provide a mechanistic link among chronic stress, prefrontal cortical hypofunction, and behavioral dysfunction.

Mouse handling limits the impact of stress on metabolic endpoints.

Studies focused on end-points that are confounded by stress are best performed under minimally stressful conditions. The objective of this study was to demonstrate the impact of handling designed to reduce animal stress on measurements of glucose tolerance. A cohort of mice (CD1.C57BL/6) naïve to any specific handling was subjected to either a previously described "cup" handling method, or a "tail-picked" method in which the animals were picked up by the tail (as is common for metabolic studies). Following training, an elevated plus maze (EPM) test was performed followed by measurement of blood glucose and plasma corticosterone. A second cohort (CD1.C57BL/6) was rendered obese by exposure to a high fat diet, handled with either the tail-picked or cup method and subjected to an intraperitoneal glucose tolerance test. A third cohort of C57BL/6 mice was exposed to a cup regimen that included a component of massage and was subjected to tests of anxiety-like behavior, glucose homeostasis, and corticosterone secretion. We found that the cup mice showed reduced anxiety-like behaviors in the EPM coupled with a reduction in blood glucose levels compared to mice handled by the tail-picked method. Additionally, cup mice on the high fat diet exhibited improved glucose tolerance compared to tail-picked controls. Finally, we found that the cup/massage group showed lower glucose levels following an overnight fast, and decreased anxiety-like behaviors associated with lower stress-induced plasma corticosterone concentration compared to tail-picked controls. These data demonstrate that application of handling methods that reduce anxiety-like behaviors in mice mitigates the confounding contribution of stress to interpretation of metabolic endpoints (such as glucose tolerance).

Diet-induced obese mice retain endogenous leptin action.

Obesity is characterized by hyperleptinemia and decreased response to exogenous leptin. This has been widely attributed to the development of leptin resistance, a state of impaired leptin signaling proposed to contribute to the development and persistence of obesity. To directly determine endogenous leptin activity in obesity, we treated lean and obese mice with a leptin receptor antagonist. The antagonist increased feeding and body weight (BW) in lean mice, but not in obese models of leptin, leptin receptor, or melanocortin-4 receptor deficiency. In contrast, the antagonist increased feeding and BW comparably in lean and diet-induced obese (DIO) mice, an increase associated with decreased hypothalamic expression of Socs3, a primary target of leptin. These findings demonstrate that hyperleptinemic DIO mice retain leptin suppression of feeding comparable to lean mice and counter the view that resistance to endogenous leptin contributes to the persistence of DIO in mice.

The role of ß cell glucagon-like peptide-1 signaling in glucose regulation and response to diabetes drugs.

Glucagon-like peptide-1 (GLP-1), an insulinotropic gut peptide released after eating, is essential for normal glucose tolerance (GT). To determine whether this effect is mediated directly by GLP-1 receptors (GLP1R) on islet ß cells, we developed mice with ß cell-specific knockdown of Glp1r. ß cell Glp1r knockdown mice had impaired GT after intraperitoneal (i.p.) glucose and did not secrete insulin in response to i.p. or intravenous GLP-1. However, they had normal GT after oral glucose, a response that was impaired by a GLP1R antagonist. ß cell Glp1r knockdown mice had blunted responses to a GLP1R agonist but intact glucose lowering with a dipeptidylpeptidase 4 (DPP-4) inhibitor. Thus, in mice, ß cell Glp1rs are required to respond to hyperglycemia and exogenous GLP-1, but other factors compensate for reduced GLP-1 action during meals. These results support a role for extraislet GLP1R in oral glucose tolerance and paracrine regulation of ß cells by islet GLP-1.

Alpha- and beta-synucleins mRNA expression in lymphocytes of schizophrenia patients.

Alpha-synuclein is largely, but not entirely, expressed in the central nervous system. A high concentration of alpha-synuclein in presynaptic terminals can mimic the normal function of endogenous alpha-synuclein in regulating synaptic vesicle mobilization at nerve terminals. Beta-synuclein protein is seen primarily in brain tissue and it is suggested that beta-synuclein acts as an inhibitor of alpha-synuclein aggregation, which occurs in neurodegenerative diseases. With respect to the role of synucleins in neurologic diseases such as Parkinson's disease, we decided to study the changes of alpha- and beta-synucleins in schizophrenia patients in relation to a control group. For this purpose, total RNA was extracted from the lymphocytes of patients and controls and then cDNA was synthesized and used for real-time polymerase chain reaction. Calculation of the relative expression of alpha- and beta-synucleins showed downregulation in patients in comparison to the control group. Independent two-tailed t-test showed that beta-synuclein mRNA expression in the control group was significantly higher than that in the patient group (p < 0.01), but downregulation of alpha-synuclein gene was not significant. Therefore, a significant downregulation of beta-synuclein mRNA expression appears to be a suitable biomarker for the diagnosis of schizophrenia.