Whole‐Cell Recording In Vivo

Michael R. DeWeese1

1 Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 6.22
DOI:  10.1002/0471142301.ns0622s38
Online Posting Date:  January, 2007
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

In vivo whole‐cell patch‐clamp recording provides a means for measuring membrane currents and potentials from individual cells in the intact animal. Patch‐clamp methods have largely been developed in vitro. This body of work has contributed enormously to the understanding of many important phenomena in excitable cells—including synaptic plasticity in the mammalian central nervous system, and the behavior of individual protein channels. In recent years, an increasing number of groups have applied whole‐cell recording techniques in the intact animal. Such in vivo studies offer the tantalizing possibility of uncovering the underlying principles and mechanisms of neural interactions within the natural context of fully intact biological networks. This unit focuses on strategies for overcoming the specific technical challenges posed by in vivo whole‐cell recording. A straightforward procedure is described for obtaining whole‐cell records from the cortex of the anesthetized rat; this procedure has also been applied successfully to awake animals and other rodent species with minor modifications.

Keywords: whole‐cell; patch clamp; in vivo; intracellular recording; cortex; neuron; rat

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Strategic Planning
  • Basic Protocol 1: In Vivo Whole‐Cell Patch‐Clamp Recording
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: In Vivo Whole‐Cell Patch‐Clamp Recording

  Materials
  • Physiological buffer (see recipe)
  • Normal saline: 0.9% (w/v) NaCl
  • Rats (male or female, post‐natal day 17 to 30; Sprague‐Dawley)
  • General anesthetic (e.g., 60 mg ketamine/0.5 mg medetomedine per kg)
  • Internal solution (potassium‐ or cesium‐based, see reciperecipes)
  • Agarose solution (1% to 2% agarose by weight in physiological buffer; Type III‐A, A9793, Sigma), melted and kept up to 4 hr at ∼40°C
  • Electrode puller (e.g., Narishige 2‐stage vertical puller)
  • Electrode glass (e.g., filamented, fire‐polished, thin‐walled, borosilicate electrode glass 3 in. (75 mm) length, 1.5 mm o.d., World Precision Instruments)
  • Dissecting microscope
  • Patch pipet storage container with cover
  • Disposable 1‐ml and 30‐ml syringes (for anesthesia and pipet pressure control, respectively)
  • Disposable syringe needles: 25‐G (for rats) or 27‐G (for mice)
  • Temperature controller with heating pad and rectal thermometer
  • Stereotaxic frame (for rats) that allows access to desired cortical region
  • Cotton swabs
  • High speed pneumatic dental drill
  • Gel foam sponges
  • Dural hook
  • Recording chamber, electrically shielded
  • Computer‐based data acquisition/analysis system, including A/D board and software (see units 6.1& 6.6)
  • Silver ground wire coated with AgCl at tip (e.g., model E201Ag‐AgCl pellet; Axon Instruments)
  • Amplifier with headstage (e.g., Axopatch 200B from Axon Instruments)
  • Micromanipulater for headstage (e.g., MP‐285 model, Sutter Instruments)
  • Small plastic alligator clip
  • Pipet holder with silver electrode wire coated with AgCl at tip
  • Tubing for pressure control (made of hard plastic, ∼3 mm o.d.)
  • Three‐way valve
  • Pressure gauge (e.g., DPM‐1B model, Bio Tek Instruments)
  • Additional reagents and equipment for injection of rodents ( appendix 4F) and patch‐clamp techniques (units 6.1, 6.3, 6.6, 6.7, 6.10, & 6.16)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Aksay, E., Gamkrelidze, G., Seung, H.S., Baker, R., and Tank, D.W. 2001. In vivo intracellular recording and perturbation of persistent activity in a neural integrator. Nat. Neurosci. 4:184‐193.
   Blanton, M.G., Lo Turco, J.J., and Kriegstein, A.R. 1989. Whole cell recording from neurons in slices of reptilian and mammalian cerebral cortex. J. Neurosci. Methods 30:203‐210.
   Borg‐Graham, L.J., Monier, C., and Fregnac, Y. 1998. Visual input evokes transient and strong shunting inhibition in visual cortical neurons. Nature 393:369‐373.
   Bureau, I., Shepherd, G.M., and Svoboda, K. 2004. Precise development of functional and anatomical columns in the neocortex. Neuron 42:789‐801.
   Chung, S., Li, X., and Nelson, S.B. 2002. Short‐term depression at thalamocortical synapses contributes to rapid adaptation of cortical sensory responses in vivo. Neuron 34:437‐446.
   Covey, E., Kauer, J.A., and Casseday, J.H. 1996. Whole‐cell patch‐clamp recording reveals subthreshold sound‐evoked postsynaptic currents in the inferior colliculus of awake bats. J. Neurosci. 16:3009‐3018.
   DeWeese, M.R. and Zador, A.M. 2004. Shared and private variability in the auditory cortex. J. Neurophysiol. 92:1840‐1855.
   DeWeese, M.R., Wehr, M., and Zador, A.M. 2003. Binary spiking in auditory cortex. J. Neurosci. 23:7940‐7949.
   Falke, L.C., Gillis, K.D., Pressel, D.M., and Misler, S. 1989. ‘Perforated patch recording‘ allows long‐term monitoring of metabolite‐induced electrical activity and voltage‐dependent Ca2+ currents in pancreatic islet B cells. FEBS Lett. 251:167‐172.
   Fee, M.S. 2000. Active stabilization of electrodes for intracellular recording in awake behaving animals. Neuron 27:461‐468.
   Ferster, D. and Jagadeesh, B. 1992. EPSP‐IPSP interactions in cat visual cortex studied with in vivo whole‐ cell patch recording. J. Neurosci. 12:1262‐1274.
   Gray, C.M. and McCormick, D.A. 1996. Chattering cells: Superficial pyramidal neurons contributing to the generation of synchronous oscillations in the visual cortex. Science 274:109‐113.
   Hamill, O.P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F.J. 1981. Improved patch‐clamp techniques for high‐resolution current recording from cells and cell‐free membrane patches. Pflugers Arch. 391:85‐100.
   Hirsch, J.A., Gallagher, C.A., Alonso, J.M., and Martinez, L.M. 1998. Ascending projections of simple and complex cells in layer 6 of the cat striate cortex. J. Neurosci. 18:8086‐8094.
   Larkum, M.E. and Zhu, J.J. 2002. Signaling of layer 1 and whisker‐evoked Ca2+ and Na+ action potentials in distal and terminal dendrites of rat neocortical pyramidal neurons in vitro and in vivo. J. Neurosci. 22:6991‐7005.
   Las, L., Stern, E.A., and Nelken, I. 2005. Representation of tone in fluctuating maskers in the ascending auditory system. J. Neurosci. 25:1503‐1513.
   Lee, A.K., Manns, I.D., Sakmann, B., and Brecht, M. 2006. Whole‐cell recordings in freely moving rats. Neuron 51:399‐407.
   Margrie, T.W., Brecht, M., and Sakmann, B. 2002. In vivo, low‐resistance, whole‐cell recordings from neurons in the anaesthetized and awake mammalian brain. Pflugers Arch. 444:491‐498.
   Neher, E. and Sakmann, B. 1976. Single‐channel currents recorded from membrane of denervated frog muscle fibres. Nature 260:799‐802.
   Pinault, D. 1996. A novel single‐cell staining procedure performed in vivo under electrophysiological control: morpho‐functional features of juxtacellularly labeled thalamic cells and other central neurons with biocytin or Neurobiotin. J. Neurosci. Methods 65:113‐136.
   Spruston, N. and Johnston, D. 1992. Perforated patch‐clamp analysis of the passive membrane properties of three classes of hippocampal neurons. J. Neurophysiol. 67:508‐529.
   Steriade, M., Timofeev, I., and Grenier, F. 2001. Natural waking and sleep states: A view from inside neocortical neurons. J. Neurophysiol. 85:1969‐1985.
   Stern, E.A., Jaeger, D., and Wilson, C.J. 1998. Membrane potential synchrony of simultaneously recorded striatal spiny neurons in vivo. Nature 394:475‐478.
   Svoboda, K., Denk, W., Kleinfeld, D., and Tank, D.W. 1997. In vivo dendritic calcium dynamics in neocortical pyramidal neurons. Nature 385:161‐165.
   Wehr, M. and Zador, A.M. 2003. Balanced inhibition underlies tuning and sharpens spike timing in auditory cortex. Nature 426:442‐446.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library