Spatial and temporal activation of brain regions in hibernation: c‐fos expression during the hibernation bout in thirteen‐lined ground squirrel

Hibernation occurs in some mammalian species as a natural adaptation to seasonal cold and limited food supply. Hibernating mammals, like the 13-lined ground squirrel, do not reside continuously in deep torpor throughout a winter season, they intersperse brief euthermic periods and return to torpor on an intermittent basis. During entrance into torpor the heart and respiratory rate decreases (Kayser, 1961), whole body metabolism drops to less than 5% (Storey, 2003), body temperature drops dramatically to some degrees above ambient temperature, and cerebral blood flow falls to severely oligemic levels (Frerichs et al., 1994). Yet despite these profound changes, hibernators are able to arouse from torpor by means of internal heat production and return to a euthermic state without any tissue damage. Their ability to regularly interrupt the torpor and return to an active interbout state implies a precise coordination of numerous functions orchestrated by the central nervous system (CNS). This precise neurophysiological regulation of hibernation allows ground squirrels to survive extreme environmental conditions. The potential for the hibernating state to induce tolerance against hypoxia and hypoglycemia (Frerichs and Hallenbeck, 1998) may propel studies to develop novel treatments for stroke. In order to guide future experiments that examine mechanisms regulating the hibernation bout and confer its cytoprotective properties, it is of great importance to identify the brain areas that participate in hibernation. During deep torpor the hibernator’s brain temperature can drop close to the freezing point of water and the brain becomes electrically quiescent to surface EEG (Heller and Ruby, 2004); specific brain regions, however, still remain active (Heller, 1979). The hypothalamus has been shown to play an important role in arousal (O’Hara et al., 1999). Lesions of the preoptic thermoregulatory center resulted in abnormal torpor (Satinoff, 1967). Recent studies revealed the essential importance of the suprachiasmatic nucleus in order to maintain adequate timing of hibernation and induce arousal (Kilduff et al., 1989; Bitting et al., 1994; Ruby et al., 2002). Numerous attempts have been made to reveal the localization and characterization of brain areas that may control entrance, maintenance, or arousal during the hibernation bout (Kilduff et al., 1990); however, the participating brain areas and the mechanisms of neurophysiological regulation are still obscure. Reportedly, some of the neurotransmitters may have specific roles in the hibernation cycle. The serotoninergic raphe nuclei might be involved in inducing and regulating entrance into the hibernation cycle (Canguilhem et al., 1986; Haak et al., 1991). Injection of histamine in the hippocampus has been reported to prolong hibernation (Sallmen et al., 2003a), and indeed, upregulation of hippocampal H1 and H2 receptors during torpor suggests that histamine may have a crucial role in maintaining hibernation (Sallmen et al., 2003b). In the present study we aimed to reveal and systematically describe the activation of brain regions throughout six phases of the hibernation bout. To detect activated neurons, we used a highly sensitive method, c-fos proto-oncogene in situ hybridization immunohistochemistry (Bullitt, 1990; Morgan and Curran, 1991; Bitting et al., 1994) with c-fos cDNA from the 13-lined ground squirrel. We identified certain areas being active during deep torpor, while others were activated at arousal or even after returning to normothermia. Our results lead us to conclude that functional aspects of the activated nuclei can guide future studies examining regulatory mechanisms of the hibernation bout.