Genetic Diversity of a Heat Activated Channel-TRPV1 in Two Desert Gerbil Species with Different Heat Sensitivity.

Heat sensation and tolerance are crucial for determining species' survival and distribution range of small mammals. As a member of the transmembrane proteins, transient receptor potential vanniloid 1 (TRPV1) is involved in the sensation and thermoregulation of heat stimuli; however, the associations between animal's heat sensitivity and TRPV1 in wild rodents are less studied. Here, we found that Mongolian gerbils (Meriones unguiculatus), a rodent species living in Mongolia grassland, showed an attenuated sensitivity to heat compared with sympatrically distributed mid-day gerbils (M. meridianus) based on a temperature preference test. To explain this phenotypical difference, we measured the TRPV1 mRNA expression of two gerbil species in the hypothalamus, brown adipose tissue, and liver, and no statistical difference was detected between two species. However, according to the bioinformatics analysis of TRPV1 gene, we identified two single amino acid mutations on two TRPV1 orthologs in these two species. Further Swiss-model analyses of two TRPV1 protein sequences indicated the disparate conformations at amino acid mutation sites. Additionally, we confirmed the haplotype diversity of TRPV1 in both species by expressing TRPV1 genes ectopicly in Escherichia coli system. Taken together, our findings supplemented genetic cues to the association between the discrepancy of heat sensitivity and the functional differentiation of TRPV1 using two wild congener gerbils, promoting the comprehension of the evolutionary mechanisms of the TRPV1 gene for heat sensitivity in small mammals.

Effect of Skin Ion Channel TRPM8 Activation by Cold and Menthol on Thermoregulation and the Expression of Genes of Thermosensitive TRP Ion Channels in the Hypothalamus of Hypertensive Rats.

ISIAH (inherited stress-induced arterial hypertension) rats are characterized by high blood pressure and decreased Trpm8 gene expression in the anterior hypothalamus. Thermosensitive ion channel TRPM8 plays a critical role in the transduction of moderately cold stimuli that give rise to cool sensations. In normotensive animals, the activation of skin TRPM8 is known to induce changes in gene expression in the hypothalamus and induce alterations of thermoregulatory responses. In this work, in hypertensive rats, we studied the effects of activation of the peripheral TRPM8 by cooling and by application of a 1% menthol suspension on (1) the maintenance of body temperature balance and (2) mRNA expression of thermosensitive TRP ion channels in the hypothalamus. In these hypertensive animals, (1) pharmacological activation of peripheral TRPM8 did not affect the thermoregulatory parameters either under thermoneutral conditions or during cold exposure; (2) the expression of Trpm8 in the anterior hypothalamus approximately doubled (to the level of normotensive animals) under the influence of (a) slow cooling and (b) at pharmacological activation of the peripheral TRPM8 ion channel. The latter fact seems the quite important because it allows the proposal of a tool for correcting at least some parameters that distinguish a hypertensive state from the normotensive one.

Conditional gene targeting using UCP1-Cre mice directly targets the central nervous system beyond thermogenic adipose tissues.

OBJECTIVE: Uncoupling protein 1 (UCP1) is a mitochondrial protein critical for adaptive thermogenesis in adipose tissues, and it is typically believed to be restricted to thermogenic adipose tissues. UCP1-Cre transgenic mice are utilized in numerous studies to provide "brown adipose-specific" conditional gene targeting. Here, we examined the distribution of Cre and UCP1 throughout the body in UCP1-Cre reporter mice. METHODS: UCP1-Cre mice crossed to Ai14-tdTomato and Ai9-tdTomato reporter mice were used to explore the tissue distribution of Cre recombinase and Ucp1 mRNA in various tissues. UCP1-Cre mice were independently infected with either a Cre-dependent PHP.eB-tdTomato virus or a Cre-dependent AAV-tdTomato virus to determine whether and where UCP1 is actively expressed in the adult central nervous system. In situ analysis of the deposited single cell RNA sequencing data was used to evaluate Ucp1 expression in the hypothalamus. RESULTS: As expected, Ucp1 expression was detected in both brown and inguinal adipose tissues. Ucp1 expression was also detected in the kidney, adrenal glands, thymus, and hypothalamus. Consistent with detectable Ucp1 expression, tdTomato expression was also observed in brown adipose tissue, inguinal white adipose tissue, kidney, adrenal glands, and hypothalamus of both male and female UCP1-Cre; Ai14-tdTomato and UCP1-Cre; Ai9-tdTomato mice by fluorescent imaging and qPCR. Critically, expression of tdTomato, and thus UCP1, within the central nervous system was observed in regions of the brain critical for the regulation of energy homeostasis, including the ventromedial hypothalamus (VMH). CONCLUSIONS: TdTomato expression in UCP1-Cre; tdTomato mice is not restricted to thermogenic adipose tissues. TdTomato was also expressed in the kidneys, adrenal glands, and throughout the brain, including brain regions and cell types that are critical for multiple aspects of central regulation of energy homeostasis. Collectively, these data have important implications for the utility of UCP1-Cre mice as genetic tools to investigate gene function specifically in brown adipose tissue.

Activation of Kappa Opioid Receptor Regulates the Hypothermic Response to Calorie Restriction and Limits Body Weight Loss.

Mammals maintain a nearly constant core body temperature (Tb) by balancing heat production and heat dissipation. This comes at a high metabolic cost that is sustainable if adequate calorie intake is maintained. When nutrients are scarce or experimentally reduced such as during calorie restriction (CR), endotherms can reduce energy expenditure by lowering Tb [1-6]. This adaptive response conserves energy, limiting the loss of body weight due to low calorie intake [7-10]. Here we show that this response is regulated by the kappa opioid receptor (KOR). CR is associated with increased hypothalamic levels of the endogenous opioid Leu-enkephalin, which is derived from the KOR agonist precursor dynorphin [11]. Pharmacological inhibition of KOR, but not of the delta or the mu opioid receptor subtypes, fully blocked CR-induced hypothermia and increased weight loss during CR independent of calorie intake. Similar results were seen with DIO mice subjected to CR. In contrast, inhibiting KOR did not change Tb in animals fed ad libitum (AL). Chemogenetic inhibition of KOR neurons in the hypothalamic preoptic area reduced the CR-induced hypothermia, whereas chemogenetic activation of prodynorphin-expressing neurons in the arcuate or the parabrachial nucleus lowered Tb. These data indicate that KOR signaling is a pivotal regulator of energy homeostasis and can affect body weight during dieting by modulating Tb and energy expenditure.

The GCN2 inhibitor IMPACT contributes to diet-induced obesity and body temperature control.

IMPACT, a highly conserved protein, is an inhibitor of the eIF2α kinase GCN2. In mammals, it is preferentially expressed in neurons. Knock-down of IMPACT expression in neuronal cells increases basal GCN2 activation and eIF2α phosphorylation and decreases translation initiation. In the mouse brain, IMPACT is particularly abundant in the hypothalamus. Here we describe that the lack of IMPACT in mice affects hypothalamic functions. Impact-/- mice (Imp-KO) are viable and have no apparent major phenotypic defect. The hypothalamus in these animals shows increased levels of eIF2α phosphorylation, as expected from the described role of IMPACT in inhibiting GCN2 and from its abundance in this brain region. When fed a normal chow, animals lacking IMPACT weight slightly less than wild-type mice. When fed a high-fat diet, Imp-KO animals gain substantially less weight due to lower food intake when compared to wild-type mice. STAT3 signaling was depressed in Imp-KO animals even though leptin levels were identical to the wild-type mice. This finding supports the observation that Imp-KO mice have defective thermoregulation upon fasting. This phenotype was partially dependent on GCN2, whereas the lean phenotype was independent of GCN2. Taken together, our results indicate that IMPACT contributes to GCN2-dependent and -independent mechanisms involved in the regulation of autonomic functions in response to energy availability.

Altered hypothalamic inflammatory gene expression correlates with heat stroke severity in a conscious rodent model.

It has been suggested that heat-induced hypothalamic damage mediates core temperature (Tc) disturbances during heat stroke (HS) recovery; this is significant as hypothermia and/or fever have been linked to severity and overall pathological insult. However, to date there has been a lack of histological evidence in support of these claims. We hypothesized that local hypothalamic cytokines and/or chemokines, known regulators of Tc, are mediating the elevation in Tc during HS recovery even in the absence of histological damage. In experiment 1, the hypothalamus of Fischer 344 rats was examined for 84 cytokine/chemokine genes (real-time PCR) at multiple time points (Tc,Max, 1, 3, and 10 days) during mild HS recovery. In experiment 2, the hypothalamus of three different HS severities (MILD, moderate [MOD], and severe [SEV]) in rats were examined for the same genes as experiment 1 as well as six oxidative damage markers, at a single intermediate time point (1 day). Systemic cytokines were also analyzed in experiment 2 across the three severities. There were significant alterations in 25 cytokines/chemokines expression at Tc,Max, but little or no changes in expression at longer time points in experiment 1. In experiment 2 there were significant changes in gene expression in SEV rats only, with MILD and MOD rats showing baseline expression at 1 day, despite an absence of systemic cytokine expression in any severity. There was also no change in any oxidative marker of damage at 1 day, regardless of severity. In conclusion, we show only limited changes during long term recovery from HS, but demonstrate differences in hypothalamic gene expression patterns that may be driving HS pathology and morbidity. These findings contribute to our overall understanding of HS pathology in the CNS, as well as providing avenues for future pharmacological intervention.

The evolutionary adaptation of the C282Y mutation to culture and climate during the European Neolithic.

OBJECTIVES: The C282Y allele is the major cause of hemochromatosis as a result of excessive iron absorption. The mutation arose in continental Europe no earlier than 6,000 years ago, coinciding with the arrival of the Neolithic agricultural revolution. Here we hypothesize that this new Neolithic diet, which originated in the sunny warm and dry climates of the Middle East, was carried by migrating farmers into the chilly and damp environments of Europe where iron is a critical micronutrient for effective thermoregulation. We argue that the C282Y allele was an adaptation to this novel environment. MATERIALS AND METHODS: To address our hypothesis, we compiled C282Y allele frequencies, known Neolithic sites in Europe and climatic data on temperature and rainfall for statistical analysis. RESULTS: Our findings indicate that the geographic cline for C282Y frequency in Europe increases as average temperatures decrease below 16°C, a critical threshold for thermoregulation, with rainy days intensifying the trend. DISCUSSION: The results indicate that the deleterious C282Y allele, responsible for most cases of hemochromatosis, may have evolved as a selective advantage to culture and climate during the European Neolithic.

Heat Shock Factor 1 Deficiency Affects Systemic Body Temperature Regulation.

INTRODUCTION: Heat shock factor 1 (HSF1) is a ubiquitous heat-sensitive transcription factor that mediates heat shock protein transcription in response to cellular stress, such as increased temperature, in order to protect the organism against misfolded proteins. In this study, we analysed the effect of HSF1 deficiency on core body temperature regulation. MATERIALS AND METHODS: Body temperature, locomotor activity, and food consumption of wild-type mice and HSF1-deficient mice were recorded. Prolactin and thyroid-stimulating hormone levels were measured by ELISA. Gene expression in brown adipose tissue was analysed by quantitative real-time PCR. Hypothalamic HSF1 and its co-localisation with tyrosine hydroxylase was analysed using confocal laser scanning microscopy. RESULTS: HSF1-deficient mice showed an increase in core body temperature (hyperthermia), decreased overall locomotor activity, and decreased levels of prolactin in pituitary and blood plasma reminiscent of cold adaptation. HSF1 could be detected in various hypothalamic regions involved in temperature regulation, suggesting a potential role of HSF1 in hypothalamic thermoregulation. Moreover, HSF1 co-localises with tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, suggesting a potential role of HSF1 in the hypothalamic control of prolactin release. In brown adipose tissue, levels of prolactin receptor and uncoupled protein 1 were increased in HSF1-deficient mice, consistent with an up-regulation of heat production. CONCLUSION: Our data suggest a role of HSF1 in systemic thermoregulation.

Thyroid hormone status affects expression of daily torpor and gene transcription in Djungarian hamsters (Phodopus sungorus).

Thyroid hormones (TH) play a key role in regulation of seasonal as well as acute changes in metabolism. Djungarian hamsters (Phodopus sungorus) adapt to winter by multiple changes in behaviour and physiology including spontaneous daily torpor, a state of hypometabolism and hypothermia. We investigated effects of systemic TH administration and ablation on the torpor behaviour in Djungarian hamsters adapted to short photoperiod. Hyperthyroidism was induced by giving T4 or T3 and hypothyroidism by giving methimazole (MMI) and sodium perchlorate via drinking water. T3 treatment increased water, food intake and body mass, whereas MMI had the opposite effect. Continuous recording of body temperature revealed that low T3 serum concentrations increased torpor incidence, lowered Tb and duration, whereas high T3 serum concentrations inhibited torpor expression. Gene expression of deiodinases (dio) and uncoupling proteins (ucp) were analysed by qPCR in hypothalamus, brown adipose tissue (BAT) and skeletal muscle. Expression of dio2, the enzyme generating T3 by deiodination of T4, and ucps, involved in thermoregulation, indicated a tissue specific response to treatment. Torpor per se decreased dio2 expression irrespective of treatment or tissue, suggesting low intracellular T3 concentrations during torpor. Down regulation of ucp1 and ucp3 during torpor might be a factor for the inhibition of BAT thermogenesis. Hypothalamic gene expression of neuropeptide Y, propopiomelanocortin and somatostatin, involved in feeding behaviour and energy balance, were not affected by treatment. Taken together our data indicate a strong effect of thyroid hormones on torpor, suggesting that lowered intracellular T3 concentrations in peripheral tissues promote torpor.

Long-term high-soybean oil feeding alters regulation of body temperature in rats.

We investigated whether body temperature (BT) regulatory mechanisms are influenced by dietary fatty acids (FA). Male Wistar rats were fed a high-fat diet containing fish oil (HFD), soybean oil (HSD) or lard (HLD). At the 20-week intervention, the BT of the HSD and HLD groups were lower than that of the normal diet (ND) group in the light and dark periods. The intracerebroventricular injections of interleukin-1ß and bombesin in the HSD group induced greater hyperthermia and weaker hypothermia, respectively, than in the ND group. The HSD differentially affected BT under both physiological and pharmacological conditions. In the hypothalamus, the ratio of n-6/n-3 FAs was higher in the HSD group compared with the ND group. DNA microarrays revealed increased expression of thyroid-stimulating hormone ß-subunit, and decreased expression of several genes in the hypothalamus of the HSD group compared with the ND group. The HSD feeding increased several adipokine concentrations in the plasma. However, there were no adipokines or gene expressions that changed in only the HSD and HLD groups showing significant hypothermia under the physiological condition. These findings suggested that long-term HSD intake produces abnormal BT regulation. It is less likely that adipokines or proteins/peptides are involved in abnormal BT regulation under the physiological conditions after HSD feeding.

Hypothalamic plasticity of neuropeptide Y is lacking in brain-type creatine kinase double knockout mice with defective thermoregulation.

The neural substrate of adaptive thermoregulation in mice lacking both brain-type creatine kinase isoforms is further investigated. The cytosolic brain-type creatine kinase (CK-B) and mitochondrial ubiquitous creatine kinase (UbCKmit) are expressed in neural cells throughout the central and peripheral nervous system, where they have an important role in cellular energy homeostasis. Several integral functions appear altered when creatine kinases are absent in the brain (Jost et al., 2002; Streijger et al., 2004, 2005), which has been explained by inefficient neuronal transmission. The CK--/-- double knockout mice demonstrate every morning a body temperature drop of ~1.0 °C, and they have impaired thermogenesis, as revealed by severe hypothermia upon cold exposure. This defective thermoregulation is not associated with abnormal food intake, decreased locomotive activity, or increased torpor sensitivity. Although white and brown adipose tissue fat pads are diminished in CK--/-- mice, intravenous norepinephrine infusion results in a normal brown adipose tissue response with increasing core body temperatures, indicating that the sympathetic innervation functions correctly (Streijger et al., 2009). This study revealed c-fos changes following a cold challenge, and that neuropeptide Y levels were decreased in the paraventricular nucleus of wildtype, but not CK--/--, mice. A reduction in hypothalamic neuropeptide Y is coupled to increased uncoupling protein 1 expression in brown adipose tissue, resulting in thermogenesis. In CK--/-- mice the neuropeptide Y levels did not change. This lack of hypothalamic plasticity of neuropeptide Y might be the result of inefficient neuronal transmission or can be explained by the previous observation of reduced circulating levels of leptin in CK--/-- mice.

Neonatal leptin exposure specifies innervation of presympathetic hypothalamic neurons and improves the metabolic status of leptin-deficient mice.

The paraventricular nucleus of the hypothalamus (PVH) consists of distinct functional compartments regulating neuroendocrine, behavioral, and autonomic activities that are involved in the homeostatic control of energy balance. These compartments receive synaptic inputs from neurons of the arcuate nucleus of the hypothalamus (ARH) that contains orexigenic agouti-related peptide (AgRP) and anorexigenic pro-opiomelanocortin (POMC) neuropeptides. The axon outgrowth from the ARH to PVH occurs during a critical postnatal period and is influenced by the adipocyte-derived hormone leptin, which promotes its development. However, little is known about leptin's role in specifying patterns of cellular connectivity in the different compartments of the PVH. To address this question, we used retrograde and immunohistochemical labeling to evaluate neuronal inputs onto sympathetic preautonomic and neuroendocrine neurons in PVH of leptin-deficient mice (Lep(ob)/Lep(ob)) exposed to a postnatal leptin treatment. In adult Lep(ob)/Lep(ob) mice, densities of AgRP- and α-melanocortin stimulating hormone (αMSH)-immunoreactive fibers were significantly reduced in neuroendocrine compartments of the PVH, but only AgRP were reduced in all regions containing preautonomic neurons. Moreover, postnatal leptin treatment significantly increased the density of AgRP-containing fibers and peptidergic inputs onto identified preautonomic, but not onto neuroendocrine cells. Neonatal leptin treatment neither rescued αMSH inputs onto neuroendocrine neurons, nor altered cellular ratios of inhibitory and excitatory inputs. These effects were associated with attenuated body weight gain, food intake and improved physiological response to sympathetic stimuli. Together, these results provide evidence that leptin directs cell type-specific patterns of ARH peptidergic inputs onto preautonomic neurons in the PVH, which contribute to normal energy balance regulation.

Regulation of hypothalamic signaling by tuberoinfundibular peptide of 39 residues is critical for the response to cold: a novel peptidergic mechanism of thermoregulation.

Euthermia is critical for mammalian homeostasis. Circuits within the preoptic hypothalamus regulate temperature, with fine control exerted via descending GABAergic inhibition of presympathetic motor neurons that control brown adipose tissue (BAT) thermogenesis and cutaneous vascular tone. The thermoregulatory role of hypothalamic excitatory neurons is less clear. Here we report peptidergic regulation of preoptic glutamatergic neurons that contributes to temperature regulation. Tuberoinfundibular peptide of 39 residues (TIP39) is a ligand for the parathyroid hormone 2 receptor (PTH2R). Both peptide and receptor are abundant in the preoptic hypothalamus. Based on PTH2R and vesicular glutamate transporter 2 (VGlut2) immunolabeling in animals with retrograde tracer injection, PTH2R-containing glutamatergic fibers are presynaptic to neurons projecting from the median preoptic nucleus (MnPO) to the dorsomedial hypothalamus. Transneuronal retrograde pathway tracing with pseudorabies virus revealed connectivity between MnPO VGlut2 and PTH2R neurons and BAT. MnPO injection of TIP39 increased body temperature by 2°C for several hours. Mice lacking TIP39 signaling, either because of PTH2R-null mutation or brain delivery of a PTH2R antagonist had impaired heat production upon cold exposure, but no change in basal temperature and no impairment in response to a hot environment. Thus, TIP39 appears to act on PTH2Rs present on MnPO glutamatergic terminals to regulate their activation of projection neurons and subsequent sympathetic BAT activation. This excitatory mechanism of heat production appears to be activated on demand, during cold exposure, and parallels the tonic inhibitory GABAergic control of body temperature.

[Central regulation of body temperature by RANKL/RANK pathway].

Receptor-activator of NF-κB ligand (RANKL) and its specific receptor RANK are key regulators of bone remodeling, lymph node formation, establishment of the thymic microenviroment, mammary gland development during pregnancy, bone metastasis in cancer and sex-hormone, progestin, -driven breast cancer. RANKL and RANK are also expressed in the central nervous systems (CNS) especially existed in the main region of thermoregulation. Central RANKL injection to the rodents induces fever via PGE(2)/EP3R pathway. This pathway is related with inflammation related fever. On the other hand, female mice with RANK gene deletion in neuron and astrocytes show increased their basal body temperature at the dark phase, which suggests RANKL/RANK system also regulates physiological thremoregulation in female. Not only in rodents but also in human, two children with a homozygous RANK mutation exhibit an abrogated fever response in pneumonia compare with the age-matched children with pneumonia. Thus, the central RANKL/RANK pathway has an important role for thermoregulation.

Genomics and proteomics of heat acclimation.

Plasticity of the thermoregulatory system is a key factor for the induction of heat acclimation. Temperature-adaptive shifts in gene expression play an essential role in the processes involved. This review attempts to bridge the gap between the classical physiological heat acclimation profile and the molecular/cellular mechanisms underlying the evolution of the acclimated phenotype. Essential acclimatory modifications linked with thermal tolerance are (i) neuronal plasticity (ii) cytoprotection. Leftward and rightward threshold shifts in these respective functional categories expand the dynamic thermoregulatory range of the acclimated phenotype. Neural plasticity depends on changes in hypothalamic warm/cold sensitive neuron ratio and excitability. Over the course of acclimation, there is marked upregulation of transcripts encoding voltage dependent K+ and Ca2+ channels, neurotransmitters and/or their receptors. Temperature threshold for thermal injury is associated with progressive enhancement of inducible cytoprotective networks including the essential acclimatory components HSP70, HSF1 and HIF-1. Via cross-tolerance, achieved through shared on-call cytoprotective networks, acclimation also renders protection against novel stressors. Collectively, heat acclimation is a within life evolutionarily beneficial phenomenon with a memory, imprinted via epigenetic mechanisms.

The psychosocial genomics of therapeutic hypnosis, psychotherapy, and rehabilitation.

This paean composed on the occasion of the inaugural Bernauer W. Newton Trust presentation celebrates the personal and professional culture of 50 years of mentorship, teaching, and research by the American Society for Clinical Hypnosis (ASCH). This review of current neuroscience concepts of therapeutic hypnosis and psychotherapy is made possible by the cooperation and dedication of all members of our society. Emerging pathways of psychosocial genomic research, which will lead to new directions for our society, are highlighted for their impact on our professional practice in the present and future.

Analysis of the hypothalamus in a case of X-linked lissencephaly with abnormal genitalia (XLAG).

X-linked lissencephaly with abnormal genitalia (XLAG) is characterized by lissencephaly, absent corpus callosum and ambiguous genitalia. We examined hypothalamic dysfunctions in a XLAG case with a novel mutation of the ARX gene, and performed immunohistochemical evaluation of the diencephalons in autopsy brain. A 1-year-old boy showed intractable epilepsy, persistent diarrhea and disturbed temperature regulation. This case had abnormalities in circadian rhythms and pituitary hormone reserve test. He died of pneumonia. The globus pallidus and subthalamic nucleus was not identified, and the putamen and thalamus were dysplasic. The suprachiasmatic nucleus was absent. A few neurons immunoreactive for vasopressin seemed to form the ectopic supraoptic-like nucleus. The diencephalons were disturbed differently in each sub-region, and the changes may be related to various hypothalamic dysfunctions.

Dynamic changes in DNA methylation during thermal control establishment affect CREB binding to the brain-derived neurotrophic factor promoter.

Thermal control establishment develops during a critical period by alterations in cellular properties in the frontal hypothalamus. These alterations may be modulated by the epigenetic code that determines the repertoire of transcribed proteins. Here we demonstrate transient changes in the expression of brain-derived neurotrophic factor (Bdnf) during both thermal conditioning and re-exposure of conditioned chicks to heat stress, relative to their age-matched naive counterparts. These changes coincide with changes in CpG methylation pattern in the avian Bdnf promoter region. Reduction in methylation during heat conditioning was observed at a cAMP response element-binding (CREB) site which coincided with both elevation in phospho-CREB levels and its binding to the Bdnf promoter. At the same time, an increase in methylation was observed at two other CpG sites, accompanied by elevation of the DNA methyltransferase 3a (DNMT3a) expression. DNMT3a was also found to bind to the two elevated methyl CpG sites, but not to the CREB binding site. These data suggest that complex and dynamic changes in DNA methylation are involved in the regulation of Bdnf expression during thermotolerance acquisition.