Impaired Thermoregulatory Ability of Oxytocin-Deficient Mice during Cold-Exposure

We analyzed temperature homeostasis in oxytocin-deficient (Oxt^?/?) mice and found that Oxt^?/? mice exhibited lower body temperatures than wild-type animals when they were exposed to cold. Oxt^?/? mice also showed slightly more weight gain, but there were no obvious differences in the morphology of white and brown adipose tissues as between wild-type and Oxt^?/? mice. In cold-exposed conditions, oxytocin neurons containing c-Fos immunoreactivity existed in the paraventricular nucleus of the hypothalamus.

These results suggest that the central oxytocin neurons constitute part of the thermoregulatory system involved in maintaining body temperature in cold environments.     

Oxytocinergic circuit from paraventricular and supraoptic nuclei to arcuate POMC neurons in hypothalamus

Intracerebroventricular injection of oxytocin (Oxt), a neuropeptide produced in hypothalamic paraventricular (PVN) and supraoptic nuclei (SON), melanocortin-dependently suppresses feeding. However, the underlying neuronal pathway is unclear. This study aimed to determine whether Oxt regulates propiomelanocortin (POMC) neurons in the arcuate nucleus (ARC) of the hypothalamus. Intra-ARC injection of Oxt decreased food intake. Oxt increased cytosolic Ca²⁺ in POMC neurons isolated from ARC. ARC POMC neurons expressed Oxt receptors and were contacted by Oxt terminals. Retrograde tracer study revealed the projection of PVN and SON Oxt neurons to ARC. These results demonstrate the novel oxytocinergic signaling from PVN/SON to ARC POMC, possibly regulating feeding.     

Heightened aggressive behavior in mice with lifelong versus postweaning knockout of the oxytocin receptor

Previous work implicating the neuropeptide oxytocin (Oxt) in the neural regulation of aggression in males has been limited. However, there are reports of heightened aggression in Oxt knockout and Oxt receptor (Oxtr) knockout male mice when they are born to null mutant mothers; suggesting that intrauterine exposure to Oxt may be important to normal aggression in adulthood. To explore this, we examined aggression in two lines of Oxtr mice, a total knockout (Oxtr-/-), in which the Oxtr gene is absent from the time of conception, and a predominantly forebrain specific knockout (Oxtr FB/FB), in which the Oxtr gene is not excised until approximately 21-28days postnatally. Aggression was measured in males from both lines, as well as control littermates, using a resident-intruder behavioral test. Consistent with previous reports, male Oxtr-/- mice had elevated levels of aggression relative to controls. Oxtr FB/FB mice on the other hand displayed levels of aggression similar to control animals. In addition, following a resident-intruder test, Oxtr+/+ mice that displayed aggression had less c-fos immunoreactivity in the ventral portion of the lateral septum than those that did not. Further, Oxtr-/- mice had increased c-fos immunoreactivity in the medial amygdala relative to controls. These data suggest that Oxt may play an important role during development in the organization of the neural circuits that underlie aggressive behavior in adulthood, with its absence resulting in heightened aggression.     

Deficiency of PTP1B in POMC neurons leads to alterations in energy balance and homeostatic response to cold exposure

The adipose tissue-derived hormone leptin regulates energy balance through catabolic effects on central circuits, including proopiomelanocortin (POMC) neurons. Leptin activation of POMC neurons increases thermogenesis and locomotor activity. Protein tyrosine phosphatase 1B (PTP1B) is an important negative regulator of leptin signaling. POMC neuron-specific deletion of PTP1B in mice results in reduced high-fat diet-induced body weight and adiposity gain due to increased energy expenditure and greater leptin sensitivity. Mice lacking the leptin gene (ob/ob mice) are hypothermic and cold intolerant, whereas leptin delivery to ob/ob mice induces thermogenesis via increased sympathetic activity to brown adipose tissue (BAT). Here, we examined whether POMC PTP1B mediates the thermoregulatory response of CNS leptin signaling by evaluating food intake, body weight, core temperature (T(C)), and spontaneous physical activity (SPA) in response to either exogenous leptin or 4-day cold exposure (4°C) in male POMC-Ptp1b-deficient mice compared with wild-type controls. POMC-Ptp1b(-/-) mice were hypersensitive to leptin-induced food intake and body weight suppression compared with wild types, yet they displayed similar leptin-induced increases in T(C). Interestingly, POMC-Ptp1b(-/-) mice had increased BAT weight and elevated plasma triiodothyronine (T(3)) levels in response to a 4-day cold challenge, as well as reduced SPA 24 h after cold exposure, relative to controls. These data show that PTP1B in POMC neurons plays a role in short-term cold-induced reduction of SPA and may influence cold-induced thermogenesis via enhanced activation of the thyroid axis.     

Dehydration anorexia is attenuated in oxytocin-deficient mice

Evidence in rats suggests that central oxytocin (OT) signaling pathways contribute to suppression of food intake during dehydration (i.e., dehydration anorexia). The present study examined water deprivation-induced dehydration anorexia in wild-type and OT -/- mice. Mice were deprived of food alone (fasted, euhydrated) or were deprived of both food and water (fasted, dehydrated) for 18 h overnight. Fasted wild-type mice consumed significantly less chow during a 60-min refeeding period when dehydrated compared with their intake when euhydrated. Conversely, fasting-induced food intake was slightly but not significantly suppressed by dehydration in OT -/- mice, evidence for attenuated dehydration anorexia. In a separate experiment, mice were deprived of water (but not food) overnight for 18 h; then they were anesthetized and perfused with fixative for immunocytochemical analysis of central Fos expression. Fos was elevated similarly in osmo- and volume-sensitive regions of the basal forebrain and hypothalamus in wild-type and OT -/- mice after water deprivation. OT-positive neurons expressed Fos in dehydrated wild-type mice, and vasopressin-positive neurons were activated to a similar extent in wild-type and OT -/- mice. Conversely, significantly fewer neurons within the hindbrain dorsal vagal complex were activated in OT -/- mice after water deprivation compared with activation in wild-type mice. These findings support the view that OT-containing projections from the hypothalamus to the hindbrain are necessary for the full expression of compensatory behavioral and physiological responses to dehydration.     

Low Sympathetic Tone and Obese Phenotype in Oxytocin‐deficient Mice

Oxytocin (Oxt) is secreted both peripherally and centrally and is involved in several functions including parturition, milk let‐down reflex, social behavior, and food intake. Recently, it has been shown that mice deficient in Oxt receptor develop late‐onset obesity. In this study, we characterized a murin model deficient in Oxt peptide (Oxt^?/?) to evaluate food intake and body weight, glucose tolerance and insulin tolerance, leptin and adrenaline levels. We found that Oxt^?/? mice develop late‐onset obesity and hyperleptinemia without any alterations in food intake in addition to having a decreased insulin sensitivity and glucose intolerance. The lack of Oxt in our murin model also results in lower adrenalin levels which led us to hypothesize that the metabolic changes observed are associated with a decreased sympathetic nervous tone. It has been shown that Oxt neurons in the paraventricular nucleus (PVN) are a component of a leptin‐sensitive signaling circuit between the hypothalamus and caudal brain stem for the regulation of food intake and energy homeostasis. Nevertheless, the lack of Oxt in these mice does not have a direct impact on feeding behavior whose regulation is probably dependent on the complex interplay of several factors. The lack of hyperphagia evident in the Oxt^?/? mice may, in part, be attributed to the developmental compensation of other satiety factors such as cholecystokinin or bombesin‐related peptides which merits further investigation. These findings identify Oxt as an important central regulator of energy homeostasis.     

Thyroid-stimulating hormone receptor in brown adipose tissue is involved in the regulation of thermogenesis

C.RF- Tshr(hyt/hyt) mice have a mutated thyroid-stimulating hormone receptor (TSHR), and, without thyroid hormone supplementation, these mice develop severe hypothyroidism. When hypothyroid Tshr(hyt/hyt) mice were exposed to cold (4 degrees C), rectal temperature rapidly dropped to 23.9 +/- 0.40 degrees C at 90 min, whereas the wild-type mice temperatures were 37.0 +/- 0.15 degrees C. When we carried out functional rat TSHR gene transfer in the brown adipose tissues by plasmid injection combined with electroporation, there was no effect on the serum levels of thyroxine, although rectal temperature of the mice transfected with pcDNA3.1/Zeo-rat TSHR 90 min after cold exposure remained at 34.6 +/- 0.34 degrees C, which was significantly higher than that of Tshr(hyt/hyt) mice. Transfection of TSHR cDNA increased mRNA and protein levels of uncoupling protein-1 (UCP-1) in brown adipose tissues, and the weight ratio of brown adipose tissue to overall body weight also increased. Exogenous thyroid hormone supplementation to Tshr(hyt/hyt) mice restored rectal temperature 90 min after exposure to cold (36.8 +/- 0.10 degrees C). These results indicate that not only thyroid hormone but also thyroid-stimulating hormone (TSH)/TSHR are involved in the expression mechanism of UCP-1 in mouse brown adipose tissue. TSH stimulates thermogenesis and functions to protect a further decrease in body temperature in the hypothyroid state.     

Ghrelin induces time-dependent modulation of thermoregulation in the cold

Fasted mice show torpor-like hypothermia in the cold in their inactive phase. The aim of the present study was to elucidate whether leptin and/or ghrelin are involved in this reaction and to identify its neurophysiological mechanisms. In ob/ob mice, which lack leptin, metabolic heat production (oxygen consumption, Vo(2)) was suppressed in 20°C cold in both the light and dark phases, resulting in hypothermia. When wild-type mice received a systemic injection of 8 μg ghrelin in the early light phase, followed by a 2-h cold exposure to 10°C, their core body temperature (T(b)) decreased by 1.7°C, and they displayed a less marked increase in Vo(2) compared with vehicle-injected mice. However, ghrelin injection in the early dark phase resulted in the maintenance of T(b) and increased Vo(2) in the mice, which was similar to the result observed in the vehicle-injected mice. The number of doubly labeled neurons with cFos and neuropeptide Y (NPY) in the suprachiasmatic nucleus was greater in the light phase in the ghrelin-injected mice, which may suggest that ghrelin activates NPY neurons. On the contrary, in the paraventricular nucleus, the counts became greater only when they were exposed to the cold in the dark phase. These results indicate that ghrelin plays an important role in inducing time-dependent changes in thermoregulation in the cold via hypothalamic pathways.     

Oxytocin receptor knockout mice display deficits in the expression of autism-related behaviors

A wealth of studies has implicated oxytocin (Oxt) and its receptors (Oxtr) in the mediation of social behaviors and social memory in rodents. It has been suggested that failures in this system contribute to deficits in social interaction that characterize autism spectrum disorders (ASD). In the current analyses, we investigated the expression of autism-related behaviors in mice that lack the ability to synthesize the oxytocin receptor itself, Oxtr knockout (KO) mice, as compared to their wild-type (WT) littermates. In the visible burrow system, Oxtr KO mice showed robust reductions in frontal approach, huddling, allo-grooming, and flight, with more time spent alone, and in self-grooming, as compared to WT. These results were corroborated in the three-chambered test: unlike WT, Oxtr KO mice failed to spend more time in the side of the test box containing an unfamiliar CD-1 mouse. In the social proximity test, Oxtr KO mice showed clear reductions in nose to nose and anogenital sniff behaviors oriented to an unfamiliar C57BL/6J (B6) mouse. In addition, our study revealed no differences between Oxtr WT and KO genotypes in the occurrence of motor and cognitive stereotyped behaviors. A significant genotype effect was found in the scent marking analysis, with Oxtr KO mice showing a decreased number of scent marks, as compared to WT. Overall, the present data indicate that the profile for Oxtr KO mice, including consistent social deficits, and reduced levels of communication, models multiple components of the ASD phenotype. This article is part of a Special Issue entitled Oxytocin, Vasopressin, and Social Behavior.     

Npvf: Hypothalamic Biomarker of Ambient Temperature Independent of Nutritional Status

The mechanism by which mice, exposed to the cold, mobilize endogenous or exogenous fuel sources for heat production is unknown. To address this issue we carried out experiments using 3 models of obesity in mice: C57BL/6J+/+ (wild-type B6) mice with variable susceptibility to obesity in response to being fed a high-fat diet (HFD), B6. Ucp1-/- mice with variable diet-induced obesity (DIO) and a deficiency in brown fat thermogenesis and B6. Lep-/- with defects in thermogenesis, fat mobilization and hyperphagia. Mice were exposed to the cold and monitored for changes in food intake and body composition to determine their energy balance phenotype. Upon cold exposure wild-type B6 and Ucp1-/- mice with diet-induced obesity burned endogenous fat in direct proportion to their fat reserves and changes in food intake were inversely related to fat mass, whereas leptin-deficient and lean wild-type B6 mice fed a chow diet depended on increased food intake to fuel thermogenesis. Analysis of gene expression in the hypothalamus to uncover a central regulatory mechanism revealed suppression of the Npvf gene in a manner that depends on the reduced ambient temperature and degree of exposure to the cold, but not on adiposity, leptin levels, food intake or functional brown fat.     

An Obligate Role of Oxytocin Neurons in Diet Induced Energy Expenditure

Oxytocin neurons represent one of the major subsets of neurons in the paraventricular hypothalamus (PVH), a critical brain region for energy homeostasis. Despite substantial evidence supporting a role of oxytocin in body weight regulation, it remains controversial whether oxytocin neurons directly regulate body weight homeostasis, feeding or energy expenditure. Pharmacologic doses of oxytocin suppress feeding through a proposed melanocortin responsive projection from the PVH to the hindbrain. In contrast, deficiency in oxytocin or its receptor leads to reduced energy expenditure without feeding abnormalities. To test the physiological function of oxytocin neurons, we specifically ablated oxytocin neurons in adult mice. Our results show that oxytocin neuron ablation in adult animals has no effect on body weight, food intake or energy expenditure on a regular diet. Interestingly, male mice lacking oxytocin neurons are more sensitive to high fat diet-induced obesity due solely to reduced energy expenditure. In addition, despite a normal food intake, these mice exhibit a blunted food intake response to leptin administration. Thus, our study suggests that oxytocin neurons are required to resist the obesity associated with a high fat diet; but their role in feeding is permissive and can be compensated for by redundant pathways.     

TRPV1 gene required for thermosensory transduction and anticipatory secretion from vasopressin neurons during hyperthermia

Increases in core body temperature promote thermoregulatory cooling by stimulating sweat production and preemptive renal water reabsorption through the release of vasopressin (VP, antidiuretic hormone). The mechanism by which the hypothalamus orchestrates this anticipatory VP release during hyperthermia is unknown but has been linked to a central thermosensory mechanism. Here, we report that thermal stimuli spanning core body temperatures activate a calcium-permeable, ruthenium red- and SB366791-sensitive nonselective cation conductance in hypothalamic VP neurons. This response is associated with a depolarizing receptor potential and an increase in action potential firing rate, indicating that these neurons are intrinsically thermosensitive. The thermosensitivity of VP neurons isolated from trpv1 knockout (Trpv1(-/-)) mice was significantly lower than that of wild-type counterparts. Moreover, Trpv1(-/-) mice showed an impaired VP response to hyperthermia in vivo. Channels encoded by the trpv1 gene thus confer thermosensitivity in central VP neurons and contribute to the thermal control of VP release in vivo.     

Postweaning, forebrain-specific perturbation of the oxytocin system impairs fear conditioning

Oxytocin (Oxt) and vasopressin (Avp) are important for a wide variety of behaviors and the use of transgenic mice lacking the peptides or their receptors, particularly when their loss is spatially and temporally manipulated, offers an opportunity to closely examine their role in a particular behavior. We used a cued fear conditioning paradigm to examine associative learning in three lines of transgenic mice: mice that constitutively lack vasopressin 1a (Avpr1a(-/-)) or Oxt receptors (Oxtr(-/-)) and mice that have Oxt receptor loss restricted to the forebrain that begins postweaning (Oxtr(FB/FB)). Oxtr(-/-) and Avpr1a(-/-) mice have normal conditioned freezing. Oxtr(FB/FB) mice have a reduction in freezing behavior during acquisition, as well as during context and cue retention. In addition to reduction of Oxtr in the central nucleus of the amygdala, in vitro receptor autoradiography showed that the Oxtr(FB/FB) mice have significantly reduced levels of Avpr1a only in that structure. Our results show that postweaning alteration of the distribution of Oxtr receptors is critically important for fear behavior, an effect mirrored in the neural structures that mediate it. While constitutive knockouts of Oxtr and Avpr1a are useful for identifying the neural underpinnings of some behaviors, compensatory mechanisms within some circuits may obscure other behavioral roles.     

Reduced stress- and cold-induced increase in energy expenditure in interleukin-6-deficient mice

Interleukin-6 (IL-6) deficient (-/-) mice develop mature onset obesity. Pharmacological studies have shown that IL-6 has direct lipolytic effects and when administered centrally increases sympathetic outflow. However, the metabolic functions of endogenous IL-6 are not fully elucidated. We aimed to investigate the effect of IL-6 deficiency with respect to cold exposure and cage-switch stress, that is, situations that normally increase sympathetic outflow. Energy metabolism, core temperature, heart rate, and activity were investigated in young preobese IL-6-/- mice by indirect calorimetry together with telemetry. Baseline measurements and the effect of cage-switch stress were investigated at thermoneutrality (30 degrees C) and at room temperature (20 degrees C). The effect of cold exposure was investigated at 4 degrees C. At 30 degrees C, the basal core temperature was 0.6 +/- 0.24 degrees C lower in IL-6-/- compared with wild-type mice, whereas the oxygen consumption did not differ significantly. The respiratory exchange ratio at 20 degrees C was significantly higher and the calculated fat utilization rate was lower in IL-6-/- mice. In response to cage-switch stress, the increase in oxygen consumption at both 30 and 20 degrees C was lower in IL-6-/- than in wild-type mice. The increase in heart rate was lower in IL-6-/- mice at 30 degrees C. At 4 degrees C, both the oxygen consumption and core temperature were lower in IL-6-/- compared with wild-type mice, suggesting a lower cold-induced thermogenesis in IL-6-/- mice. The present results indicate that endogenous IL-6 is of importance for stress- and cold-induced energy expenditure in mice.     

Enhanced corticosterone concentrations and attenuated Fos expression in the medial amygdala of female oxytocin knockout mice exposed to psychogenic stress

Centrally released oxytocin (OT) is believed to attenuate the response of the hypothalamic-pituitary-adrenal (HPA) axis to psychogenic stress. To test this hypothesis, we measured plasma corticosterone concentrations and Fos-immunoreactive protein in the paraventricular nucleus of the hypothalamus (PVN) and limbic brain areas of female wild-type and OT knockout mice that were exposed to a shaker platform, a predominantly psychogenic stress. Plasma corticosterone concentrations after shaker stress were higher in female OT knockout mice than wild-type mice. Genotypic differences in the corticosterone response after shaker stress persisted across all stages of the estrous cycle and when mice were conditioned to repeated shaker stress. Shaker stress activated Fos in OT-positive neurons of wild-type mice and corticotropin-releasing hormone-positive, but not vasopressin-positive, neurons within the PVN of wild-type and OT knockout mice. Fos expression was also increased after shaker stress in the bed nucleus of the stria terminalis, medial and central nuclei of the amygdala, medial preoptic area, and the paraventricular nucleus of the thalamus of wild-type and OT knockout mice. However, Fos expression in the medial amygdala was significantly lower in female OT knockout mice than wild-type mice. Our findings indicate heightened stress-induced corticosterone release in female OT knockout mice. Therefore, the results suggest that OT pathways play a role in attenuating the HPA axis response to psychogenic stress in female mice.