Tracing tools to resolve neural circuits

Abstract

In this paper we summarize neuroanatomical tracing methods, in particular combinations of methods designed to achieve the combined goals of tracing connectivity and extracting extra information from the projection or the target neurons. These combinations include techniques that identify projection fibres together with the neurons from which they originate, methods which establish the morphological or chemical identity of the target neurons and techniques to verify the presence of contacts between the terminal boutons on fibres and prospective target neurons. In the second part of this paper we describe a method recently developed in our laboratory, which consists of fluorescence tracing (introducing marker #1) combined with immunofluorescence with different fluorochromes (markers #2 and #3). This method enables us to observe the distribution of terminals of a particular set of projection fibres in contact with neurons belonging to two chemically different populations of interneuron. Multifluorescence confocal laser scanning is used for image acquisition of fibres and processes of presumed target neurons, and follow-up is by three-dimensional computer reconstruction. These reconstructions are used to determine that the contacts between the differentially labelled structures are real, i.e. with no optically empty space in between regardless of the angle of inspection. The specific experiments reported in this paper comprise the tracing with biotinylated dextran amine of fibres in the rat brain running from the presubiculum to layer III of the medial division of the entorhinal cortex. We studied the possibility of contacts in the latter area between the terminal boutons of these projecting fibres in association with presumed target neurons, i.e. parvalbumin and calretinin expressing interneurons.

Neuroanatomical Projections of the Species-Specific Tyrosine Hydroxylase-Immunoreactive Cells of the Male Prairie Vole Bed Nucleus of the Stria Terminalis and Medial Amygdala

“…It remains possible that future studies may demonstrate that even in prairie voles the ventral BSTpr (which contains few TH-ir cells) does project to these sites. Another difference among these anterograde tracing studies is that previous investigations in rats and hamsters used phytohemagglutinin-L rather than BDA, but this is probably only of minor consequence because these tracers have repeatedly been compared and produce very similar patterns of labeled fibers [Reiner et al, 2000;Wouterlood et al, 2002;Raju and Smith, 2006]. It may also be relevant for evaluating the possible species differences in BST projections that Wood and Swann [2005] proposed, namely, that the hamster BST has only three divisions, as opposed to the four described in rats [Aldheid et al, 1995], while the cytoarchitecture of the BST in prairie voles remains unknown.…”

In Vivo Mapping of Fiber Pathways in the Rhesus Monkey Brain

“…A popular strategy for studying fiber pathways in non-human primates is to apply a neuronal tracer such as horseradish peroxidase or lucifer yellow to single fibers or multi-fiber systems in vivo or in vitro. Active intraneuronal transport or passive diffusion spread the marker molecule within the neurons up to the fine ramifications of axons and dendrites, and thus allow for a detailed anatomical delineation of the target neurons (reviewed in [1]). Of course, tracer methods are invasive and therefore not suitable for healthy humans in vivo.…”

Advanced microscopy techniques for quantitative analysis in neuromorphology and neuropathology research: current status and requirements for the future

“…Filling individual cells with a tracer, and searching for tracer infusion into another neuron through gap junctions (tracer coupling), imaged in fixed tissue with brightfield microscopy (Vandecasteele et al, 2005) -The application of this technique allows visualization of electrical synapses between neurons. Filling individual cells with a fluorescent dye, and searching for colocalization with neurochemical markers by immunohistochemistry, imaged in fixed tissue with CLSM (Wouterlood et al, 2002b;Benavides-Piccione et al, 2005) -This has become an important technology to analyze neuronal circuitry on a large scale, with the possibility to determine whether neurochemical systems show target specificity in their innervation patterns. Combinatorial transgenic expression of fluorescent proteins (Brainbow transgenes), imaged in fixed tissue with CLSM (Livet et al, 2007) -The Brainbow system is essentially based on (i) the possibility to mutate GFP for generating variants with altered spectral properties (these proteins were termed collectively ''XFPs'' by Feng et al, 2000), and (ii) the use of the Cre/lox recombination system to create a stochastic choice of expression between three or more XFPs (the Cre/lox system can switch on gene expression by DNA excision, inversion, or interchromosomal recombination; for details see, e.g., Zong et al, 2005).…”