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Tweak Your Technique Electrophysiology 101

Tweak Your Technique Electrophysiology 101. August 19, 2011 Kevin Lee. What can be studied with electrophysiology?. Single channel behaviour in expression systems: HEK293, CHO’s, X. Laevis oocytes Intrinsic properties of excitable cells neurons, myocytes Synaptic transmission

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Tweak Your Technique Electrophysiology 101

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  1. Tweak Your TechniqueElectrophysiology 101 August 19, 2011 Kevin Lee

  2. What can be studied with electrophysiology? • Single channel behaviour • in expression systems: HEK293, CHO’s, X. Laevisoocytes • Intrinsic properties of excitable cells • neurons, myocytes • Synaptic transmission • neuromuscular junction, neuronal synapses

  3. The hippocampus

  4. Field recordings

  5. LTP/LTD

  6. Whole-cell Recordings

  7. Patch-Recording Methods

  8. Whole-cell recordingsCurrent-clamp vs Voltage-clamp “EPSP” “EPSC”

  9. Whole-cell recordingsCurrent-clamp vs Voltage-clamp

  10. Voltage-clamp

  11. mEPSCs (minis) mEPSC amplitude reflects synaptic AMPAR content Beique et al. (PNAS, 2006)

  12. mEPSCs (minis) mEPSC frequency can reflect: - total number of synapses - presynaptic function Beique et al. (PNAS, 2006)

  13. Release probability (Pr) of glutamate Changes in frequency of mEPSCs is generally believed to reflect changes in Pr. “Paired-Pulse” electrical stimulation can also provide information about Pr. Beique et al. (PNAS, 2006)

  14. What about Molecular interventions?

  15. Biolistic transfection of cortical neurons in organotypic slices Beique & Andrade (J. Physiol, 2002; cover photo)

  16. Biolistic transfection of cortical neurons in organotypic slices

  17. Biolistic transfection of cortical neurons in organotypic slices Cull-Candy et al. (Science STKE, 2004) Barria & Malinow (Neuron, 2002)

  18. Drawbacks to electrical stimulation: • Difficult to isolate individual synaptic events • Spatial Parameters left unknown

  19. 2P-Uncaging of MNI-Glutamate

  20. 2P-Uncaging of MNI-Glutamate Uncaging of MNI-Glu activates both AMPA and NMDA receptors High spatial resolution of glutamate uncaging Béïque et al., PNAS 2006

  21. Single Synapse Ca2+ Imaging Simultaneous Glutamate Uncaging and Calcium Imaging Uncage 1 2 Time  1 2

  22. Fundamental dendritic properties probed by glutamate uncaging and calcium imaging Losonczy & Magee (Neuron, 2006)

  23. Fundamental dendritic properties probed by glutamate uncaging and calcium imaging Losonczy & Magee (Neuron, 2006)

  24. Experimental Wizardry to Envy Kravitz et al. (Nature, 2009)

  25. Experimental Wizardry to Envy Kravitz et al. (Nature, 2009)

  26. Multiple Simultaneous Recordings in Slices Olah et al. (Nature, 2009)

  27. The End

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