Counting contacts between neurons in 3D in confocal laser scanning images

A half century of experimental neuroanatomical tracing

“…This characteristic allows extremely precise mapping of fiber tracts, the analysis of the a half century of experimental neuroanatomical tracing compartmentation of large fascicles and association bundles and the study of terminal projection patterns. Quantitative estimation of the numbers and densities of labeled axon terminals can be attempted (Wouterlood et al, 2007(Wouterlood et al, , 2008. In the electron microscope the label generated by BDAprocessing can be seen inside the nuclei of neurons and throughout the cytoplasmic compartments including that of the main dendrites and their branches down to the most distal branchlets (at least when dealing with BDA-filled neurons in the vicinity of the injection sites) and in fibers with their varicosities and axon terminals.…”

“…Most exemplary in this respect are brain areas wherein the neurons possess long apical dendrites, e.g., in cerebral and cerebellar cortex and in hippocampus where fibers of afferent projections may terminate in a stratified way, far away from the cell bodies of the neurons that receive synaptic contacts. In field CA1 of the hippocampus for example a 500 μm ‗gap' exists between stratum lacunosum-moleculare where entorhinal cortical afferents end and stratum pyramidale that host the cell bodies of the CA1 pyramidal neurons (Kajiwara et al, 2008). One way to visualize entire, individual neurons, apart from procedures described in previous sections, is via intracellular injection of a dye in living neurons in situ or in vitro slice preparations, or in slices of lightly fixed brain tissue (the latter pioneered in 1986 by Rho and Sidman and refined in 1989 by Buhl and Lübke).…”

“…The triple-immunofluorescence sections were investigated at high magnification (63x immersion objective) in a three-channel confocal laser scanning microscope equipped wit the appropriate lasers and filtering (details in Kajiwara et al, 2008). A striking example of a contact between a a half century of experimental neuroanatomical tracing BDA labeled striatonigral fiber and a LY filled dendrite of a tyrosine hydroxylase positive, FGlabeled nigrostriatal neuron, is shown in Figure 10.…”

“…The first is when we suspect that the terminal field of a fiber projection to an area of interest is located at a dendrite's length away (the ‗previously discussed ‗gap') from the perikarya of the neurons that project out of that area of interest. The second situation occurs when we are interested in the distribution of contacts of afferent fibers on the dendritic tree of neurons of a particular morphological type, and the third situation is when we are interested in the convergence of fibers that originate from two sources onto the dendrites of individual neurons in a particular area (e.g., Kajiwara et al, 2008).…”

Confocal Laser Scanning: of Instrument, Computer Processing, and Men

“…It can do this in two related 3D pixel matrices (a two-channel Z-image series), and next the program can calculate for each aggregate in each channel the number of appositions it has with aggregates in the other channel's dataset and reject those appositions that do not fulfill the minimum of 100 voxel overlap criterion. For this purpose we use dedicated software written in SCIL_Image that can be called using scripts that can be run unattended (Wouterlood et al, 2007(Wouterlood et al, , 2008a. The program provides two readouts: the number of recognized contacts of 3D objects present in the first channel versus those in the second (or third) channel, and vice versa.…”