This article details a method for recording postsynaptic responses in neurons by establishing whole-cell configurations in both presynaptic and postsynaptic neurons. The technique involves using patch pipettes to achieve loose-seal configurations and monitor action potentials.
Secure a brain slice with the target region in a recording chamber.
Position a recording pipette containing an internal solution over a postsynaptic neuron and achieve a whole-cell configuration.
Introduce a searching patch pipette near a presynaptic neuron.
This pipette is filled with a sodium-based solution to prevent neuron depolarization while searching for the presynaptic cell.
Use gentle suction to establish a loose-seal configuration with the searching pipette.
Hold the membrane potential and apply large current pulses to trigger an action potential in the presynaptic neuron.
Monitor the postsynaptic response to this stimulation.
If no response is detected, patch another presynaptic neuron and repeat stimulation.
Continue this process until a response is observed in the postsynaptic neuron.
Replace the searching pipette with a recording pipette and establish a whole-cell configuration in the presynaptic neuron.
Apply current pulses to elicit action potentials in the presynaptic neuron and record the postsynaptic response.
After preparing slices of the target region, allow them to incubate. During this time, pull the patch pipettes, which should have a long and slender shank. After securing a brain slice with a platinum harp in an experimental chamber, fill a recording pipette with internal solution.
Place the pipette in the preamplifier and patch a putative postsynaptic neuron in whole-cell mode. Using a searching patch pipette of 8 to 10 megaohm resistance filled with an internal solution in which potassium salts is replaced by sodium salts, apply gentle suction to the searching pipette to patch a potential presynaptic neuron in a loose-cell attached configuration.
Hold the membrane potential under the loose seal to between negative 30 and negative 60 millivolts in current clamp mode. Then, apply large current pulses of 0.2 to 2 nanoamps to elicit an action potential in the potential presynaptic cell.
Observe this action potential as a small spike lit on the voltage response. Set the stimulation frequency to 0.1 Hertz to prevent rundown of the postsynaptic response. If the postsynaptic neuron does not respond to stimulation of the tested potentially presynaptic neuron, patch a new potential presynaptic neuron in loose-seal mode.
Test up to 30 potentially presynaptic neurons in this way, continuing to use the same searching patch electrode as long as a seal with a resistance of greater than 30 megaohm can be established.
If stimulation in the loose-seal mode results in an EPSP or IPSP with a latency less than five milliseconds in the postsynaptic neuron, remove the searching pipette from the presynaptic neuron.
Using a recording patch electrode filled with biocytin containing regular internal solution with a resistance of four to eight megaohm, patch the presynaptic neuron and record in whole-cell current clamp mode. Elicit action potentials by current injection in the presynaptic neurons and record the postsynaptic response.
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