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Review
. 2019 Aug 27:13:897.
doi: 10.3389/fnins.2019.00897. eCollection 2019.

A Student's Guide to Neural Circuit Tracing

Christine Saleeba  1   2 , Bowen Dempsey  3 , Sheng Le  1 , Ann Goodchild  1 , Simon McMullan  1
Affiliations
Review

A Student's Guide to Neural Circuit Tracing

Christine Saleeba et al. Front Neurosci. .

Erratum in

Abstract

The mammalian nervous system is comprised of a seemingly infinitely complex network of specialized synaptic connections that coordinate the flow of information through it. The field of connectomics seeks to map the structure that underlies brain function at resolutions that range from the ultrastructural, which examines the organization of individual synapses that impinge upon a neuron, to the macroscopic, which examines gross connectivity between large brain regions. At the mesoscopic level, distant and local connections between neuronal populations are identified, providing insights into circuit-level architecture. Although neural tract tracing techniques have been available to experimental neuroscientists for many decades, considerable methodological advances have been made in the last 20 years due to synergies between the fields of molecular biology, virology, microscopy, computer science and genetics. As a consequence, investigators now enjoy an unprecedented toolbox of reagents that can be directed against selected subpopulations of neurons to identify their efferent and afferent connectomes. Unfortunately, the intersectional nature of this progress presents newcomers to the field with a daunting array of technologies that have emerged from disciplines they may not be familiar with. This review outlines the current state of mesoscale connectomic approaches, from data collection to analysis, written for the novice to this field. A brief history of neuroanatomy is followed by an assessment of the techniques used by contemporary neuroscientists to resolve mesoscale organization, such as conventional and viral tracers, and methods of selecting for sub-populations of neurons. We consider some weaknesses and bottlenecks of the most widely used approaches for the analysis and dissemination of tracing data and explore the trajectories that rapidly developing neuroanatomy technologies are likely to take.

Keywords: anterograde tracer; connectome analysis; neuroanatomy; retrograde tracers; synaptic contacts; viral tracers.

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Figures

FIGURE 1
FIGURE 1
Macro-, meso-, and microscale connectomics. Schematic diagram illustrating connectome analysis at macroscale (top), mesoscale (purple inset), and nanoscale resolutions (blue inset). Macroscale approaches (top) examine communication between regions of the brain on a global level, using approaches such as diffusion-weighted magnetic resonance imaging. Mesoscale connectomics interrogates neural circuitry at the cellular level, employing light microscopy to map the distribution of synaptically linked neurons, in this case monosynaptic inputs to putative sympathetic premotor neurons (see Menuet et al., 2017, modified with permission). Nanoscale connectomics assesses individual synaptic contacts using electron microscopy. Nanoscale image shows electron micrograph of synapses and polysialyic acid immunoreactivity: see Bokiniec et al. (2017).
FIGURE 2
FIGURE 2
Anterograde and retrograde labeling with static and trans-synaptic tracers. Tracers are categorized as anterograde or retrograde based on their direction of travel within neurons. Anterograde tracers (green) are taken up by neuronal cell bodies at the injection site and travel down the axon to terminal processes. Retrograde tracers (blue) are taken up by terminals and travel back to the cell body. Static tracers remain within the first neurons they enter, while other tracers can spread trans-synaptically and may or may not be monosynaptically restricted. CTb: cholera toxin B subunit; H129ΔTK: tyrosine kinase-deleted H129 herpes; PHA-L: Phaseolus vulgaris-leucoagglutinin; PRV: pseudorabies virus; SADΔG(EnvA): glycoprotein-deleted EnvA-pseudotyped rabies.
FIGURE 3
FIGURE 3
H129 tracing of a polysynaptic pathway. Demonstration of H129 infection of a polysynaptic pathway from the retina, through the deep superior colliculus (dSC), to the locus coeruleus (LC). (A) Intravitreal injections of H129-HCMV-loxP-EGFP-HCMVpA-loxP-tdTomato-SV40pA initially infected retinal ganglion cells, driving EGFP expression in retinorecipient nuclei and downstream neurons. When AAV-Cre was injected into the dSC prior to H129 infection (yellow boundary in B) the genomes of H129 virions passing through the dSC were cleaved, resulting in tdTomato expression in post-synaptic neurons. Red neurons in downstream regions, such as LC (Low power micrograph in C, magnified view in D’), can therefore be interpreted as being part of a pathway passing through the dSC. Co-expression of tdTomato and EGFP was common (see merged image, D”) and may indicate incomplete H129 cleavage or multiple pathways converging on the same region. LGN: lateral geniculate nucleus; sSC: superficial layer of the superior colliculus.
FIGURE 4
FIGURE 4
Monosynaptically restricted retrograde tracing using glycoprotein-deleted rabies. (A) Experimental strategy: the starting point is co-expression of genes that encode TVA and the rabies glycoprotein in target neurons, in this case delivered by a lentiviral vector that targets putative sympathetic premotor neurons in the ventrolateral medulla (Lv-PRSx8-YFP-TVA-G). Subsequent infection of TVA/G-expressing neurons by SADΔG(EnvA) microinjection leads to trans-synaptic infection of pre-synaptic (“input”) neurons. Panel (B) shows low power micrograph with infected neurons concentrated in the ventrolateral medulla (boxed region, enlarged in C) “Seed” neurons may be distinguished from “input” neurons by co-expression of the reporters contained in the lentiviral (C’) and rabies constructs (C”), which appear white in the merged image (C”’, denoted by blue arrowheads). Modified with permission from Menuet et al. (2017).
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References

    1. Akli S., Caillaud C., Vigne E., Stratford-Perricaudet L. D., Poenaru L., Perricaudet M., et al. (1993). Transfer of a foreign gene into the brain using adenovirus vectors. Nat. Genet. 3 224–228. 10.1038/ng0393-224 - DOI - PubMed
    1. Andrushko V. A., Verano J. W. (2008). Prehistoric trepanation in the Cuzco region of Peru: a view into an ancient Andean practice. Am. J. Phys. Anthropol. 137 4–13. 10.1002/ajpa.20836 - DOI - PubMed
    1. Angelucci A., Clascá F., Sur M. (1996). Anterograde axonal tracing with the subunit B of cholera toxin: a highly sensitive immunohistochemical protocol for revealing fine axonal morphology in adult and neonatal brains. J. Neurosci. Methods 65 101–112. 10.1016/0165-0270(95)00155-7 - DOI - PubMed
    1. Archin N. M., Atherton S. S. (2002). Rapid spread of a neurovirulent strain of HSV-1 through the CNS of BALB/c mice following anterior chamber inoculation. J. Neurovirol. 8 122–135. 10.1080/13550280290049570 - DOI - PubMed
    1. Aston-Jones G., Card J. P. (2000). Use of pseudorabies virus to delineate multisynaptic circuits in brain: opportunities and limitations. J. Neurosci. Methods 103 51–61. 10.1016/s0165-0270(00)00295-8 - DOI - PubMed