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Outline

Outline. Cell-attached vs. whole cell patch Ohm’s Law Current Clamp Voltage Clamp Current-Voltage Relationships Components/ conductances of an action potential Single channel recordings. Ohm’s Law.  V = IR.

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Outline

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  1. Outline Cell-attached vs. whole cell patch Ohm’s Law Current Clamp Voltage Clamp Current-Voltage Relationships Components/ conductances of an action potential Single channel recordings

  2. Ohm’s Law V = IR The potential difference between two points (A, B) linked by current path with the conductance (G) and current (I) is as follows: A B

  3. Cell-attached Patch Whole-cell Patch GOhm Seal between pipet and membrane Pipet and cell are contiguous Record single channels Monitor spiking Measure “macro” currents Monitor synaptic events

  4. Current Clamp Monitors the potential of the cell - units will be in volts By convention V= Einside – Eoutside Upward deflections are depolarizing; downward are hyperpolarizing -55 mV

  5. Current Clamp Monitors the potential of the cell- therefore units will be in volts By convention V= Einside – Eoutside Upward deflections are depolarizing; downward are hyperpolarizing -55 mV Membrane time constant = RmC

  6. Current Clamp Action potentials can be measured V I

  7. Current Clamp There are many types of action potentials Cells have different properties for firing action potentials Silent Tonic Na+ spike Ca2+ spike Bursting

  8. = 1/g g C Current Clamp Current-Voltage Relationship & Measuring conductance (g): -55 mV “Ohmic” I-V curve Slope = I/Vm = 1/R = g (conductance)

  9. Voltage Clamp V-clamp Battery: imposes a voltage drop across the cell membrane I-clamp Measures the amount of current needed to hold the cell at a given potential. Itotal = IC + Iionic where IC = C(dV/dt) when clamping the cell at a certain voltage, at steady state, dV/dt=0, thus Itotal = II Vhold I

  10. Voltage Clamp V-clamp Can compensate for the capacitive current with amplifier. (ie. can force the Ic=0) By injecting an equal and opposite amount of current I-clamp Iinj Thus have full control over the membrane potential of the cell. Vhold Vm Thus, can measure the conductance of the cell due to Iionic at any voltage

  11. Voltage Clamp vs. Current Clamp I-clamp V-clamp glut Upward deflections are depolarizing; downward are hyperpolarizing Downward (negative deflections) are inward currents; upward are outward

  12. non-Ohmic Current-Voltage Relationships Ohmic Slope = I/Vm = g (conductance); x-intercept: Erev

  13. Action Potentials The Hodgkin-Huxley Model The squid giant axon action potential had only sodium and potassium currents…. Other cells’ action potentials are shaped by a number of other conductances.

  14. Squid Axon Action Potentials Current clamp Voltage clamp Assymetrical currents w/ depol or hyperpol V-steps ie. non-Ohmic I-V relationship

  15. Squid Axon Action Potentials Family of voltage clamp currents I-V relationship reveal voltage-dependence of Na and K channels

  16. Squid Axon Action Potentials (blocks Na channels) (blocks K channels) Family of voltage clamp currents Specific blockade of ion channels: confirms two separate channels: Na and K underlying the action potential

  17. Single Channel Recordings Single channel currents Whole cell ‘macro’ currents Single channel currents Single channel currents

  18. Single Channel Recordings a single channel flickers open and close stocastically according to an open probability, and inactivation or closing probability. => these all depend on the rate constants of the channel

  19. Single Channel Recordings Single channel currents Avg current • Inward current • Na channel: • fast activating • fast inactivating • Outward current • K channel: • slow activating • slow inactivating

  20. Things to look for… Current clamp or voltage clamp Concentration of ions in the internal vs. external solution (determines Eion) Pharmacology Temperature of recording

  21. Supplement: Voltage Clamp Series resistance (Rs) is due to the resistance of the intracellular solution (Ohms) and the access through the pipet tip (MOhms). Voltage Clamp errors: Current across Rs causes a voltage drop. (voltage error) Rs in series with C forms a low-pass filter. (temporal error) current voltage of cell  = [(Rs*Rm)/(Rs+Rm)]Cm; Rm >>Rs, thus  = RsCm

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