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Most of the animal studies using inflammation-induced cognitive change have relied on behavioral testing without objective and biologically solid methods to quantify the severity of cognitive disturbances. We have developed a bispectral EEG (BSEEG) method using a novel algorithm in clinical study. This method effectively differentiates between patients with and without delirium, and predict long-term mortality. In the present study, we aimed to apply our bispectral EEG (BSEEG) method, which can detect patients with delirium, to a mouse model of delirium with systemic inflammation induced by lipopolysaccharides (LPS) injection. We recorded EEG after LPS injection using wildtype early adulthood mice (2~3-month-old) and aged mice (18-19-month-old). Animal EEG recordings were converted for power spectral density to calculate BSEEG score using the similar BSEEG algorithm previously developed for our human study. The BSEEG score was relatively stable and slightly high during the day. Alternatively, the BSEEG score was erratic and low in average during the night. LPS injection increased the BSEEG score dose-dependently and diminished the diurnal changes. The mean BSEEG score increased much more in the aged mice group as dosage increased. Our results suggest that BSEEG method can objectively "quantify" level of neuro-Inflammation induced by systemic inflammation (LPS), and that this BSEEG method can be useful as a model of delirium in mice.

Estrogen receptor alpha (ERα)-mediated estrogen signaling play a pivotal role in both reproductive and non-reproductive functions. Transcriptional regulation of ERα gene is highly complex, with multiple transcript variants being differentially produced across the tissues. However, tissue-specific variation and physiological specificity of the ERα variants are not yet fully understood. In an attempt to generate a Cre-dependently restorable ERα-null mice for functional investigation of the genetic sufficiency, we unexpectedly produced ERα hypomorphic mice with biased downregulation of a previously unappreciated long ERα isoform that is enriched in the female reproductive organs (uterus and ovaries) and the pituitary but minimally expressed in the brain. Female homozygous mutant mice were capable of pregnancy but displayed irregular estrus cycle and rarely maintained alive newborns without significant morphological and pathological changes in reproductive system and the disruption of body weight homeostasis, indicating the vital role of this long isoform in female reproductive function. Collectively, our results define a tissue-specifically enriched long ERα isoform and its preferential role in female reproductive function over body weight homeostasis.

Objective The paraventricular nucleus of hypothalamus (PVN), an integrative center in the brain, orchestrates a wide range of physiological and behavioral responses. While the PVN melanocortin 4 receptor (MC4R) signaling (PVNMC4R+) is involved in feeding regulation, the neuroanatomical organization of PVNMC4R+ connectivity and its role in other physiological regulations are incompletely understood. Here we aimed to better characterize the input–output organization of PVNMC4R+ neurons and test their physiological functions beyond feeding. Methods Using a combination of viral tools, we mapped PVNMC4R+ circuits and tested the effects of chemogenetic activation of PVNMC4R+ neurons on thermoregulation, cardiovascular control, and other behavioral responses beyond feeding. Results We found that PVNMC4R+ neurons innervate many different brain regions that are known to be important not only for feeding but also for neuroendocrine and autonomic control of thermoregulation and cardiovascular function, including but not limited to the preoptic area, median eminence, parabrachial nucleus, pre-locus coeruleus, nucleus of solitary tract, ventrolateral medulla, and thoracic spinal cord. Contrary to these broad efferent projections, PVNMC4R+ neurons receive monosynaptic inputs mainly from other hypothalamic nuclei (preoptic area, arcuate and dorsomedial hypothalamic nuclei, supraoptic nucleus, and premammillary nucleus), the circumventricular organs (subfornical organ and vascular organ of lamina terminalis), the bed nucleus of stria terminalis, and the parabrachial nucleus. Consistent with their broad efferent projections, chemogenetic activation of PVNMC4R+ neurons not only suppressed feeding but also led to an apparent increase in heart rate, blood pressure, and brown adipose tissue temperature. These physiological changes accompanied acute transient hyperactivity followed by hypoactivity and resting-like behavior. Conclusions Our results elucidate the neuroanatomical organization of PVNMC4R+ circuits and shed new light on the roles of PVNMC4R+ pathways in autonomic control of thermoregulation, cardiovascular function, and biphasic behavioral activation., Highlights • PVNMC4R+ neurons innervate brain regions involved in feeding and autonomic regulation. • Activation of PVNMC4R+ neurons increases BAT thermogenesis. • Activation of PVNMC4R+ neurons increases blood pressure and heart rate. • Activation of PVNMC4R+ neurons lead to transient hyperactivity followed by hypoactivity and resting-like behavior.

Significance Sleep loss disrupts long-term storage of episodic and spatial memories that require activity in the hippocampus. We find that sleep deprivation in mice leads to selective disruption of activity-dependent phosphorylation of ribosomal protein S6 in the hippocampus, which is normally increased in the hippocampus after learning. We used an unbiased approach to profile messenger RNAs associated with phosphorylated ribosomes and found evidence that cholinergic hippocampal inputs and somatostatin-containing hippocampal neurons are selectively activated with sleep deprivation. We find functional evidence supporting the idea that during sleep loss, activity in these two cell populations may act as an inhibitory gate, reducing the activity of dentate gyrus neurons. We find that this inhibitory gating mechanism is capable of disrupting memory consolidation., Sleep loss disrupts consolidation of hippocampus-dependent memory. To characterize effects of learning and sleep loss, we quantified activity-dependent phosphorylation of ribosomal protein S6 (pS6) across the dorsal hippocampus of mice. We find that pS6 is enhanced in dentate gyrus (DG) following single-trial contextual fear conditioning (CFC) but is reduced throughout the hippocampus after brief sleep deprivation (SD; which disrupts contextual fear memory [CFM] consolidation). To characterize neuronal populations affected by SD, we used translating ribosome affinity purification sequencing to identify cell type–specific transcripts on pS6 ribosomes (pS6-TRAP). Cell type–specific enrichment analysis revealed that SD selectively activated hippocampal somatostatin-expressing (Sst+) interneurons and cholinergic and orexinergic hippocampal inputs. To understand the functional consequences of SD-elevated Sst+ interneuron activity, we used pharmacogenetics to activate or inhibit hippocampal Sst+ interneurons or cholinergic input from the medial septum. The activation of either cell population was sufficient to disrupt sleep-dependent CFM consolidation by gating activity in granule cells. The inhibition of either cell population during sleep promoted CFM consolidation and increased S6 phosphorylation among DG granule cells, suggesting their disinhibition by these manipulations. The inhibition of either population across post-CFC SD was insufficient to fully rescue CFM deficits, suggesting that additional features of sleeping brain activity are required for consolidation. Together, our data suggest that state-dependent gating of DG activity may be mediated by cholinergic input and local Sst+ interneurons. This mechanism could act as a sleep loss–driven inhibitory gate on hippocampal information processing.

ObjectiveThe paraventricular nucleus of hypothalamus (PVN) is an integrative center in the brain orchestrating a wide range of physiological and behavioral responses. While the PVN melanocortin 4 receptor (MC4R) signaling (PVNMC4R+) is undoubtedly involved in feeding regulation, the neuroanatomical organization of PVNMC4R+ pathway and its role in diverse physiological and behavioral regulations have not been fully understood. Here we aimed to better characterize the input-output organization of PVNMC4R+ neurons and further test their potential functional roles beyond feeding.MethodsUsing a combination of viral tools, we performed a comprehensive mapping of PVNMC4R+ circuits and tested the effects of chemogenetic activation of PVNMC4R+ neurons on thermogenesis, cardiovascular control and other behavioral regulations beyond feeding.ResultsWe found that PVNMC4R+ neurons broadly innervate many different brain regions known to be important not only for feeding but also for neuroendocrine and autonomic control of thermogenesis and cardiovascular function, including but not limited to preoptic area, median eminence, parabrachial nucleus, locus coeruleus, nucleus of solitary tract, ventrolateral medulla and thoracic spinal cord. Contrary to broad efferent projections, PVNMC4R+ neurons receive monosynaptic inputs from limited brain regions, including medial preoptic nucleus, arcuate and dorsomedial hypothalamic nuclei, and supraoptic nucleus. Consistent with broad efferent projections, chemogenetic activation of PVNMC4R+ neurons not only suppressed feeding but also led to an apparent increase in heart rate, blood pressure and brown adipose tissue thermogenesis. Strikingly, these physiological changes accompanied an unexpected repetitive bedding-removing behavior followed by hypoactivity and resting-like behavior.ConclusionsOur results clarify the neuroanatomical organization of PVNMC4R+ circuits and shed new light on the roles of PVNMC4R+ pathways in autonomic control of thermogenesis, cardiovascular function and other behavioral regulations.

Eating disorders (EDs) are serious mental illnesses thought to arise from the complex gene-environment interactions. DNA methylation patterns in histone deacetylase 4 (HDAC4) locus have been associated with EDs and we have previously identified a missense mutation in the HDAC4 gene (HDAC4 A786T ) that increases the risk of developing an ED. In order to evaluate the biological consequences of this variant and establish a useful mouse model of EDs, here we performed behavioral characterization of mice homozygous for Hdac4 A778T (corresponding to human HDAC4 A786T ) that were further backcrossed onto C57BL/6 background. When fed high-fat diet, male, but not female, homozygous mice showed a trend toward decreased weight gain compared to their wild-type littermates. Behaviorally, male, but not female, homozygous mice spent less time in eating and exhibited reduced motivation to work for palatable food and light phase-specific decrease in locomotor activity. Additionally, homozygous Hdac4 A778T female, but not male, mice display social subordination when subjected to a tube dominance test. Collectively, these results reveal a complex sex- and circadian-dependent role of ED-associated Hdac4 A778T mutation in affecting mouse behaviors. Homozygous Hdac4 A778T mice could therefore be a useful animal model to gain insight into the neurobiological basis of EDs.

Obesity is commonly associated with sympathetic overactivity and elevated blood pressure, and accumulating evidence suggests that an adipocyte-derived metabolic hormone, leptin, may plays an important role in these pathological processes, likely through a mechanism of so-called ‘selective leptin resistance’. However, underlying neural circuits through which leptin causes sympathoexcitation and increases blood pressure remain incompletely understood. Here we hypothesized that leptin may acts on a subset of lateral hypothalamic area (LHA) neurons to affect cardiovascular functions. We show that stereotaxic microinfusion of leptin into the LHA dose-dependently increases renal sympathetic nerve activity (RSNA) (% changes from baseline at 4 th hour: vehicle -25.03 ± 7.09 % vs leptin 100.23 ± 26.94 %, p

Leptin resistance is a hallmark of obesity with unclear etiology. Celastrol, a compound found in the roots of the Tripterygium wilfordii and known to reduce endoplasmic reticulum (ER) stress, has recently emerged as a promising candidate to treat obesity by improving leptin sensitivity. However, the underlying neural mechanisms by which celastrol reduces obesity remain unclear. Using three different mouse models of obesity—diet-induced obesity (DIO), leptin receptor (LepR)-null, and melanocortin 4 receptor (MC4R)-null mice—in this study, we show that systemic celastrol administration substantially reduces food intake and body weight in MC4R-null comparable to DIO, proving the MC4R-independent antiobesity effect of celastrol. Body weight reduction was due to decreases in both fat and lean mass, and modest but significant body weight reduction was also observed in nonobese wild-type and LepR-null mice. Unexpectedly, celastrol upregulated proinflammatory cytokines without affecting genes involved in ER stress. Importantly, celastrol steadily increased sympathetic nerve activity to the brown fat and kidney with concordant increases of resting metabolic rate and arterial pressure. Our results suggest a previously unappreciated mechanism of action of celastrol in the regulation of energy homeostasis and highlight the need for careful consideration of its development as a safe antiobesity medication.

It is well established that the central melanocortin system is critical for autonomic functions and energy homeostasis mainly via signaling at melanocortin-4 receptor (MC4R). Importantly, while obesity is commonly associated with elevated sympathetic tone and blood pressure, severely obese humans and rodents due to genetic MC4R deficiency exhibit normal to low sympathetic tone and blood pressure, suggesting a significant role of MC4R pathway in mediating obesity-associated sympathoexcitation and hypertension. MC4R is widely expressed in the brain including hypothalamic paraventricular nucleus (PVN) which regulates feeding and sympathetic traffic. However, the neuroanatomical basis of PVN MC4R neurons for sympathetic regulation is unclear. The goal of current study is to map the PVN MC4R neural circuits affecting sympathetic tone in mice. To this end, we injected Cre-dependent AAV driving eYFP-fused channel rhodopsin-2 expression into the PVN of MC4R-t2a-Cre knock-in mice, which allow targeted anterograde tract-tracing of PVN MC4R neurons throughout the brain. In addition to known brain region for feeding behavior (i.e. parabrachial nucleus), we found broad innervations of PVN MC4R neurons to various brain regions important for autonomic-cardiovascular control, including, but not limited to, nucleus of solitary tractus, dorsomotor nucleus of vagus, and ventrolateral medulla. Considerable innervation was also evident in spinal cord, which is further confirmed by Fluoro-gold (FG)-mediated retrograde tracing in the spinal cord (thoracic T6-10) of MC4R-GFP transgenic mouse. Double immunofluorescence labeling of GFP and FG revealed that ~50% (58 out of 116) MC4R neurons in the posterior parvocellular subdivision of PVN project to thoracic spinal cord. Furthermore, microinjection of synthetic MC4R agonist (MTII) into the PVN evokes ~42% increase (from baseline) in renal sympathetic nerve activity in anesthetized mice. These results provide important insights into understanding the divergent neural circuits by which PVN MC4R signaling differentially regulates metabolic and cardiovascular functions. Functional dissection of these diverging neural pathways using optogenetic/chemogenetic approaches is ongoing.