lucifer-yellow and tetramethylrhodamine

This report describes a combined retrograde tracing, intracellular injection and anterograde fluorescence labeling method using the application of confocal laser scanning microscopy. By simultaneously viewing the morphology of identified projection neurons and the distribution of anterogradely labeled fibers and terminals, this approach allows accurate characterization of the anatomical relationships between these two elements. To demonstrate this approach, the retrograde tracer Fast Blue was injected into the bed nucleus of stria terminalis (BNST) and the anterograde tracer tetramethylrhodamine-conjugated dextran was injected into the insular cortex in adult rats. After one week survival time, the brains were fixed and sectioned on a vibratome. Individual BNST projecting neurons identified in the amygdaloid complex on 120 microns thick sections were intracellularly injected with Lucifer Yellow under visual control and analyzed with confocal laser scanning microscopy. The 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 optical sections show, in very great detail, the morphology of projection neurons in three-dimensions. Compared to other methodological combinations, the present method provides a more simple and efficient means to trace three successive components of a putative neuron chain.

Topics: Amidines; Animals; Axonal Transport; Cerebral Cortex; Fluorescent Dyes; Image Processing, Computer-Assisted; Isoquinolines; Lasers; Male; Microscopy; Neurons; Rats; Rats, Sprague-Dawley; Rhodamines; Staining and Labeling

We have examined five conjugated 10,000 mol. wt. dextrans as potential anterograde tract tracers: Lucifer Yellow, Texas Red, fluorescein, Cascade Blue and tetramethylrhodamine. Pressure injections were made into the brain, dorsal root ganglia or footpads of adult rats. The retrograde tracer Fluoro-Gold was injected alone or mixed with the dextrans before injection. Three-14 days after injection, animals were perfused and sections cut with a freezing microtome. Texas Red-, fluorescein- and tetramethylrhodamine-conjugated dextrans produced intense labeling of neuronal cell bodies, axons and dendritic processes at the injection site and were transported by neurons predominantly in an anterograde direction to yield terminal and preterminal labeling. Relative to the fluorescein and tetramethylrhodamine conjugates, the quality and intensity of the anterograde labeling produced by Texas Red was variable. Results with Lucifer Yellow and Cascade Blue conjugates were negative. Optimal results were produced by slow-pressure injections via glass micropipettes. In comparison with Fluoro-Gold, retrograde transport by the dextran conjugates was present, but limited in its extent. Injections of the tetramethylrhodamine conjugate into dorsal root ganglia produced anterograde labeling of afferent fibers in visceral organs and injections into the nucleus ambiguous labeled motor fibers in the esophagus. Double/triple labeling was observed in the brain and spinal cord following multiple injections of fluorescein, tetramethylrhodamine and Fluoro-Gold. Also, Fluoro-Gold could be mixed with one of the dextrans in order to produce specific retrograde and anterograde labeling from the same injection site. The conjugates were compatible with fluorescent immunocytochemical procedures, but proved unsuitable for peripheral injections.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Brain; Dextrans; Female; Fluorescein; Fluoresceins; Fluorescent Dyes; Hypothalamus; Immunohistochemistry; Isoquinolines; Male; Phytohemagglutinins; Rats; Rats, Inbred Strains; Rhodamines; Staining and Labeling; Stereotaxic Techniques; Stilbamidines; Xanthenes