A Student’s Guide to Neural Circuit Tracing


doi: 10.3389/fnins.2019.00897.

eCollection 2019.

A Student’south Guide to Neural Circuit Tracing


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A Student’s Guide to Neural Excursion Tracing

Christine Saleeba
 et al.

Front Neurosci.


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The mammalian nervous organisation 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 construction that underlies encephalon function at resolutions that range from the ultrastructural, which examines the system of individual synapses that impinge upon a neuron, to the macroscopic, which examines gross connectivity between large brain regions. At the mesoscopic level, afar 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, informatics and genetics. As a consequence, investigators now savour an unprecedented toolbox of reagents that can be directed against selected subpopulations of neurons to place 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 information drove to analysis, written for the novice to this field. A brief history of neuroanatomy is followed by an assessment of the techniques used past contemporary neuroscientists to resolve mesoscale arrangement, such as conventional and viral tracers, and methods of selecting for sub-populations of neurons. Nosotros consider some weaknesses and bottlenecks of the most widely used approaches for the analysis and dissemination of tracing information and explore the trajectories that rapidly developing neuroanatomy technologies are likely to take.

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anterograde tracer; connectome assay; neuroanatomy; retrograde tracers; synaptic contacts; viral tracers.


Effigy 1

Macro-, meso-, and microscale connectomics. Schematic diagram illustrating connectome analysis at macroscale (peak), mesoscale (purple inset), and nanoscale resolutions (blue inset). Macroscale approaches (top) examine communication betwixt 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 (meet Menuet et al., 2017, modified with permission). Nanoscale connectomics assesses individual synaptic contacts using electron microscopy. Nanoscale prototype shows electron micrograph of synapses and polysialyic acid immunoreactivity: encounter Bokiniec et al. (2017).


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 upwards by neuronal jail cell bodies at the injection site and travel downwardly the axon to terminal processes. Retrograde tracers (blueish) are taken up past terminals and travel back to the jail cell body. Static tracers remain within the outset neurons they enter, while other tracers can spread trans-synaptically and may or may not exist monosynaptically restricted. CTb: cholera toxin B subunit; H129ΔTK: tyrosine kinase-deleted H129 canker; PHA-L:
Phaseolus vulgaris-leucoagglutinin; PRV: pseudorabies virus; SADΔG(EnvA): glycoprotein-deleted EnvA-pseudotyped rabies.


Effigy three

H129 tracing of a polysynaptic pathway. Sit-in of H129 infection of a polysynaptic pathway from the retina, through the deep superior colliculus (dSC), to the locus coeruleus (LC).
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. Cherry-red neurons in downstream regions, such as LC (Depression power micrograph in
C, magnified view in
D’), can therefore be interpreted as beingness office of a pathway passing through the dSC. Co-expression of tdTomato and EGFP was common (see merged paradigm,
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.



Monosynaptically restricted retrograde tracing using glycoprotein-deleted rabies.
Experimental strategy: the starting point is co-expression of genes that encode TVA and the rabies glycoprotein in target neurons, in this instance delivered by a lentiviral vector that targets putative sympathetic premotor neurons in the ventrolateral medulla (Lv-PRSx8-YFP-TVA-G). Subsequent infection of TVA/Chiliad-expressing neurons by SADΔG(EnvA) microinjection leads to trans-synaptic infection of pre-synaptic (“input”) neurons. Panel
shows low ability micrograph with infected neurons concentrated in the ventrolateral medulla (boxed region, enlarged in C) “Seed” neurons may be distinguished from “input” neurons past co-expression of the reporters contained in the lentiviral
and rabies constructs
(C”), which appear white in the merged image (C”’, denoted by bluish arrowheads). Modified with permission from Menuet et al. (2017).

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