Dendritic patch-clamp recording

Abstract

The patch-clamp technique allows investigation of the electrical excitability of neurons and the functional properties and densities of ion channels. Most patch-clamp recordings from neurons have been made from the soma, the largest structure of individual neurons, while their dendrites, which form the majority of the surface area and receive most of the synaptic input, have been relatively neglected. This protocol describes techniques for recording from the dendrites of neurons in brain slices under direct visual control. Although the basic technique is similar to that used for somatic patching, we describe refinements and optimizations of slice quality, microscope optics, setup stability and electrode approach that are required for maximizing the success rate for dendritic recordings. Using this approach, all configurations of the patch-clamp technique (cell-attached, inside-out, whole-cell, outside-out and perforated patch) can be achieved, even for relatively distal dendrites, and simultaneous multiple-electrode dendritic recordings are also possible. The protocol--from the beginning of slice preparation to the end of the first successful recording--can be completed in 3 h.

Figure 1. Patch-clamp setup.

(a) Composite photograph of a typical patch-clamp setup (in a Faraday cage) used for dendritic recording. (b) Schematic sketch of the image in a. Red, imaging equipment; blue, electrode manipulators and pressure controlling equipment; black, perfusion system; green, vibration isolation table. (1) an upright microscope equipped with a 40x objective and IR-DIC optics, mounted on an XY stage; (2) magnifier; (3) video camera; (4) black and white video monitor; (5) three micromanipulators (oriented so that each pipette can be changed independently); (6) micromanipulator remote control panels, mounted on a bench which is well separated from the vibration isolation table (boxes containing micromanipulator controller electronics are below the vibration isolation table); (7) manometers; (8) switchable pressure valves; (9) reservoir of carbogen-bubbled ACSF; (10) oxygen-impermeable Teflon tubing providing inflow to the recording chamber (heating jacket prior to chamberinflow not visible); (11) dripper, interrupting solution inflow; (12) outflow from chamber, connected to suction via a collection reservoir; (13) temperature monitor (connected to a thermocouple element placed in the recording chamber, not illustrated); (14) vibration isolation table.

Figure 2. Imaging dendrites using videomicroscopy.

(a) Composite DIC image of a Purkinje cell with one of the main dendritic branches shown (white arrowheads). Same cell as in Supplementary Video 1, showing the process of dendritic patching. (b) Healthy dendritic branch of a Purkinje cell (white arrowheads). Also shown is the soma of a damaged interneuron (blue asterisk). Note that the healthy dendrite appears convex compared to the damaged interneuron, which has a concave, hole-like appearance. (c) The same image as in b but inverted digitally, reproducing the effect of rotating the 1/4 wave plate or biasing the upper DIC prism in the opposite direction. Note the inverted appearance of healthy and damaged structure. (d) Image of blood vessels, which sometimes can be mistaken for dendrites. Key identifying features are red blood cells in the lumen (red asterisk; these will be absent if cardiac perfusion has been used to cool the brain before slicing) and thicker edges. (e) Dead Purkinje cell dendrites. Compare the crisper, more contrasted appearance with a and b. White arrowheads show thin, presumably spiny dendrites, which are not usually visible when alive. (f) Example of an IR-DIC image of the soma and apical dendrite of a layer V pyramidal neuron in somatosensory cortex which has a high probability for sealing, with a low contrast, smooth appearance (white arrowheads). (g) An unhealthy L5 pyramidal cell in the same area with a low probability for high-resistance sealing. Note high contrast of dendrite and “collapsed”, dimpled appearance of soma.

Figure 3

Steps in dendritic patching. A schematic illustration showing the basic steps involved in making a dendritic patch-clamp recording. Top: a section of the slice, containing a healthy, connected dendrite and soma, together with a patch pipette. Middle: oscilloscope displaying the command current for a negative voltage test pulse (offset so as to be centered on the screen). Bottom: the pressure applied to the patch pipette (dotted line: 0 mbar). (a) A patch pipette, with ~30 mbar of pressure blowing external solution from its tip, is lowered into position above a clearly visible dendrite. (b) With increased pressure, such that slice debris is cleared from in front of the pipette tip, the pipette is moved in the diagonal ‘in and out’ direction (red arrow) through the slice and onto the dendrite. (c) When good contact is made with the surface of the dendrite, a ‘dimple’ starts to form in the dendritic membrane. (d) Immediately, the pressure behind the pipette is released, gentle suction applied and the holding potential lowered to ~–75 mV. The steady-state current required to clamp the patch at this potential (observable on the oscilloscope and on the patch-clamp amplifier display) indicates a giga-ohm seal has formed. If your patch-clamp amplifier allows it, compensate now for the charging of the pipette capacitance (remove the capacitive transients). (e) Brief pulses of suction are applied until the membrane under the pipette tip is ruptured, as seen by a change in the test-pulse current (now reflecting both the charging of the cell’s membrane and the local input resistance).