Brain energy metabolism: A roadmap for future research.

Although we have learned much about how the brain fuels its functions over the last decades, there remains much still to discover in an organ that is so complex. This article lays out major gaps in our knowledge of interrelationships between brain metabolism and brain function, including biochemical, cellular, and subcellular aspects of functional metabolism and its imaging in adult brain, as well as during development, aging, and disease. The focus is on unknowns in metabolism of major brain substrates and associated transporters, the roles of insulin and of lipid droplets, the emerging role of metabolism in microglia, mysteries about the major brain cofactor and signaling molecule NAD+, as well as unsolved problems underlying brain metabolism in pathologies such as traumatic brain injury, epilepsy, and metabolic downregulation during hibernation. It describes our current level of understanding of these facets of brain energy metabolism as well as a roadmap for future research.

Characterization and Seasonal Modulation of Adenosine A1 Receptors in the Arctic Ground Squirrel Brain.

Hibernation is an adaptation that allows animals such as the Arctic ground squirrel (AGS) to survive the absence of food or water during the winter season. Understanding mechanisms of metabolic suppression during hibernation torpor promises new therapies for critical care. The activation of the Adenosine A1 receptor (A1AR) has been shown to be necessary and sufficient for entrance into hibernation with a winter season sensitization to the agonist, but the role of the A1AR in seasonal sensitization is unknown. In the current study, we characterize the A1AR in the forebrain, hippocampus and hypothalamus of summer and torpid AGS. For the first time, we define the pharmacological characteristics of the A1AR agonist, N6-cyclohexyladenosine and the A1AR antagonist dipropylcyclopentylxanthine (DPCPX) in the AGS brain. In addition, we test the hypothesis that increased A1AR agonist efficacy is responsible for sensitization of the A1AR during the torpor season. The resulting 35S-GTPγS binding data indicate an increase in agonist potency during torpor in two out of three brain regions. In addition to 35S-GTPγS binding, [3H]DPCPX saturation and competition assays establish for the first-time pharmacological characteristics for the A1AR agonist, N6-cyclohexyladenosine and the A1AR antagonist dipropylcyclopentylxanthine (DPCPX) in AGS brain.

Red Blood Cell Metabolic Responses to Torpor and Arousal in the Hibernator Arctic Ground Squirrel.

Arctic ground squirrels provide a unique model to investigate metabolic responses to hibernation in mammals. During winter months these rodents are exposed to severe hypothermia, prolonged fasting, and hypoxemia. In the light of their role in oxygen transport/off-loading and owing to the absence of nuclei and organelles (and thus de novo protein synthesis capacity), mature red blood cells have evolved metabolic programs to counteract physiological or pathological hypoxemia. However, red blood cell metabolism in hibernation has not yet been investigated. Here we employed targeted and untargeted metabolomics approaches to investigate erythrocyte metabolism during entrance to torpor to arousal, with a high resolution of the intermediate time points. We report that torpor and arousal promote metabolism through glycolysis and pentose phosphate pathway, respectively, consistent with previous models of oxygen-dependent metabolic modulation in mature erythrocytes. Erythrocytes from hibernating squirrels showed up to 100-fold lower levels of biomarkers of reperfusion injury, such as the pro-inflammatory dicarboxylate succinate. Altered tryptophan metabolism during torpor was here correlated to the accumulation of potentially neurotoxic catabolites kynurenine, quinolinate, and picolinate. Arousal was accompanied by alterations of sulfur metabolism, including sudden spikes in a metabolite putatively identified as thiorphan (level 1 confidence)-a potent inhibitor of several metalloproteases that play a crucial role in nociception and inflammatory complication to reperfusion secondary to ischemia or hemorrhage. Preliminary studies in rats showed that intravenous injection of thiorphan prior to resuscitation mitigates metabolic and cytokine markers of reperfusion injury, etiological contributors to inflammatory complications after shock.

Seasonal decrease in thermogenesis and increase in vasoconstriction explain seasonal response to N6 -cyclohexyladenosine-induced hibernation in the Arctic ground squirrel (Urocitellus parryii).

Hibernation is a seasonal phenomenon characterized by a drop in metabolic rate and body temperature. Adenosine A1 receptor agonists promote hibernation in different mammalian species, and the understanding of the mechanism inducing hibernation will inform clinical strategies to manipulate metabolic demand that are fundamental to conditions such as obesity, metabolic syndrome, and therapeutic hypothermia. Adenosine A1 receptor agonist-induced hibernation in Arctic ground squirrels is regulated by an endogenous circannual (seasonal) rhythm. This study aims to identify the neuronal mechanism underlying the seasonal difference in response to the adenosine A1 receptor agonist. Arctic ground squirrels were implanted with body temperature transmitters and housed at constant ambient temperature (2°C) and light cycle (4L:20D). We administered CHA (N6 -cyclohexyladenosine), an adenosine A1 receptor agonist in euthermic-summer phenotype and euthermic-winter phenotype and used cFos and phenotypic immunoreactivity to identify cell groups affected by season and treatment. We observed lower core and subcutaneous temperature in winter animals and CHA produced a hibernation-like response in winter, but not in summer. cFos-ir was greater in the median preoptic nucleus and the raphe pallidus in summer after CHA. CHA administration also resulted in enhanced cFos-ir in the nucleus tractus solitarius and decreased cFos-ir in the tuberomammillary nucleus in both seasons. In winter, cFos-ir was greater in the supraoptic nucleus and lower in the raphe pallidus than in summer. The seasonal decrease in the thermogenic response to CHA and the seasonal increase in vasoconstriction, assessed by subcutaneous temperature, reflect the endogenous seasonal modulation of the thermoregulatory systems necessary for CHA-induced hibernation. Cover Image for this issue: doi: 10.1111/jnc.14528.

Arctic ground squirrel hippocampus tolerates oxygen glucose deprivation independent of hibernation season even when not hibernating and after ATP depletion, acidosis, and glutamate efflux.

Cerebral ischemia/reperfusion (I/R) triggers a cascade of uncontrolled cellular processes that perturb cell homeostasis. The arctic ground squirrel (AGS), a seasonal hibernator resists brain damage following cerebral I/R caused by cardiac arrest and resuscitation. However, it remains unclear if tolerance to I/R injury in AGS depends on the hibernation season. Moreover, it is also not clear if events such as depletion of ATP, acidosis, and glutamate efflux that are associated with anoxic depolarization are attenuated in AGS. Here, we employ a novel microperfusion technique to test the hypothesis that tolerance to I/R injury modeled in an acute hippocampal slice preparation in AGS is independent of the hibernation season and persists even after glutamate efflux. Acute hippocampal slices were harvested from summer euthermic AGS, hibernating AGS, and interbout euthermic AGS. Slices were subjected to oxygen glucose deprivation (OGD), an in vitro model of I/R injury to determine cell death marked by lactate dehydrogenase (LDH) release. ATP was assayed using ENLITEN ATP assay. Glutamate and aspartate efflux was measured using capillary electrophoresis. For acidosis, slices were subjected to pH 6.4 or ischemic shift solution (ISS). Acute hippocampal slices from rats were used as a positive control, susceptible to I/R injury. Our results indicate that when tissue temperature is maintained at 36°C, hibernation season has no influence on OGD-induced cell death in AGS hippocampal slices. Our data also show that tolerance to OGD in AGS hippocampal slices occurs despite loss of ATP and glutamate release, and persists during conditions that mimic acidosis and ionic shifts, characteristic of cerebral I/R. Read the Editorial Comment for this article on page 10.

Optimization of Thermolytic Response to A1 Adenosine Receptor Agonists in Rats.

Cardiac arrest is a leading cause of death in the United States, and, currently, therapeutic hypothermia, now called targeted temperature management (TTM), is the only recent treatment modality proven to increase survival rates and reduce morbidity for this condition. Shivering and subsequent metabolic stress, however, limit application and benefit of TTM. Stimulating central nervous system A1 adenosine receptors (A1AR) inhibits shivering and nonshivering thermogenesis in rats and induces a hibernation-like response in hibernating species. In this study, we investigated the pharmacodynamics of two A1AR agonists in development as antishivering agents. To optimize body temperature (Tb) control, we evaluated the influence of every-other-day feeding, dose, drug, and ambient temperature (Ta) on the Tb-lowering effects of N6-cyclohexyladenosine (CHA) and the partial A1AR agonist capadenoson in rats. The highest dose of CHA (1.0 mg/kg, i.p.) caused all ad libitum-fed animals tested to reach our target Tb of 32°C, but responses varied and some rats overcooled to a Tb as low as 21°C at 17.0°C Ta Dietary restriction normalized the response to CHA. The partial agonist capadenoson (1.0 or 2.0 mg/kg, i.p.) produced a more consistent response, but the highest dose decreased Tb by only 1.6°C. To prevent overcooling after CHA, we studied continuous i.v. administration in combination with dynamic surface temperature control. Results show that after CHA administration control of surface temperature maintains desired target Tb better than dose or ambient temperature.

Neural Signaling Metabolites May Modulate Energy Use in Hibernation.

Despite an epidemic in obesity and metabolic syndrome limited means exist to effect adiposity or metabolic rate other than life style changes. Here we review evidence that neural signaling metabolites may modulate thermoregulatory pathways and offer novel means to fine tune energy use. We extend prior reviews on mechanisms that regulate thermogenesis and energy use in hibernation by focusing primarily on the neural signaling metabolites adenosine, AMP and glutamate.

Proteomic Profiling Reveals Adaptive Responses to Surgical Myocardial Ischemia-Reperfusion in Hibernating Arctic Ground Squirrels Compared to Rats.

BACKGROUND: Hibernation is an adaptation to extreme environments known to provide organ protection against ischemia-reperfusion (I/R) injury. An unbiased systems approach was utilized to investigate hibernation-induced changes that are characteristic of the hibernator cardioprotective phenotype, by comparing the myocardial proteome of winter hibernating arctic ground squirrels (AGS), summer active AGS, and rats subjected to I/R, and further correlating with targeted metabolic changes. METHODS: In a well-defined rodent model of I/R by deep hypothermic circulatory arrest followed by 3 or 24 h of reperfusion or sham, myocardial protein abundance in AGS (hibernating summer active) and rats (n = 4 to 5/group) was quantified by label-free proteomics (n = 4 to 5/group) and correlated with metabolic changes. RESULTS: Compared to rats, hibernating AGS displayed markedly reduced plasma levels of troponin I, myocardial apoptosis, and left ventricular contractile dysfunction. Of the 1,320 rat and 1,478 AGS proteins identified, 545 were differentially expressed between hibernating AGS and rat hearts (47% up-regulated and 53% down-regulated). Gene ontology analysis revealed down-regulation in hibernating AGS hearts of most proteins involved in mitochondrial energy transduction, including electron transport chain complexes, acetyl CoA biosynthesis, Krebs cycle, glycolysis, and ketogenesis. Conversely, fatty acid oxidation enzymes and sirtuin-3 were up-regulated in hibernating AGS, with preserved peroxisome proliferator-activated receptor-α activity and reduced tissue levels of acylcarnitines and ceramides after I/R. CONCLUSIONS: Natural cardioprotective adaptations in hibernators involve extensive metabolic remodeling, featuring increased expression of fatty acid metabolic proteins and reduced levels of toxic lipid metabolites. Robust up-regulation of sirtuin-3 suggests that posttranslational modifications may underlie organ protection in hibernating mammals.

No oxygen? No problem! Intrinsic brain tolerance to hypoxia in vertebrates.

Many vertebrates are challenged by either chronic or acute episodes of low oxygen availability in their natural environments. Brain function is especially vulnerable to the effects of hypoxia and can be irreversibly impaired by even brief periods of low oxygen supply. This review describes recent research on physiological mechanisms that have evolved in certain vertebrate species to cope with brain hypoxia. Four model systems are considered: freshwater turtles that can survive for months trapped in frozen-over lakes, arctic ground squirrels that respire at extremely low rates during winter hibernation, seals and whales that undertake breath-hold dives lasting minutes to hours, and naked mole-rats that live in crowded burrows completely underground for their entire lives. These species exhibit remarkable specializations of brain physiology that adapt them for acute or chronic episodes of hypoxia. These specializations may be reactive in nature, involving modifications to the catastrophic sequelae of oxygen deprivation that occur in non-tolerant species, or preparatory in nature, preventing the activation of those sequelae altogether. Better understanding of the mechanisms used by these hypoxia-tolerant vertebrates will increase appreciation of how nervous systems are adapted for life in specific ecological niches as well as inform advances in therapy for neurological conditions such as stroke and epilepsy.

Pre-hibernation diet alters skeletal muscle relaxation kinetics, but not force development in torpid arctic ground squirrels.

During the hibernation season, Arctic ground squirrels (AGS) experience extreme temperature fluctuations (body temperature, Tb, as low as - 3 °C), during which they are mostly physically inactive. Once Tb reaches ~ 15 °C during interbout arousals, hibernators recruit skeletal muscle (SkM) for shivering thermogenesis to reach Tb of ~ 35 °C. Polyunsaturated fatty acids (PUFA) in the diet are known to influence SkM function and metabolism. Recent studies in the cardiac muscle of hibernators have revealed that increased levels of ω-6 and the ω-6:ω-3 PUFA ratio correlate with sarco/endoplasmic reticulum calcium ATPase (SERCA) activity and hibernation status. We hypothesized that diet (increased ω-6:ω-3 PUFA ratio) and torpor status are important in the regulation of the SERCA pump and that this may improve SkM performance during hibernation. Ex vivo functional assays were used to characterize performance changes in SkM (diaphragm) from AGS fed the following diets. (1) Standard rodent chow with an ω-6:ω-3 ratio of 5:1, or (2) a balanced diet with an ω-6:ω-3 ratio of 1:1 that roughly mimics wild diet. We collected diaphragms at three different stages of hibernation (early torpor, late torpor, and arousal) and evaluated muscle function under hypothermic temperature stress at 4 °C, 15 °C, 25 °C, and 37 °C to determine functional resilience. Our data show that torpid animals fed standard rodent chow have faster SkM relaxation when compared to the balanced diet animals. Furthermore, we discovered that standard rodent chow AGS during torpor has higher SkM relaxation kinetics, but this effect of torpor is eliminated in balanced diet AGS. Interestingly, neither diet nor torpor influenced the rate of force development (rate of calcium release). This is the first study to show that increasing the dietary ω-6:ω-3 PUFA ratio improves skeletal muscle performance during decreased temperatures in a hibernating animal. This evidence supports the interpretation that diet can change some functional properties of the SkM, presumably through membrane lipid composition, ambient temperature, and torpor interaction, with an impact on SkM performance.

Remodelling of Skeletal Muscle Myosin Metabolic States in Hibernating Mammals.

Hibernation is a period of metabolic suppression utilized by many small and large mammal species to survive during winter periods. As the underlying cellular and molecular mechanisms remain incompletely understood, our study aimed to determine whether skeletal muscle myosin and its metabolic efficiency undergo alterations during hibernation to optimize energy utilization. We isolated muscle fibers from small hibernators, Ictidomys tridecemlineatus and Eliomys quercinus and larger hibernators, Ursus arctos and Ursus americanus. We then conducted loaded Mant-ATP chase experiments alongside X-ray diffraction to measure resting myosin dynamics and its ATP demand. In parallel, we performed multiple proteomics analyses. Our results showed a preservation of myosin structure in U. arctos and U. americanus during hibernation, whilst in I. tridecemlineatus and E. quercinus, changes in myosin metabolic states during torpor unexpectedly led to higher levels in energy expenditure of type II, fast-twitch muscle fibers at ambient lab temperatures (20°C). Upon repeating loaded Mant-ATP chase experiments at 8°C (near the body temperature of torpid animals), we found that myosin ATP consumption in type II muscle fibers was reduced by 77-107% during torpor compared to active periods. Additionally, we observed Myh2 hyper-phosphorylation during torpor in I. tridecemilineatus, which was predicted to stabilize the myosin molecule. This may act as a potential molecular mechanism mitigating myosin-associated increases in skeletal muscle energy expenditure during periods of torpor in response to cold exposure. Altogether, we demonstrate that resting myosin is altered in hibernating mammals, contributing to significant changes to the ATP consumption of skeletal muscle. Additionally, we observe that it is further altered in response to cold exposure and highlight myosin as a potentially contributor to skeletal muscle non-shivering thermogenesis.

Remodeling of skeletal muscle myosin metabolic states in hibernating mammals.

Hibernation is a period of metabolic suppression utilized by many small and large mammal species to survive during winter periods. As the underlying cellular and molecular mechanisms remain incompletely understood, our study aimed to determine whether skeletal muscle myosin and its metabolic efficiency undergo alterations during hibernation to optimize energy utilization. We isolated muscle fibers from small hibernators, Ictidomys tridecemlineatus and Eliomys quercinus and larger hibernators, Ursus arctos and Ursus americanus. We then conducted loaded Mant-ATP chase experiments alongside X-ray diffraction to measure resting myosin dynamics and its ATP demand. In parallel, we performed multiple proteomics analyses. Our results showed a preservation of myosin structure in U. arctos and U. americanus during hibernation, whilst in I. tridecemlineatus and E. quercinus, changes in myosin metabolic states during torpor unexpectedly led to higher levels in energy expenditure of type II, fast-twitch muscle fibers at ambient lab temperatures (20 °C). Upon repeating loaded Mant-ATP chase experiments at 8 °C (near the body temperature of torpid animals), we found that myosin ATP consumption in type II muscle fibers was reduced by 77-107% during torpor compared to active periods. Additionally, we observed Myh2 hyper-phosphorylation during torpor in I. tridecemilineatus, which was predicted to stabilize the myosin molecule. This may act as a potential molecular mechanism mitigating myosin-associated increases in skeletal muscle energy expenditure during periods of torpor in response to cold exposure. Altogether, we demonstrate that resting myosin is altered in hibernating mammals, contributing to significant changes to the ATP consumption of skeletal muscle. Additionally, we observe that it is further altered in response to cold exposure and highlight myosin as a potentially contributor to skeletal muscle non-shivering thermogenesis.

Many animals use hibernation as a tactic to survive harsh winters. During this dormant, inactive state, animals reduce or limit body processes, such as heart rate and body temperature, to minimise their energy use. To conserve energy during hibernation, animals can use different approaches. For example, garden dormice undergo periodic states of extremely low core temperatures (down to 4­8^oC); whereas Eurasian brown bears see milder temperature drops (down to 23­25^oC). An important organ that changes during hibernation is skeletal muscle. Skeletal muscle typically uses large amounts of energy, making up around 50% of body mass. To survive, hibernating animals must change how their skeletal muscle uses energy. Traditionally, active myosin ­ a protein found in muscles that helps muscles to contract ­ was thought to be responsible for most of the energy use by skeletal muscle. But, more recently, resting myosin has also been found to use energy when muscles are relaxed. Lewis et al. studied myosin and skeletal muscle energy use changes during hibernation and whether they could impact the metabolism of hibernating animals. Lewis et al. assessed myosin changes in muscle samples from squirrels, dormice and bears during hibernation and during activity. Experiments showed changes in resting myosin in squirrels and dormice (whose temperature drops to 4­8^oC during hibernation) but not in bears. Further analysis revealed that cooling samples from non-hibernating muscle to 4­8^oC increased energy use in resting myosin, thereby generating heat. However, no increase in energy use was found after cooling hibernating muscle samples to 4­8^oC. This suggest that resting myosin generates heat at cool temperatures ­ a mechanism that is switched off in hibernating animals to allow them to cool their body temperature. These findings reveal key insights into how animals conserve energy during hibernation. In addition, the results show that myosin regulates energy use in skeletal muscles, which indicates myosin may be a potential drug target in metabolic diseases, such as obesity.

Near infrared spectroscopy detection of hemispheric cerebral ischemia following middle cerebral artery occlusion in rats.

Timely and sensitive in vivo estimation of ischemic stroke-induced brain infarction are necessary to guide diagnosis and evaluation of treatments' efficacy. The gold standard for estimation of the cerebral infarction volume is magnetic resonance imaging (MRI), which is expensive and not readily accessible. Measuring regional cerebral blood flow (rCBF) with Laser Doppler flowmetry (LDF) is the status quo for confirming reduced blood flow in experimental ischemic stroke models. However, rCBF reduction following cerebral artery occlusion often does not correlate with subsequent infarct volume. In the present study, we employed the continuous-wave near infrared spectroscopy (NIRS) technique to monitor cerebral oxygenation during 90 min of the intraluminal middle cerebral artery occlusion (MCAO) in Sprague-Dawley rats (n = 8, male). The NIRS device consisted of a controller module and an optical sensor with two LED light sources and two photodiodes making up two parallel channels for monitoring left and right cerebral hemispheres. Optical intensity measurements were converted to deoxyhemoglobin (Hb) and oxyhemoglobin (HbO2) changes relative to a 2-min window prior to MCAO. Area under the curve (auc) for Hb and HbO2 was calculated for the 90-min occlusion period for each hemisphere (ipsilateral and contralateral). To obtain a measure of total ischemia, auc of the contralateral side was subtracted from the ipsilateral side resulting in ΔHb and ΔHbO2 parameters. Infarct volume (IV) was calculated by triphenyl tetrazolium chloride (TTC) staining at 24h reperfusion. Results showed a significant negative correlation (r = -0.81, p = 0.03) between ΔHb and infarct volume. In conclusion, our results show feasibility of using a noninvasive optical imaging instrument, namely NIRS, in monitoring cerebral ischemia in a rodent stroke model. This cost-effective, non-invasive technique may improve the rigor of experimental models of ischemic stroke by enabling in vivo longitudinal assessment of cerebral oxygenation and ischemic injury.

Cardiac Rhythms and Variation in Hibernating Arctic Ground Squirrels.

AbstractThe dramatic decrease in heart rate (HR) during entrance into hibernation is not a mere response to the lowering of core body temperature (Tb) but a highly regulated fall, as the decrease in HR precedes the drop in Tb. This regulated fall in HR is thought to be mediated by increased cardiac parasympathetic activity. Conversely, the sympathetic nervous system is thought to drive the increase of HR during arousal. Despite this general understanding, we lack temporal information on cardiac parasympathetic regulation throughout a complete hibernation bout. The goal of this study was to fill this gap in knowledge by using Arctic ground squirrels implanted with electrocardiogram/temperature telemetry transmitters. Short-term HR variability (root mean square of successive differences [RMSSD]), an indirect measure of cardiac parasympathetic regulation, was calculated in 11 Arctic ground squirrels. RMSSD, normalized as RMSSD/RR interval (RRI), increased fourfold during early entrance (from 0.2±0.1 to 0.8±0.2, P<0.05). RMSSD/RRI peaked after HR dropped by over 90% and Tb fell by 70%. Late entrance was delineated by a decline in RMSSD/RRI while Tb continued to decrease. During arousal, HR started to increase 2 h before Tb, with a concurrent decrease in RMSSD/RRI to a new minimum. As Tb increased to a maximum during interbout arousal, HR declined, and RMSSD/RRI increased. These data suggest that activation of the parasympathetic nervous system initiates and regulates the HR decrease during entrance into hibernation and that withdrawal of parasympathetic activation initiates arousal. We conclude that cardiac parasympathetic regulation persists throughout all phases of a hibernation bout-a feature of the autonomic nervous system's regulation of hibernation that was not appreciated previously.

Opportunities and barriers to translating the hibernation phenotype for neurocritical care.

Targeted temperature management (TTM) is standard of care for neonatal hypoxic ischemic encephalopathy (HIE). Prevention of fever, not excluding cooling core body temperature to 33°C, is standard of care for brain injury post cardiac arrest. Although TTM is beneficial, HIE and cardiac arrest still carry significant risk of death and severe disability. Mammalian hibernation is a gold standard of neuroprotective metabolic suppression, that if better understood might make TTM more accessible, improve efficacy of TTM and identify adjunctive therapies to protect and regenerate neurons after hypoxic ischemia brain injury. Hibernating species tolerate cerebral ischemia/reperfusion better than humans and better than other models of cerebral ischemia tolerance. Such tolerance limits risk of transitions into and out of hibernation torpor and suggests that a barrier to translate hibernation torpor may be human vulnerability to these transitions. At the same time, understanding how hibernating mammals protect their brains is an opportunity to identify adjunctive therapies for TTM. Here we summarize what is known about the hemodynamics of hibernation and how the hibernating brain resists injury to identify opportunities to translate these mechanisms for neurocritical care.

Beyond the symptom: the biology of fatigue.

A workshop titled "Beyond the Symptom: The Biology of Fatigue" was held virtually September 27-28, 2021. It was jointly organized by the Sleep Research Society and the Neurobiology of Fatigue Working Group of the NIH Blueprint Neuroscience Research Program. For access to the presentations and video recordings, see: https://neuroscienceblueprint.nih.gov/about/event/beyond-symptom-biology-fatigue. The goals of this workshop were to bring together clinicians and scientists who use a variety of research approaches to understand fatigue in multiple conditions and to identify key gaps in our understanding of the biology of fatigue. This workshop summary distills key issues discussed in this workshop and provides a list of promising directions for future research on this topic. We do not attempt to provide a comprehensive review of the state of our understanding of fatigue, nor to provide a comprehensive reprise of the many excellent presentations. Rather, our goal is to highlight key advances and to focus on questions and future approaches to answering them.

Season primes the brain in an arctic hibernator to facilitate entrance into torpor mediated by adenosine A(1) receptors.

Torpor in hibernating mammals defines the nadir in mammalian metabolic demand and body temperature that accommodates seasonal periods of reduced energy availability. The mechanism of metabolic suppression during torpor onset is unknown, although the CNS is a key regulator of torpor. Seasonal hibernators, such as the arctic ground squirrel (AGS), display torpor only during the winter, hibernation season. The seasonal character of hibernation thus provides a clue to its regulation. In the present study, we delivered adenosine receptor agonists and antagonists into the lateral ventricle of AGSs at different times of the year while monitoring the rate of O(2) consumption and core body temperature as indicators of torpor. The A(1) antagonist cyclopentyltheophylline reversed spontaneous entrance into torpor. The adenosine A(1) receptor agonist N(6)-cyclohexyladenosine (CHA) induced torpor in six of six AGSs tested during the mid-hibernation season, two of six AGSs tested early in the hibernation season, and none of the six AGSs tested during the summer, off-season. CHA-induced torpor within the hibernation season was specific to A(1)AR activation; the A(3)AR agonist 2-Cl-IB MECA failed to induce torpor, and the A(2a)R antagonist MSX-3 failed to reverse spontaneous onset of torpor. CHA-induced torpor was similar to spontaneous entrance into torpor. These results show that metabolic suppression during torpor onset is regulated within the CNS via A(1)AR activation and requires a seasonal switch in the sensitivity of purinergic signaling.

Inhibition of NMDA-type glutamate receptors induces arousal from torpor in hibernating arctic ground squirrels (Urocitellus parryii).

Hibernation is an adaptation to overcome periods of resource limitation often associated with extreme climatic conditions. The hibernation season consists of prolonged bouts of torpor that are interrupted by brief interbout arousals. Physiological mechanisms regulating spontaneous arousals are poorly understood, but may be related to a need for gluconeogenesis or elimination of metabolic wastes. Glutamate is derived from glutamine through the glutamate-glutamine cycle and from glucose via the pyruvate carboxylase pathway when nitrogen balance favors formation of glutamine. This study tests the hypothesis that activation of NMDA-type glutamate receptors (NMDAR) maintains torpor in arctic ground squirrel (arctic ground squirrel (AGS); Urocitellus parryii). Administration of NMDAR antagonists MK-801 (5 mg/kg, i.p.) that crosses the blood-brain barrier and AP5 (5 mg/kg, i.p.) that does not cross the blood-brain barrier induced arousal in AGS. Central administration of MK-801 (0.2, 2, 20 or 200 µg; icv) to hibernating AGS failed to induce arousal. Results suggest that activation of NMDAR at a peripheral or circumventricular site is necessary to maintain prolonged torpor and that a decrease in glutamate at these sites may contribute to spontaneous arousal in AGS.

Translating PUFA omega 6:3 ratios from wild to captive hibernators (Urocitellus parryii) enhances sex-dependent mass-gain without increasing physiological stress indicators.

Omega 3 polyunsaturated fatty acids (PUFAs) are well-documented for their influence on health and weight loss. Recent studies indicate omega 3 PUFAs may exert a negative impact on cellular stress and physiology in some hibernators. We asked if physiological stress indicators, lipid peroxidation and mass gain in Arctic Ground Squirrels (AGS) were negatively influenced by naturally occurring dietary omega 3 PUFA levels compared to omega 3 PUFA levels found in common laboratory diets. We found plasma fatty acid profiles of free-ranging AGS to be high in omega 3 PUFAs with balanced omega 6:3 ratios, while standard laboratory diets and plasma of captive AGS are high in omega 6 and low in omega 3 PUFAs with higher omega 6:3 ratios. Subsequently, we designed a diet to mimick free-range AGS omega 6:3 ratios in captive AGS. Groups of wild-caught juvenile AGS were either fed: (1) Mazuri Rodent Chow (Standard Rodent chow, 4.95 omega 6:3 ratio), or (2) balanced omega 6:3 chow (Balanced Diet, 1.38 omega 6:3). AGS fed the Balanced Diet had plasma omega 6:3 ratios that mimicked plasma profiles of wild AGS. Balanced Diet increased female body mass before hibernation, but did not influence levels of cortisol in plasma or levels of the lipid peroxidation product 4-HNE in brown adipose tissue. Overall, as the mass gain is critical during pre-hibernation for obligate hibernators, the results show that mimicking a fatty acid profile of wild AGS facilitates sex-dependent mass accumulation without increasing stress indicators.

Thermoregulation in hibernating mammals: The role of the "thyroid hormones system".

Hibernation is a unique evolutionary adaptation to conserve energy. During the pre-hibernation (i.e. fall) season, a progressive decline in core body temperature and further decrease in metabolism underlie a seasonal modulation in thermoregulation. The onset of hibernation requires marked changes in thermoregulatory attributes including adjustment in body temperature and tissue specific increases in thermogenic capacity. The hibernation season is characterized by a regulated suppression in thermogenesis allowing the onset of torpor interrupted by periodic activation of thermogenesis to sustain interbout arousals. Thyroid hormones are known to regulate both body temperature and metabolism, and for this reason, the hypothalamic-pituitary-thyroid axis and thyroid hormones have been investigated as modulators of thermogenesis in the phenomenon of hibernation, but the mechanisms remain poorly understood. In this review, we present an overview of what is known about the thermogenic roles of thyroid hormones in hibernating species across seasons and within the hibernating season (torpor-interbout arousal cycle). Overall, the hypothalamic-pituitary-thyroid axis and thyroid hormones play a role in the pre-hibernation season to enhance thermogenic capacity. During hibernation, thermogenesis is attenuated at the level of sympathetic premotor neurons within the raphe pallidus and by deiodinase expression in the hypothalamus. Further, as recent work highlights the direct effect of thyroid hormones within the central nervous system in activating thermogenesis, we speculate how similar mechanisms may occur in hibernating species to modulate thermogenesis across seasons and to sustain interbout arousals. However, further experiments are needed to elucidate the role of thyroid hormones in hibernation, moving towards the understanding that thyroid hormones metabolism, transport and availability within tissues may be the most telling indicator of thyroid status.