Equipment and Reagents

Slice and Patch specific equipment (Vendor lists are not exhaustive): * Vibration isolation table (Newport, Kinetic Systems, Technical Manufacturing Corp.) * Pipette Puller (Sutter, Narashige, Warner) * Upright fixed-stage microscope (Zeiss, Nikon, Olympus) * Hot air gun * Binocular (stereo) dissecting microscope * Tissue slicer and light source (Leica, Pella, Camden, FHC) * Recording amplifier (Axon/Molecular Devices, Heka, Dagan, NPI, ALembic Instruments) * Micromanipulator - Sutter, Burleigh, Luigs and Neumann * CCD Camera * Oscillscope (needed for troubleshooting; > 10 MHz bandwidth, 2 channels) * Slice chamber (Warner Instruments, Scientific Systems Design, Cell MicroControls) * Stage temperature controller (Warner Instruments) * Translation Stage (Sutter, Burleigh, custom designs) * Faraday cage (optional but recommended - can be purchased with vibration table or homemade) * Computer * Data acquisition system or cards (Molecular Devices Digidata for use with pClamp, National Instruments, Data Translation, Cambridge Electronic Design).

Standard Laboratory Equipment: * Balance * pH meter * Osmometer * Pipettors (10, 100, 200, 1000 μl) * Refrigerators, freezers (-20 non-defrosting and -80 °C) * Centrifuge (Eppendorf, tabletop). * Waterbath (electronic control, room temp - to 40 °C)

  • General Glassware (flasks, beakers, graduated cylinders)
  • Sintered glass gas dispersion tubes (Corning)
  • Tygon and teflon tubing (We use Teflon tubing for all solution lines, and Tygon for gas distribution)
  • Silastic tubing (for flexible joints between Teflon sections and glass, or for to the suction line to the electrode holder).
  • Polyethylene tubing (pipette suction line)
  • Teflon valves (Cole Parmer)
  • Pipette Glass (Garner Glass Co. KG33 or N51, Sutter Instruments 1.2 mm pre-polished, WPI, Dagan, AM Systems).
  • Holding chamber (small glass beaker with insert to hold slices)

Data acquisition software (pClamp, Cambridge Electronic Design, or custom, such as Acq4 (Campagnola et al., 2014)

Data analysis software (Igor Pro (Wavemetrics, Inc. Oswego, OR) with TaroTools (https://sites.google.com/site/tarotoolsregister/) or Neuromatic (http://www.neuromatic.thinkrandom.com), pClamp (Molecular Devices), AxoGraph (http://www.axograph.com), MATLAB (The Mathworks, Natick, MA)

Reagents:
Salts should be purchased from a reliable vendor, and should be at least ACS grade or better. In particular, pay attention to the level of impurities in the salts. Storage should be according to the vendor’s recommendations. We have indicated our preferred storage below (D = dessicator, R = refrigerated at 4 °C, F = freezer at -20 °C, F80 = freezer at -80 °C). Dessication may not be a problem if you live in a dry area such as the Southwestern desert, but in the American South, it can be important.
  • NaCl (D)
  • KCl (D)
    • KH2PO4 (D)
  • MgSO4 (D)
  • CaCl2(D)
  • Glucose (D)
  • Sucrose (D)
  • N-Methyl-D-Glucamine (B)
  • Ascorbic Acid (R)
  • Myo-inositol (R)
  • Sodium pyruvate (R)
    • HEPES (D)
  • EGTA (D)
  • K-gluconate (D)
  • Mg-ATP (F80)
  • Na-GTP (F80)
  • phosphocreatine (F80)
  • Alexa-fluor 488 or similar dye, hydrazide salt (F)
  • Lucifer Yellow (K+ salt). (F)

List of Definitions * ACSF - Artificial Cerebrospinal fluid * Internal solution - the solution that is used in the patch pipette, usually similar to the internal salt contents of a cell. * Electrode solution - see Internal solution * Dissection buffer - A variation of ACSF that is used during tissue dissection and cutting. May have substitutions of ions (e.g., * NMDG for sodium chloride), and different pH buffer systems. * Amplifier - An electronic unit that connects to a headstage, and provides filtering, current and voltage command adjustments, electrode resistance compensation. The amplifier may be computer controlled or may only provide analog signals to the computer. * Headstage - the portion of the Amplifier that is placed close to the preparation and usually also holds the electrode. * Patch Pipette - A small diameter glass capillary that has been pulled to have a rapid taper to a small tip (1-2 μm diameter) that is fire polished. * Recording chamber - A polycarbonate or plexiglass chamber on the microscope stage, usually with a glass bottom. The chamber can be heated, has inlet and outlet connections for solution exchange, and provisions for positioning a reference electrode.

Further Reading

  • The Axon Guide - This is a dated, but still very useful book. It can be found on the Molecular Devices website, and also on various laboratory websites.
  • Brown, K.T. Flaming, D.G. Advanced Micropipette Techniques for Cell Physiology. IBRO Handbook Series: Methods in the Neurosciences, V9. 1995.
  • Sakmann, B. and Neher, E. Single channel recording. Springer, 2nd ed. 2009.

References

Blanton, M.G., Lo Turco, J.J., Kriegstein, A. Whole cell recording from neurons in slices of reptilian and mammalian cerebral cortex J. Neurosci. Meth., 30: 203–210, 1989.

Campagnola, L., Kratz, M. B., & Manis, P. B.. ACQ4: an open-source software platform for data acquisition and analysis in neurophysiology research. Frontiers in Neuroinformatics, 8, 3. 2014

Clements JD, Bekkers JM. Detection of spontaneous synaptic events with an optimally scaled template. Biophys J. 73:220-229. 1997

Hamill O.P., Marty A., Neher E., Sakmann B., Sigworth F.J. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 391: 85-100, 1981

Lorteije J.A., Rusu S.I., Kushmerick C., Borst J.G. Reliability and precision of the mouse calyx of Held synapse. J Neurosci. 2009 Nov 4;29(44):13770-84

Magistretti J., Mantegazza M., de Curtis M., Wanke E. Modalities of distortion of physiological voltage signals by patch-clamp amplifiers: a modeling study. Biophys J. 74:831-42, 1988.

Neher E., Sakmann B., Steinbach JH. The extracellular patch clamp: a method for resolving currents through individual open channels in biological membranes. Pflugers Arch. 375: 219-28, 1978.

Richardson MJ, Silberberg G. Measurement and analysis of postsynaptic potentials using a novel voltage-deconvolution method. J Neurophysiol. 99:1020-31, 2008.

Rothman J.S., Manis P.B. The roles potassium currents play in regulating the electrical activity of ventral cochlear nucleus neurons. J Neurophysiol.89: 3097-3113, 2003

Shao X.M., Feldman J.L. Micro-agar salt bridge in patch-clamp electrode holder stabilizes electrode potentials. J Neurosci Methods. 159:108-15, 2007.

Snyder K.V., Kriegstein A.M., Sachs F. A convenient electrode holder for glass pipettes to stabilize electrode potentials. Pflugers Arch. 438: 405-411, 1999.

Smith, T.G., Lecar, H., Redman, S.J., Gage, P.W. Voltage and Patch Clamping with Microelectrodes. American Physiological Society, Bethesda, MD, 1985.

Spruston N., Jaffe D.B., S.H., Johnston D. Voltage- and space-clamp errors associated with the measurement of electrotonically remote synaptic events. J Neurophysiol. 70: 781-802, 1993.

Tanaka, Y., Tanaka, Y., Furuta, T., Yanagawa, Y., Kaneko, T. The effects of cutting solutions on the viability of GABAergic interneurons in cerebral cortical slices of adult mice. J. Neurosci. Methods, 171: 118-125, 2008.

Waldron, T. A practical Interference Free Audio System (parts 1 and 2). Web resource: http://www.nutwooduk.co.uk/archive/Old_Archive/020918.htm

Williams S.R., Mitchell S.J. Direct measurement of somatic voltage clamp errors in central neurons. Nat Neurosci. 11: 790-798, 2008

Yamamoto C., McIlwain H. Potentials evoked in vitro in preparations from the mammalian brain. Nature. 210: 1055-1056, 1966.

Figure Legends

Figure 1. Schematic of glass electrode patched onto cell with equivalent circuit diagram. Vp: Voltage inside pipette; this is the voltage controlled or measured by the amplifier, less the electrochemical junction potential. Cp: Pipette capacitance; typically a few picofarads. Rs: Series (or access) resistance; this is the resistance separating the pipette from the cell body and is due mainly to the narrow pipette tip and organelles that may be blocking it. Rseal: Seal resistance; the resistance of the region of contact between the pipette and the membrane. To make quality recordings, this must be > 1 GΩ. Vm: Membrane voltage; the voltage of the interior of the neuron relative to the bath. Cm: Cell membrane capacitance. Rm: Cell membrane resistance; also called input resistance. Vb: Bath voltage, as measured by the ground electrode.

Figure 2. A minimal patch electrophysiology rig. Left to right: Oxygenated ACSF is siphoned through a fluid heater and into the recording chamber where it continuously washes over the brain slice. Fluid is then aspirated out of the recording chamber and into a waste flask. A patch clamp amplifier headstage is mounted to a micromanipulator and holds the patch pipette, which currently impales the brain slice (detailed in Figure 3). The headstage ouput is amplified, digitized, and finally recorded on a computer.

Figure 3. Patch recording equipment. A) 63x ceramic, water-immersion objective. B) Silver chloride wire connected to headstage ground output. C) Heated aluminum holder for recording chamber. D) Plastic recording chamber with glass coverslip on the bottom. E) Brain slice bathed in warm, oxygenated ACSF. F) Glass patch pipette filled with electrode solution. G) Electrode holder. H) AgCl electrode wire. This wire fits inside the patch pipette and makes electrical contact with the electrode solution as well as the I) gold pin which conducts electrode potential into the amplifier headstage. J) Pressure control tube. This allows the experimenter to increase or decrease the pressure inside the patch pipette. K) amplifier headstage.

Figure 4. Ideal patch pipette shape. The pipette is pulled in multiple stages. The first stage is a long, narrow pull which thins the tip to help it fit under the objective. The following stages produce a rapid taper (about 15°) to reduce resistance and end with a 1.5μm tip.

Figure 5. Making patch pipette fillers from disposable pipette tips. A) Heating a 100 μm disposable pipette tip over a small flame. The tip should be rotated to produce even heating and care should be taken to avoid burning the plastic. B) As soon as the tip has melted through, remove it from the heat and pull into a thin tube (this takes some practice). Cut the tube with a sharp blade to avoid crushing it. C) Filler made from tip of pipette inserted into 1 ml syringe. D) Filler made from base of pipette attached to 1 ml syringe and a low-volume, 0.2 μm-pore syringe filter.

Figure 6. Neuron examples in a cortical brain slice under gradient illumination. Black arrows indicate unhealthy or dead cells, white arrows indicate healthy cells, and a grey arrow indicates a borderline cell. (This figure is a composite of multiple images from different regions of a slice.)

Figure 7. Voltage-clamp recording from cell-attached pipette before (dashed line) and after (solid line) adjusting the pipette capacitance compensation. The seal resistance has increased to 1.6 GΩ. A) Photo of patch pipette in bath. B) Voltage clamp command and current recording from patch pipette in bath. The voltage clamp requires 2.4 nA of current to effect a 10 mV pulse, indicating a pipette resistance of 4.2 MΩ.

Figure 8. Patch procedure. A) Approach the cell with positive pressure in the pipette. The surface of the cell should form a visible dimple. B) Release pressure on pipette, then apply gentle suction to seal the membrane against the pipette. This is the cell-attached configuration. C) Apply sharp suction to the pipette to rupture the membrane, granting electrical access to the cell interior. This is the whole-cell configuration. D) From whole-cell, pull the pipette very gently away from the cell until E) the membrane separates and re-closes. This is the outside-out configuration.

Figure 9. Left: A ‘dimpled’ cell immediately before being patched. Right: voltage-clamp recording shortly after releasing pipette pressure. The resistance at the pipette tip has increased to 66MΩ.

Figure 10. Voltage-clamp recording from cell-attached pipette before (dashed line) and after (solid line) adjusting the pipette capacitance compensation. The seal resistance has increased to 1.6 GΩ.

Figure 11. A) Whole-cell patched neuron filled with fluorescent dye. B) Voltage-clamp recording from the same neuron. The steady-state current is about 80 pA, indicating an input resistance of 125 MΩ. The peak of the charging transient is 900 pA past the steady-state, indicating an access resistance of 11 MΩ.

Figure 12. The effects of pipette capacitance and series resistance compensation (0%, 70%, and 95%) on a simulated neuron (soma, 20 μm in diameter, dendrites 200 μm long and 1.5 μm in diameter). A) Schematic of neuron; a single-compartment soma with four dendrites. The somatic electrode is used to voltage clamp the cell, while the dendritic electrode records voltage (in current clamp) at a mid-dendritic position 100 μm from the soma to illustrate the inadequate space clamp. B) Clamp currents (somatic electrode) in whole-cell recording mode, for 3 different levels of compensation for voltage steps from -60 to -70 mV. Compensation increases the amplitude of the capacitive charging transient, as well as the steady-state current. Inset: initial transient on an expanded time scale. C) Comparison of voltage at the soma for different amounts of compensation, compared to the command voltage (short dashed lines). Longer dashed lines indicate voltage recorded at the mid-dendrite position. Note that until compensation approaches 95%, the somatic voltage differs from the command voltage. The dendritic voltage control is not improved by increasing compensation, and at best the voltage step is only about half of the command step. D) Currents in response to depolarization from -60 to -20 mV. With no compensation, the sodium conductance leads to an oscillatory current, corresponding to unclamped partial action potentials. Increasing compensation brings the currents under better control (inset), and also increases the outward potassium current. E) Command voltage and actual voltage at the soma and dendrites. Note the action-potential like waveforms with no compensation. Even with 95% compensation the voltage in the dendrite (long dashed lines) varies with time and does not reach the command level. B-E: Insets show the first 1 msec of the current traces (B,D) or voltage (C, E) for each compensation level. Model: “Type I” neuron (Rothman and Manis, 2003), with sodium, delayed rectifier, hyperpolarization-activated cation conductance, and leak conductance in soma. Dendrites have a delayed rectifier, Ih current and leak conductance, with Ri = 150 Ω-cm.