Sex- and age-dependent contribution of System xc- to cognitive, sensory, and social behaviors revealed by comprehensive behavioral analyses of System xc- null mice.

Background: System xc- (Sxc-) is an important heteromeric amino acid cystine/glutamate exchanger that plays a pivotal role in the CNS by importing cystine into cells while exporting glutamate. Although certain behaviors have been identified as altered in Sxc- null mutant mice, our understanding of the comprehensive impact of Sxc- on behavior remains incomplete. Methods: To address this gap, we compared motor, sensory and social behaviors of male and female mice in mice null for Sxc- (SLC7A11sut/sut) with wildtype littermates (SLC7A11+/+) in a comprehensive and systematic manner to determine effects of genotype, sex, age, and their potential interactions. Results: Motor performance was not affected by loss of Sxc- in both males and females, although it was impacted negatively by age. Motor learning was specifically disrupted in female mice lacking Sxc- at both 2 and 6 months of age. Further, female SLC7A11sut/sut mice at both ages exhibited impaired sociability, but normal spatial and recognition memory, as well as sensorimotor gating. Finally, pronounced open-space anxiety was displayed by female SLC7A11sut/sut when they were young. In contrast, young SLC7A11sut/sut male mice demonstrated normal sociability, delayed spatial learning, increased open-space anxiety and heightened sensitivity to noise. As they aged, anxiety and noise sensitivity abated but hyperactivity emerged. Discussion: We find that the behavioral phenotypes of female SLC7A11sut/sut are similar to those observed in mouse models of autism spectrum disorder, while behaviors of male SLC7A11sut/sut resemble those seen in mouse models of attention deficit hyperactivity disorder. These results underscore the need for further investigation of SLC7A11 in neurodevelopment. By expanding our understanding of the potential involvement of Sxc-, we may gain additional insights into the mechanisms underlying complex neurodevelopmental conditions.

Nitrogen recycling buffers against ammonia toxicity from skeletal muscle breakdown in hibernating arctic ground squirrels.

Hibernation is a state of extraordinary metabolic plasticity. The pathways of amino acid metabolism as they relate to nitrogen homeostasis in hibernating mammals in vivo are unknown. Here we show, using pulse isotopic tracing, evidence of increased myofibrillar (skeletal muscle) protein breakdown and suppressed whole-body production of metabolites in vivo throughout deep torpor. As whole-body production of metabolites is suppressed, amino acids with nitrogenous side chains accumulate during torpor, while urea cycle intermediates do not. Using 15N stable isotope methodology in arctic ground squirrels (Urocitellus parryii), we provide evidence that free nitrogen is buffered and recycled into essential amino acids, non-essential amino acids and the gamma-glutamyl system during the inter-bout arousal period of hibernation. In the absence of nutrient intake or physical activity, our data illustrate the orchestration of metabolic pathways that sustain the provision of essential and non-essential amino acids and prevent ammonia toxicity during hibernation.

Sexually dimorphic and brain region-specific transporter adaptations in system xc- null mice.

System xc- is a heterodimeric amino acid antiporter that, in the central nervous system, is best known for linking the import of L-cystine (CySS) with the export of L-glutamate for the production and maintenance of cellular glutathione (GSH) and extracellular glutamate levels, respectively. Yet, mice that are null for system xc- are healthy, fertile, and, morphologically, their brains are grossly normal. This suggests other glutamate and/or cyst(e)ine transport mechanisms may be upregulated in compensation. To test this, we measured the plasma membrane expression of Excitatory Amino Acid Transporters (EAATs) 1-3, the Alanine-Serine-Cysteine-Transporter (ASCT) 1, the sodium-coupled neutral amino acid transporter (SNAT) 3 and the L Amino Acid Transporter (LAT) 2 in striatum, hippocampus and cortex of male and female mice using Western Blot analysis. Present results demonstrate brain region and transporter-specific changes occurs in female system xc- null mice with increased expression of EAAT1 and ASCT1 occurring in the striatum and cortex, respectively, and decreased SNAT 3 expression in cortex. In male system xc- null brain, only SNAT3 was altered significantly - increasing in the cortex, but decreasing in the striatum. Total levels of GSH and CyS were similar to that found in age and sex-matched littermate control mice, however, reductions in the ratio of reduced to oxidized GSH (GSH/GSSG) - a hallmark of oxidative stress - were found in all three brain regions in female system xc- null mice, whereas this occurred exclusively in the striatum of males. Protein levels of Superoxide dismutase (SOD) 1 were reduced, whereas SOD2 was enhanced in the hippocampus of male xc- null mice only. Finally, striatal vulnerability to 3-nitropropionic acid (3-NP)-mediated oxidative stress in either sex showed no genotype difference, although 3-NP was more toxic to female mice of either genotype, as evidenced by an increase in moribundity as compared to males.

Iodine Redistribution During Trauma, Sepsis, and Hibernation: An Evolutionarily Conserved Response to Severe Stress.

OBJECTIVE: We performed these studies to learn how iodine in the form of free iodide behaves during stress. DESIGN: Prospective observational trial using samples obtained from human trauma patients and retrospective observational study using remnant samples from human sepsis patients and arctic ground squirrels. Preclinical interventional study using hind-limb ischemia and reperfusion injury in mice. SETTING: Level I trauma center emergency room and ICU and animal research laboratories. SUBJECTS: Adult human sepsis and trauma patients, wild-caught adult arctic ground squirrels, and sexually mature laboratory mice. INTERVENTIONS: Ischemia and reperfusion injury was induced in mice by temporary application of tourniquet to one hind-limb. Iodide was administered IV just prior to reperfusion. MEASUREMENTS AND MAIN RESULTS: Free iodide was measured using ion chromatography. Relative to iodide in plasma from normal donors, iodide was increased 17-fold in plasma from trauma patients and 26-fold in plasma from sepsis patients. In arctic ground squirrels, iodide increases over three-fold during hibernation. And during ischemia/reperfusion injury in mice, iodide accumulates in ischemic tissue and reduces both local and systemic tissue damage. CONCLUSIONS: Iodide redistributes during stress and improves outcome after injury. Essential functions of iodide may have contributed to its evolutionary selection and be useful as a therapeutic intervention for human patients.

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.

The Raphe Pallidus and the Hypothalamic-Pituitary-Thyroid Axis Gate Seasonal Changes in Thermoregulation in the Hibernating Arctic Ground Squirrel (Urocitellus parryii).

Thermoregulation is necessary to maintain energy homeostasis. The novel discovery of brown adipose tissue (BAT) in humans has increased research interests in better understanding BAT thermogenesis to restore energy balance in metabolic disorders. The hibernating Arctic ground squirrel (AGS) offers a novel approach to investigate BAT thermogenesis. AGS seasonally increase their BAT mass to increase the ability to generate heat during interbout arousals. The mechanisms promoting the seasonal changes in BAT thermogenesis are not well understood. BAT thermogenesis is regulated by the raphe pallidus (rPA) and by thyroid hormones produced by the hypothalamic-pituitary-thyroid (HPT) axis. Here, we investigate if the HPT axis and the rPA undergo seasonal changes to modulate BAT thermogenesis in hibernation. We used histological analysis and tandem mass spectrometry to assess activation of the HPT axis and immunohistochemistry to measure neuronal activation. We found an increase in HPT axis activation in fall and in response to pharmacologically induced torpor when adenosine A1 receptor agonist was administered in winter. By contrast, the rPA neuronal activation was lower in winter in response to pharmacologically induced torpor. Activation of the rPA was also lower in winter compared to the other seasons. Our results suggest that thermogenic capacity develops during fall as the HPT axis is activated to reach maximum capacity in winter seen by increased free thyroid hormones in response to cooling. However, thermogenesis is inhibited during torpor as sympathetic premotor neuronal activation is lower in winter, until arousal when inhibition of thermogenesis is relieved. These findings describe seasonal modulation of thermoregulation that conserves energy through attenuated sympathetic drive, but retains heat generating capacity through activation of the HPT axis.

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.

Membrane phospholipid fatty acid composition regulates cardiac SERCA activity in a hibernator, the Syrian hamster (Mesocricetus auratus).

Polyunsaturated fatty acids (PUFA) have strong effects on hibernation and daily torpor. Increased dietary uptake of PUFA of the n-6 class, particularly of Linoleic acid (LA, C18:2 n-6) lengthens torpor bout duration and enables animals to reach lower body temperatures (T(b)) and metabolic rates. As previously hypothesized, this well-known influence of PUFA may be mediated via effects of the membrane fatty acid composition on sarcoplasmic reticulum (SR) Ca(2+-)ATPase 2a (SERCA) in the heart of hibernators. We tested the hypotheses that high proportions of n-6 PUFA in general, or specifically high proportions of LA (C18:2 n-6) in SR phospholipids (PL) should be associated with increased cardiac SERCA activity, and should allow animals to reach lower minimum T(b) in torpor. We measured activity of SERCA from hearts of hibernating and non-hibernating Syrian hamsters (Mesocricetus auratus) in vitro at 35 °C. Further, we determined the PL fatty acid composition of the SR membrane of these hearts. We found that SERCA activity strongly increased as the proportion of LA in SR PL increased but was negatively affected by the content of Docosahexaenoic acid (DHA; C22:6 n-3). SR PL from hibernating hamsters were characterized by high proportions of LA and low proportions of DHA. As a result, SERCA activity was significantly higher during entrance into torpor and in torpor compared to inter-bout arousal. Also, animals with increased SERCA activity reached lower T(b) during torpor. Interestingly, a subgroup of hamsters which never entered torpor but remained euthermic throughout winter displayed a phenotype similar to animals in summer. This was characterized by lower proportions of LA and increased proportions of DHA in SR membranes, which is apparently incompatible with torpor. We conclude that the PUFA composition of SR membranes affects cardiac function via modulating SERCA activity, and hence determines the minimum T(b) tolerated by hibernators.