Improvements for the anatomical characterization of insect neurons in whole mount: the use of cyanine-derived fluorophores and laser scanning confocal microscopy

The optical sectioning capability of the laser scanning confocal microscope was utilized to image dye-filled neurons within whole-mounted insect ganglia. Specific pterothoracic interneurons, in the moth Manduca sexta, were retrogradely filled with Neurobiotin and subsequently visualized with a monoclonal anti-biotin conjugated with one of the following fluorophores: fluorescein, and the newly developed cyanines, Cy3.18 (Cy3) and Cy5.18 (Cy5). Overall, the Cy5 fluorophore was best suited for imaging insect neurons within ganglia. This new methodology allowed us to identify and characterize morphologically a collection of descending multisegmental interneurons with large or small diameter somata. A variety of larger molecular weight (10000 daltons) tracers was also used to examine the possibility of nonselective filling of neurons with Neurobiotin, possibly through gap junctions. We also investigated the usefulness of Cy3 and Cy5 as fluorophores for transmitter immunostaining of neurons in whole mount. Neurons immunoreactive for serotonin and the neuropeptides, FMR Famide and SCP b, were imaged in the brain and the pterothoracic ganglion. The central projections of some of these immunoreactive neurons were imaged in their entirety.