Juxtacellular labelling of single neurons

After the extracellular recordings, wich were performed as described in the "In vivo electrophysiology" part of the methods, juxtacellular labelling was performed as follows: 

1) apply positive current pulses (1-10 nA, 200 ms long square on/off pulses, ELC-03M, NPI Electronics) via the recording electrode with careful and continuous monitoring of the single-unit activity

2) increase pulse currents slowly until successful electroporation (maximum: 10nA) while correcting electrode resistance and capacitance artifacts with the bridge balance and capacitance compensation circuits of the amplifier

3) immediately identify successful electroporation by an increase in membrane noise and modulation of firing rate

4) once electroporation is achieved, quickly reduce the current by approximately half to avoid cellular damage, and monitor the modulated activity for at least 20 seconds

5) after successful modulation, reduce the current amplitude slowly and monitor the cell activity for another minute

If electroporation does not occur, the pipette is lowered by <5μm and the process repeated.

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