Dorsomedial and preoptic hypothalamic circuits control torpor.

Endotherms can survive low temperatures and food shortage by actively entering a hypometabolic state known as torpor. Although the decrease in metabolic rate and body temperature (Tb) during torpor is controlled by the brain, the specific neural circuits underlying these processes have not been comprehensively elucidated. In this study, we identify the neural circuits involved in torpor regulation by combining whole-brain mapping of torpor-activated neurons, cell-type-specific manipulation of neural activity, and viral tracing-based circuit mapping. We find that Trpm2-positive neurons in the preoptic area and Vgat-positive neurons in the dorsal medial hypothalamus are activated during torpor. Genetic silencing shows that the activity of either cell type is necessary to enter the torpor state. Finally, we show that these cells receive projections from the arcuate and suprachiasmatic nucleus and send projections to brain regions involved in thermoregulation. Our results demonstrate an essential role of hypothalamic neurons in the regulation of Tb and metabolic rate during torpor and identify critical nodes of the torpor regulatory network.

Optogenetic induction of hibernation-like state with modified human Opsin4 in mice.

We recently determined that the excitatory manipulation of Qrfp-expressing neurons in the preoptic area of the hypothalamus (quiescence-inducing neurons [Q neurons]) induced a hibernation-like hypothermic/hypometabolic state (QIH) in mice. To control the QIH with a higher time resolution, we develop an optogenetic method using modified human opsin4 (OPN4; also known as melanopsin), a G protein-coupled-receptor-type blue-light photoreceptor. C-terminally truncated OPN4 (OPN4dC) stably and reproducibly induces QIH for at least 24 h by illumination with low-power light (3 µW, 473 nm laser) with high temporal resolution. The high sensitivity of OPN4dC allows us to transcranially stimulate Q neurons with blue-light-emitting diodes and non-invasively induce the QIH. OPN4dC-mediated QIH recapitulates the kinetics of the physiological changes observed in natural hibernation, revealing that Q neurons concurrently contribute to thermoregulation and cardiovascular function. This optogenetic method may facilitate identification of the neural mechanisms underlying long-term dormancy states such as sleep, daily torpor, and hibernation.

Neurons in the Dorsomedial Hypothalamus Promote, Prolong, and Deepen Torpor in the Mouse.

Torpor is a naturally occurring, hypometabolic, hypothermic state engaged by a wide range of animals in response to imbalance between the supply and demand for nutrients. Recent work has identified some of the key neuronal populations involved in daily torpor induction in mice, in particular, projections from the preoptic area of the hypothalamus to the dorsomedial hypothalamus (DMH). The DMH plays a role in thermoregulation, control of energy expenditure, and circadian rhythms, making it well positioned to contribute to the expression of torpor. We used activity-dependent genetic TRAPing techniques to target DMH neurons that were active during natural torpor bouts in female mice. Chemogenetic reactivation of torpor-TRAPed DMH neurons in calorie-restricted mice promoted torpor, resulting in longer and deeper torpor bouts. Chemogenetic inhibition of torpor-TRAPed DMH neurons did not block torpor entry, suggesting a modulatory role for the DMH in the control of torpor. This work adds to the evidence that the preoptic area of the hypothalamus and the DMH form part of a circuit within the mouse hypothalamus that controls entry into daily torpor.SIGNIFICANCE STATEMENT Daily heterotherms, such as mice, use torpor to cope with environments in which the supply of metabolic fuel is not sufficient for the maintenance of normothermia. Daily torpor involves reductions in body temperature, as well as active suppression of heart rate and metabolism. How the CNS controls this profound deviation from normal homeostasis is not known, but a projection from the preoptic area to the dorsomedial hypothalamus has recently been implicated. We demonstrate that the dorsomedial hypothalamus contains neurons that are active during torpor. Activity in these neurons promotes torpor entry and maintenance, but their activation alone does not appear to be sufficient for torpor entry.

Neurons that regulate mouse torpor.

The advent of endothermy, which is achieved through the continuous homeostatic regulation of body temperature and metabolism1,2, is a defining feature of mammalian and avian evolution. However, when challenged by food deprivation or harsh environmental conditions, many mammalian species initiate adaptive energy-conserving survival strategies-including torpor and hibernation-during which their body temperature decreases far below its homeostatic set-point3-5. How homeothermic mammals initiate and regulate these hypothermic states remains largely unknown. Here we show that entry into mouse torpor, a fasting-induced state with a greatly decreased metabolic rate and a body temperature as low as 20 °C6, is regulated by neurons in the medial and lateral preoptic area of the hypothalamus. We show that restimulation of neurons that were activated during a previous bout of torpor is sufficient to initiate the key features of torpor, even in mice that are not calorically restricted. Among these neurons we identify a population of glutamatergic Adcyap1-positive cells, the activity of which accurately determines when mice naturally initiate and exit torpor, and the inhibition of which disrupts the natural process of torpor entry, maintenance and arousal. Taken together, our results reveal a specific neuronal population in the mouse hypothalamus that serves as a core regulator of torpor. This work forms a basis for the future exploration of mechanisms and circuitry that regulate extreme hypothermic and hypometabolic states, and enables genetic access to monitor, initiate, manipulate and study these ancient adaptations of homeotherm biology.

Neuronal plasticity in the forebrain of the male red-sided garter snake: Effect of season, low temperature dormancy, and hormonal status on dendritic spine density.

Numerous studies have reported seasonal variations in regional morphology in the brains of seasonally breeding vertebrates. These alterations of neuronal morphology and dendritic spine density appear to be an active process within specific brain nuclei that regulate seasonal behaviors. In many cases, this neural plasticity has been found to be in response to changes in circulating sex steroid hormone levels and occur within pathways essential for the control of reproductive behaviors. Male red-sided garter snakes (Thamnophis sirtalis parietalis) (RSGS) exhibit a dissociated reproductive pattern where mating is initiated at a time when the gonads and steroidogenesis are inactive. And, although circulating levels of sex steroid hormones are elevated at the initiation of courtship and mating, the only known cue found to initiate courtship behavior and mating, is an extended period of low temperature dormancy (LTD) followed by exposure to warm temperatures. This study was designed to examine the role of seasons, sex steroid hormones, and LTD on neuronal and dendritic spine density within the anterior hypothalamus-preoptic area (AHPOA), a region shown to be critical for the regulation of reproductive behaviors. In the male RSGS, the density of dendritic spines on neurons in the AHPOA was significantly greater in spring, actively courting animals, than summer or fall, non-courting animals. Animals maintained under conditions of LTD exhibited significantly increasing spine density as time maintained in LTD increased. Animals receiving either testosterone or estradiol had a significantly greater density of dendritic spines than control animals. This study offers evidence suggesting that the "set up" of the pathways controlling courtship behavior and mating in the male RSGS, is not due solely to an extended period of LTD, but that an extended period of LTD in conjunction with circulating sex steroid hormones are critical for the initiation of reproductive behavior.

Sex-specific effects of food supplementation on hibernation performance and reproductive timing in free-ranging common hamsters.

Hibernation is characterized by reduced metabolism and body temperature during torpor bouts. Energy reserves available during winter play an important role for hibernation and some species respond to high energy reserves with reduced torpor expression. Common hamsters are food-storing hibernators and females hibernate for shorter periods than males, probably related to larger food stores. In this study, we provided free-ranging common hamsters with sunflower seeds shortly before winter and recorded body temperature using subcutaneously implanted data loggers. We compared hibernation patterns and body mass changes between individuals with and without food supplements and analysed reproductive onset in females. Supplemented males delayed hibernation onset, hibernated for much shorter periods, and emerged in spring with higher body mass than unsupplemented ones. Additional food did not affect hibernation performance in females, but supplemented females emerged earlier and preceded those without food supplements in reproductive onset. Thus, males and females differently responded to food supplementation: access to energy-rich food stores enabled males to shorten the hibernation period and emerge in better body condition, probably enhancing mating opportunities and reproductive success. Females did not alter hibernation patterns, but started to reproduce earlier than unsupplemented individuals, enabling reproductive benefits by an extended breeding period.

Hypothalamic control systems show differential gene expression during spontaneous daily torpor and fasting-induced torpor in the Djungarian hamster (Phodopus sungorus).

Djungarian hamsters are able to use spontaneous daily torpor (SDT) during the winter season as well as fasting-induced torpor (FIT) at any time of the year to cope with energetically challenging environmental conditions. Torpor is a state of severely reduced metabolism with a pronounced decrease in body temperature, which enables animals to decrease their individual energy requirements. Despite sharing common characteristics, such as reduced body mass before first torpor expression and depressed metabolism and body temperature during the torpid state, FIT and SDT differ in several physiological properties including torpor bout duration, minimal body temperature, fuel utilization and circadian organization. It remains unclear, whether SDT and FIT reflect the same phenomenon or two different physiological states. The hypothalamus has been suggested to play a key role in regulating energy balance and torpor. To uncover differences in molecular control mechanisms of torpor expression, we set out to investigate hypothalamic gene expression profiles of genes related to orexigenic (Agrp/Npy), circadian clock (Bmal1/Per1) and thyroid hormone (Dio2/Mct8) systems of animals undergoing SDT and FIT during different torpor stages. Orexigenic genes were mainly regulated during FIT and remained largely unaffected by SDT. Expression patterns of clock genes showed disturbed circadian clock rhythmicity in animals undergoing FIT, but not in animals undergoing SDT. During both, SDT and FIT, decreased Dio2 expression was detected, indicating reduced hypothalamic T3 availability in both types of torpor. Taken together, our results provide evidence that SDT and FIT also differ in certain central control mechanisms and support the observation that animals undergoing SDT are in energetical balance, whereas animals undergoing FIT display a negative energy balance. This should be carefully taken into account when interpreting data in torpor research, especially from animal models of fasting-induced hypometabolism such as mice.

Effects of food store quality on hibernation performance in common hamsters.

Hibernating animals can adjust torpor expression according to available energy reserves. Besides the quantity, the quality of energy reserves could play an important role for overwintering strategies. Common hamsters are food-storing hibernators and show high individual variation in hibernation performance, which might be related to the quality of food hoards in the hibernacula. In this study, we tested the effects of food stores high in fat content, particularly polyunsaturated fatty acids (PUFAs), on hibernation patterns under laboratory conditions. Control animals received standard rodent pellets only, while in the other group pellets were supplemented with sunflower seeds. We recorded body temperature during winter using subcutaneously implanted data loggers, documented total food consumption during winter, and analysed PUFA proportions in white adipose tissue (WAT) before and after the winter period. About half of the individuals in both groups hibernated and torpor expression did not differ between these animals. Among the high-fat group, however, individuals with high sunflower seeds intake strongly reduced the time spent in deep torpor. PUFA proportions in WAT decreased during winter in both groups and this decline was positively related to the time an individual spent in deep torpor. Sunflower seeds intake dampened the PUFA decline resulting in higher PUFA levels in animals of the high-fat group after winter. In conclusion, our results showed that common hamsters adjusted torpor expression and food intake in relation to the total energy of food reserves, underlining the importance of food hoard quality on hibernation performance.

Gene expression analysis and microdialysis suggest hypothalamic triiodothyronine (T3) gates daily torpor in Djungarian hamsters (Phodopus sungorus).

Thyroid hormones play an important role in regulating seasonal adaptations of mammals. Several studies suggested that reduced availability of 3,3',5-triiodothyronine (T3) in the hypothalamus is required for the physiological adaptation to winter in Djungarian hamsters. We have previously shown that T3 is involved in the regulation of daily torpor, but it remains unclear, whether T3 affects torpor by central or peripheral mechanisms. To determine the effect of T3 concentrations within the hypothalamus in regulating daily torpor, we tested the hypothesis that low hypothalamic T3 metabolism would favour torpor and high T3 concentrations would not. In experiment 1 gene expression in torpid hamsters was assessed for transporters carrying thyroid hormones between cerebrospinal fluid and hypothalamic cells and for deiodinases enzymes, activating or inactivating T3 within hypothalamic cells. Gene expression analysis suggests reduced T3 in hypothalamic cells during torpor. In experiment 2, hypothalamic T3 concentrations were altered via microdialysis and torpor behaviour was continuously monitored by implanted body temperature transmitters. Increased T3 concentrations in the hypothalamus reduced expression of torpor as well as torpor bout duration and depth. Subsequent analysis of gene expression in the ependymal layer of the third ventricle showed clear up-regulation of T3 inactivating deiodinase 3 but no changes in several other genes related to photoperiodic adaptations in hamsters. Finally, serum analysis revealed that increased total T3 serum concentrations were not necessary to inhibit torpor expression. Taken together, our results are consistent with the hypothesis that T3 availability within the hypothalamus significantly contributes to the regulation of daily torpor via a central pathway.

Dietary Supplementation with n-3 Polyunsaturated Fatty Acids Reduces Torpor Use in a Tropical Daily Heterotherm.

Polyunsaturated fatty acids (PUFAs) are involved in a variety of physiological mechanisms, including heterothermy preparation and expression. However, the effects of the two major classes of PUFAs, n-6 and n-3, can differ substantially. While n-6 PUFAs enhance torpor expression, n-3 PUFAs reduce the ability to decrease body temperature. This negative impact of n-3 PUFAs has been revealed in temperate hibernators only. Yet because tropical heterotherms generally experience higher ambient temperature and exhibit higher minimum body temperature during heterothermy, they may not be affected as much by PUFAs as their temperate counterparts. We tested whether n-3 PUFAs constrain torpor use in a tropical daily heterotherm (Microcebus murinus). We expected dietary n-3 PUFA supplementation to induce a reduction in torpor use and for this effect to appear rapidly given the time required for dietary fatty acids to be assimilated into phospholipids. n-3 PUFA supplementation reduced torpor use, and its effect appeared in the first days of the experiment. Within 2 wk, control animals progressively deepened their torpor bouts, whereas supplemented ones never entered torpor but rather expressed only constant, shallow reductions in body temperature. For the rest of the experiment, the effect of n-3 PUFA supplementation on torpor use remained constant through time. Even though supplemented animals also started to express torpor, they exhibited higher minimum body temperature by 2°-3°C and spent two fewer hours in a torpid state per day than control individuals, on average. Our study supports the view that a higher dietary content in n-3 PUFAs negatively affects torpor use in general, not only in cold-acclimated hibernators.

Effects of unsaturated fatty acids on torpor frequency and diet selection in Djungarian hamsters (Phodopus sungorus).

Essential polyunsaturated fatty acids (PUFA) have been shown to play a beneficial role in hibernating mammals. High amounts of dietary PUFA led to an earlier hibernation onset, deeper and longer hibernation bouts and a higher proportion of hibernating animals in several species. In contrast, the relevance of dietary PUFA for daily heterotherms exhibiting only brief and shallow torpor bouts is less well studied. Therefore, diets differing in PUFA composition were used to examine the effects on the frequency of spontaneous daily torpor in Djungarian hamsters (Phodopus sungorus). In contrast to earlier studies, we were interested in whether the ratio of n-6 to n-3 PUFA affects torpor expression, and in comparison with a diet rich in monounsaturated fatty acids (MUFA). Although we found a positive effect on torpor frequency in hamsters fed a diet rich in n-6 PUFA compared with the groups fed diets either rich in n-3 PUFA or MUFA, the latter two groups did not show unusually low torpor frequencies. The results of the additional diet choice experiment indicated that hamsters in short photoperiod select food with only a slight excess of n-6 PUFA compared with n-3 PUFA (ratio of 1 to 1.5). However, there was no significant difference in torpor frequency between the diet choice group and hamsters fed on standard chow with a sevenfold excess of n-6 PUFA. In summary, the present data strongly indicate that the dietary composition of unsaturated fatty acids plays a minor role in the occurrence of spontaneous daily torpor in Djungarian hamsters.

The effects of poly-unsaturated fatty acids on the physiology of hibernation in a South American marsupial, Dromiciops gliroides.

Many mammals hibernate, which is a profound lethargic state of several weeks or months during winter, that represents a transitory episode of hetherothermy. As with other cases of dormancy, the main benefit of hibernation seems to be energy saving. However, the depth and duration of torpor can be experimentally modified by the composition of food, especially by fattyacid composition. In eutherians, diets rich in unsaturated fatty acids (i.e., fatty acids with at least one double bond) lengthen torpor, reduce metabolism and permit hibernation at lower temperatures. Here we studied whether diets varying in fatty acid composition have an effect on the physiology of hibernation in a South American marsupial, Dromiciops gliroides. We designed a factorial experiment where thermal acclimation (two levels: natural versus constant temperature) was combined with diet acclimation: saturated (i.e., diets with high concentration of saturated fatty acids) versus unsaturated (i.e., diets with high concentration of unsaturated fatty acids). We measured energy metabolism in active and torpid individuals, as well as torpor duration, and a suite of 12 blood biochemical parameters. After a cafeteria test, we found that D. gliroides did not show any preference for a given diet. Also, we did not find effects of diet on body temperature during torpor, or its duration. However, saturated diets, combined with high temperatures provoked a disproportionate increase in fat utilization, leading to body mass reduction. Those animals were more active, and metabolized more fats than those fed with a high proportion of unsaturated fatty acids (="unsaturated diets"). These results contrast with previous studies, which showed a significant effect of fatty acid composition of diets on food preferences and torpor patterns in mammals.

Changes in expression of appetite-regulating hormones in the cunner (Tautogolabrus adspersus) during short-term fasting and winter torpor.

Feeding in vertebrates is controlled by a number of appetite stimulating (orexigenic, e.g., orexin and neuropeptide Y, NPY) and appetite suppressing (anorexigenic, e.g., cholecystokinin, CCK and cocaine- and amphetamine-regulated transcript, CART) hormones. Cunners (Tautogolabrus adspersus) survive the winter in shallow coastal waters by entering a torpor-like state, during which they forgo feeding. In order to better understand the mechanisms regulating appetite/fasting in these fish, quantitative real-time PCR was used to measure transcript expression levels of four appetite-regulating hormones: NPY, CART, orexin and CCK in the forebrain (hypothalamus and telencephalon) and CCK in the gut of fed, short-term summer fasted, and natural winter torpor cunners. Summer fasting induced a decrease in hypothalamic orexin levels and telencephalon NPY, CART and CCK mRNA levels. All brain hormone mRNA levels decreased during natural torpor as compared to fed summer fish. In the gut, CCK expression levels decreased during summer fasting. These results indicate that, in cunner, orexin, NPY, CART and CCK may play a role in appetite regulation and might mediate different physiological responses to short-term summer fasting and torpor-induced long-term fasting.