Control experiments reveal a leak of “invisible” TVA

To verify that not just our mouse line but also our viruses have high specificity, we performed several important control experiments. First, we injected RV-EGFP without prior injection of the AAVs (n=4 hemispheres) to verify that the transduction of RV-EGFP exclusively depends on the interaction with TVA. We did not detect EGFP labeled cells indicating that RV-EGFP failed to transduce TVA-negative cells (Figure 7A). Because we made sure that this particular batch of RV-EGFP was not contaminated (C) Wildtype oG (left) but not fragments used for exon 1 or exon 2 (center, right) encodes functional rabies glycoprotein, as assayed by antibody staining in HEK293 cells.

(D) Con/Fon-oG only encodes functional protein in the presence of both Cre and Flp while neither Cre nor Flp in isolation is sufficient to produce functional glyco¬protein, as assayed by flow cytometry on HEK293 cells co-transfected with Con/Fon-oG and indicated recombinases (scale bars: big panel: 50 μm, small panels: 5 μm).

(E) Molecular design (top) of Cre- (yellow) and Flp-dependent (purple) exons of TVA-mCherry created through the introduction of two introns (open boxes) and (bottom) primers used for PCR and RT-PCR.

(F) PCR (lanes 1 and 3) and RT-PCR (lanes 2 and 4) using noted primers of wild-type TVA-mCherry (left) and Con/Fon-TVA-mCherry (right), showing expected band for Con/Fon-TVA-mCherry cDNA and confirming proper exon re-orientation after recombinase activity and intron splicing. Splicing was further validated by sequencing of the WT- and Con/Fon-TVA-mCherry cDNA bands, including minor ones (bottom). The major band represents ideal splicing of the intron and recombinase components, while the smaller bands in both the wild-type and INTRSECT versions are either non-specific or represent inherent splicing separate from the introns introduced during INTRSECT molecular engineering.

(G) Cultured neurons express functional Con/Fon-TVA-mCherry (red) only when co-transfected with Cre (blue) and Flp (green) (scale bars: big panel: 50 μm, small panels: 5 μm).

The figure was made by Lief Fenno. The data shown in this figure as well as the viral constructs were gen-erated by Lief Fenno, Charu Ramakrishnan, Yoon S. Kim and Karl Deisseroth and shared with us in the framework of a collaboration.

Figure 5: Experimental workflow of intersectional RV-tracing of GABAergic PV cells

On day 1, AAV8-Con/Fon-TVA-mCherry and AAV8-Con/Fon-oG are co-injected into the barrel cortex of Vgat-Cre/PV-Flp mice. The expression of TVA-mCherry and oG is conditional on the activity of both Cre and Flp.

Cutting by recombinases prompts the correct orientation of the constructs. Subsequent splicing of artificial introns (black boxes) enables the assembly of the constructs. Because in Vgat-Cre/PV-Flp mice co-expres-sion of Cre and Flp is exclusive to the intersectional population of GABAergic PV cells, TVA-mCherry and oG expression is restricted to this cell-type.

RV-SAD-ΔG-EGFP (EnvA) (RV-EGFP) is injected 14 days later at the same location. EnvA protein conditions RV transduction to TVA-exposing cells. Upon uptake into GABAergic PV cells, it incorporates the oG into its envelope, which allows the spread to first-order presynaptic neurons. The absence of a glycoprotein in these neurons prohibits further spread. RV-EGFP expression mixes with AAV-mCherry expression to label starter cells yellow, while the presynaptic neurons exclusively express RV-EGFP and thus appear green.

with RV particles coated by the endogenous glycoprotein, we used only this batch for subsequent ex-periments.

Second, we investigated if the recombination of the AAVs only happens in the presence of both recombinases. We injected BL6 mice, which express neither Cre nor Flp, with experimental titers of AAV-TVA-mCherry and AAV-oG before injection of RV-EGFP (n=2 mice). We observed weakly mCher-ry-positive cells as well as a low number of EGFP-positive cells at the injection site (Figure 7B). This was also the case when we injected the same virus combination in either Vgat-Cre (n=2 mice) or

PV-Cre

Flp

Helper Virus

Rabies Virus

Cre

Flp

EGFP

Day 1: Helper virus Day 15: Rabies virus AAV8-Con/Fon-TVA-mCherry

AAV8-Con/Fon-oG RV-SAD-ΔG-EGFP (EnvA)

TVA

[N] WPRE

N P M EGFP L

nEF ^[C] mCh TVA[C]- mCh[N]

EF1a ^[N] oG ^[C] oG WPRE

Cre-dependent Flp-dependent Cre-dependent Flp-dependent

TVA (EnvA-R) optimized rabies glycoprotein

Day 22: Analysis

Flp (n=2 mice) mice, where one recombinase was present, except that the number of EGFP-positive cells was slightly higher (Figure 7C, D). However, we never observed EGFP-positive cells outside of the injection site in any mouse line. These results lead us to the following considerations:

(I) TVA-mCherry can be expressed in the absence of one or two recombinases at a very low lev-el. However, when we injected AAV-TVA-mCherry into Vgat-Cre/PV-Flp animals, we found that about 97% (n=4 mice, 11 sections) of TVA-mCherry positive cells were co-labeled with PV-immunostaining, indicating a high specificity of the virus (Figure 7E). We do not have a clear explanation for this dis-crepancy but assume that under our experimental conditions TVA-mCherry labeling is mostly directed to PV cells.

(II) Our virally delivered oG did not have a fluorescent tag. An indicator that oG is expressed in the absence of recombinases would be transsynaptic spread of RV-EGFP. We saw EGFP-positive, mCherry-negative cells at the injection site but never outside of it. We closely looked at the ventral posteromedial nucleus of the thalamus (VPM), which provides reliable long-range input to the barrel cortex (Figure 7B-D). We never found any labeling there. The cells at the injection site could be trans-synaptically labeled but then we would expect at least a few cells in distant areas as well. Instead, the restriction to the injection site could be due to a local leak of TVA (Do et al., 2016; Faget et al., 2016;

Kim et al., 2018; Mijamichi et al., 2013; Schwarz et al., 2015; Watabe-Uchida et al., 2012; Weissbourd et al., 2014). EnvA-pseudotyped RV is highly sensitive to TVA (Mijamichi et al., 2013). Therefore, a small number of TVA molecules can already permit RV entry into cells. In case that TVA is expressed in a recombinase-independent way, RV-EGFP could enter these cells directly making them appear as

A1

Figure 6: Mapping the C2 whisker representation in barrel cortex

(A) In a proof-of-principle experiment we showed that intrinsic signal optical imaging can be used to target injections to a defined column of the barrel cortex. We illuminated the exposed cortical surface above the barrel cortex with red light, while stimulating the C2 whisker and recording the reflectance with a CCD-cam-era. 30 frames were recorded and averaged. Repetitive whisker stimulation led to a localized change in blood flow, which induced a change in light reflectance visible as a dark dot.

(B) Surface vasculature was overlaid with image A and the location of the highest change in reflectance was marked with a red dot. The blood vessels were used as landmarks to guide the injection pipette.

(C) Tangential section through the barrel cortex of a Scnn1a-Tge-Cre/tdTomato mouse. Injection of DiO crystals was guided by the intrinsic signal elicited upon C2 whisker stimulation shown in A. This experiment demonstrated the accuracy of the injection procedure (scale bars: 200 μm).

A

(III) To investigate the extent of possible TVA leak, we injected AAV-TVA-mCherry without AAV-oG into Vgat-Cre/PV-Flp mice and two weeks later RV-EGFP (n=4 hemispheres, Figure 7F). About 15.7

± 6.1% of the transduced cells were exclusively EGFP-positive, mCherry-negative cells (Figure 7G).

Because no oG was provided, RV must have transduced them directly due to leaky expression of TVA.

These cells displayed no mCherry signal making the TVA “invisible”. This suggests that RV-EGFP-pos-itive, mCherry-negative cells at the injection site in our control experiments result most likely from direct RV uptake. Therefore, recombinase-independent expression of “invisible” TVA and not oG is responsi-ble for the exclusively green cells at the injection site in our control experiments.

In sum, these control experiments indicate that our intersectional viruses allow the specific tracing of long-range projections to GABAergic PV cells. Tracing of local circuits is slightly confounded be-cause transsynaptically labeled, RV-EGFP positive cells are indistinguishable from cells transduced by RV-EGFP directly due to “invisible” TVA leak.