Microiontophoresis electrode location by neurohistological marking: Comparison of four native dyes applied from current balancing electrode channels

Introduction: The re-establishment of electrode position after microiontophoresis with multi-barrelled electrodes has always been a complicated and mostly inaccurate procedure. The present report describes a new method for 'on line' neurohistological marking of each recording position during experimental sessions in which in vivo microiontophoresis was combined with extracellular single unit recording. Methods: We tested the effectiveness of four native dyes: pontamine sky blue (PSB), methylene blue (MB), cresyl violet (CV) and toluidine blue (TB). They were ejected from the continuous balancing channels of multi-barrelled microelectrodes during iontophoresis in place of the usual sodium chloride solution. Results: The present results are based on observations obtained from 66 neuronal recordings and 42 labelled sites in the rat neocortex. Results indicate that none of the tested native dyes altered the spontaneous firing rate of the neurons, nor did they have any influence on the applied bioactive compounds. Recording positions were detected as small, labelled spots in brain slices (2-12 μm in diameter) at the end of the recording tracks. Labelling was successful with MB and PSB, but not with CV or TB. In addition, MB provided good labelling with a low net ejection current. Discussion: The present results suggest that using native dyes, especially MB, instead of sodium chloride in current balancing channels during in vivo microiontophoresis provides fast and accurate confirmation of electrode placement. This method saves the experimenter the time consuming further application of secondary labelling procedures after such experiments. Moreover, the MB-labelling we describe here is focal, enabling a more accurate verification of the site of recording and/or application of various neuroactive compounds. In addition, MB labelled accurately and reliably and with very low ejection currents, presumably due to its smaller molecular weight and higher relative mobility.