SCN

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Jihwan Myung http//www-ph.postech.ac.kr/myung jh
myung_at_postech.ac.kr
October 5, 2002 Biophys Lab., POSTECH
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Ca2i oscillation of SCN in brain slice

• Ca2i shows circadian oscillation
• Overall Ca2i slightly higher during the day
• TTX stops the oscillation
• Methoxyverapamil also kills the rhythm

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Slice Prep

• SD rats btn 10 14 days
• Cold aCSF 130 NaCl, 26 NaHCO3, 3 KCl, 5 MgCl2,
  1.25 NaH2PO4, 1 CaCl2, 10 glucose (pH
  7.2-7.4, 348.5 mOsm)
• Incubating aCSF 130 NaCl, 26 NaHCO3, 3 KCl, 2
  MgCl2, 1.25 NaH2PO4, 2 CaCl2, 10
  glucose, 4 lactate
• Incubation at 25oC for at least an hour
• Coronal slices 350 um thick
• 1320 cells from 65 animals

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Ratiometry

• Fura-2 AM bulk loading 50 uM at 35oC for 10 min
• Lambda DG-4 (ours 10-2), Princeton Microview
  MetaFlour
• Intensity (18 uW) sampling freq (0.5 Hz)
  minimized to reduce bleaching
• Calibration High 10 Ca2, low 10 EGTA in vitro
• 5 min for one cell
• NADH autofluorescence 5 of signalintensity, no
  circadian oscillation

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Ca oscillation
Resting Ca2ILarger for the day
More variation among the day group
More time variation for the day cells
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Freerun Block
Freerun The rhythm is circadian (Excitation due
to photic input?)
TTX prevents depol Methoxyverapamil blocks L-type
Ca channel
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Discussion

• Ca2 oscillation driven by Vm?
• Rhythm not observed in rats younger than 7 days.
  Why?

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Reference

• Colwell, CS (2000). Euro. J. Neurosci. 12571-576.