Semi-loose seal Neurobiotin electroporation for combined structural and functional analysis of neurons.
Intracellular sharp-electrode, whole-cell patch clamp and juxtacellular labeling methods have previously been developed for combined analysis of neuronal structure and function. We describe a novel electroporation technique for labeling neurons with Neurobiotin, using patch electrodes in a semi-loose seal configuration (R = 100-300 MOmega) with very small amplitude pulses (50 mV). The addition of 2% Neurobiotin to the intracellular solution in the patch electrode reduces the dielectric membrane breakdown voltage threshold by about threefold. The resulting pore formation allows for (1) the stable recording of spontaneous and light-evoked postsynaptic potentials without significant cytoplasmic washout and (2) the passage of dye without spillover. The efficiency and reliability of the method makes it particularly suitable for the serial recording and labeling of multiple neurons in a small area of tissue.
Kanjhan
Refik
R
Queensland Brain Institute, The University of Queensland, Brisbane, 4072, Queensland, Australia. r.kanjhan@uq.edu.au
Vaney
David I
DI
D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives*; D004396:Coloring Agents; D017097:Electric Impedance; D018274:Electroporation; D008839:Microelectrodes; D010775:Photic Stimulation; D011817:Rabbits; D015203:Reproducibility of Results; D055351:Retinal Neurons / Q000502:physiology*; D013194:Staining and Labeling / Q000295:instrumentation / Q000379:methods*; D055366:Synaptic Potentials; D013997:Time Factors; D014785:Vision, Ocular
D004396:Coloring Agents; C070614:neurobiotin; D001710:Biotin