This project tests hypotheses about the role of
neurotransmitters and neuromodulators in mediating CNS and metabolic suppression
during hibernation. Neurotransmitters
in the brains of freely moving arctic grounds squirrels are sampled using in vivo microdialysis and analyzed using
capillary electrophoresis with laser-induced fluorescence detection.
Background and Significance:
Thermal or Q10 effects resulting
from dramatic drops in body temperature are thought by some investigators to
account for metabolic arrest during hibernation (Snapp and Heller, 1981). Moderate reductions in metabolic rates in rats and other endotherms have
been achieved through hypothermia and other means. Mechanisms in addition to
hypothermia, such as decreases in intracellular pH, are thought to contribute to
metabolic arrest during hibernation (Malan, 1988). Recent studies conducted at UAF found pronounced discrepancies between
core body temperature and metabolic rate (O2 consumption) measured during steady
state torpor over a range of ambient temperatures (Buck and Barnes, 2000).
These results strongly support the notion that mechanisms in addition to
Q10 effects contribute to metabolic suppression in hibernating ground squirrels.
One focus of our research is to investigate non-thermal mechanisms of
metabolic and central nervous system suppression in hibernating ground
squirrels.
Role
of ADENOSINE: Adenosine is a
metabolic depressor that is thought to function as a retaliatory, inhibitory
neuromodulator in situations where energy reserves are limiting and neuronal
inhibition is advantageous; such as during hypoxia and ischemia and during early
stages of anoxia in anoxia-tolerant species (Dragunow and Faull, 1988; Nilsson
and Lutz, 1992). Adenosine, a
metabolite of adenylates (AMP, ADP and ATP) increases both intra- and
extracellularly when energy reserves decrease. If high energy phosphates are
limiting at any time during hibernation adenosine would be expected to increase
and inhibit neuronal activity. Finally, if energy reserves exceed demand (as a
consequence of extreme metabolic suppression), interstitial concentrations of
adenosine could decrease. Thus,
monitoring adenosine throughout a torpor bout will provide information regarding
energy balance throughout entrance, maintenance and exit from torpor and will
test the hypothesis that increases in adenosine during periods of negative
energy balance contributes to metabolic suppression during hibernation.
One hypothesis currently under investigation is that adenosine release is
associated with entrance into torpor and initial suppression of energy demand.
ROLE OF GLUTAMATE: Glutamate is the primary excitatory neurotransmitter in the CNS. Glu increases neuronal activity throughout the brain including regions thought to be involved in the regulation of sleep and hibernation (Heller, 1979; O’Hara et al., 1995; Cape and Jones, 2000; Azuma et al., 1996; Shinohara et al., 2000; Carre and Harley, 2000). Preliminary data suggest that extracellular fluid concentrations of glutamate ([glu]ecf) increase throughout prolonged torpor such that low concentrations during early torpor may result in decreased neuronal activity and promote subsequent metabolic suppression. As extracellular glutamate accumulates over a torpor bout it may contribute to increased irritability (Lyman, 1948; Beckman and Stanton, 1976) and eventual arousal from torpor. Furthermore, evidence suggests glutamatergic transmission accounts for 80-90% of glucose oxidation in brain (Sibson et al., 1998). Glucose utilization in brain is severely depressed during hibernation (Frerichs et al., 1995) consistent with a global decrease in glutamatergic transmission during early torpor. We are currently monitoring extracellular glutamate in brains of hibernating arctic ground squirrels using in vivo microdialysis coupled with capillary electrophoresis and laser-induced fluorescence detection.
Role
of Hypoxia: Recent
evidence from our lab suggests that arctic ground squirrels are hypoxic during
arousal from torpor. Secondary
effects of hypoxia, such as a decrease in pH or increase in GABA or adenosine
release may facilitate metabolic suppression. Another research focus is on the
role hypoxia plays in regulating energy demand during entrance and exit from
torpor.
Microdialysis
Capillary electrophoresis with laser-induced fluorescence detection (CE-LIFD): A limitation of microdialysis in the past has been poor temporal resolution that is limited by the perfusion rate through the probe and the sample volume (often microliters) required by off-line HPLC separation; and by the detection limit of the assay. These disadvantages have largely been overcome by on-line coupling of the microdialysis probe to capillary electrophoresis (CE) which requires nanoliter injection volumes (Lada et al, 1997) and gives attomole detection limits (Lada et al., 1997; Tseng et al., 1994). These limits of detection are more than 1000 fold better than the most sensitive, microbore HPLC assays. Temporal resolution on the order of 10-14 s for glutamate and aspartate has been achieved using on-line in vivo analysis. Our research maximizes time resolution of neurotransmitter release using on-line CE-LIFD analysis. High temporal resolution is particularly relevant for studies of glutamate where rapid sampling intervals are necessary to observe physiologically significant, action potential dependent release (Lada et al., 1997; 1998; Timmerman and Westerink, 1997). Resolution is also relevant for adenosine where rapid fluctuations in energy balance may produce physiologically significant fluctuations in extracellular adenosine and ATP.