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Transcript
Principles of patch-‐clamp electrical recording and optogene4cs Karl Iremonger Centre for Neuroendocrinology Department of Physiology University of Otago The brain is an electrical organ 1) Be able to record the electrical ac4vity of neurons 2) Have techniques to precisely perturb electrical ac4vity to determine the effect on physiology Patch-‐clamp recordings: tells us what is going on inside the neuron (electrically speaking) Spiking Underlying synap4c poten4als How to record membrane current and voltage in a neuron The basic circuit The basic circuit Bath ground Ag wire Amplifier Ag pellet Pipette holder pipette AD converter bath Brain slice Computer “if you can see it, you can patch it” IR - sensitive video camera magnification Patch pipette camera controller video monitor analyser DIC prism objective patch pipette brain slice condenser DIC prism polariser IR filter Differen4al interference contrast (DIC) image “if you can see it, you can patch it” GFP can be used to iden4fy neurons of a par4cular gene4c iden4ty Epifluorescence and DIC image overlaid Patch pipette Differen4al interference contrast (DIC) image Approaching the neuron Glass micropipette! Posi4ve pressure Channel or receptor! neuron Forming a loose seal on-cell recording Release of posi4ve pressure Used to record spiking activity! without rupturing cell membrane! ! neuron! Low resistance (10-50 MΩ) seal! is formed! Forming a tight seal on-cell recording ! Used to record activity of single! ion channels in the cell membrane! ! Nega4ve pressure (mild suc4on) neuron! High resistance (>1GΩ) seal! is formed after suction applied! Establishing a whole-cell recording Strong suction! neuron! Establishing a whole-cell recording Strong suction! -recording membrane potential! -membrane currents! -synaptic currents! K! Na! Current clamp spiking neuron! Voltage clamp synap4c currents Current clamp vs voltage clamp recording Hold voltage or current constant, measure the other parameter. • 1) Current clamp (IC) – measure membrane poten4al (voltage) while controlling amount of injected current • 2) Voltage clamp (VC) – measure membrane current while clamping the membrane voltage • These are opera4onal modes of a single amplifier-‐ therefore within an experiment, you can switch back and forward between IC and VC with the click of a buVon. Resources • Axon Guide, Third Edi4on – available for download online (just google “axon guide”) Part 2: Optogene4cs Optogene4cs Combining gene*cs and op*cs to achieve gain-‐ or loss-‐of-‐func*on of well-‐defined events within specific cells of living *ssue. CRH neuron Oxytocin neuron Nature Methods 8, 26–29 (2011) doi:10.1038/nmeth.f.324 hVp://neurobyn.blogspot.se/2011/01/controlling-‐brain-‐with-‐lasers.html Light gated ion channels, pumps and pathways Channelrhodosin2 (ChR2)-‐light sensi4ve ca4on channel Channelrhodopsin (VChR1) from Volvox carteri-‐ red shieed variant of ChR2 Halorhodopsin (NpHR)-‐ light sensi4ve Cl-‐ pump (hyperpolarising) Archaerhodopsin (Arch) -‐ light sensi4ve H+ pump (hyperpolarising) OptoXR-‐ light sensi4ve ac4va4on of G-‐protein pathways (modified bovine rhodospins) Channelrhodospin 2 (ChR2) Zhang et al 2007 • Blue laser – (oeen diode lasers, 473nm, 100mW) • Blue Light emijng diode (LED), 470nm Applying optogene4cs in vitro Laser scanning microscope Laser or LED light focused via objec4ve Laser or LED light via op4c fiber Op4c fiber 50-‐200 um internal diameter Accurate, but ac4va4on is inefficient Efficient ac4va4on, and some ability to control spot size Efficient ac4va4on, But difficult to precisely control area of illumina4on Optogene4cs in vivo Deisseroth Lab Resources hVp://openoptogene4cs.org/ hVp://web.stanford.edu/group/dlab/optogene4cs/ Neuron, 2011 (great review ar4cle) ChR2 is expressed in soma, dendrite and axons OR Yizhar et al, Neuron 2011 Op4c fiber + electrode = optrode op4c fiber 4p recording electrodes Anikeeva et al Nature Neurosci 2011 Royer et al EJN, 2010 Caveats • High laser powers with long pulse dura4ons can induce hea4ng and 4ssue damage. • ChR2 ac4va4on of many neurons induces an “ar4ficial synchroniza4on” of the neural network. • Changes in ionic gradients occur when using light gated ion pumps. • Non-‐specific targe4ng-‐ leaky expression or retrograde transport of AAV • Direct s4mula4on of nerve terminals evokes abnormal paVerns of transmiVer release (ChR2 is also a Ca channel)-‐ see regehr paper How to get light sensi4ve ion channel into your neuron? • Adeno-‐associated virus (AAV) or len4virus • hVp://www.med.upenn.edu/gtp/vectorcore/ • hVp://www.med.unc.edu/genetherapy/ vectorcore (other facili4es exist-‐ also can ask a researcher directly if they have made an interes4ng one) • In utero electropora4on (see Petreanu et al., 2007 Nature Neuroscience) • Gene4cally modified mouse (either ChR2 gene inserted or use cre-‐loxp transgenic) Channelrhodopsin-‐2 is ac4vated by photoisomeriza4on of all-‐trans re4nal to 13-‐cis re4nal at wavelengths of 470 nm. Aeer photoisomeriza4on, the covalently bound re4nal spontaneously relaxes to all-‐trans in the dark, providing closure of the ion channel and regenera4on of the chromophore. Wong et al 2012, Journal of the Mechanics and Physics of Solids Current clamp While the amount of injected current is controlled, the cell may open ion channels that lead to other currents flowing into the cell This nega4ve res4ng membrane poten4al is rela4ve to the bath ground The membrane poten4al of the neuron does not change instantaneously, but charges and uncharges with a par4cular 4me constant (τ). -‐60mV 0 pA Measured voltage 100 pA 0 pA Injected current (command current) Voltage clamp Amp Vcommand Outward current Spontaneous synap4c currents Current trace (pA) Inward current Membrane voltage (clamp at -‐60 mV) -‐ The command voltage is set by the experimenter (ie – 60mV). -‐ The difference between the membrane voltage (Vm) and the command voltage (Vc) is measured by the amplifier. -‐ If Vm is not equal to Vc, then the amplifier generates a current that is injected into the neuron to make Vm = Vc -‐This feedback circuit keeps Vm as close to Vc as possible and operates almost instantaneously -‐The amount of current required to keep Vm = Vc can be measured and recorded
