Biomedical Science Honours

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Biomedical Science (Honours)

• Research Presentation
• 2004

Electrophysiological evidence for the involvement
of TRP channels in neuronal cell death during
acute ischemia
Thomas (In Hyeup) Park
Supervisor Prof. J. Lipski Neurophysiol
ogy Laboratory
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What is STROKE?

• Neuronal injury or death resulting from decrease
  in cerebral blood flow through parts of the brain
• This can arise from
• Occlusion of cerebral blood vessels by embolus or
  local thrombosis
• Rupture of cerebral arteries (Hemorrhagic stroke)

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Neuronal cell death during ischemia
Severe hypoxia/hypoglycemia
Decrease in cellular energy (ATP)
Malfunction of ATP dependant ion
channels/transporters
Cell Swelling, Excitotoxicity, Free Radical
production, Caspase activation
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In vitro model of ischemic neuronal cell death

• Hippocampal brain slices
• CA1 neurons of stratum pyramidale
• Perfuse them with hypoxic/aglycemic solution
• (in vitro ischemia)

CA1
Fiala, J.C. et al. 2001
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Electrophysiological characteristics of rat CA1
neurons during ischemia

• Patch or intracellular recordings of hypoxic
  SD-like depolarization in rat CA1 neurons show
• Distinct triphasic electrophysiological recording
• Initial hyperpolarization
• Slow depolarization
• Fast depolarization (Hypoxic Spreading Depression)

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• Control period pre-hypoxia shows neurons resting
  membrane potential at about -65mV.
• Initial hyperpolarization due to hypoxia
• Slow depolarization leading onto the fast
  depolarization
• Fast depolarization (Hypoxic SD)

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Hypoxic Spreading Depression

• What causes this depression
• Cationic currents
• Voltage-gated channels
• Ligand-gated channels
• Evidence of voltage-gated channel involvement

Somjen et al. 2000
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The Slow Depolarization
Slow Depolarization
Cocktail of all known blockers cationic channels
Removal of extracellular cations
Abolished Slow Depolarization
Attenuated BUT failed to abolish depolarization
X
HSD and Cell Death

• Removal of cations are more effective in
  attenuating HSD then blocking currently known
  cationic channels

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Unidentified Cation Channel

• So are there other unidentified cation channels?
• I HOPE SO!
• Our lab group is proposing a contribution by
  relatively newly discovered group of non-specific
  cation channels
• TRP Channels!!
• The transient receptor potential (TRP) channels
  - non-voltage gated
• - non-glutamate gated
• cation channels

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What are TRP Channels?

• Characteristics of TRP channels
• 3 main families TRPC, TRPV and TRPM
• Non-specific cation channels
• Some families expressed in neurons
• Operated by
• Internal Ca2 store depletion (Capacitive Ca2
  currents) - TRPCs
• Mechanical Stress (cell swelling) TRPV4
• Oxidative Stress TRPM2,7
• Temperature Changes TRPV14

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Aims

• To test the hypothesis that
• Ischemia induced metabolic and oxidative stress
  could activate TRP channels and contribute to the
  disruption of ionic homeostasis
• Neuroprotective effects of hypothermia could be
  due to the inactivation of temperature sensitive
  TRP channels at lower temperatures

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Methods

• In vitro studies conducted in CA1 neurons of live
  rat hippocampal slices
• Extracellular Recordings
• Field Excitatory Post-Synaptic Potential
• Negative DC shifts
• Whole Cell Patch-Clamp Recording (Blind)
• Voltage-Clamp
• Current-Clamp

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Electrophysiological characteristics of CA1
neurons during hypoxia

• Extracellular Recordings (36ºC)

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Effects of non-specific TRP channel blocker and
lowering temperature on the occurrence of HSD
P lt 0.01
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Whole Cell Patch-Clamp Recordings
Voltage-Clamp (36ºC)
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Whole Cell Patch-Clamp Recordings
Voltage-Clamp (Lanthanum, 36ºC)
Black Control Recordings Red Lanthanum
Recordings
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Whole Cell Patch-Clamp Recordings
Current-Clamp (36ºC)
Black Control Recordings Red Lanthanum
Recordings
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Proposed Mechanism
During Hypoxia/Aglycemia
Other Cationic Channels/Transporters
TRP Channels
Slow Depolarization Occurrence of HSD
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Proposed Mechanism
During Hypoxia/Aglycemia TRP Channel Blockers
Other Cationic Channels/Transporters
TRP Channels
2-ABP
X
RR
Na, Ca2, K
Na, Ca2, K
X
Cations
X
Lanthanum
Delayed occurrence of Hypoxic SD
Lowering Temperature
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Summary

• All of the administrated non-specific TRP channel
  blockers
• Attenuated slow depolarization
• Delayed occurrence of HSD
• Lowering temperature also delayed occurrence of
  HSD
• Lanthanum and lowering temperature had an
  occlusion effect

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Conclusions

• TRP channels are involved in conduction of
  cationic currents during in vitro ischemia.
• Lanthanum and lowering temperature are
  inactivating similar TRP channels, thus acting in
  a similar neuroprotective pathway.

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Possible Clinical Applications

• TRP channels
• Potential target for therapeutic interventions
  for stroke and acute brain injury
• Can be regarded as a specific target mimicking
  neuroprotective effects of hypothermia

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Acknowledgements
Molecular Neurophysiology Laboratory
Prof. J. Lipski
Supervisor
Lab Members
Dr Hanne Skiri
Dr D. de Castro
Dong Li
Our trusty PhD Students
John Lin
Kenny Chung
Pete Freestone
My Fellow Honours Student and Friend
Stanley Lee
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Thanks For Listening!
Any Questions?