Anterograde transneuronal tracing and genetic control with engineered yellow fever vaccine YFV-17D

Abstract

Transneuronal viruses are powerful tools for tracing neuronal circuits or delivering genes to specific neurons in the brain. While there are multiple retrograde viruses, few anterograde viruses are available. Further, available anterograde viruses often have limitations such as retrograde transport, high neuronal toxicity or weak signals. We developed an anterograde viral system based on a live attenuated vaccine for yellow fever-YFV-17D. Replication- or packaging-deficient mutants of YFV-17D can be reconstituted in the brain, leading to efficient synapse-specific and anterograde-only transneuronal spreading, which can be controlled to achieve either monosynaptic or polysynaptic tracing. Moreover, inducible transient replication of YFV-17D mutant is sufficient to induce permanent transneuronal genetic modifications without causing neuronal toxicity. The engineered YFV-17D systems can be used to express fluorescent markers, sensors or effectors in downstream neurons, thus providing versatile tools for mapping and functionally controlling neuronal circuits.

Extended Data Figure 1

(a) Schematics showing the PFC-striatum-SN pathway. (b) Expression of mVenus after injection of YFV-mVenus into the PFC. Brains were collected 3, 6 or 9 days after injection. All images are tile scans of brain sections. The blue color is counterstaining with DAPI. (c) Quantification of images in b: Density of mVenus-positive neurons in each brain region on day 3, 6, and 9. The bars are mean±SEM, n=10–12 sections from 3 mice for each brain region.

Extended Data Figure 2

(a)The dentate gyrus (DG)-CA3-CA1-subiculum (SUB)-striatum pathway. (b) Expression of mVenus after injection of YFV-mVenus into the DG. Brains were collected 3, 6 or 10 days after injection. (c-e) YFV-mVenus spread from the CA1 to SUB and dorsal striatum along polysynaptic pathways. mVenus expression after YFV-mVenus injection into dorsal CA1. Brains were collected 6 (c), 9 (d) or 11 (e) days after injection. (e) Images of brain sections containing the striatum from a mouse 11 days after YFV-mVenus injection at dorsal CA1 that were immunostained with MOR, a marker for striosomes. The experiments were repeated 3 times with similar results. All images are tile scans of brain sections. The blue color is counterstaining with DAPI.

Extended Data Figure 3

(a) Images of brain sections from mice infected with YFV-mVenus that were immunostained with antibody for YFV protein E. The green fluorescence was from native mVenus without immunostaining. The arrowheads indicate cells positive for E but not mVenus. (b) Quantification of mVenus-positive cells expressing E, and E-positive cells expressing mVenus. (c) Images of brain sections from mice infected with YFV-mVenus that were immunostained with antibody for NS1. The arrowheads indicate cells positive for NS1 but not mVenus. (d) Quantification of mVenus-positive cells expressing NS1, or NS1-positive cells expressing mVenus. n=14 brain sections from 3 mice for “E” and n=14 brain sections from 4 mice for “NS1”. All images are tile scans of brain sections. The blue color is counterstaining with DAPI.

Extended Data Figure 4

Expression of the neuronal marker NeuN in brain sections from mice infected with YFV-mVenus. The green fluorescence was from native mVenus without immunostaining. The arrowheads indicate a non-neuronal cell expressing mVenus. n=22 brain sections from 3 mice. The images are tile scans of brain sections. The blue color is counterstaining with DAPI.

Extended Data Figure 5

(a) NG2 (Neuron-glial antigen 2) expression in brain sections from mice infected with YFV-mVenus. NG2 is a marker of NG2 cells (also referred to as oligodendrocyte precursor cells). The green fluorescence was from native mVenus without immunostaining. (b) High-resolution images of the areas indicated by arrowheads in a, showing a NG2-positive cell next to a mVenus-positive cell. (n=25 brain sections from 3 mice). The images are tile scans of brain sections. The blue color is counterstaining with DAPI.

Extended Data Figure 6

(a) Olig2 (a marker of oligodendrocytes) expression in brain sections from mice infected with YFV-mVenus. The green fluorescence was from native mVenus without immunostaining. (b) High-resolution images of the cropped areas in a. CC: corpus callosum. n=26 brain sections from 3 mice. The images are tile scans of brain sections. The blue color is counterstaining with DAPI.

Extended Data Figure 7

(a) Expression of mVenus in multiple brain regions after injection of YFV-mVenus into the PFC and fixation of the brains 15 days later. (b) Expression of mVenus in a cerebellar Purkinje cell. The experiments were repeated 6 times with similar results. The images are tile scans of brain sections. The blue color is counterstaining with DAPI.

Extended Data Figure 8

Expression of mVenus and tdTomato after Injection of AAVDJ-Syn-NS1 into the PFC and the striatum, and injection of YFV^ΔNS1-mVenus into the striatum. The brains were fixed 6 (a) or 12 (b) days after YFV^ΔNS1-mVenus injection. The experiments were repeated 3 times with similar results. The images are tile scans of brain sections. The blue color is counterstaining with DAPI.

Extended Data Figure 9

(a) The experimental design was the same as in Fig. 4b–d except that no AAVs for NS1 expression were injected into the striatum. We fixed the brains 15 days after YFV^ΔNS1-Cre injections. (b) jGCaMP7f-positive neurons in the striatum. The image is a tile scan of the brain section. The blue color is counterstaining with DAPI. (c) The density of labeled cells in mice with or without NS1 in the striatum (n=15 sections from 4 mice for “with NS1” and n=14 sections from 4 mice “without NS1”, *** P=0.000000026, Mann-Whitney test, two tailed). The data of the group “with NS1” were also shown in Fig. 4d.

Extended Data Figure 10

(a) Image of a brain section after injection of AAV-tTA, AAV-TRE-C-prM-E-NS1 and YFV^ΔCME-mVenus into pontine nucleus and adjacent reticular tegmental nucleus. (b) mVenus-positive cells in the granular layer of the cerebellar cortex. The sections were counterstained with a marker for Purkinje cells, PCP4 (red), and DAPI (Blue). The experiments were repeated 4 times with similar results. The images are tile scans of brain sections. The blue color is counterstaining with DAPI.

Figure 1.. Controlled anterograde transneuronal spreading of YFV-17D.

(a) Construction of YFV-mVenus and YFV^ΔNS1-mVenus. (b) AAVs mediating constitutive (AAV-Syn-NS1) or conditional (AAV-DIO-NS1) expression of NS1. (c, d) Expression of mVenus and tdTomato after injection of YFV-mVenus and AAVDJ-tdTomato into the PFC. (d) Top: mVenus-positive neurons and tdTomato-positive axons in the striatum. Bottom: contacts between mVenus-positive neurons and tdTomato-positive axonal terminals are indicated by arrowheads. (e, f) Expression of mVenus and tdTomato (representing Cre) after injection of YFV-mVenus and rAAV2-retro-Cre into the striatum of an Ai9 mouse. (f) mVenus and tdTomato (representing Cre) in the PFC (top) and SN (bottom). (g) Expression of NS1 after injection of AAVDJ-Syn-NS1 and YFV^ΔNS1-mVenus into the striatum and the PFC, respectively. (h) Expression of NS1 and mVenus after injection of AAVDJ-Syn-NS1 and YFV^ΔNS1-mVenus into the PFC. (i) Expression of NS1 and mVenus after injection of AAVDJ-Syn-NS1 into the PFC and striatum, and YFV^ΔNS1-mVenus into the PFC. (j) Expression of NS1 after injection of AAVDJ-Syn-NS1 into the PFC and YFV^ΔNS1-mVenus into the striatum. (k) Expression of NS1 after injection of AAVDJ-DIO-NS1 and YFV^ΔNS1-mVenus into the PFC, and AAVDJ-NS1 into the striatum. (l) Expression of NS1 and mVenus after injection of AAVDJ-DIO-NS1, AAV-Cre and YFV^ΔNS1-mVenus into the PFC, and AAVDJ-NS1 into the striatum. All images except Fig. 1d are tile scans of brain sections. The blue color is counterstaining with DAPI. The experiments in Fig. 1c–d, e–f, g, j, k and l were repeated 3 times with similar results. The experiments in Fig. 1h and iv were repeated 4 times with similar results.

Figure 2.. Anterograde-only tracing by inducible replication of YFV^ΔNS1.

(a) Schematics showing viral vectors mediating inducible expression of NS1 and replication of YFV^ΔNS1-mVenus. (b) Quantification of the genomic copies of YFV^ΔNS1-mVenus in PFC after injection of the indicated AAVs and YFV^ΔNS1-mVenus into the PFC. Data are presented as scatter and mean. n=4–7 mice in each group, * P=0.029, ** P=0.0061, Mann-Whitney test, two tailed, compared to mice receiving YFV^ΔNS1-mVenus alone. (c) Experimental procedures for d and e. (d) Expression of tdTomato and mVenus after injection of AAVDJ-rtTA and AAVDJ-TRE-NS1/dTomato into both the PFC and the striatum and YFV^ΔNS1-mVenus into the PFC. (e) Expression of tdTomato and mVenus after injection of AAVDJ-rtTA and AAVDJ-TRE-NS1/dTomato into both the PFC and the striatum, YFV^ΔNS1-mVenus into the striatum. The images in d-e are tile scans of brain sections. The blue color is counterstaining with DAPI. (f) Percentage of mVenus-positive cells after the mice underwent the same procedure as in d with either concentrated or 5 to 10-fold diluted YFV^ΔNS1-mVenus injected into the PFC. We counted mVenus-positive cells and DAPI-stained nuclei in the striatum to calculate the tracing efficiency. Data are presented as scatter and mean±standard error of the mean (SEM), n=12 and 16 brain sections from 4 mice per group for “diluted” and “concentrated”, respectively. *** P=0.000004, Mann-Whitney test, two tailed. The experiments in Fig. 2d and e were repeated 4 times with similar results.

Figure 3.. Dual fluorescence tracing of parallel circuits.

(a-c) Tracing long-range projections from the cortical regions to the midbrain with YFV^ΔNS1. (a) Schematics showing the neuronal pathways and viral injections. The AAVs include AAVDJ-rtTA and AAVDJ-TRE-NS1. (b, c) Representative images showing mVenus- or mCherry- positive neurons or axons in the cortex, striatum and midbrain nuclei. (d-f) Tracing polysynaptic projections from the cortical regions via the striatum to the midbrain with YFV^ΔNS1. (d) The mice received the same viral injections as those in a except that the AAVs were also injected into the striatum. (e, f) Representative images showing mVenus- or mCherry- positive neurons in the cortex, striatum and midbrain nuclei. All images are tile scans of brain sections. The blue color is counterstaining with DAPI. The experiments in Fig. 3a–c, Fig. 3d–f were repeated 3 times with similar results.

Figure 4.. Transneuronal genetic control by YFV^ΔNS1-Cre.

(a) Schematic of transneuronal genetic modification with YFV^ΔNS1-Cre. (b-d) Injection of YFV^ΔNS1-Cre into PFC to turn on a reporter in the striatum. (b) Viral injection scheme. Brains were fixed 15 or 30 days after YFV^ΔNS1-Cre injection. (c) Expression of jGCaMP7f in the striatum. (d) Quantification of the densities (left) or percentage (right) of jGCaMP7f-labeled striatal neurons. Data are presented as scatter and mean±SEM, n=15–18 brain sections from 4 mice per group. P=0.26 for “Density” and P=0.13 for “Percentage”, Mann-Whitney test, two tailed. (e-g) Electrophysiological recording of striatal neurons with optogenetic stimulation of axons from the PFC. (e) Viral injection scheme. (f) Top: jGCaMP7f-positive striatal neurons and tdTomato-positive axons. Bottom: a representative trace of postsynaptic currents. The blue bars indicate optogenetic stimulation. (g) The amplitudes (n=12 cells from 3 mice, left) and synaptic delays (n=12 and 8 cells for jGCaMP7f- positive and -negative cells, respectively, right) of synaptic currents. Data are presented as scatter and mean±SEM ** P=0.0011 for “EPSC amplitude” and P=0.0019 for “Synaptic delay”, Mann-Whitney test, two tailed. (h-j) Imaging neuronal calcium activities in freely moving mice. (h) Viral injections and lens implantation. (i) Individual image frames. (j) Neuronal calcium activities of a mouse recorded on day 42 and day 49 after YFV^ΔNS1-Cre injection, respectively. Calcium activity (ΔF/F) of individual neurons was plotted together with the locomotion speed of the mouse. Images in 4c and f are tile scans of brain sections. The blue color is counterstaining with DAPI. The experiments in Fig. 4h–j were repeated 3 times with similar results.

Figure 5.. Mapping monosynaptic projectomes with YFV^ΔCME.

(a) Construction of YFV^ΔCME-mVenus by removing the coding sequences of three structural proteins (C-prM-E). (b) Mapping axonal and synaptic projections of neurons with AAV-SynaptoTAG2. (c) Distribution of tdTomato and Syb2-EGFP after injection of AAVDJ-SynaptoTAG2 into the PFC. (d, e) Distribution of mVenus after injection of YFV^ΔCME-mVenus, AAVDJ-tTA and AAVDJ-C-prM-E-NS1 into the PFC. Abbreviations: CLA, claustrum; MD, mediodorsal nucleus of the thalamus; VM, ventromedial thalamic nucleus; Re, nucleus reuniens; ZI, zona incerta; SC, superior colliculus; MRN, midbrain reticular nucleus; PAG, periaqueductal grey area. (f) Left: Axons (red) and synaptic terminals (green) labeled by SynaptoTAG2. Right: Postsynaptic neurons traced by YFV^ΔCME-mVenus. The arrowheads and insert show the mossy tuft-like structures on the dendrite. (g) Quantification of the density of neurons traced by YFV^ΔCME-mVenus. Data are presented as scatter and mean+SEM, n=20 brain sections from 4 mice for each region. Ipsi and contra: ipsilateral or contralateral to the injection site. Images in c and d are tile scans of brain sections. The blue color is counterstaining with DAPI. The experiments in Fig. 5c–f were repeated 4 times with similar results.

Figure 6.. Monosynaptic transneuronal genetic control with YFV^ΔCMENS1.

(a) Construction of YFV^ΔCMENS1-Cre by removing the coding sequences of C-prM-E and NS1. (b) Schematic showing monosynaptic transneuronal genetic control with YFV^ΔCMENS1-Cre. (c) The scheme of viral injections and Dox diet. The AAVs include AAVDJ-tTA and AAVDJ-TRE-C-prM-E-NS1 or AAVDJ-TRE-NS1. (d-g) Injection of YFV^ΔCMENS1–Cre into the PFC to turn on the reporter in the striatum. (d) jGCaMP7f expression after injection of AAVDJ-tTA, AAVDJ-TRE-C-prM-E-NS1 and YFV^ΔCMENS1–Cre into the PFC and AAVDJ-tTA, AAVDJ-TRE-NS1 and AAV9-DIO-jGCaMP into the striatum. (e) jGCaMP7f-positve neurons in the striatum. (f) Expression of jGCaMP7f in striatal neurons. The insert shows the spines on a segment of a dendrite. (g) Quantification of the traced neurons in the striatum. Data are presented as scatter and mean±SEM, n=15–19 brain sections from 3–4 mice. P=0.26 for “density” and 0.16 for “percentage”, Mann-Whitney test, two tailed. Images in 6d-e are tile scans of brain sections. The blue color is counterstaining with DAPI.