Inhibition of NMDA-type glutamate receptors induces arousal from torpor in hibernating arctic ground squirrels (Urocitellus parryii)

Abstract

Hibernation is an adaptation to overcome periods of resource limitation often associated with extreme climatic conditions. The hibernation season consists of prolonged bouts of torpor that are interrupted by brief interbout arousals. Physiological mechanisms regulating spontaneous arousals are poorly understood, but may be related to a need for gluconeogenesis or elimination of metabolic wastes. Glutamate is derived from glutamine through the glutamate-glutamine cycle and from glucose via the pyruvate carboxylase pathway when nitrogen balance favors formation of glutamine. This study tests the hypothesis that activation of NMDA-type glutamate receptors (NMDAR) maintains torpor in arctic ground squirrel (arctic ground squirrel (AGS); Urocitellus parryii). Administration of NMDAR antagonists MK-801 (5 mg/kg, i.p.) that crosses the blood-brain barrier and AP5 (5 mg/kg, i.p.) that does not cross the blood-brain barrier induced arousal in AGS. Central administration of MK-801 (0.2, 2, 20 or 200 μg; icv) to hibernating AGS failed to induce arousal. Results suggest that activation of NMDAR at a peripheral or circumventricular site is necessary to maintain prolonged torpor and that a decrease in glutamate at these sites may contribute to spontaneous arousal in AGS.

Fig. 1. NMDAR activation is necessary for maintenance of torpor in AGS

MK-801 (5mg/kg, ip) or saline was administered to hibernating AGS habituated to ip saline injections. Arousal from hibernation was quantified using an arousal index scale. MK-801, but not saline induced arousal in all AGS tested (n=3 AGS). Arrow represents the time point of injections. Data shown are the arousal index for individual animals. Presence or absence of arousal was combined with data shown in Fig. 2a for statistical analysis; 0424 (♂), 0425(♂), and 0433(♀) are animal identification numbers.

Fig. 2. Arousal induced by inhibiting NMDAR and by handling-induced arousal

MK-801 (5mg/kg; ip) induced an increase in the rate of O₂ consumption (Vo₂) and core body temperature (T_b) in all of the animals tested (a) while vehicle had no effect (n=3) (b). Handling-induced arousal (HIA) is shown for comparison (n=3, c). AP5 (5mg/kg; ip) induced arousal from torpor in all of the animals tested while vehicle had no effect in any of the animals tested (n=5) (d). Arrow head at 0.5h represents the time point where either drug (a,d) or vehicle (b,d) was delivered or arousal was initiated by handling (c).

Fig. 3. Inhibiting peripheral 5-HT₃R does not induce arousal from torpor in hibernating AGS

Mean rate of O₂ consumption measured for 2.5h following MDL-72222 (5mg/kg; ip) or vehicle (n=3) shows that MDL-72222 does not induce arousal from torpor in AGS when compared with HIA.

Fig.4. Administering NMDAR antagonist MK-801 by icv injection does not induce arousal from torpor in hibernating AGS

Mean rate of O₂ consumption for a period of 2.5h following MK-801, icv [0.2μg (n=5), 2.0μg (n=4), 20μg (n=3) or 200μg (n=3) does not differ from mean rate of O₂ consumption following saline injection (icv) (n=5)]. Insert shows that handling-induced arousal (HIA) (n=3) produces a significant increase in O₂ consumption compared to MK-801 (200μg, icv) (*p<0.05, 2-way ANOVA with repeated measures).