Viral Vectors for Neural Circuit Mapping and Recent Advances in Trans-synaptic Anterograde Tracers

Abstract

Viral tracers are important tools for neuroanatomical mapping and genetic payload delivery. Genetically modified viruses allow for cell-type-specific targeting and overcome many limitations of non-viral tracers. Here, we summarize the viruses that have been developed for neural circuit mapping, and we provide a primer on currently applied anterograde and retrograde viral tracers with practical guidance on experimental uses. We also discuss and highlight key technical and conceptual considerations for developing new safer and more effective anterograde trans-synaptic viral vectors for neural circuit analysis in multiple species.

Figure 1.. Illustration of major families of animal viruses, distinguished by their genome type and viral structure, and gene expression and replication mode.

This figure is adapted and rendered based on relevant information from the book chapters in S.J. Flint et al. (2015) Principles of Virology (4th edition, ASM Press, Washington, DC), and Knipe & Howley (2013) Fields Virology (6^th edition, Wolters Kluwer / Lippincott Williams & Wilkins). The virion images are newly reconstructed based on S.J. Flint et al. (2015). The virus family (bolded) belongs to the viruses often used in neuroscience research. # denotes that the Baculoviridae family does not naturally infect vertebrates. ## denotes that the Hepadnaviridae family uses a unique reverse-transcription process which is not shown in the illustration for simplicity.

Figure 2.. Experimental planning flowcharts are provided to aid experimental choices of viral reagents.

The bold font indicates the viral reagent often used in neural circuit studies. Please see the main text for related references. rAAV, recombinant AAV; rAAV-Retro, retrograde transporting AAV; LV-RVG: lentivirus pseudotyped with rabies virus glycoprotein protein (RVG); SINV, Sindbis virus; SFV, Semliki forest virus; AdV5, adenovirus type 5; RVΔG, rabies virus with glycoprotein gene deletion; CAV2, canine adenovirus type 2; PRV-Bartha: pseudorabies virus strain Bartha; PRV-ΔIE: PRV-Bartha with IE gene deletion; RV: rabies virus; VSVΔG: vesicular stomatitis virus with the glycoprotein coding gene deletion; VSVΔG (EnvA + RVG): G-deleted VSV pseudotyped with EnvA and RVG; HSV1-H129: herpes simplex virus 1 strain H129; H129ΔTK-TT: recombinant H129 with Cre dependent expression of TK and tdTomato; H129-ΔTK: recombinant H129 with TK gene deletion.

Figure 3.. Retrograde-transporting viruses and specific illustrations of labeling projection cell types through axonal retrograde transport.

(A) Schematic illustration of retrograde labeling of neuronal cell bodies via axonal uptake of a retrograde transporting virus. The listed viruses are replication deficient, and they cannot spread out of the initially infected neuron. (B1-B3) rAAV2-retro-mediated labeling demonstrates a new CRH+ input pathway from the amygdalar complex to the nucleus of accumbens (NAc). (B1) Schematic of the retrograde-labeling virus and the location of the NAc injection site in CRH-IRES-Cre mice. To reduce clutter, only one of the retrogradely labeled neurons (red) in the input-mapped region is illustrated with its axonal terminal in the NAc (the AAV2-retro injection site). There is local AAV2-retro labeling of neurons (red) at the NAc injection site. (B2) Endogenous CRH+ cells in the NAc are infected by the rAAV2-retro. (B3) A significant portion of the brain-wide CRH+ projections originate from the amygdala nuclei including the basolateral amygdala (BLA). Images are modified with permission from our published work (Itoga et al., 2019). (C1-3) Corticothalamic neurons in mouse primary somatosensory cortex labeled via the injection of glycoprotein gene-deleted rabies virus expressing mCherry (RVΔG-mCherry) in somatosensory thalamus. In C1, only two of the retrogradely labeled corticothalamic neurons are illustrated with their axonal projections toward the thalamus (the RVΔG injection site). Images are based on unpublished data from the Xu lab.

Figure 4.. Schematic illustrations of transneuronal or trans-synaptic viral tracing.

(A) Retrograde tracing follows the synaptic connection from the postsynaptic starter neuron to its presynaptic partners. (B) Anterograde tracing follows the synaptic connection from the presynaptic starter neuron to its postsynaptic partners.

Figure 5.. Specific circuit mapping applications by retrograde monosynaptic rabies virus tracing and anterograde herpes simplex virus (H129) tracing.

(A-D) Direct subiculum (SUB)-CA1 back-projections are shown by monosynaptic retrograde rabies tracing. This experiment was independently repeated in 12 mice, each with similar results. (A) The scheme for our Cre-dependent, monosynaptic rabies tracing approach. Using Camk2a-Cre; TVA mice, we mapped direct presynaptic input connections onto Camk2a-Cre expressing excitatory neurons in hippocampal CA1 in the intact brain. Starter neurons in dorsal hippocampal CA1 are shown (B, top panel), labeled by both EGFP and dsRed expression from both AAV and rabies infection (B, bottom panels). Their presynaptic partners (e.g., local interneurons and CA3 neurons) are labeled with the red fluorescent protein dsRed from the rabies virus infection. (C-D) Retrogradely labeled Subiculum (SUB) neurons presynaptic to CA1 excitatory neurons are seen in sections of dorsal SUB at different anterior-posterior positions (C, AP: −2.92 mm; D, AP: −3.40 mm). (E-H) Time-limited anterograde-directed HSV tracing supports SUB-CA1 projections. We used the conservative time control of 48 h post-injection to limit labeling to directly connected postsynaptic neurons. This experiment was independently repeated in 5 mice, each with similar results. (E) The scheme for anterograde tracing by combined use of CAV2-Cre injection in CA1 and the injection of Cre-dependent H129 (H129ΔTK-tdTomato) in SUB to map projections of CA1-projecting SUB excitatory neurons. Note that the combined use of different viruses is becoming more appreciated in the field. (F) H129 infected neurons at the injection site in the SUB are shown in red; DAPI staining in blue. (G-H) Postsynaptic neuronal labeling is robustly seen in hippocampal CA1 ipsilaterally at 48 hours post H129 viral injection. (I-L) Besides CA1, postsynaptic neuronal labeling by H129 is seen in the perirhinal cortex (PRh) ipsilaterally. This experiment was independently repeated in 5 mice, each with similar results. (I) An example of PRh labeling, with a white arrow pointing to the atlas aligned brain structure. (J) An enlarged view of perirhinal neuronal labeling in (I). (K-L) Perirhinal labeling from a different animal. Abbreviation: DG, dentate gyrus. Images are modified from our published work (Sun et al., 2019) with Springer Nature permission.

Figure 6.. H129-G4 is an anterograde, polysynaptic tracer that drives strong EGFP expression, which allows for visualization of detailed neuronal morphological features without immunostaining enhancement.

H129-G4 was injected into the primary motor cortex of a C57BL/6 mouse. A coronal brain section image (A) was obtained at 4 days post-infection. The boxed areas in (A) are presented in the right panels (B-C) at higher magnification. A representative H129-G4 labeled single neuron is shown in (D). High magnification images of the dendritic segments with individual spines (E and F) and the axon (G) are presented in the right panels. Images are modified from our published work (Zeng et al., 2017) under the Creative Commons license with Springer Nature.

Figure 7.. Illustration of the anterograde monosynaptic viral tracing system. (A-C) A conceptual framework for our development of deletion mutant H129-based anterograde monosynaptic viral tracers.

(A-B) Genetic engineering of the thymidine kinase (TK) deletion mutant H129 (H129-ΔTK) that is replication defective in non-proliferating cells such as neurons. The mutants are constructed by deleting the TK gene and adding the tdTomato (tdT) or 4xEGFP gene to the H129 genome. (C1) H129-ΔTK can initially infect and enter neurons, but the deletion mutant virus is replication incompetent. (C2) The mutant virus can replicate in neurons co-expressing exogenous TK by way of a TK-expressing helper AAV. As indicated, this conditional replication results in tdTomato expression from the recombinant H129 genome, along with EGFP expression from the helper AAV genome in initially infected neurons. (C3) H129-ΔTK/AAV-TK targeted co-expression limits viral replication to initial infected starter neurons from which viral progeny propagate to label direct postsynaptic neurons. As labeled postsynaptic neurons lack TK expression, the viral label cannot spread beyond anterograde monosynaptically connected cells. (D-G) H129-ΔTK with Cre-dependent helper AAV enables mapping of output connections of specific neuron types, as illustrated by tracing the monosynaptic projection targets of parvalbumin-expressing neurons in the reticular nucleus of thalamus (nRT) of PV-Cre mice. The schematic (E) shows the timeline of injection of AAV-DIO-TK-GFP, and H129-ΔTK-tdT (injected to the same site 21 days apart). The animals were perfused at 10 days post-injection of H129-ΔTK-tdT, brains were extracted and tdTomato label was enhanced with immunostaining. Example brain section images are shown in (F-G). The image of the injection site in nRT is shown in (F1), and a small region (label “2”) is shown in the inset of (F1) and at higher magnification in (F2). The initially infected starter neurons express both tdTomato and GFP, indicated with the white arrows. Note that this section image was acquired at day 3 after H129-ΔTK-tdT injection. (G1) Monosynaptic anterograde label of nRT-PV neurons in a brain section. (G2-G4) Representative regions mapped by H129-ΔTK-tdT, including ventral posterolateral (VP), ventral posteromedial (VM), posterior nucleus (Po) of the thalamus. See Zeng et al. (2017) for postsynaptic neuronal labels in long-range projection targets. Images are modified from our published work (Zeng et al., 2017) under the Creative Commons license with Springer Nature.