Mechanism of Metabolic Suppression in Hibernators

1
Mechanism of Metabolic Suppression in
Hibernators

• Thomas K. Green, PI
• Kelly L. Drew, Collaborator
• Department of Chemistry Biochemistry
• Institute of Arctic Biology
• University of Alaska Fairbanks

2
Significance of Research

• Ischemic stroke is a leading cause of death
  and long term disability
• in the United States.
• Neurons in the ischemic penumbra, the region
  that surrounds the
• immediate damage, are potentially
  protectable. However, glutamate
• reuptake fails and excess glutamate
  overstimulates the neurons
• causing cell death. Lee, JM Nature 1999.
• The drug MK-801, a glutamate antagonist,
  improves an animals
• recovery if given soon after the stroke, but
  impairs recovery if given
• later.
• The most effective laboratory method so far is
  to the cool the brain.
• The cooled brain has less activity, lower
  energy needs, and less risk
• of overstimulation.

3
Hypotheses

• Glutamate release in the suprachiasmatic
• nucleus (SCN) of hibernating animals
  coordinates
• immergence and emergence from torpor.
• There is an increase in glutamatergic
  transmission
• in the SCN upon entry into torpor.

4
Support for Hypotheses

• Glucose utilization in the SCN of golden-mantled
  ground squirrels increases relative to other
  brain structures during the hibernation cycle.
  Kilduff TS et al 1989, 1982.
• Systemic administration of MK-801, a
  noncompetitive, NMDA antagonist induces arousal
  in golden-mantled ground squirrels Harris MJ and
  Milsom WK, 2000.

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Aims

• Aim 1 Determine whether MK-801, a glutamate
  antagonist found to arouse hibernating golden
  mantled ground squirrels, also arouses
  hibernating ground squirrels (AGS).
• Aim 2 Monitor in vivo glutamate release in the
  SCN in parallel with whole-animal oxygen
  consumption and heart rate (early indicators of
  entrance and arousal) as well as core body
  temperature.
• Aim 3 Administer MK-801 via microdialysis
  locally to the SCN to confirm or deny the
  hypothesis that glutamate overflow specifically
  in the SCN coordinates hibernation.

6
Methods

• In vivo monitoring of glutamate will be
  accomplished using
• a combination of microdialysis and capillary
  electrophoresis coupled
• with laser induced fluorescence detection
  (CE/LIF).
• Oxygen comsumption with be measured by
  open-flow respirometry.
• Body temperature and heart rate will also be
  monitored.
• Administration of MK-801 will be accomplished
  by microdialysis

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What is Microdialysis?
Perfusion fluid
To separation and detection
tissue analyte
Hollow fiber membrane
Inner cannula
from CMA/Microdialysis
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Microdialysis

• Developed by U. Ungerstedt and C. Pycock, mid
  70s
• Used in neurochemistry, pharmokinetics,
  biotechnology
• Used for both sampling and drug delivery

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Microdialysis

• Probe Designs
• Concentric probe
• Commercially available in 240 mm o.d.
• Side by side construction (200 mm o.d.)
• Flow through design
• Smaller is better
• Most probe designs have o.d.s of 200-300 mm
  

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UAF Microdialsysis Probe
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Capillary Electrophoresis
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Derivatization Chemistry


NDANaphthalene-2,3-dicarboxaldehyde
Glutamate
?abs 419 nm, 440 nm ?em 493 nm
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CE/LIF Detection
Switching Solenoid Computer Valve

Crossflow Buffer
Photometer
NDA
Microscope
NaCN
Separation Capillary
Reaction Capillary
Laser, 405nm
ACSF
0.1 µM glutamate in ACSF
Microdialysis Probe
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Gated Flow Injection
Crossflow
_
Separation Capillary 20 µm
Reaction Capillary 75 µm
Electrokinetic Injection achieved by stopping
crossflow for 0.3 sec, drawing analyte into
separation capillary by electroosmotic flow.
Gap set to 10-30 ?m

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Gated Flow Injection
Buffer in
5 cm
Glutamate NDA NaCN
Detection Window
Buffer out
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CE/LIF Instrument
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Initial Results on Standard Solutions
glu glutamate asp aspartate fluor
fluorescein internal standard aa aminoadipic
acid
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Probe Response to Changes in glutamate