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by: Merlin Hyatt


Merlin Hyatt
GPA 3.51

Frederick Rieke

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Frederick Rieke
Class Notes
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This 13 page Class Notes was uploaded by Merlin Hyatt on Wednesday September 9, 2015. The Class Notes belongs to NEUBEH 532 at University of Washington taught by Frederick Rieke in Fall. Since its upload, it has received 29 views. For similar materials see /class/192530/neubeh-532-university-of-washington in Neurobiology And Behavior at University of Washington.

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Date Created: 09/09/15
PBIO NeuBeh 532 Electrical Recording Techniques Goals for today Quick summary of different techniques to measure electrical signals Current and voltageclamp recording Series resistance issues in patchclamp recording Conductanceclamp recordings SOME ESSENTIAL PHYSICS sorry No absolute voltage Only measure voltage differences do you know what your ground is doing Conservation of Charge Current flows in loops unless there are locations where charge accumulates I gt Capacitors Q CV C I C dVdt Capacitors oppose voltage changes I gt Resistors and Ohm s Law V I R Thermal noise Any resistor at finite temperate generates voltage fluctuations SUMMARY OF RECORDING TECHNIQUES extracellular recording good choice if all you want is action potentials occasionally can see synaptic potentials as well permits multiunit recording tetrodes electrode arrays isolating spikes from single cell takes some work cannot control currentvoltage sharp intracellular recording good choice for blind intracellular recordings can damage cell due to leak at site of impalement hard to control currentvoltage patch clamp recording WHOLECELL PATCH CLAMP RECORDING CURRENT CLAMP VOLTAGE CLAMP amp provides current required to keep voltage inside electrode surrogate for intracellular voltage equal to command voltage ie cancels cell current measure measure 47 0 voltage current V command PROS CONS often easier on cell than sharp dilute cell contents except electrode perforated patch Isolates single cell cleanly clear access to cell is helpful good voltagecurrent control ability to alter cell contents ONE APPLICATION OF VOLTAGE CLAMP ISOLATING EXCITATORY AND INHIBITORY SYNAPTIC INPUTS l MW Jaw V12 M W 90 gtlt V Vexc 200 ms 1 aw 905 HUV Vexc gechym ms 100 ms I How could this go wrong LIMITATIONS OF VOLTAGE CLAMP DUE TO SERIES RESISTANCE time constant measure RS X Cm current current V command Vm V command shortly after step well after step lots of current flowing to voltage drop across charge Cm hence large Rs and Rm If voltage drop across Rs RsltltRm then Vm close to V command Vm does not equal V command Vm does not change instantaneously SERIES RESISTANCE S MORE THAN AN ESOTERIC CONCERN 2 nA 200 ms 45 actual voltage can differ E 3950quot from holding voltage by 55 1015 mV even with good 60 access eg 10 M0 here What can you do try to get better access decrease size of currents increase cell resistance use compensation circuitry BUT39H4EREIS SOME HOPE good access 75 o compensation poor access no compensation 1 nA 1 nAV 1 mV 20 mV 500 ms 500 ms SPACE CLAMP what can you do not much decrease size of currents increase dendritic resistance study synapses close to soma search for best voltage DYNAMIC CONDUCTANCE CLAMP mimicking a real conductance with injected current fails to account for voltage dependence dynamic clamp is an alternative 1 measure voltage 2 compute current I geXCV exc ginhV Huh 3 inject current SINGLE ELECTRODE VOLTAGE CLAMP Problem series resistance errors which are nuisance for patch recordings are about 100x worse for sharp intracellular electrodes This makes voltage clamp difficult measure current Solution Rapidly switch between injecting current and measuring voltage Rely on membrane RC time constant to V command keep voltage fixed when not injecting current current T Rs 100 MOhm measure voltage while no injected current membrane Rm100 Cm10pF MOhm command voHage LIMITATIONS IMPOSED BY THOSE PESKY THERMAL FLUCTUATIONS 6V V4kTBR time Prob Thermal movement of ions through resistor generates voltage noise RMS voltage noise eg current clamp 6V V4 kT B R k Boltzman39s constant T 2 temperature in Kelvin B bandwidth R 2 Resistance RMS current noise eg voltage clamp 51 q 4kT B R This noise is a fundamental limit to what you can measure THERMAL NOISE IS ALSO NOT AN ENTIRELY ESOTERIC CONCERN 16 mM CAMP 20 pAl Sundermcm and Zagotta 1999 200 ms 51 x4kTBR 04 pA Thermal noise can be similar in magnitude to signals of interest eg small single channel currents


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