With super SDMs (machine learning, open access big data, and the cloud) towards more holistic global squirrel hotspots and coldspots.

Species-habitat associations are correlative, can be quantified, and used for powerful inference. Nowadays, Species Distribution Models (SDMs) play a big role, e.g. using Machine Learning and AI algorithms, but their best-available technical opportunities remain still not used for their potential e.g. in the policy sector. Here we present Super SDMs that invoke ML, OA Big Data, and the Cloud with a workflow for the best-possible inference for the 300 + global squirrel species. Such global Big Data models are especially important for the many marginalized squirrel species and the high number of endangered and data-deficient species in the world, specifically in tropical regions. While our work shows common issues with SDMs and the maxent algorithm ('Shallow Learning'), here we present a multi-species Big Data SDM template for subsequent ensemble models and generic progress to tackle global species hotspot and coldspot assessments for a more inclusive and holistic inference.

COVID-19 prediction using Caviar Squirrel Jellyfish Search Optimization technique in fog-cloud based architecture.

In the pandemic of COVID-19 patients approach to the hospital for prescription, yet due to extreme line up the patient gets treatment after waiting for more than one hour. Generally, wearable devices directly measure the preliminary data of the patient stored in capturing mode. In order to store the data, the hospitals require large storage devices that make the progression of data more complex. To bridge this gap, a potent scheme is established for COVID-19 prediction based fog-cloud named Caviar Squirrel Jellyfish Search Optimization (CSJSO). Here, CSJSO is the amalgamation of CAViar Squirrel Search Algorithm (CSSA) and Jellyfish Search Optimization (JSO), where CSSA is blended by the Conditional Autoregressive Value-at-Risk (CAViar) and Squirrel Search Algorithm (SSA). This architecture comprises the healthcare IoT sensor layer, fog layer and cloud layer. In the healthcare IoT sensor layer, the routing process with the collection of patient health condition data is carried out. On the other hand, in the fog layer COVID-19 detection is performed by employing a Deep Neuro Fuzzy Network (DNFN) trained by the proposed Remora Namib Beetle JSO (RNBJSO). Here, RNBJSO is the combination of Namib Beetle Optimization (NBO), Remora Optimization Algorithm (ROA) and Jellyfish Search optimization (JSO). Finally, in the cloud layer, the detection of COVID-19 employing Deep Long Short Term Memory (Deep LSTM) trained utilizing proposed CSJSO is performed. The evaluation measures utilized for CSJSO_Deep LSTM in database-1, such as Mean Squared Error (MSE) and Root Mean Squared Error (RMSE) observed 0.062 and 0.252 in confirmed cases. The measures employed in database-2 are accuracy, sensitivity and specificity achieved 0.925, 0.928 and 0.925 in K-set.

Stress associated ovarian dysfunctions in a seasonal breeder Funambulus pennanti: Role of glucocorticoids and possible amelioration by melatonin.

Studies have shown that stress caused by lack of physical activity disrupts the normal pattern of glucocorticoid secretion which adversely affects the reproductive axis. We studied the effect of chronic movement restriction on ovarian responses in the Indian Palm Squirrel Funambulus pennanti, a highly active diurnal rodent. Physical restraint of squirrels induced stress that led to a significant increase in plasma cortisol, corticosterone and decreased 17ß-estradiol level leading to follicular atresia. Ovarian Reactive Oxygen Species (ROS) content, lipid peroxidation (LPO), activities of superoxide dismutase (SOD) and catalase (CAT) enzymes increased in restrained squirrels. Elevated ROS increased the oxidative load that led to ovarian cell death as evidenced by increased Bax and decreased Bcl2 expression causing further decline in Aromatase and ERα proteins. To elaborate the mechanism(s) involved in stress induced glucocorticoid mediated oxidative damages to the ovary we extended our study by exposing ovaries in vitro to the synthetic glucocorticoid dexamethasone (200 µM). We observed that glucocorticoid receptor (GR) expression was significantly increased in dexamethasone treated ovaries in vitro with a decrease in expression of Nrf2 and HO-1 proteins. Melatonin supplementation (10 nM) along with dexamethasone significantly decreased ovarian ROS production, lipid peroxidation and increased antioxidant enzyme activities by improving the expression of Nrf2 and HO-1, reinstating the cellular redox homeostasis. Therefore, it can be suggested that physical restraint induced glucocorticoid and its receptor activation interfered with the ovarian antioxidant defense mechanism. Melatonin via its receptor MT1 significantly alleviated ovarian damages acting as a cytoprotective agent.

Phylogenetic and morphological significance of an overlooked flying squirrel (Pteromyini, Rodentia) from the eastern Himalayas with the description of a new genus.

The flying squirrels (Pteromyini, Rodentia) are the most diverse and widely distributed group of gliding mammals. Taxonomic boundaries and relationships within flying squirrels remain an area of active research in mammalogy. The discovery of new specimens of Pteromys ( Hylopetes) leonardi Thomas, 1921 previously considered a synonym of Hylopetes alboniger, in Yunnan Province, China allowed a morphological and genetic reassessment of the status of this taxon. Phylogenetic reconstruction was implemented using sequences of two mitochondrial (12S ribosomal DNA and 16S ribosomal DNA) and one nuclear (interphotoreceptor retinoid-binding protein) gene fragments. Morphological assessments involved examinations of features preserved on skins, skulls, and penises of museum specimens, supplemented with principal component analysis of craniometric data. Together these assessments revealed that this taxon should be recognized not only as a distinct species, and should also be placed within a new genus, described here as Priapomys.

Estimating included animal species in mixed crude drugs derived from animals using massively parallel sequencing.

We developed a method that can detect each animal species of origin for crude drugs derived from multiple animal species based on massively parallel sequencing analysis of mitochondrial genes. The crude drugs derived from animals investigated in this study were Cervi Parvum Cornu and Trogopterorum feces, which are derived from a mix of different animal species, two chopped cicada sloughs, and two commercial Kampo drugs. The mitochondrial 12S rRNA, 16S rRNA, and cytochrome oxidase subunit I gene regions were amplified and sequenced using MiSeq. The ratios of haplotype to total number of sequences reads were calculated after sequence extraction and trimming. Haplotypes that exceeded the threshold were defined as positive haplotypes, which were compared with all available sequences using BLAST. In the Cervi Parvum Cornu and Trogopterorum feces samples, the haplotype ratios corresponded roughly to the mixture ratios, although there was a slight difference from mixture ratios depending on the gene examined. This method could also roughly estimate the compositions of chopped cicada sloughs and Kampo drugs. This analysis, whereby the sequences of several genes are elucidated, is better for identifying the included animal species. This method should be useful for quality control of crude drugs and Kampo drugs.

Seasonal changes in adenosine kinase in tanycytes of the Arctic ground squirrel (Urocitellus parryii).

Hibernation is a seasonal strategy to conserve energy, characterized by modified thermoregulation, an increase in sleep pressure and drastic metabolic changes. Glial cells such as astrocytes and tanycytes are the brain metabolic sensors, but it remains unknown whether they contribute to seasonal expression of hibernation. The onset of hibernation is controlled by an undefined endogenous circannual rhythm in which adenosine plays a role through the activation of the A1 adenosine receptor (A1AR). Seasonal changes in brain levels of adenosine may contribute to an increase in A1AR sensitivity leading to the onset of hibernation. The primary regulator of extracellular adenosine concentration is adenosine kinase, which is located in astrocytes. Using immunohistochemistry to localize and quantify adenosine kinase in Arctic ground squirrels' brain collected during different seasons, we report lower expression of adenosine kinase in the third ventricle tanycytes in winter compared to summer; a similar change was not seen in astrocytes. Moreover, for the first time, we describe adenosine kinase expression in tanycyte cell bodies in the hypothalamus and in the area postrema, both brain regions involved in energy homeostasis. Next we describe seasonal changes in tanycyte morphology in the hypothalamus. Although still speculative, our findings contribute to a model whereby adenosine kinase in tanycytes regulates seasonal changes in extracellular concentration of adenosine underling the seasonal expression of hibernation.

Cold exposure increased hypothalamic orexigenic neuropeptides but not food intake in fattening Daurian ground squirrels.

Energy balance and thermoregulation in many fat-storing seasonal hibernators show a circannual rhythm. To understand the physiological mechanisms of the seasonal pre-hibernation fattening related to the regulation of energy expenditure and thermogenesis, we cold-exposed fattening Daurian ground squirrels (Spermophilus dauricus) in late summer for 3 weeks. We predicted that cold-exposed squirrels would increase food intake rather than express torpor to accommodate both fattening and thermoregulatory fuel allocation. Food intake and body mass were quantified. After 3 weeks, body compositions, serum leptin concentration, expression of hypothalamic neuropeptides related to regulation of energy balance and uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) were measured. There was no change in body mass after 3-weeks of cold exposure. Hypothalamic orexigenic neuropeptides and UCP1 levels in BAT were up-regulated after cold exposure. Food intake, serum leptin concentration and the expression of leptin signal suppressors, suppressor of cytokine signaling 3 and protein tyrosine phosphatase 1B, in hypothalamus showed no differences compared with controls. The core body temperature was unaffected by cold exposure. Our data suggest that cold exposure affected fattening mainly because of the increased heat loss, whereas energy balance and thermoregulation are under control of a strong circannual rhythm in the Daurian ground squirrels.

Simultaneous Determination of Prostaglandin and Hormones in Excreta of Trogopterus xanthipes.

The excreta of Trogopterus xanthipes (also called Wulingzhi in Chinese, WLZ) is a well-known traditional Chinese medicine used for the treatment of irregular menstruation in clinic. Few reports are available on the chemical profiling of WLZ. In this work, qualitative and quantitative analyses of endogenous prostaglandin and hormones in WLZ were performed using UHPLC-orbitrap-MSn. In total, 48 compounds were identified in urine of T. xanthipes. Furthermore, the contents of four target compounds were simultaneously quantitated in 20 batches of samples by UPLC-MS/MS. The quantitative method showed a good linear correlation (R > 0.995) in a wide range for each compound. The method had a high sensitivity with LOD (0.5-1.0 ng/mL) and LOQ (1.0-2.5 ng/mL). The intra- and inter-day precisions were < 9.17 (RSD %), and repeatability and stability were < 6.14 (RSD %). The recovery of the analytes varied between 85.8% and 97.3% at three different concentrations. The present integrated qualitative and quantitative assessment of WLZ provides an evaluation strategy to assess the constituent in traditional Chinese medicine.

CNGA3 acts as a cold sensor in hypothalamic neurons.

Most mammals maintain their body temperature around 37°C, whereas in hibernators it can approach 0°C without triggering a thermogenic response. The remarkable plasticity of the thermoregulatory system allowed mammals to thrive in variable environmental conditions and occupy a wide range of geographical habitats, but the molecular basis of thermoregulation remains poorly understood. Here we leverage the thermoregulatory differences between mice and hibernating thirteen-lined ground squirrels (Ictidomys tridecemlineatus) to investigate the mechanism of cold sensitivity in the preoptic area (POA) of the hypothalamus, a critical thermoregulatory region. We report that, in comparison to squirrels, mice have a larger proportion of cold-sensitive neurons in the POA. We further show that mouse cold-sensitive neurons express the cyclic nucleotide-gated ion channel CNGA3, and that mouse, but not squirrel, CNGA3 is potentiated by cold. Our data reveal CNGA3 as a hypothalamic cold sensor and a molecular marker to interrogate the neuronal circuitry underlying thermoregulation.

Perineuronal net expression in the brain of a hibernating mammal.

During hibernation, mammals like the 13-lined ground squirrel cycle between physiological extremes. Most of the hibernation season is spent in bouts of torpor, where body temperature, heart rate, and cerebral blood flow are all very low. However, the ground squirrels periodically enter into interbout arousals (IBAs), where physiological parameters return to non-hibernating levels. During torpor, neurons in many brain regions shrink and become electrically quiescent, but reconnect and regain activity during IBA. Previous work showed evidence of extracellular matrix (ECM) changes occurring in the hypothalamus during hibernation that could be associated with this plasticity. Here, we examined expression of a specialized ECM structure, the perineuronal net (PNN), in the forebrain of ground squirrels in torpor, IBA, and summer (non-hibernating). PNNs are known to restrict plasticity, and could be important for retaining essential connections in the brain during hibernation. We found PNNs in three regions of the hypothalamus: ventrolateral hypothalamus, paraventricular nucleus (PVN), and anterior hypothalamic area. We also found PNNs throughout the cerebral cortex, amygdala, and lateral septum. The total area covered by PNNs within the PVN was significantly higher during IBA compared to non-hibernating and torpor (P < 0.01). Additionally, the amount of PNN coverage area per Nissl-stained neuron in the PVN was significantly higher in hibernation compared to non-hibernating (P < 0.05). No other significant differences were found across seasons. The PVN is involved in food intake and homeostasis, and PNNs found here could be essential for retaining vital life functions during hibernation.

First record of Leptospira and Blastocystis infections in captive flying squirrels (Trogopterus xanthipes) from Enshi County, China.

In traditional Chinese medicine, the feces of flying squirrels have long been used to promote blood circulation and relieve bodily stasis. However, the excrement of flying squirrels may harbor zoonotic agents that could be hazardous to public health. To understand the occurrence of bacterial and parasitic infections in this species, we investigated selected zoonotic pathogens including Leptospira and Blastocystis in the urine and feces of flying squirrels in China. Urine and fecal samples from flying squirrels were collected from a family-owned flying squirrel farm located in Enshi County, Hubei Province in China. Leptospira and Blastocystis DNA was extracted from the urine and feces of flying squirrels, and used as targets for PCR amplification, using different specific primers. PCR amplification and DNA sequencing showed that 4.4% (3/69) of flying squirrels were positive for Leptospira, while 30.4% (21/69) of the animals were positive for Blastocystis. Notably, 1.4% (1/69) of flying squirrels were found to be co-infected with Leptospira and Blastocystis. Sequence analyses allowed for the detection of 3 Blastocystis subtypes (ST1, ST3 and ST13), and mixed infections of Blastocystis subtype 1 and subtype 3 were found in 4.4% (3/69) of flying squirrels. Phylogenetic analysis of the 16S ribosomal RNA gene (rrs2), the flagellin B gene (flaB), and outer membrane lipoprotein lipL32 gene (LipL32) sequences indicated that the Leptospira species detected in the study was L. interrogans. We concluded that flying squirrels from central China were infected with Leptospira and Blastocystis, suggesting that these animals can be a source of infection for their owners, and using fresh excrement from this animal as traditional medicine could be risky to human health. To the best of our knowledge, this is the first report of Leptospira and Blastocystis infection in flying squirrels from Enshi County, China. Our findings provide new data on the epidemiology of these pathogens in this region.

Preclinical pharmacokinetic evaluation to facilitate repurposing of tyrosine kinase inhibitors nilotinib and imatinib as antiviral agents.

BACKGROUND: Several tyrosine kinase inhibitors (TKIs) developed as anti-cancer drugs, also have anti-viral activity due to their ability to disrupt productive replication and dissemination in infected cells. Consequently, such drugs are attractive candidates for "repurposing" as anti-viral agents. However, clinical evaluation of therapeutics against infectious agents associated with high mortality, but low or infrequent incidence, is often unfeasible. The United States Food and Drug Administration formulated the "Animal Rule" to facilitate use of validated animal models for conducting anti-viral efficacy studies. METHODS: To enable such efficacy studies of two clinically approved TKIs, nilotinib, and imatinib, we first conducted comprehensive pharmacokinetic (PK) studies in relevant rodent and non-rodent animal models. PK of these agents following intravenous and oral dosing were evaluated in C57BL/6 mice, prairie dogs, guinea pigs and Cynomolgus monkeys. Plasma samples were analyzed using an LC-MS/MS method. Secondarily, we evaluated the utility of allometry-based inter-species scaling derived from previously published data to predict the PK parameters, systemic clearance (CL) and the steady state volume of distribution (Vss) of these two drugs in prairie dogs, an animal model not tested thus far. RESULTS: Marked inter-species variability in PK parameters and resulting oral bioavailability was observed. In general, elimination half-lives of these agents in mice and guinea pigs were much shorter (1-3 h) relative to those in larger species such as prairie dogs and monkeys. The longer nilotinib elimination half-life in prairie dogs (i.v., 6.5 h and oral, 7.5 h), facilitated multiple dosing PK and safety assessment. The allometry-based predicted values of the Vss and CL were within 2.0 and 2.5-fold, respectively, of the observed values. CONCLUSIONS: Our results suggest that prairie dogs and monkeys may be suitable rodent and non-rodent species to perform further efficacy testing of these TKIs against orthopoxvirus infections. The use of rodent models such as C57BL/6 mice and guinea pigs for assessing pre-clinical anti-viral efficacy of these two TKIs may be limited due to short elimination and/or low oral bioavailability. Allometry-based correlations, derived from existing literature data, may provide initial estimates, which may serve as a useful guide for pre-clinical PK studies in untested animal models.

Retinoid isomerase inhibitors impair but do not block mammalian cone photoreceptor function.

Visual function in vertebrates critically depends on the continuous regeneration of visual pigments in rod and cone photoreceptors. RPE65 is a well-established retinoid isomerase in the pigment epithelium that regenerates rhodopsin during the rod visual cycle; however, its contribution to the regeneration of cone pigments remains obscure. In this study, we use potent and selective RPE65 inhibitors in rod- and cone-dominant animal models to discern the role of this enzyme in cone-mediated vision. We confirm that retinylamine and emixustat-family compounds selectively inhibit RPE65 over DES1, the putative retinoid isomerase of the intraretinal visual cycle. In vivo and ex vivo electroretinography experiments in Gnat1-/- mice demonstrate that acute administration of RPE65 inhibitors after a bleach suppresses the late, slow phase of cone dark adaptation without affecting the initial rapid portion, which reflects intraretinal visual cycle function. Acute administration of these compounds does not affect the light sensitivity of cone photoreceptors in mice during extended exposure to background light, but does slow all phases of subsequent dark recovery. We also show that cone function is only partially suppressed in cone-dominant ground squirrels and wild-type mice by multiday administration of an RPE65 inhibitor despite profound blockade of RPE65 activity. Complementary experiments in these animal models using the DES1 inhibitor fenretinide show more modest effects on cone recovery. Collectively, these studies demonstrate a role for continuous RPE65 activity in mammalian cone pigment regeneration and provide further evidence for RPE65-independent regeneration mechanisms.

[Research on compatibility of prescriptions including Ginseng Radix et Rhizoma and Trogopterus Dung based on complex network analysis].

The applications of prescriptions including Ginseng Radix et Rhizoma and Trogopterus Dung in contemporary literatures from 1949 to 2016 are compiled and the data mining techniques containing scale-free complex network method are utilized to explore its practical characteristics, with comparison between modern and ancient ones. The results indicate that malignant neoplasms, coronary heart disease which present Qi deficiency and blood stasis type are the main diseases treated by prescriptions including Ginseng Radix et Rhizoma and Trogopterus Dung according to the reports during 1949 to 2016. The complex network connection shows that Glycyrrhizae Radixet Rhizoma, Angelicae Sinensis Radix, Astragali Radix, Typhae Pollen, Salviae Miltiorrhizae Radix et Rhizoma are the primary drugs related to Ginseng Radix et Rhizoma and Trogopterus Dung. The next are Paeoniae Radix Alba, Atractylodis Macrocephalae Rhizoma, Persicae Semen, Foria, et al. Carthami Flos, Notoginseng Radix et Rhizoma, Cyperi Rhizoma, Bupleuri Radix are the peripheral ones. Also, Ginseng Radix et Rhizoma-Glycyrrhizae Radixet Rhizoma, Trogopterus Dung-Glycyrrhizae Radixet Rhizoma, Ginseng Radix et Rhizoma-Angelicae Sinensis Radix, Trogopterus Dung-Angelicae Sinensis Radix, Ginseng Radix et Rhizoma-Astragali Radix, Trogopterus Dung-Astragali Radix are the main paired drugs. The paired drugs including Ginseng Radix et Rhizoma-Trogopterus Dung-Glycyrrhizae Radixet Rhizoma, Ginseng Radix et Rhizoma-Trogopterus Dung-Angelicae Sinensis Radix, Ginseng Radix et Rhizoma-Trogopterus Dung-Astragali Radix, Ginseng Radix et Rhizoma-Trogopterus Dung-Typhae Pollen have a higher support degree. The main compatible drugs are different in ancient and modern prescriptions including Ginseng Radix et Rhizoma and Trogopterus Dung. Notoginseng Radix et Rhizoma, Typhae Pollen, Salviae Miltiorrhizae Radix et Rhizoma, Astragali Radix are utilized frequently in modern prescriptions while less used in ancient ones. It is also shown that more attentions are paid to the drugs contributing to invigorating Qi and promoting blood circulation in modern times with comparative results between modern and ancient prescriptions.

Lipid emulsion enhances cardiac performance after ischemia-reperfusion in isolated hearts from summer-active arctic ground squirrels.

Hibernating mammals, like the arctic ground squirrel (AGS), exhibit robust resistance to myocardial ischemia/reperfusion (IR) injury. Regulated preference for lipid over glucose to fuel metabolism may play an important role. We tested whether providing lipid in an emulsion protects hearts from summer-active AGS better than hearts from Brown Norway (BN) rats against normothermic IR injury. Langendorff-prepared AGS and BN rat hearts were perfused with Krebs solution containing 7.5 mM glucose with or without 1% Intralipid™. After stabilization and cardioplegia, hearts underwent 45-min global ischemia and 60-min reperfusion. Coronary flow, isovolumetric left ventricular pressure, and mitochondrial redox state were measured continuously; infarct size was measured at the end of the experiment. Glucose-only AGS hearts functioned significantly better on reperfusion than BN rat hearts. Intralipid™ administration resulted in additional functional improvement in AGS compared to glucose-only and BN rat hearts. Infarct size was not different among groups. Even under non-hibernating conditions, AGS hearts performed better after IR than the best-protected rat strain. This, however, appears to strongly depend on metabolic fuel: Intralipid™ led to a significant improvement in return of function in AGS, but not in BN rat hearts, suggesting that year-round endogenous mechanisms are involved in myocardial lipid utilization that contributes to improved cardiac performance, independent of the metabolic rate decrease during hibernation. Comparative lipid analysis revealed four candidates as possible cardioprotective lipid groups. The improved function in Intralipid™-perfused AGS hearts also challenges the current paradigm that increased glucose and decreased lipid metabolism are favorable during myocardial IR.

Regulation of gene expression by NFAT transcription factors in hibernating ground squirrels is dependent on the cellular environment.

Calcineurin is a calmodulin-stimulated phosphatase that regulates the nuclear translocation of nuclear factor of activated T cell (NFAT) c1-4 through dephosphorylation. We believe that this mechanism plays various roles in the remodeling and maintenance of Ictidomys tridecemlineatus skeletal muscle. During hibernation, bouts of torpor and arousal take place, and squirrels do not lose muscle mass despite being inactive. Protein expression of Ca(2+) signaling proteins were studied using immunoblotting. A DNA-protein interaction ELISA technique was created to test the binding of NFATs in the nucleus to DNA probes containing the NFAT response element under environmental conditions reflective of those during hibernation. Calcineurin protein levels increased by 3.08-fold during torpor (compared to euthermic control), whereas calpain1 levels also rose by 3.66-fold during torpor. Calmodulin levels were elevated upon entering torpor. NFATc4 binding to DNA showed a 1.4-fold increase during torpor, and we found that this binding was further enhanced when 600 nM of Ca(2+) was supplemented. We also found that decreasing the temperature of ELISAs resulted in progressive decreases in the binding of NFATs c1, c3, and c4 to DNA. In summary, calmodulin and calpain1 appear to activate calcineurin and NFATc4 during torpor. NFAT binding to target promoters is affected by intranuclear [Ca(2+)] and environmental temperatures. Therefore, Ca(2+) signaling and temperature changes play key roles in regulation of the NFAT-calcineurin pathway in skeletal muscle of hibernating 13-lined ground squirrels over the torpor-arousal cycle, and they may contribute to the avoidance of disuse-induced muscle atrophy that occurs naturally in these animals.

Leptin regulates energy intake but fails to facilitate hibernation in fattening Daurian ground squirrels (Spermophilus dauricus).

Body fat storage before hibernation affects the timing of immergence in Daurian ground squirrels (Spermophilus dauricus). Leptin is an adipose signal and plays vital role in energy homeostasis mainly by action in brain. To test the hypothesis that leptin plays a role in facilitating the process of hibernation, squirrels were administrated with recombinant murine leptin (1µg/day) through intracerebroventricular (ICV) injection for 12 days during fattening. From day 7 to 12, animals were moved into a cold room (5±1°C) with constant darkness which functioned as hibernaculum. Energy intake, body mass and core body temperature (Tb) were continuously monitored throughout the course of experiment. Resting metabolic rate (RMR) was measured under both warm and cold conditions. At the end of leptin administration, we measured the serum concentration of hormones related to energy regulation, mRNA expression of hypothalamic neuropeptides and uncoupling protein 1 (UCP1) levels in brown adipose tissue (BAT). Our results showed that during leptin administration, the cumulative food intake and increase of body mass were suppressed while Tb and RMR were unaltered. The proportion of torpid squirrels was not different between two groups. At the end of leptin administration, the expressions of hypothalamic neuropeptide Y and agouti gene-related protein were suppressed. There were no differences in UCP1 mRNA expression or protein content in BAT between groups. Our data suggest that leptin can affect energy intake via hypothalamic neuropeptides, but is not involved in the initiation of hibernation in fattening Daurian ground squirrels.

Hypothalamic gene expression underlying pre-hibernation satiety.

Prior to hibernation, 13-lined ground squirrels (Ictidomys tridecemlineatus) enter a hypophagic period where food consumption drops by an average of 55% in 3 weeks. This occurs naturally, while the ground squirrels are in constant environmental conditions and have free access to food. Importantly, this transition occurs before exposure to hibernation conditions (5°C and constant darkness), so the ground squirrels are still maintaining a moderate level of activity. In this study, we used the Illumina HiSeq 2000 system to sequence the hypothalamic transcriptomes of ground squirrels before and after the autumn feeding transition to examine the genes underlying this extreme change in feeding behavior. The hypothalamus was chosen because it is known to play a role in the control and regulation of food intake and satiety. Overall, our analysis identified 143 genes that are significantly differentially expressed between the two groups. Specifically, we found five genes associated with feeding behavior and obesity (VGF, TRH, LEPR, ADIPOR2, IRS2) that are all upregulated during the hypophagic period, after the feeding transition has occurred. We also found that serum leptin significantly increases in the hypophagic group. Several of the genes associated with the natural autumnal feeding decline in 13-lined ground squirrels show parallels to signaling pathways known to be disrupted in human metabolic diseases, like obesity and diabetes. In addition, many other genes were identified that could be important for the control of food consumption in other animals, including humans.

The expression of leptin, hypothalamic neuropeptides and UCP1 before, during and after fattening in the Daurian ground squirrel (Spermophilus dauricus).

The Daurian ground squirrel (Spermophilus dauricus) accumulates large amounts of body fat during pre-hibernation fattening. Leptin, an adipose-derived hormone, plays important roles in energy balance and thermogenesis. We predicted that body fat accumulation would lead to the elevation of leptin concentration while its effect on satiety would be suppressed in hypothalamus during fattening. In addition, the uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) would increase and correlated positively with leptin concentration before hibernation. Here, we measured serum leptin concentration and leptin mRNA in white adipose tissue (WAT), hypothalamic neuropeptides involved in energy regulation and UCP1 in BAT before, during and after fattening in squirrels. The fat mass gradually increased during fattening but serum leptin increased mainly in the late phase of fattening, which was consistent with leptin mRNA expression in WAT. During fattening, the mRNA of hypothalamic leptin receptor was up-regulated and correlated positively with serum leptin. Orexigenic neuropeptide Y mRNA increased by 67%; however agouti-related peptide remained unchanged before hibernation. There was no significant change in anorexigenic neuropeptide mRNA. No change in suppressor of cytokine signaling-3 and protein tyrosine phosphatase-1B was detected. UCP1 mRNA expression and protein content in BAT increased significantly after fattening. These changes were independent of environmental conditions and serum leptin concentration. Our results suggest that the dissociation of leptin production and adiposity during fattening may facilitate fat accumulation. No evidence of suppressed leptin signal was found in fattening squirrels. The UCP1 recruitment in post-fattening squirrels could occur without winter-like acclimation and increased leptin.

Seasonal and regional differences in gene expression in the brain of a hibernating mammal.

Mammalian hibernation presents a unique opportunity to study naturally occurring neuroprotection. Hibernating ground squirrels undergo rapid and extreme physiological changes in body temperature, oxygen consumption, and heart rate without suffering neurological damage from ischemia and reperfusion injury. Different brain regions show markedly different activity during the torpor/arousal cycle: the cerebral cortex shows activity only during the periodic returns to normothermia, while the hypothalamus is active over the entire temperature range. Therefore, region-specific neuroprotective strategies must exist to permit this compartmentalized spectrum of activity. In this study, we use the Illumina HiSeq platform to compare the transcriptomes of these two brain regions at four collection points across the hibernation season: April Active, October Active, Torpor, and IBA. In the cerebral cortex, 1,085 genes were found to be differentially expressed across collection points, while 1,063 genes were differentially expressed in the hypothalamus. Comparison of these transcripts indicates that the cerebral cortex and hypothalamus implement very different strategies during hibernation, showing less than 20% of these differentially expressed genes in common. The cerebral cortex transcriptome shows evidence of remodeling and plasticity during hibernation, including transcripts for the presynaptic cytomatrix proteins bassoon and piccolo, and extracellular matrix components, including laminins and collagens. Conversely, the hypothalamic transcriptome displays upregulation of transcripts involved in damage response signaling and protein turnover during hibernation, including the DNA damage repair gene RAD50 and ubiquitin E3 ligases UBR1 and UBR5. Additionally, the hypothalamus transcriptome also provides evidence of potential mechanisms underlying the hibernation phenotype, including feeding and satiety signaling, seasonal timing mechanisms, and fuel utilization. This study provides insight into potential neuroprotective strategies and hibernation control mechanisms, and also specifically shows that the hibernator brain exhibits both seasonal and regional differences in mRNA expression.