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 describe d, which results in labeling of viable cells from which electrophysiol ogical or optical recordings can be obtained. The dye-coated surface o f a glass microelectrode tip is used to apply anatomical tracers or ca lcium sensitive probes with spatial precision. A total of three types of dyes have been applied in this way to find and record from olfactor y interneurons in the terrestrial mollusc Limax maximus. Crystals of 1 '-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) fo rmed on the tips of glass microelectrodes were placed in the procerebr al lobe, the major olfactory processing center of Limax. Somata in buc cal and pedal ganglia with processes extending several 100 mu m 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. C ross correlograms of the oscillatory field potential in the procerebra l lobe and spontaneous action potentials in P-PC or B-PC show that P-P C activity is weakly coupled to the oscillation in the procerebral lob e, while B-PC activity is clearly coupled to the oscillation. Stimulat ion of the procerebral lobe with nitric oxide activated P-PC cells but suppressed activity in B-PC cells. Calcium green-10gdextran coated el ectrodes were used to place calcium green in the cell body layer of th e procerebral lobe. Bursting and nonbursting procerebral neurons incor porated and transported the calcium green-dextran. Optical recordings of changes in fluorescence signals from several bursting cells recorde d simultaneously were used to test alternative mechanisms of bursting cell coupling. Application of biotin-3Kdextran to the midline of the c erebral ganglion revealed a group of cells in each procerebral lobe wi th processes crossing the midline of the cerebral ganglion. These cell s may couple right and left procerebral lobe activity during odor proc essing. (C) 1997 Elsevier Science B.V.