Transcript M. Woodin
Electrophysiology the science and branch of physiology that pertains to the flow of ions in biological tissues and, in particular, to the electrical recording techniques that enable the measurement of this flow and the potential changes related to them Intracellular Recording Intracellular Recording: A recording electrode is inserted into a cell, so that the intracellular potential can be measured against the extracellular potential cell The Nobel Prize in Physiology or Medicine 1963 Sir John Carew Eccles Alan Lloyd Hodgkin Andrew Fielding Huxley "for their discoveries concerning the ionic mechanisms involved in excitation and inhibition in the peripheral and central portions of the nerve cell membrane" http://nobelprize.org/medicine/laureates/1963/ Membrane potentials in squid axons: The first experiments examining how changes in ion concentrations affect the membrane potential were done on squid. Why squid axons? Voltage Clamp Allows you to ‘clamp’ the membrane potential and record the ionic current Normally, the voltage is ‘stepped; and the resulting current is measured Difficulties with Voltage Clamp and sharp intracellular recordings? E. Neher B. Sakmann 1991 Nobel Prize in Medicine For the development of the Patch clamp Small patch of membrane is sealed to the tip of a micropipette The high resistance seal (called a gigaohm seal)ensures that currents flow through the amplifier rather than escaping through the rim of the patch Recording Configurations • Why select cellattached vs. wholecell recording? Cell-attached recording • Allows the recording from a single ion channel located in the area of the patch under the pipette • As the ion channel opens or closes there will be an abrupt increase or decrease in the conductance of the patch of membrane • Ion channels can be characterized by their conductance, their open time, and probability of channel opening (in addition to pharmacological inhibition and ion substitution) Figure 4.6 Effect of Potential on Currents Single spontaneously active K+ channel; 150 mM KCl on both sides Channel current as a function of voltage; the slope of the line indicates the channel conductance; the ability of a channel to pass current A linear IV curve indicates no voltage-dependent gating of the channel Permeability = open channel Conductance = permeability + ions Equilibirum Potential for K+ Currents [K+]i = 90mM [K+]o = 3mM; (B) with no electrical gradient K+ flows out (C) with 20mV applied to the cell the flow of K+ out of the cell increases (D) -50mV inside the cell reduces the current amplitude Non-linear curve indicates the channel is voltage-dependent What is the difference between Patch Clamp and Voltage Clamp? Can they be used simultaneously? Recording Configurations • Why select cellattached vs. wholecell recording? • How do you decide what solution to include in your whole cell solution? • Electrical access vs. dialysis? Figure 7.1 Membrane Currents Produced by Depolarization • Ion substitution experiments Figure 7.3 Dependence of Early and Late Currents on Potential How can we find synaptically connected neurons? EPSPs IPSPs Figure 11.8 Reversal Potential for Synaptic Currents Measured by Voltage Clamp Recording STDP Fiumelli & Woodin 2007 Current Opinion in Neurobiology Perforated Patch Clamp Recording