SIMULTANEOUS CHARACTERIZATION OF EFFERENT AND AFFERENT CONNECTIVITY, NEUROACTIVE SUBSTANCES, AND MORPHOLOGY OF NEURONS

We present a method for establishing in a single experiment four chara cteristics of individual neurons: the efferent and afferent connectivi ty, the morphology, and the content of a particular neuroactive substa nce. The connectivity of the neurons is determined by retrograde fluor escent tracing with Fast Blue and anterograde tracing with the lectin Phaseolus vulgaris leucoagglutinin (PHA-L). After fixation, the brain is cut into 300-mu-m thick slices. Neurons containing retrogradely tra nsported Fast Blue are intracellularly injected with the fluorescent d ye Lucifer Yellow to fill their dendritic trees. The slices are then r esectioned at 20-40-mu-m. One section through the soma of a Lucifer Ye llow-filled neuron is selected for the detection of a neuroactive subs tance contained by this cell [immunofluorescence, secondary antiserum conjugated to tetramethylrhodamine (TRITC)]. Using appropriate filteri ng, it can be determined in the fluorescence microscope whether a Luci fer Yellow-containing cell body has also been labeled with TRITC, i.e. , whether it is immunoreactive for this neuroactive substance. The adj acent sections are subjected to dual peroxidase immunocytochemistry wi th different chromogens to visualize the PHA-L-labeled afferent fibers (nickel-enhanced diaminobenzidine, blue-black reaction product) and t o stabilize the Lucifer Yellow (diaminobenzidine, brown reaction produ ct) in the dendrites of the intracellular injected cells. The other se ctions are used for electron microscopic visualization of the transpor ted PHA-L. The relationships between the PHA-L-labeled afferent fibers (blue color) and the dendrites of the intracellularly Lucifer Yellow- injected, retrogradely Fast Blue-labeled cells (brown color) are studi ed by light microscopy. The electron microscope supplies ultrastructur al data on the PHA-L-labeled axon terminals.