COMBINATION OF INTRACELLULAR STAINING OF RETROGRADELY LABELED NEURONSAND ANTEROGRADE FLUORESCENT TRACING - USE OF THE CONFOCAL LASER SCANNING MICROSCOPE
Cj. Shi et Md. Cassell, COMBINATION OF INTRACELLULAR STAINING OF RETROGRADELY LABELED NEURONSAND ANTEROGRADE FLUORESCENT TRACING - USE OF THE CONFOCAL LASER SCANNING MICROSCOPE, Journal of neuroscience methods, 47(1-2), 1993, pp. 23-31
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.