Vital staining from dye-coated microprobes identifies new olfactory interneurons for optical and electrical recording

A versatile technique for dye application in living tissue is described, which results in labeling of viable cells from which electrophysiological or optical recordings can be obtained. The dye-coated surface of a glass microelectrode tip is used to apply anatomical tracers or calcium sensitive probes with spatial precision. A total of three types of dyes have been applied in this way to find and record from olfactory interneurons in the terrestrial mollusc Limax maximus. Crystals of 1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) formed on the tips of glass microelectrodes were placed in the procerebral lobe, the major olfactory processing center of Limax. Somata in buccal and pedal ganglia with processes extending several 100 microm to the procerebral lobe were stained within 4-6 h. Intracellular recordings from DiI stained buccal (B(PC)) and pedal (P(PC)) cells were obtained. Cross correlograms of the oscillatory field potential in the procerebral lobe and spontaneous action potentials in P(PC) or B(PC) show that P(PC) activity is weakly coupled to the oscillation in the procerebral lobe, while B(PC) activity is clearly coupled to the oscillation. Stimulation of the procerebral lobe with nitric oxide activated P(PC) cells but suppressed activity in B(PC) cells. Calcium green-10Kdextran coated electrodes were used to place calcium green in the cell body layer of the procerebral lobe. Bursting and nonbursting procerebral neurons incorporated and transported the calcium green-dextran. Optical recordings of changes in fluorescence signals from several bursting cells recorded simultaneously were used to test alternative mechanisms of bursting cell coupling. Application of biotin 3Kdextran to the midline of the cerebral ganglion revealed a group of cells in each procerebral lobe with processes crossing the midline of the cerebral ganglion. These cells may couple right and left procerebral lobe activity during odor processing.