Activation of oxytocinergic neurons enhances torpor in mice.

Mus musculus enters a torpid state in response to caloric restriction in sub-thermoneutral ambient temperatures. This torpid state is characterized by an adaptive and controlled decrease in metabolic rate, heart rate, body temperature, and activity. Previous research has identified the paraventricular nucleus (PVN) within the hypothalamus, a region containing oxytocin neurons, as a location that is active during torpor onset. We hypothesized that oxytocin neurons within the PVN are part of this neural circuit and that activation of oxytocin neurons would deepen and lengthen torpor bouts. We report that activation of oxytocin neurons alone is not sufficient to induce a torpor-like state in the fed mouse, with no significant difference in body temperature or heart rate upon activation of oxytocin neurons. However, we found that activation of oxytocin neurons prior to the onset of daily torpor both deepens and lengthens the subsequent bout, with a 1.7 ± 0.4 °C lower body temperature and a 135 ± 32 min increase in length. We therefore conclude that oxytocin neurons are involved in the neural circuitry controlling daily torpor in the mouse.

Health Effects of Alternate Day Fasting Versus Pair-Fed Caloric Restriction in Diet-Induced Obese C57Bl/6J Male Mice.

Alternate day fasting (ADF) induces weight loss and improves various markers of health in rodents and humans. However, it is unclear whether the benefits of ADF are derived from the lower caloric intake of ADF or from the 24-h fasting period. Therefore, this study directly compared selected markers for health - such as glucose control, body weight, liver triglycerides, T cell frequencies, and others - in high-fat (60% calories from fat) diet-induced obese mice subjected to either ADF or caloric restriction (CR). Obese mice were randomly assigned to one of four groups: (1) ADF: remained on the high-fat diet, but fed on alternate days (n = 5), (2) PF: remained on the high-fat diet, but pair-fed to the ADF group (n = 5), (3) LF: moved to a chow ad libitum diet (n = 5; 17% calories from fat), and (4) HF: remained on the high-fat ad libitum diet (n = 5). An additional group of non-obese mice maintained on a chow diet since weaning were used as controls (CON: n = 5). After 10 weeks, ADF, PF, and LF mice ate fewer kcals, had a lower body mass, had smaller epididymal fat pads, improved glucose tolerance, and had a lower hepatic triglyceride content relative to HF mice (p < 0.05), but none reached that of CON mice in these measures. T cell frequencies of the spleen, blood, and mesenteric lymph nodes were reduced in ADF, PF, and HF compared to the CON group. Importantly, there were no significant differences between the ADF and PF groups in any of the measurements made in the current study. These data suggest that ADF, PF, and LF diets each lead to improved markers of health relative to high-fat diet-induced obese mice, and that the caloric restriction associated with ADF is the major factor for the noted improvements.

Behavioral thermoregulation in the fasted C57BL/6 mouse.

Under relatively cool ambient temperatures and a caloric deficit, mice will undergo daily torpor - a short-term regulated reduction in metabolic rate with a concomitant drop in body temperature. Mice can alternatively achieve metabolic savings by utilizing behavioral changes, such as seeking a warmer environment. However, there is a lack of knowledge about the behavioral interaction between torpor utilization and thermotaxis. That is, if a fasted mouse is faced with a choice between a warm environment not conducive for torpor, and a cool environment that will induce torpor, which scenario will the fasting mouse choose? Here, the temperature preferences of fasted mice were studied using a temperature gradient device that allows a mouse to freely move along a gradient of temperatures. C57BL/6 mice were implanted with temperature telemeters that recorded location, core temperature (Tb), and activity concurrently over a 23-h period in the thermal gradient. When the gradient was on, mice preferred the warm end of the gradient when fed (71 ± 4% of the time) and even more so when fasted (84 ± 2%). When the gradient was on, the fasted minimum Tb was significantly higher (34.4 ± 0.3 °C) than when the gradient was off (27.7 ± 1.6 °C). Further, fasted mice lost significantly more weight when the gradient was off despite maintenance of a metabolically favorable lower minimum Tb in this condition. These results indicate that fasted mice not only prefer warm ambient temperatures when given the choice, but that it is also the pathway with more favorable metabolic outcomes in a period of reduced caloric intake.

Physiological response to the odorant TMT in fully fed and calorically restricted laboratory mice.

2,3,5-trimethyl-3-thiazoline (TMT) is a chemical compound that is extracted from red fox urine and can be used to artificially simulate the presence of a predator. The purpose of this study was to test the hypothesis that TMT would block entry into torpor in the calorically restricted C57Bl/6 mouse. We first demonstrated that TMT induced fear in the mouse. Exposure to TMT induced an acute freeze response (67.2 ± 6.7% of time), as compared to 6.7 ± 1.7% when exposed to water. Further, exposure to TMT for 30 min led to elevated circulating corticosterone levels, 377 ± 33 ng/ml, as compared to 29 ± 4 ng/ml when exposed to water. When mice were exposed to TMT during the dark or light phase, body temperature (Tb) dropped by 1.7 ± 0.9 °C and 0.7 ± 1.1 °C, respectively, over the first 110 min after exposure. To determine whether TMT influences daily torpor, mice were calorically restricted and exposed to either water or TMT. Mice were exposed 30 min before the start of torpor, determined by the bout of the previous day. Exposure to TMT significantly (p < 0.01) blunted the fall in the minimum Tb from 28.8 ± 0.3 °C (water) to 30.1 ± 0.6 °C (TMT) and significantly (p < 0.05) decreased the amount of time Tb was under 32 °C, from 431 ± 48 min (water) to 292 ± 78 min (TMT). These results establish that mice perceived the scent of TMT as a physiologically stressful stimulus and that Tb response is modestly blunted in the presence of that stressor. Our experiment highlights the intricate interplay between predation risk and energy conservation.

Effect of stevia on the gut microbiota and glucose tolerance in a murine model of diet-induced obesity.

Artificial sweeteners have been shown to induce glucose intolerance by altering the gut microbiota; however, little is known about the effect of stevia. Here, we investigate whether stevia supplementation induces glucose intolerance by altering the gut microbiota in mice, hypothesizing that stevia would correct high fat diet-induced glucose intolerance and alter the gut microbiota. Mice were split into four treatment groups: low fat, high fat, high fat + saccharin and high fat + stevia. After 10 weeks of treatment, mice consuming a high fat diet (60% kcal from fat) developed glucose intolerance and gained more weight than mice consuming a low fat diet. Stevia supplementation did not impact body weight or glucose intolerance. Differences in species richness and relative abundances of several phyla were observed in low fat groups compared to high fat, stevia and saccharin. We identified two operational taxonomic groups that contributed to differences in beta-diversity between the stevia and saccharin groups: Lactococcus and Akkermansia in females and Lactococcus in males. Our results demonstrate that stevia does not rescue high fat diet-induced changes in glucose tolerance or the microbiota, and that stevia results in similar alterations to the gut microbiota as saccharin when administered in concordance with a high fat diet.

The physiological signature of daily torpor is not orexin dependent.

Under conditions of scarce food availability and cool ambient temperature, the mouse (Mus Musculus) enters into torpor, a state of transient metabolic suppression mediated in part by the autonomic nervous system. Hypothalamic orexins are involved in the coordination of behaviors and autonomic function. We tested whether orexins are necessary for the coordinated changes in physiological variables, which underlie torpor and represent its physiological signature. We performed simultaneous measurements of brain temperature, electroencephalographic, and electromyographic activity allowing objective assessment of wake-sleep behavior, and cardiovascular, respiratory, and metabolic variables in orexin knockout mice (ORX-KO) and wild-type mice (WT) during torpor bouts elicited by caloric restriction and mild cold stress. We found that torpor bouts in WT are characterized by an exquisitely coordinated physiological signature. The characteristics of torpor bouts in terms of duration and rate of change of brain temperature and electromyographic activity at torpor entrance and exit did not differ significantly between ORX-KO and WT, and neither did the cardiovascular, respiratory, and metabolic characteristics of torpor. ORX-KO and WT also had similar wake-sleep state changes associated with torpor bouts, with the exception of a significantly higher rapid-eye movement sleep time in ORX-KO at torpor entrance. Our results demonstrate that orexins are not necessary either for the normal physiological adaptations occurring during torpor in mice or for their coordination, suggesting that mechanisms different from orexin peptide signaling may be involved in the regulation and the coordination of these physiological responses.

A robust diving response in the laboratory mouse.

The diving response is a coordinated physiological response to submersion under water and has been documented amongst all mammals tested to date. The physiological response consists of three primary reflexes: an immediate bradycardia, apnea, and selective constriction of peripheral blood vessels. We hypothesized that mice would exhibit a diving response upon voluntary submersion into water typically seen in other mammals. In this study, telemeters that measure arterial pressure were implanted into male and female C57Bl/6J mice. These mice were trained to voluntarily dive underwater for a distance of 40 cm over a 4-6 s period. Just before the dive, the interbeat interval (IBI) was 87 ± 6 ms (mean ± SD) and diastolic pressure was 99 ± 14 mmHg. Underwater submersion caused (1) a dramatic bradycardia immediately at the onset of each dive, as IBI increased to 458 ± 104 ms, and (2) a large drop in diastolic pressure, to 56 ± 16 mmHg despite the elevation in peripheral resistance. Mice experienced a short bout (~ 2 s) of hypertension (diastolic pressure rose to 131 ± 17 mmHg) upon emergence. The bradycardia and hypotension appeared to be vagally mediated, since both these responses were blocked with atropine pre-treatment. These data demonstrate that the mouse exhibits a robust diving response upon voluntary submersion into water.

Alternate-day feeding leads to improved glucose regulation on fasting days without significant weight loss in genetically obese mice.

Alternate-day fasting (ADF) is effective for weight loss and increases insulin sensitivity in diet-induced obese rodents. However, the efficacy of ADF in genetic models of obesity has not been comprehensively studied. Mice that are deficient in leptin (ob/ob mice) are obese, diabetic, and prone to deep bouts of torpor when fasted. We tested the hypotheses that an ADF protocol in ob/ob mice would result in 1) induction of torpor on fasted days, 2) minimal body weight loss if the mice experienced torpor, and 3) no improvement in glucose control in the absence of weight loss. Female ob/ob mice and littermate controls were assigned to 1) an ad libitum regimen or 2) an ADF regimen, consisting of fasting every other day with ad libitum feeding between fasts. Over a 19-day period, littermate control mice on the ADF regimen consumed the same amount of food as littermate control mice on the ad libitum regimen, whereas the ADF ob/ob mice consumed 37% less food than ad libitum ob/ob mice. Fasting days, but not fed days, led to torpor in both genotypes. Fasting days, but not fed days, led to weight loss in both genotypes relative to ad libitum controls. Fasting days, but not fed days, produced enhanced insulin sensitivity in both genotypes and normalized circulating glucose in ob/ob mice. These data demonstrate improved glucose control on fasting days with the use of ADF in a genetic model of obesity in the face of minimal weight loss.

The impact of deep breathing and alternate nostril breathing on heart rate variability: a human physiology laboratory.

An increase in the beat-to-beat variability of heart rate (HRV) is a robust marker of enhanced parasympathetic activity and of a calm and relaxed state. The purpose of this laboratory activity was to introduce the concept of HRV to our students, while having them address a novel question of whether two yogic breathing techniques, namely alternate nostril breathing (ANB) and standard deep breathing (DB), impact the SD of instantaneous heart rate (SDHR), a measure of HRV. Fifty-five undergraduates enrolled in a physiology course designed for nonscience majors were tasked with analyzing HR and SDHR from electrocardiograms recorded during normal breathing, DB, and ANB. A repeated-measures ANOVA showed that HR was significantly, albeit slightly, elevated from normal (74.5 ± 13.4 beats/min; means ± SD) during DB (76.5 ± 11.2 beats/min), but not during ANB (75.7 ± 10.1 beats/min). Analysis of SDHR showed significant differences between conditions (normal: 5.5 ± 2.1, DB: 8.6 ± 3.0, ANB: 7.8 ± 2.8 beats/min). The instructors further analyzed the same data set using more robust measures of HRV (SD of sequential N-N intervals, root mean square of successive differences, and high-frequency domain of HRV) to determine whether SDHR during a 2-min epoch is a sufficient measure for HRV in the undergraduate course setting. Statistical analysis for these measures showed a near identical pattern of magnitude and significance among the groups as SDHR. Our students developed a greater appreciation for the effects of breathing patterns on HRV and HR, using the simple measure of SDHR.

The heat is on: A device that reduces cold stress-induced tachycardia in laboratory mice.

Mouse vivaria are typically maintained at an ambient temperature (Ta) of 20-26 °C which is comfortable for human researchers. However, as this Ta is well below the mouse thermoneutral zone (TNZ) of 30-32 °C, typical vivarium temperatures result in cold stress for mice. Recently, a cage has been developed that provides variable cage floor heating, allowing mice to behaviorally regulate body temperature through thermotaxis. A hand warmer provides supplemental heat, elevating cage floor surface temperature for 13 + hours up to 30 °C. This provides a heated surface for the entirety of the light phase. Here, we test the ability of these local heat sources to remove physiological signs of cold stress in mice housed at room temperature by analyzing heart rate (HR), activity, and body temperature in three experimental conditions: 23 °C, 23 °C + heated surface, or 30 °C. The location of C57Bl/6 J mice within the cage was recorded using an infrared camera. In the presence of supplemental heat at a Ta of 23 °C, mice resided atop of the area of the heated surface 85 ±â€¯3% of the 12-h light phase, as compared to 7 ±â€¯2% in the absence of supplemental heat. Further, addition of supplemental heat lowered light phase HR and activity to that seen at a Ta of 30 °C. These results indicate that provision of a local heat source is successful in reducing cold-induced tachycardia in mice housed at typical vivarium temperatures without increasing the ambient temperature of the entire laboratory and subjecting researchers to heat stress.

Defective daily temperature regulation in a mouse model of amyotrophic lateral sclerosis.

Current understanding of the pathogenesis of the familial form of amyotrophic lateral sclerosis has been aided by the study of transgenic mice that over-express mutated forms of the human CuZn-superoxide dismutase (SOD1) gene. While mutant SOD1 in motor neurons determines disease onset, other non-cell autonomous factors are critical for disease progression, and altered energy metabolism has been implicated as a contributing factor. Since most energy expended by laboratory mice is utilized to defend body temperature (Tb), we analyzed thermoregulation in transgenic mice carrying the G93A mutation of the human SOD1 gene, using implantable temperature data loggers to continuously record Tb for up to 85 days. At room (22 °C) ambient temperature, G93A mice exhibited a diminished amplitude of the daily Tb rhythm compared to C57BL/6J controls, secondary to decreased Tb values during the dark (behaviorally active) phase of the light-dark cycle. The defect arose at 85-99 days of age, around the age of symptom onset (as assessed by grip strength), well before observable weakness and weight loss, and could not be accounted for by decreased levels of locomotor activity or food consumption. Housing under thermoneutral (29 °C) ambient temperature partially rescued the defect, but age-dependently (only in animals >100 days of age), suggesting that the deficit in older mice was due in part to inadequate thermogenesis by "peripheral" thermogenic organs as the disease progressed. In younger mice, we found that cold-induced thermogenesis and energy expenditure were intact, hinting that an initial "central" defect might localize to the subparaventricular zone, involving neural output pathways from the circadian clock in the hypothalamic suprachiasmatic nucleus to forebrain thermoregulatory circuitry.

Neurons in ventral tegmental area tonically inhibit sympathetic outflow to brown adipose tissue: possible mediation of thermogenic signals from lateral habenula.

The lateral habenula (LHb), a nucleus involved in the response to salient, especially adverse, environmental events, is implicated in brown adipose tissue (BAT) thermogenesis caused by these events. LHb-elicited thermogenesis involves a neural pathway to the lower brain stem sympathetic control center in the medullary raphé. There are no direct connections from the LHb to the medullary raphé. LHb-mediated behavioral responses involve inhibitory control over the dopamine neurons in the ventral tegmental area (VTA), mediated via an excitatory drive from the LHb to GABAergic neurons in the tail of the VTA. We hypothesized that inhibition of the VTA is also involved in LHb-mediated BAT thermogenesis. To test this hypothesis, inhibition of neurons in the VTA with muscimol increased BAT sympathetic nerve discharge by 22.0 ± 9.2 dBµV ( n = 24, P < 0.0001) and BAT temperature by 1.2 ± 0.1°C ( P < 0.001). This response was abolished by inhibition of the medullary raphé neurons with muscimol. BAT thermogenesis initiated with focal injections of bicuculline in the LHb was reversed by subsequent blockade of GABAA receptors in the VTA with bicuculline. These results suggest that, at least in anesthetized rats, neurons in the VTA tonically inhibit BAT thermogenesis via a link, presently unknown, to the medullary raphé. Removal of this VTA-initiated inhibition is an important mechanism whereby LHb neurons activate BAT thermogenesis.

Changes in blood glucose as a function of body temperature in laboratory mice: implications for daily torpor.

Many small mammals, such as the laboratory mouse, utilize the hypometabolic state of torpor in response to caloric restriction. The signals that relay the lack of fuel to initiate a bout of torpor are not known. Because the mouse will only enter a torpid state when calorically challenged, it may be that one of the inputs for initiation into a bout of torpor is the lack of the primary fuel (glucose) used to power brain metabolism in the mouse. Using glucose telemetry in mice, we tested the hypotheses that 1) circulating glucose (GLC), core body temperature (Tb), and activity are significantly interrelated; and 2) that the level of GLC at the onset of torpor differs from both GLC during arousal from torpor and during feeding when there is no torpor. To test these hypotheses, six C57Bl/6J mice were implanted with glucose telemeters and exposed to different feeding conditions (ad libitum, fasting, limited food intake, and refeeding) to create different levels of GLC and Tb. We found a strong positive and linear correlation between GLC and Tb during ad libitum feeding. Furthermore, mice that were calorically restricted entered torpor bouts readily. GLC was low during torpor entry but did not drop precipitously as Tb did at the onset of a torpor bout. GLC significantly increased during arousal from torpor, indicating the presence of endogenous glucose production. While low GLC itself was not predictive of a bout of torpor, hyperactivity and low GLC preceded the onset of torpor, suggesting that this may be involved in triggering torpor.

Clocks and meals keep mice from being cool.

Daily torpor is used by small mammals to reduce daily energy expenditure in response to energetic challenges. Optimizing the timing of daily torpor allows mammals to maximize its energetic benefits and, accordingly, torpor typically occurs in the late night and early morning in most species. However, the regulatory mechanisms underlying such temporal regulation have not been elucidated. Direct control by the circadian clock and indirect control through the timing of food intake have both been suggested as possible mechanisms. Here, feeding cycles outside of the circadian range and brain-specific mutations of circadian clock genes (Vgat-Cre+ CK1δfl/fl εfl/+ ; Vgat-Cre+ Bmal1fl/fl ) were used to separate the roles of the circadian clock and food timing in controlling the timing of daily torpor in mice. These experiments revealed that the timing of daily torpor is transiently inhibited by feeding, while the circadian clock is the major determinant of the timing of torpor. Torpor never occurred during the early part of the circadian active phase, but was preferentially initiated late in the subjective night. Food intake disrupted torpor in the first 4-6 h after feeding by preventing or interrupting torpor bouts. Following interruption, re-initiation of torpor was unlikely until after the next circadian active phase. Overall, these results demonstrate that feeding transiently inhibits torpor while the central circadian clock gates the timing of daily torpor in response to energetic challenges by restricting the initiation of torpor to a specific circadian phase.

Is Adenosine Action Common Ground for NREM Sleep, Torpor, and Other Hypometabolic States?

This review compares two states that lower energy expenditure: non-rapid eye movement (NREM) sleep and torpor. Knowledge on mechanisms common to these states, and particularly on the role of adenosine in NREM sleep, may ultimately open the possibility of inducing a synthetic torpor-like state in humans for medical applications and long-term space travel. To achieve this goal, it will be important, in perspective, to extend the study to other hypometabolic states, which, unlike torpor, can also be experienced by humans.

Desynchrony between brain and peripheral clocks caused by CK1δ/ε disruption in GABA neurons does not lead to adverse metabolic outcomes.

Circadian disruption as a result of shift work is associated with adverse metabolic consequences. Internal desynchrony between the phase of the suprachiasmatic nuclei (SCN) and peripheral clocks is widely believed to be a major factor contributing to these adverse consequences, but this hypothesis has never been tested directly. A GABAergic Cre driver combined with conditional casein kinase mutations (Vgat-Cre+CK1δfl/flεfl/+ ) was used to lengthen the endogenous circadian period in GABAergic neurons, including the SCN, but not in peripheral tissues, to create a Discordant mouse model. These mice had a long (27.4 h) behavioral period to which peripheral clocks entrained in vivo, albeit with an advanced phase (∼6 h). Thus, in the absence of environmental timing cues, these mice had internal desynchrony between the SCN and peripheral clocks. Surprisingly, internal desynchrony did not result in obesity in this model. Instead, Discordant mice had reduced body mass compared with Cre-negative controls on regular chow and even when challenged with a high-fat diet. Similarly, internal desynchrony failed to induce glucose intolerance or disrupt body temperature and energy expenditure rhythms. Subsequently, a lighting cycle of 2-h light/23.5-h dark was used to create a similar internal desynchrony state in both genotypes. Under these conditions, Discordant mice maintained their lower body mass relative to controls, suggesting that internal desynchrony did not cause the lowered body mass. Overall, our results indicate that internal desynchrony does not necessarily lead to metabolic derangements and suggest that additional mechanisms contribute to the adverse metabolic consequences observed in circadian disruption protocols.

Heart rate dynamics in a marsupial hibernator.

The eastern pygmy possum (Cercartetus nanus) is a small marsupial that can express spontaneous short bouts of torpor, as well as multi-day bouts of deep hibernation. To examine heart rate (fH) control at various stages of torpor in a marsupial hibernator, and to see whether fH variability differs from that of deep placental hibernators, we used radiotelemetry to measure ECG and body temperature (Tb) while measuring the rate of O2 consumption and ventilation. fH and O2 consumption rate during euthermia were at a minimum (321±34 beats min-1, 0.705±0.048 ml O2 g-1 h-1) at an ambient temperature (Ta) of 31°C. fH had an inverse linear relationship with Ta to a maximum of 630±19 beats min-1 at a Ta of 20°C. During entry into torpor at a Ta of 20°C, fH slowed primarily as a result of episodic periods of cardiac activity where electrical activity of the heart occurred in groups of 3 or 4 heart beats. When Tb was stable at 24°C in these torpor bouts, the episodic nature of fH had disappeared (i.e. no asystoles) with a rate of 34±3 beats min-1 For multi-day bouts of deep torpor, Ta was lowered to 6.6±0.8°C. During these deep bouts of torpor, Tb reached a minimum of 8.0±1.0°C, with a minimum fH of 8 beats min-1 and a minimum O2 consumption rate of 0.029±0.07 ml O2 g-1 h-1 Shivering bouts occurred in deep torpor about every 8 min, during which ventilation occurred, and fH was elevated to 40 beats min-1 The duration of the QRS complex increased from 12 ms during euthermia to 69 ms at a Tb of 8°C. These findings demonstrate the dynamic functioning range of fH to be about 600 beats min-1 (∼80-fold), one of the largest known ranges in mammals. Our study shows that despite a separation of ∼160 million years, the control and function of the cardiac system seems indistinguishable in marsupial and placental hibernating mammals.

Central activation of the A1 adenosine receptor in fed mice recapitulates only some of the attributes of daily torpor.

Mice enter bouts of daily torpor, drastically reducing metabolic rate, core body temperature (T b), and heart rate (HR), in response to reduced caloric intake. Because central adenosine activation has been shown to induce a torpor-like state in the arctic ground squirrel, and blocking the adenosine-1 (A1) receptor prevents daily torpor, we hypothesized that central activation of the A1 adenosine receptors would induce a bout of natural torpor in mice. To test the hypothesis, mice were subjected to four different hypothermia bouts: natural torpor, forced hypothermia (FH), isoflurane-anesthesia, and an intracerebroventricular injection of the selective A1 receptor agonist N6-cyclohexyladenosine (CHA). All conditions induced profound hypothermia. T b fell more rapidly in the FH, isoflurane-anesthesia, and CHA conditions compared to torpor, while mice treated with CHA recovered at half the rate of torpid mice. FH, isoflurane-anesthesia, and CHA-treated mice exhibited a diminished drop in HR during entry into hypothermia as compared to torpor. Mice in all conditions except CHA shivered while recovering from hypothermia, and only FH mice shivered substantially while entering hypothermia. Circulating lactate during the hypothermic bouts was not significantly different between the CHA and torpor conditions, both of which had lower than baseline lactate levels. Arrhythmias were largely absent in the FH and isoflurane-anesthesia conditions, while skipped beats were observed in natural torpor and periodic extended (>1 s) HR pauses in the CHA condition. Lastly, the hypothermic bouts showed distinct patterns of gene expression, with torpor characterized by elevated hepatic and cardiac Txnip expression and all other hypothermic states characterized by elevated c-Fos and Egr-1 expression. We conclude that CHA-induced hypothermia and natural torpor are largely different physiological states.

Accurate discrimination of the wake-sleep states of mice using non-invasive whole-body plethysmography.

A major limitation in the study of sleep breathing disorders in mouse models of pathology is the need to combine whole-body plethysmography (WBP) to measure respiration with electroencephalography/electromyography (EEG/EMG) to discriminate wake-sleep states. However, murine wake-sleep states may be discriminated from breathing and body movements registered by the WBP signal alone. Our goal was to compare the EEG/EMG-based and the WBP-based scoring of wake-sleep states of mice, and provide formal guidelines for the latter. EEG, EMG, blood pressure and WBP signals were simultaneously recorded from 20 mice. Wake-sleep states were scored based either on EEG/EMG or on WBP signals and sleep-dependent respiratory and cardiovascular estimates were calculated. We found that the overall agreement between the 2 methods was 90%, with a high Cohen's Kappa index (0.82). The inter-rater agreement between 2 experts and between 1 expert and 1 naïve sleep investigators gave similar results. Sleep-dependent respiratory and cardiovascular estimates did not depend on the scoring method. We show that non-invasive discrimination of the wake-sleep states of mice based on visual inspection of the WBP signal is accurate, reliable and reproducible. This work may set the stage for non-invasive high-throughput experiments evaluating sleep and breathing patterns on mouse models of pathophysiology.