Abstract
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 O2 consumption and core body temperature as indicators of torpor. The A1 antagonist cyclopentyltheophylline reversed spontaneous entrance into torpor. The adenosine A1 receptor agonist N6-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 A1AR activation; the A3AR agonist 2-Cl-IB MECA failed to induce torpor, and the A2aR 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 A1AR activation and requires a seasonal switch in the sensitivity of purinergic signaling.
Introduction
Hibernation is essential for survival during seasonal deficiencies in food supply in several diverse lineages of mammals (Carey et al., 2003; Dausmann et al., 2004; Heldmaier et al., 2004). Survival is achieved by severe metabolic suppression, termed torpor, where rates of O2 consumption fall to as low as 1% of resting metabolic rate and core body temperature (Tb) falls to as low as −3°C (Barnes, 1989; Geiser, 2004; Heldmaier et al., 2004). Torpor in hibernating mammals thus defines the nadir of mammalian metabolism and Tb, but mechanisms regulating initiation of torpor have been poorly understood (Heldmaier et al., 2004; Drew et al., 2007). Transition into the torpid state has been postulated to include three processes: (1) altered CNS control of thermoregulatory processes (Heller et al., 1977) and an extension of sleep (Walker et al., 1977, 1980), (2) active inhibition of metabolism such as inhibition of mitochondrial oxidative phosphorylation (Muleme et al., 2006), and (3) temperature-dependent effects on metabolic rate, or “Q10 effects” (Geiser, 2004). For larger mammals, the debate has centered more extensively on CNS control versus active suppression of metabolism via modulation of biochemical processes within metabolically active tissues.
In seasonal (obligate) hibernators, such as the arctic ground squirrel (AGS; Urocitellus parryii), torpor depends on a circannual cycle. The circannual cycle persists under constant photoperiods with food provided ad libitum (Heller and Poulson, 1970; Pengelley et al., 1976; Lee and Zucker, 1991). The seasonal character of hibernation thus provides a clue to its regulation. Once torpor ensues animals rewarm spontaneously every 2–3 weeks for brief (12–24 h) periods of normal body temperature (termed euthermy). This cycle continues until torpor ceases to occur in the spring. A two-switch model suggests that one physiological switch initiates the onset of the hibernation season and another switch initiates the onset of torpor (Serkova et al., 2007). The role of the CNS or the nature of the signaling events involved in either of these two switches have been unknown. Central purinergic signaling via A1 adenosine receptors (A1ARs) mediates sleep drive (Benington et al., 1995; Porkka-Heiskanen and Kalinchuk, 2011) and decreases body temperature (Miller and Hsu, 1992; Barros et al., 2006), and more recently, endogenous adenosine within the CNS has been found to decrease body temperature at presumed torpor onset in hamsters (Mesocricetus auratus) (Shiomi and Tamura, 2000; Tamura et al., 2005). Here we used intracerebroventricular drug administration to test the hypothesis that a seasonal change in purinergic signaling within the CNS is necessary for the onset of spontaneous torpor in the AGS, a seasonal hibernator. We show for the first time that activation of A1AR within the CNS is necessary and sufficient to induce torpor in AGS during the hibernation season, but not during the off-season, when AGSs do not spontaneously hibernate. The CNS and purinergic signaling are therefore key regulators of torpor onset, and sensitization to the effects of endogenous adenosine serves as a seasonally regulated switch that facilitates A1AR-mediated torpor.
Materials and Methods
Animals.
Procedures were approved by the University of Alaska Fairbanks Institutional Animal Care and Use Committee and Department of Defense Animal Care and Use Review Office. AGSs were captured near 66°38′N, 149°38′W under permit from the Alaska Department of Fish and Game. Animals were fed rodent chow and housed at 20°C with natural lighting for their wild-trapped latitude until mid-August, when they were moved to environmental chambers set to 2°C and 4:20 h light:dark (L:D). AGSs remained in these conditions until the end of the study. The hibernation season was defined by the presence of spontaneous torpor. The off-hibernation season (off-season) was defined by an absence of spontaneous torpor. Torpor was monitored daily by placing shavings on the animals' backs.
Surgery.
Under sterile conditions, telemetry transmitters (model VM-FH, MiniMitter, or model CTA-F40, Data Sciences International) were implanted under isoflurane anesthesia. The head was leveled in a rat stereotaxic frame (Stoelting). Copalite (Cooley & Cooley) was applied to the skull. A target was marked at APEBZ +8.5 mm, LEBZ +3.0 mm, the arm was tilted 15°, and the cannula tip was repositioned on the target. An internal cannula extending 1.0 mm beyond the guide cannula was connected to a syringe primed with sterile saline. The cannula assembly was lowered 5.5 mm from the brain surface and retracted until CSF was withdrawn. The guide cannula was secured to anchoring screws (Stoelting) and plugged with a dummy cannula (Plastics One). Animals received enrofloxacin (Bayer Health Care) (5 mg/kg, s.c., BID for 3 d), and ketoprofen (Fort Dodge Animal Health, 1 mg/kg, QD, s.c., for 3 d total). When CTA-F40 transmitters were used, animals received buprenorphine (Hospira, 0.03 mg/kg, QD, i.m., for 3 d) and 2 weeks separated transmitter surgery and intracerebroventricular cannula surgery. Following surgery, animals were housed at 20°C 4:20 h L:D and wounds were cleaned for 10 d before returning to environmental chambers at 2°C. Surgery was performed at least 1 month before drug testing.
O2 consumption and body temperature.
A cylindrical Plexiglas metabolic chamber (diameter 28 cm, height 23 cm) on a rat-turn (Bioanalytical Systems) was positioned over a telemetric receiver and Tb was acquired using DataQuest software A.R.T.2.3 (Data Sciences International). Air was drawn from a gas-tight swivel at the bottom of the chamber, filtered, and passed through a mass flow controller at 3 L/min (Model 840, 0–5 L/min, Sierra Instruments), and a subsample was passed through a multiplexing valve system and dried by a Nafion drier used in reflux mode (model PD-50T-24-PP, Perma Pure) before passing through the O2 and CO2 analyzers (Model FC-1B and CA-2A, Sable Systems International). The automated data acquisition and analysis software (LabGraph, developed by Tøien) interpolated between calibrations. O2 consumption was corrected for respiratory volume change according to the principles of the Haldane transformation (Wagner et al., 1973; Karpovich et al., 2009). The integrity of the system was tested during and after the study period by burning 100% ethanol. Measured O2 consumption was within 4% of that calculated from the weight loss of the lamp.
For monitoring subcutaneous temperature following intraperitoneal CHA injections, animals were implanted with IPTT-300 transponders (Bio Medic Data Systems), subcutaneously between the scapula. Tb was monitored using a telemetry system (DAS-6000; Bio Medic Data Systems) in the home cage every 30–60 min for at least 1 h before drug injection and every 1 h after injection for 4 h and again 30 h after intraperitoneal injection of CHA. Because the IPTT transponders are not reliable below ∼30°C, to confirm minimal Tb at 30 h, rectal temperature was monitored with a thermocouple (Model H H21 Microprocessor Thermometer, Type J-K-T Thermocouple, OMEGA Engineering) in animals that were torpid after 30 h.
Drug administration.
Animals tested for drug-induced torpor were aroused on day 3 or 4 of a torpor bout and moved from the environmental chamber (2°C, 4:20 L:D) to a warmer room (20°C, 4:20 L:D), where they remained overnight. On the following day, they were handled as described below and placed in the metabolic chamber for baseline recordings for at least 1 h before drug administration. For intracerebroventricular administration of CHA, injection cannulae primed with CHA or vehicle by an observer unaware of treatment were connected to a perfusion pump (Harvard Apparatus). Euthermic animals were lightly anesthetized with isoflurane as described for surgery and fit with a harness and injection cannula in a way that allowed animals to move freely within the metabolic chamber. After recovery from anesthesia, baseline O2 consumption and Tb were collected for 1 h before delivering the drug (0.5 nmol CHA/10 μl, delivered over 1 min) or vehicle (10 μl, delivered over 1 min). The cannula was left in place and O2 consumption and Tb were monitored for at least 24 h or until Tb was stable. Solutions used to dissolve the drugs (vehicle) were administered in a balanced cross-over design by an observer unaware of treatment. In this way, half of the animals received drug on the first test and vehicle on the second test and the other half received vehicle on the first test and drug on the second. Drug and vehicle tests were separated by at least 1 week. In a separate group of animals, a Y-injection cannula (Plastics One) was primed with 2-Cl-IB-MECA (3 nmol/10 μl), and the secondary line was primed with CHA (0.5 nmol/10 μl). Animals were treated as above except that the injection of 2-Cl-IB MECA (10 μl, delivered over 1 min) was followed by a second injection of CHA (3.3 μl to clear the cannula of 2-Cl-IB MECA, then 10 μl of CHA at 10 μl, delivered over 1 min).
Additional animals received pentobarbital (20 mg/kg, i.p.) during the midseason or off- (nonhibernating) season and Tb and O2 consumption were monitored as described above. To ensure that the stress of intraperitoneal injections did not interfere with drug-induced torpor, a separate group of AGSs was administered CHA intraperitoneally during midseason.
For antagonist studies, torpid AGSs were habituated to handling before drug testing. During habituation, AGSs were handled daily to mimic handling necessary for the experiment until handling failed to induce arousal. At the next signs of torpor when Tb dipped to ∼34°C, AGSs were fit with a harness and an injection cannula primed with antagonist (CPT, 3.0 nmol/10 μl) or vehicle by an experimenter unaware of treatment and onset of torpor proceeded without interruption. When Tb reached 10°C, 10 μl was delivered over 1 min and the cannula was left in place for an additional 24 h. MSX-3 (3.0 nmol/10 μl) was administered in the same way to another group of animals. The 3 nmol dose of MSX-3 was considered to be equipotent to the 3 nmol dose of CPT since MSX-2 has similar affinity for the A2aAR as CPT has for the A1AR. The 3 nmol dose of 2-Cl-IB MECA was considered to be higher than an equipotent dose of 0.5 nmol of CHA since 2-Cl-IB MECA has a slighter higher affinity for A3AR than CHA has for A1AR (Bruns et al., 1986; Klotz, 2000; Sauer et al., 2000; Solinas et al., 2005).
Drugs.
N6-Cyclohexyladenosine (CHA), 8-cyclopentyltheophylline (CPT), and phosphoric acid mono-(3-{8-[2-(3-methoxyphenyl) vinyl]-7-methyl-2,6-dioxo-1-prop-2-ynyl-1,2,6,7-tetrahydropurin-3-yl}propyl) ester disodium salt (MSX-3) hydrate were purchased from Sigma-Aldrich, and 2-chloro-N6-(3-iodobenzyl) adenosine-5′-N-methyluronamide (2-Cl-IB MECA) was purchased from Tocris Bioscience. CHA was dissolved in 0.01 m phosphate buffer, CPT and 2-Cl-IB-MECA were dissolved in 1% DMSO, and MSX-3 hydrate was dissolved in water. All solutions were sterilized by 0.2 μm filtration before use except for pentobarbital sodium, which was obtained as an injectable solution (50 mg/ml) (Abbott Laboratories).
Results
Onset of torpor requires A1AR activation
To investigate whether A1AR activation by endogenous adenosine within the CNS is necessary for the onset of spontaneous torpor in AGS, the A1AR antagonist CPT (3 nmol/10 μl) was administered into the lateral ventricle during onset of spontaneous torpor, via an indwelling intracerebroventricular cannula. CPT, delivered by an investigator unaware of treatment, reversed torpor onset in all animals tested, while vehicle had no effect (Fig. 1).
Onset of torpor requires A1AR activation. a, An increase in the rate of O2 consumption (V̇O2) and an increase in Tb to euthermic levels occurred in all animals tested following administration of CPT (3 nmol, i.c.v.) during onset of spontaneous torpor. This indicates that A1AR activation is necessary for torpor onset. b, Vehicle had no effect in any of the animals tested. Results are shown as means and SEM; n = 6 AGSs.
Sensitivity to the torpor-inducing effects of the A1AR agonist CHA increases as the hibernation season progresses
We next asked whether A1AR activation within the CNS was sufficient to induce a state of torpor that mimicked spontaneous torpor both in temporal profile and in magnitude of decline in the rate of O2 consumption and Tb. We also investigated whether the sensitivity to torpor-inducing effects of CHA, an A1AR agonist, would increase as the hibernation season progressed. Six AGSs instrumented with intracerebroventricular cannula open to the lateral ventricle were administered CHA (0.5 nmol/10 μl) or vehicle in a blinded, balanced, cross-over fashion at three times of the year. These three tests commenced during the off-season when AGSs were not displaying spontaneous torpor, during the early hibernation season after all AGSs had begun to display spontaneous torpor, and during the middle of the hibernation season.
CHA administered during the off-season, induced a slight, temporary, reduction in O2 consumption and Tb in all AGSs tested (Fig. 2a,g). Early in the hibernation season the same dose of CHA delivered to the same six AGSs induced a torpor-like decline in O2 consumption and Tb in two of six animals tested (Fig. 2b,h) and an off-season-like response in the remaining four animals (Fig. 2c,i). By mid-hibernation season (midseason), the same dose of CHA induced a torpor-like response in all of these same six animals (Fig. 2d,j). The torpor-like response to CHA resembled spontaneous entry into torpor (Fig. 2e,k). Pentobarbital (20 mg/kg, i.p.) produced an off-season-like response regardless of season (Fig. 2f,l). Vehicle (phosphate buffer, i.c.v. for CHA or saline, i.p. for pentobarbital) did not produce a notable effect on Tb or rate of O2 consumption at any season tested (Fig. 3).
Sensitivity to the torpor-inducing effects of the A1AR agonist CHA increases as the hibernation season progresses. a, CHA during the off-season, when animals were not displaying spontaneous torpor, induced a slight decrease in V̇O2 and Tb in all six AGSs tested. b, Early in the hibernation season after all animals showed evidence of spontaneous torpor, CHA induced a torpor-like response in two of six animals tested. c, In the remaining four animals, the same dose of the drug did not induce torpor. d, By the middle of the hibernation season (midseason), the same dose of CHA induced torpor in all six AGSs tested. e, Spontaneous torpor in one AGS. f, Pentobarbital, regardless of season, induced a response similar to CHA during the off-season (n = 3). (The time scale on the x-axis in c applies to d and e and is a continuous 30 h.) g–l, Detail of the first 4.5 h of a–f illustrates that CHA-induced torpor resembles spontaneous torpor where a rapid drop in metabolism is followed by a slow gradual decrease in Tb. g, During the off-season CHA induces a rapid drop in Tb that begins before and at the same rate as the decline in O2 consumption. h, j, k, When CHA induces torpor (h, j) and when animals spontaneously enter torpor (k), Tb declines more slowly than O2 consumption. g, i, l, When CHA fails to induce torpor (g, i) and after pentobarbital (l), Tb and O2 consumption decline at similar rates. Data shown are means ± SEM.
None of the vehicles tested produced a notable effect on Tb or V̇O2. a–d, Vehicle (0.01 m phosphate buffer, i.c.v., for CHA; a–c); and saline (i.p., for pentobarbital; d) failed to produce any notable change in Tb or V̇O2. Data shown are means and SEM; n = 6 AGSs.
We asked whether characteristics such as body weight, sex, age, and timing or evidence of prior torpor bouts predicted the magnitude or quality of the CHA-induced response during the early hibernation season. The two animals that displayed CHA-induced torpor when tested early in the season (Early season) had exhibited slightly more bouts of spontaneous torpor before this CHA test than the other four animals (Table 1). Other variables did not predict the larger response to CHA in these animals. Data shown in Table 1 illustrate that the hibernation season was defined by the presence of spontaneous torpor. In these animals, progression of the hibernation season was evident from the number of torpor bouts noted since the onset of spontaneous torpor. The circannual cycle of obligate hibernators, such as AGS, will free run when animals are housed under constant L:D conditions (Pengelley et al., 1976; Lee and Zucker, 1991). This free-running circannual cycle explains why the first day of spontaneous torpor occurred in July in many of the animals in this study.
Characteristics of AGS treated with CHA (0.5 nmol, i.c.v) during the three test seasons
CHA-induced and spontaneous torpor is specific to A1AR
Although CHA is fairly selective for A1AR, it has some affinity for A3AR (Gao et al., 2003), leading us to ask whether A3AR activation could account for CHA-induced torpor. The A3AR agonist 2-Cl-IB-MECA (3 nmol/10 μl, i.c.v.), delivered during midseason, failed to induce torpor in any of the animals tested, although a subsequent injection of CHA (0.5 nmol/10 μl, i.c.v.) induced torpor as observed previously (Fig. 4a), indicating that A3AR activation is not sufficient to induce torpor. Both A1AR and A2aAR play a role in sleep (Porkka-Heiskanen et al., 1997; Huang et al., 2005; Oishi et al., 2008); and torpor is in part an extension of sleep (Walker et al., 1977). We therefore asked whether A2aAR receptors contribute to the onset of torpor. MSX-3, a water-soluble prodrug of the selective, high-affinity A2aAR antagonist MSX-2 (Solinas et al., 2005), failed to reverse onset of spontaneous torpor in any of the animals tested (Fig. 4b). These results indicate that A2aAR activation is not necessary for torpor onset.
CHA-induced and spontaneous torpor is specific to A1AR. a, The selective A3AR agonist 2-Cl-IB-MECA (3 nmol, i.c.v.) failed to induce torpor in any of the animals tested, while a subsequent injection of CHA (0.5 nmol, i.c.v.) induced torpor (n = 3). Top trace is Tb; bottom trace is V̇O2. b, MSX-3 (3 nmol, i.c.v.), a water-soluble prodrug of the A2AR antagonist MSX-2, failed to reverse onset of spontaneous torpor (n = 3). Data shown are means and SEM.
Pentobarbital is a positive allosteric modulator of GABAA receptors, promotes sleep, and shows seasonal-dependent changes in efficacy across the hibernation season in thirteen-lined ground squirrels (Hengen et al., 2011). To investigate whether the seasonal change in response to CHA-induced torpor was specific to an adenosine agonist, pentobarbital was administered during the midseason and off-season. Pentobarbital was administered, intraperitoneally, to two groups of animals. One group was tested during the off-season, when animals failed to demonstrate spontaneous torpor. Another group was tested during the mid-hibernation season, when the total number of bouts of spontaneous torpor ranged between 12 and 16 bouts. Figure 2, f and l, shows that pentobarbital failed to induce torpor at any season tested. Table 2 shows that the characteristics of AGSs treated with pentobarbital during these two seasons are similar to the off-season and midseason groups of AGSs treated with CHA. Injections of pentobarbital, i.p., were noted to produce a brief, but detectible, increase in V̇O2 that was not noted with intracerebroventricular administration of CHA (Fig. 2f). To ensure that intraperitoneal injections did not interfere with drug-induced torpor, separate groups of AGSs were treated with CHA (0.5 mg/kg, i.p.) during the off-season and during the midseason. Data shown in Table 3 show that intraperitoneal injections of CHA induced torpor during the midseason, but not during the off-season as seen for intracerebroventricular administration. Characteristics of AGSs were similar to other groups of animals tested during these two seasons.
Characteristics of AGSs treated with pentobarbital (20 mg/kg, i.p.) during the off-season and during the middle of the hibernation season
Characteristics of AGSs treated with CHA (0.5 mg/kg, i.p.)
Discussion
These results indicate that the CNS regulates the onset of torpor via activation of A1AR. Sensitivity to the torpor-inducing effects of the A1AR agonist CHA increases as the hibernation season progresses, and the torpor-inducing efficacy of CHA is specific to A1AR activation. It is unlikely that the seasonal response to CHA was due to differences in cold adaptation, since animals were housed at 2°C throughout the study period.
Torpor in hibernating animals represents an extreme example of decreased metabolism, but the central or peripheral sites or signaling mechanisms involved in torpor onset have been unknown (Heldmaier et al., 2004). By administering purinergic ligands specific for the adenosine A1, A2a, or A3 receptors (Table 4) into the lateral ventricle of AGSs at various times across the annual hibernation cycle, we find that adenosine within the CNS meets all of the necessary requirements for an endogenous mediator of torpor in AGS. A progressive increase in the sensitivity of AGS to A1AR-mediated signaling within the CNS parallels the seasonal transition into the hibernation phenotype and provides an example of a seasonal switch proposed in the two-switch model for obligate hibernation (Serkova et al., 2007). We show that in the context of this model, increased gain in central purinergic signaling serves as the first switch, and stimulation of central A1AR by endogenous adenosine serves as a second switch, that induces torpor (Fig. 5).
Binding affinities for adenosine ligands
Enhanced purinergic signaling turns on the seasonal switch to hibernate in arctic ground squirrels. Schematic diagram modified from the two-switch model of Serkova et al. (2007) illustrates how seasonal sensitization of purinergic signaling primes the brain for adenosine-induced torpor during the hibernation season. The off-season, commonly referred to as the “summer-active” season, is indicated by a white background. During the off-season, overflow of adenosine that occurs as part of normal purinergic signaling fails to induce torpor. Here we use homeostatic sleep drive as an example of normal purinergic signaling (Porkka-Heiskanen et al., 1997; Basheer et al., 2004). The present report shows that an increase in the gain in purinergic signaling occurs during the hibernation season. The hibernation season is indicated by a dark background and the shading from light to dark illustrates an increase in gain in purinergic signaling as the season progresses. This increased gain in purinergic signaling during the hibernation season primes the brain such that overflow of endogenous adenosine with subsequent activation of A1AR now induces torpor. The effect of endogenous adenosine is demonstrated by the ability of an A1AR antagonist (CPT) to reverse onset of spontaneous torpor.
Because A1AR activation is necessary for the homeostatic sleep response (Bjorness et al., 2009), an increased gain in purinergic signaling predicts an increase in sleep drive during the hibernation season. Although sleep drive has not been monitored in AGS, golden-mantled ground squirrels sleep more during the hibernation season (Walker et al., 1980).
Prolonged torpor in hibernating mammals is distinguished by at least three distinct processes that include onset of torpor, maintenance of torpor, and arousal from torpor (Drew et al., 2007). In hamsters (M. auratus), A1AR activation is necessary for torpor onset, as shown here for AGS, but is not necessary to maintain prolonged torpor (Tamura et al., 2005). Seasonal alterations in signaling events involved in torpor maintenance or interbout arousal are as yet unclear. Moreover, while the present results demonstrate that A1AR activation is necessary and sufficient to induce torpor in AGS, it is unlikely that adenosine is the only neuromodulator involved with torpor onset.
The present results clearly show that A1AR stimulation is sufficient to initiate torpor onset that results in a decrease in metabolic rate to levels that are below basal metabolic rate, a value determined to be within 0.40 and 0.61 ml · g−1 · h−1 for AGS (Scholander et al., 1950; Withers et al., 1979). Geiser (2004) describes a scenario in which decreased thermogenesis leads to cooling, which then via thermodynamic effects decreases oxygen consumption to torpid metabolic rates. This scenario may account for how central A1AR-induced inhibition of thermogenesis in AGS could lead to a consequent lowering of Tb that is then sufficient to account for torpid metabolic rates reported here. This explanation incorporates two of three proposed mechanisms of metabolic suppression in hibernating animals. One mechanism includes the central inhibition of thermogenesis associated with a lowering of brain temperature necessary to induce thermogenesis (Heller et al., 1977). A second mechanism involves thermodynamic effects of cooling on metabolic rate described by the Van't Hoff equation (Atkins and De Paula, 2006). In biological systems, temperature effects are often referred to as Q10 effects, where reaction rates generally double or triple with every 10°C increase in temperature (Schmidt-Nielsen, 1997). A third set of mechanisms involves “active” suppression of cellular processes such as ion channel arrest (Hochachka, 1986) or inhibition of cellular respiration (Muleme et al., 2006). It is difficult to explain how central A1AR activation could directly cause global, “active” suppression of cellular processes; however, global effects could occur downstream to torpor initiation.
Given that cooling contributes to metabolic rate reduction, cooling during onset of torpor will influence the subsequent decrease in metabolic rate. Unexpectedly, a rapid rate of cooling induced by CHA during the off-season contrasted with a significantly slower rate of cooling induced by CHA during the hibernation season. All animals had been housed at 2°C for several months, so cold adaptation is unlikely to account for differences in the rate of cooling. O2 consumption and Tb were measured in the present study as physiological parameters used to clearly distinguish torpor onset from hypothermia. Since thermoregulatory systems are independently regulated, further study of multiple thermoeffector activities is warranted to achieve a more complete understanding of torpor as a thermoregulatory response (Romanovsky, 2007; Nakamura et al., 2009).
The mechanism that increases the gain in purinergic signaling may involve changes in receptor expression or function, changes in extracellular levels of adenosine, or changes in neural circuits regulating sleep, metabolism, or body temperature. Seasonal changes in sensitivity to allosteric modulation of GABAA receptors by pentobarbital in thirteen-lined ground squirrels have been observed. These changes are restricted to cardiorespiratory neurons and are associated with altered expression of ε and δ GABAA receptor subunits (Hengen et al., 2009, 2011). In the present study, pentobarbital did not induce torpor; however, a role for altered allosteric modulation of GABAAR in adenosine-mediated torpor induction cannot be ruled out.
The capacity of an A1AR agonist to induce a torpid state may confer some of the neuroprotective aspects of hibernation noted previously (Zhou et al., 2001). Central A1AR stimulation prevents cardiac arrhythmias during cooling in hamsters (Miyazawa et al., 2008) and may offer a means to avoid cardiac arrhythmias or other side effects encountered during therapeutic hypothermia (Polderman and Herold, 2009). H2S-induced suspended animation has led to investigation of H2S as a therapeutic agent (Blackstone et al., 2005). Likewise, understanding how hibernating mammals regulate metabolic suppression has potential to translate to improved therapies for conditions in which oxygen and energy supply fail to meet demand. Such conditions include stroke, cardiac arrest, hemorrhagic shock, and trauma.
Footnotes
This work was supported by U.S. Army Research Office Grant W911NF-05-1-0280, U.S. Army Medical Research and Materiel Command Grant 05178001, National Institute of Neurological Disorders and Stroke Grants NS041069-06 and R15NS070779, and Alaska Experimental Program to Stimulate Competitive Research. The content of this report is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Neurological Disorders and Stroke or the National Institutes of Health. We thank B. Warlick, C. L. Buck, and B. M. Barnes for critical discussions or reading of the manuscript, B. Rasley for technical assistance, and J. Moore, L. Bogren, Z. Carlson, and J. Olson for support. We also thank J. Vonnahme, H. Crispell, L. Norris, C. Pylant, and V. Combs for assisting with surgeries, and J. Blake, C. Willetto, and C. Terzi for veterinary support.
The authors declare no competing financial interests.
- Correspondence should be addressed to Kelly L. Drew at the above address. kdrew{at}alaska.edu
