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.

Thalamic innervation of the direct and indirect basal ganglia pathways in the rat: Ipsi- and contralateral projections

“…While the CLSM increases the resolution by a factor of 1.4 in comparison to light microscopy (Pawley, 1995; Inoué, 1995), terminal boutons or synaptic contacts can only be properly visualized with electron microscopy. Nevertheless, we have followed the criteria suggested by Wouterlood et al (2002a, b) to ascertain whether a point of physical contract represents a “true” synapse. However, the accurate visualization of synaptic contacts between labeled fibers and dendritic shafts or spines located far away from the neuron soma is sometimes hampered by the intrinsic nature of retrograde tracing techniques.…”

Retinal afferents synapse with relay cells targeting the middle temporal area in the pulvinar and lateral geniculate nuclei

“…The resulting voxel size was 130 nm × 130 nm × 300 nm, and all images were left in grayscale for colocalization analysis and pseudocolored afterward for illustration of colocalization. Deconvolution was used to reduce background and extract statistically reliable, relevant information (Wouterlood et al, 2002a,b) from the z-stacks, which were deconvolved using the commercially available software AutoQuant X (vX2.1.1, MediaCybernetics, Inc). Colocalization of synaptophysin within retinal axons was demonstrated using the colocalization module of Imaris software (v6.3.1, Bitplane) to calculate Pearson's coefficient which measures the strength of the linear relationship between the grey values of the fluorescence intensity pixels of green and red image pairs (Bolte and Cordelières, 2006).…”

Fluorescence-Based Monitoring of In Vivo Neural Activity Using a Circuit-Tracing Pseudorabies Virus

“…Genetic tools offer complementary ways of distinguishing and targeting cell types [1], but a combination of functional imaging and circuit tracing is still missing. Conversely, commonly used chemical tracers [2] and neurotropic viruses [3] can identify neuroanatomical circuits but do not document activity. An ideal tool should simultaneously report activity and connectivity with sensitivity and reliability for long periods of time in vivo .…”