A 'puff and advance' technique for visually controlled staining of turtle retinal ganglion cells

We describe a 'puff and advance' technique for visually controlled staining of retinal ganglion cells (GCs) in the unfixed, living retina for light and electron microscopy. Glass microelectrodes are filled with rhodamine-isothiocyanate labeled horseradish peroxidase (Rh-HRP), or Lucifer yellow (LY), or a mixture of both, or with 5,6-carboxytetramethylrhodamine (5,6-Rh) and advanced tangentially through the GC layer with microscopic observation using epifluorescence. Brief "puffs" of LY or 5,6-Rh are constantly ejected from the advancing electrode tip by a train of negative current pulses. GC penetration is signaled by virtually instantaneous staining of its soma (and eventually its axon and dendrites if the electrode is not advanced further). An impaled GC can be electron densely stained with the Rh-HRP complex by switching to positive current pulses. The extent of dye filling is monitored through the microscope using a filter combination appropriate for the dye. After fixation, standard histochemical procedures reveal HRP stained GCs in wholemount views for light microscopical examination. Furthermore, the preservation of the labeled cells and the neuropil is of a quality to allow electron microscopic analysis for synaptic input. This technique can be used in combination with LY backfiling of GCs from the optic nerve and with retinas in which GCs have been prelabeled with rhodamine beads retrogradely transported from the optic tectum as well.