Monosynaptic restriction of the anterograde herpes simplex virus strain H129 for neural circuit tracing

Abstract

Identification of synaptic partners is a fundamental task for systems neuroscience. To date, few reliable techniques exist for whole brain labeling of downstream synaptic partners in a cell-type-dependent and monosynaptic manner. Herein, we describe a novel monosynaptic anterograde tracing system based on the deletion of the gene UL6 from the genome of a cre-dependent version of the anterograde Herpes Simplex Virus 1 strain H129. Given that this knockout blocks viral genome packaging and thus viral spread, we reasoned that co-infection of a HSV H129 ΔUL6 virus with a recombinant adeno-associated virus expressing UL6 in a cre-dependent manner would result in monosynaptic spread from target cre-expressing neuronal populations. Application of this system to five nonreciprocal neural circuits resulted in labeling of neurons in expected projection areas. While some caveats may preclude certain applications, this system provides a reliable method to label postsynaptic partners in a brain-wide fashion.

Figure 1:. Functional deletion of HSV UL6 in H129 LSL-TK-TT prohibits viral spread in vitro and in vivo.

(a) Illustration of HSV packaging of viral DNA into the capsid via UL6 portal. (b) UL6 deletion scheme. The UL6 locus (top) was targeted with a GFP expression cassette (middle) flanked by regions homologous to the UL6 gene, resulting in the virus HSV H129 LSL-TK-TT ΔUL6-GFP. Following purification, targeted removal of the GFP expression cassette by CRISPR/Cas9 resulted in a frameshift after 377 amino acids of the UL6 coding sequence and premature UL6 termination (bottom). (c) Insertion of the GFP expression cassette prohibits spread of the virus. Application of H129 LSL-TK-TT ΔUL6-GFP to monolayers of vero cells, vero cells constitutively expressing UL6 (31-UL6), and vero cells transfected prior to inoculation with the plasmid pNef-coUL6. (d) Brain tissue collected 5-days after injection of HSV H129 LSL-TK-TT (left column) and HSV H129 LSL-TK-TT ΔUL6 (right column) in primary visual cortex of Sim1-cre mice. Anti-HSV antibody staining in magenta, DAPI in blue.

Figure 2:. Transcomplementation of H129 LSL-TK-TT ΔUL6 with AAV in nonreciprocal circuits results in HSV labeled neurons in anterograde nuclei.

(a) Illustration of transcomplementation of H129 LSL-TK-TT ΔUL6 with AAV expression of codon-optimized UL6. (b) Experimental timeline of transcomplementation experiment. (c) Schematic of retina injection into Slc17a6:Cre mice. (d) Flat-mount retina in the right eye of a Slc17a6-cre mouse after intravitreal injection of H129 LSL-TK-TT ΔUL6. Inset shows dotted-box region of injection site; white arrows denote putative starter cells. AAV-expressed GFP in green, HSV-expressed tdTomato in red. (e) Brain slices from the same mouse showing HSV-positive neurons in visual thalamic nuclei. Example neuron shown in the inset at right. (f) Brain slices from the same mouse showing HSV-positive neurons in superior colliculus. Example neuron shown in the inset at right. (g) Schematic of experiment design targeting somatostatin positive neurons of the GPi and their postsynaptic targets in LH. (h) Injection site of AAV8-nef-2N-DIO-GFP-p2a-coUL6 and H129 LSL-TK-TT ΔUL6 in the GPi of a Som:cre mouse. White arrows denote putative starter cells. (i) Ipsilateral LH from the same mouse. (j) Schematic of experiment design targeting VGLUT2 positive neurons of the dSub and their postsynaptic targets in LM. (k) Injection site of AAV8-nef-2N-DIO-GFP-p2a-coUL6 and H129 LSL-TK-TT ΔUL6 in the dSub of a Slc17a6:Cre mouse. (l) Ispilateral LM from the same mouse. DAPI in blue, anti-HSV antibody in magenta. Tissue collected 7 days after HSV injections. dLG, dorsal lateral geniculate nucleus; vLG, ventral geniculate nucleus; LP, lateral posterior nucleus of the thalamus; SCsg, superior colliculus superficial gray layer; SCm, superior colliculus, motor related; GPe, globus pallidus external segment; GPi, globus pallidus internal segment; int, internal capsule; RT, reticular thalamus; AMY, amygdalar nuclei; MH, medial habenula; LH, lateral habenula; RSPv, ventral retrosplenial cortex; POST, postsubiculum; dSub, dorsal subiculum; fx, columns of the fornix; LM, lateral mammillary body.

Figure 3:. HSV H129 can travel by direct, but not transsynaptic, retrograde spread.

(a) Viral surgery overview. Injection of AAV8 nef-2N-DIO-GFP-p2a-coUL6 into the LGN of a CRH:Cre mouse, with restricted expression of Cre in the thalamus, was followed two weeks later by injection of HSV H129 LSL-TK-TT ΔUL6 into visual cortex. (b) Experimental overview. Direct axonal uptake of HSV infects LGN neurons that were infected directly by the AAV helper virus. HSV is injected in V1, and not LGN, to avoid direct retrograde infection of retinal ganglion cells. As such, viral spread found in the retina must come from transsynaptic retrograde spread. (c) HSV injection site in V1. (d) AAV injection site in LGN. Magenta labelling is the result of direct retrograde HSV spread. (e) The left and (f) right retinas, flat mounted. Tissue collected 7 days post-injection. HSV antibody staining in magenta, GFP in green, and DAPI in blue. V1, primary visual cortex; LGv, lateral geniculate nucleus, ventral; LGd, lateral geniculate nucleus, dorsal; LP, lateral posterior nucleus.

Figure 4:. Transcomplementation leads to rapid cell death while UL6 knockout leads to latent-like state.

(a) Whole brain slice showing condensed DAPI staining at the injection site (dotted box). 7 days post injection of H129 LSL-TK-TT ΔUL6, 21 days post injection of AAV8 nef-2N-DIO-GFP-2A-coUL6. Injection targeting GPi. (b) Example HSV labeled neurons present in LGN after transcomplementation in retina of Slc17a6-cre mouse showing a live cell (red arrow) and a dead cell (red box). Anti-HSV antibody in magenta, DAPI in blue. Tissue collected 7 days after injection. Channels split at right. (c-f) Primary visual cortex tissue following injection of H129 LSL-TK-GFP ΔUL6-mCherry. (c) Fluorescent protein expression in tissue from a Som:Cre mouse four days after injection. GFP in green, mCherry in red. Channels split below. (d) Tissue collected at 4, 8, and 16 days post injection. GFP channel not shown. mCherry in red, DAPI in blue. Channels split below. (e) Fluorescent protein expression in tissue from a Ntsr1:Cre mouse two days after injection. GFP in green, mCherry in red. Channels split below. (f) Primary visual cortex tissue following injection of H129 LSL-TK-GFP ΔUL6-mCherry in Ntsr1-cre mice. Tissue collected 2, 7, and 14 days post injection. mCherry channel not shown. DAPI in blue, GFP in green. Split channels below.

Figure 5:. Transcomplementation of H129 LSL-TK-GFP ΔUL6 with AAV in nonreciprocal circuits results in HSV labeled neurons in anterograde nuclei.

(a) Schematic of injection into the STN of Slc17a6:Cre mice. (b) Brain slice from tissue collected 8 days post-injection showing mCherry expression from the AAV8-nef-2N-DIO-mCherry-p2a-coUL6 helper virus. (c) Close up of putative starter cells expressing mCherry and GFP from the AAV and HSV viruses, respectively. (d) Brain slices from the same mouse showing GFP expression from the HSV virus in neurons of the SNr. A close-up shows example neuron in the inset at right. (e) Schematic of experiment design targeting Parvalbumin-positive neurons of the posterior Substantia Innominata (pSI) and their postsynaptic targets in S1. In this mouse, AAV helper virus and H129 LSL-TK-GFP ΔUL6 were injected in the left hemisphere, which H129 LSL-TK-GFP ΔUL6 alone was injected in the right. Tissue was collected (f) Brain slice showing the injection site of AAV8-nef-2N-DIO-mCherry-p2a-coUL6 and H129 LSL-TK-GFP ΔUL6 in the left pSI. (g) Brain slice showing GFP positive cells in left S1. (h) Brain slice showing the injection site of H129 LSL-TK-GFP ΔUL6 in the right pSI. (i) Brain slice showing no GFP positive cells in the right S1. PV, parvalbumin; STN, subthalamic nuclei; cp, cerebral peduncle; SNr, Substantia Nigra pars reticulata; pSI, posterior substantia innominata; GPi, globus pallidus internal segment; S1, somatosensory cortex.