COMBINATION OF INTRACELLULAR STAINING OF RETROGRADELY LABELED NEURONSAND ANTEROGRADE FLUORESCENT TRACING - USE OF THE CONFOCAL LASER SCANNING MICROSCOPE

This report describes a combined retrograde tracing, intracellular inj ection and anterograde fluorescence labeling method using the applicat ion of confocal laser scanning microscopy. By simultaneously viewing t he morphology of identified projection neurons and the distribution of anterogradely labeled fibers and terminals, this approach allows accu rate characterization of the anatomical relationships between these tw o elements. To demonstrate this approach, the retrograde tracer Fast B lue was injected into the bed nucleus of stria terminalis (BNST) and t he anterograde tracer tetramethylrhodamine-conjugated dextran was inje cted into the insular cortex in adult rats. After one week survival ti me, the brains were fixed and sectioned on a vibratome. Individual BNS T projecting neurons identified in the amygdaloid complex on 120 mum t hick sections were intracellularly injected with Lucifer Yellow under visual control and analyzed with confocal laser scanning microscopy. T he results demonstrate that images from very thin optical sections can clearly show potential synaptic contacts between anterograde labeling and intracellularly labeled projecting neurons. Stacked images from o ptical sections show, in very great detail, the morphology of projecti on neurons in three-dimensions. Compared to other methodological combi nations, the present method provides a more simple and efficient means to trace three successive components of a putative neuron chain.