Monosynaptic Tracing Success Depends Critically on Helper Virus Concentrations

Abstract

Monosynaptically-restricted transsynaptic tracing using deletion-mutant rabies virus (RV) has become a widely used technique in neuroscience, allowing identification, imaging, and manipulation of neurons directly presynaptic to a starting neuronal population. Its most common implementation is to use Cre mouse lines in combination with Cre-dependent "helper" adeno-associated viral vectors (AAVs) to supply the required genes to the targeted population before subsequent injection of a first-generation (ΔG) rabies viral vector. Here we show that the efficiency of transsynaptic spread and the degree of nonspecific labeling in wild-type control animals depend strongly on the concentrations of these helper AAVs. Our results suggest practical guidelines for achieving good results.

Keywords: AAV (adeno-associated virus); circuit tracing; monosynaptic tracing; rabies; virus.

Figure 1

Strategy for monosynaptic tracing with helper adeno-associated viral vectors (AAVs). Helper viruses are injected in a Cre mouse, or a Cre-negative mouse in the case of control experiments, then rabies virus (RV; ΔG, EnvA-coated, expressing mCherry) is injected in the same location subsequently. While different labs have used various intervals and survival times, we used a 7-day interval between AAV and RV injections, and another 7-day interval between RV injection and perfusion, in all experiments for this article. Brain image adapted with permission from the Allen Mouse Brain Atlas.

Figure 2

Use of helper viruses at excessive concentrations can result in near-complete failure of monosynaptic tracing. The two-AAV combination described in Liu et (; AAV1-synP-FLEX-splitTVA-EGFP-B19G mixed with AAV1-TREtight-mTagBFP2-B19G) was injected in the somatosensory cortex of PV-Cre × Ai65F (FLPo-dependent tdTomato reporter) mice, followed by RVΔG-4FLPo(EnvA) 7 days later. (A–H) Very poor results were obtained when using new preparations of these AAVs undiluted. (A) Injection site in S1. Green = anti-EGFP staining, blue = mTagBFP2, red = tdTomato. Individual channels from this field are shown in panels (E–H). (B) No labeled neurons were found in the ipsilateral secondary somatosensory cortex (S2). (C) Very few labeled neurons were found in ipsilateral thalamus (VPL and VPM). (D) No labeled neurons were found in contralateral S1. (E–H) Individual channels from the field shown in panel (A). (E) Anti-parvalbumin staining (not shown in panel A). (F) Anti-EGFP staining, indicating expression from the first, Cre-dependent AAV. (G) mTagBFP2, indicating expression from the second, tTA-dependent AAV. (H) tdTomato, reporting activity of the FLPo-encoding RV. (I,L) Injection site after using undiluted viruses in two-helper combination in Cre-negative animal: many labeled cells are seen. (I) Overlay of (J–L). (J) Anti-EGFP staining, (K) mTagBFP2 signal, (L) tdTomato marking RV labeling. Scale bar in (A): 200 μm, applies to all panels.

Figure 3

The use of insufficiently-diluted helper viruses results in excessive background labeling in Cre-negative animals. (A–H) Diluting the helper viruses to concentrations matching previously used preparations gave much better results. (A) Injection site in S1; individual channels from this field are shown in panels (E–H). (B–D) Many labeled presynaptic neurons were found in ipsilateral secondary somatosensory cortex (B), ipsilateral thalamus (VPL and VPM; C), and contralateral S1 (D). (E) Anti-parvalbumin staining (not shown in panel A). (F) Anti-EGFP staining, indicating expression from the first, Cre-dependent AAV. (G) mTagBFP2, indicating expression from the second, G-encoding AAV. (H) tdTomato, reporting activity of the FLPo-encoding RV. (I–L) Even with the AAVs diluted to match the titers of previous batches, excessive background labeling is seen at the injection site. (I) Overlay of (J–L). (J) Anti-EGFP staining, (K) mTagBFP2 signal, (L) tdTomato marking RV labeling. Scale bar in (A): 200 μm, applies to all panels.

Figure 4

Quantification of results in PV-Cre and Cre-negative mice: a pilot study with helper viruses either undiluted or diluted to titers used in previous work. Black depicts numbers of neurons labeled by RV in the contralateral cortex (in every other 50 μm section) in PV-Cre mice; red depicts numbers of RV-labeled neurons in the vicinity of the injection site, i.e., in ipsilateral cortex (in every other 50 μm section) in Cre-negative mice for all conditions. Diamonds represent cell counts from individual mice; the middle lines in the boxes represent the average count for each condition. Numbers in green represent the ratio of contralateral neurons in Cre+ mice to ipsilateral neurons in Cre− mice. “Diluted” here means diluted to the titers of other batches used previously in our laboratory; see main text for details. Excessive concentrations of helper viruses gave very poor results. Source numbers are provided in Supplementary Table S1.

Figure 5

Quantification of results in PV-Cre and Cre-negative mice: systematic dilution series. (A) Results of varying the concentrations of the two helper viruses in the tTA-TRE combination system. The highest ratio of contralateral neurons in Cre+ mice to ipsilateral neurons in Cre− mice was obtained with a 1:200 dilution of AAV1-syn-FLEX-splitTVA-EGFP-tTA and a 1:20 dilution of AAV1-TREtight-mTagBFP2-B19G (“0.005 × tTA and 0.05 × TRE” in the figure). (B) Results of varying the concentration of the single helper virus AAV1-syn-FLEX-splitTVA-EGFP-B19G. Higher dilutions (out to 1:33.3) give higher ratios of contralateral neurons in Cre+ mice to ipsilateral neurons in Cre− mice, but results with the single-helper approach were nowhere near as good as with the two-helper combination. n = 2 for all conditions. Diamonds represent cell counts from individual mice; the middle lines in the boxes represent the average count for each condition. Source numbers are provided in Supplementary Table S2.

Figure 6

Example results using recommended dilutions of two-helper combination. (A–H) Use of the two-AAV combination at 1:200 and 1:20 dilutions (see main text) labeled many presynaptic neurons. (A) Injection site in S1. Green = anti-EGFP staining, blue = mTagBFP2, red = mCherry. Individual channels from this field are shown in panels (E–H). (B) Many labeled neurons were found in ipsilateral S2. (C) Many labeled neurons were found in ipsilateral thalamus (VPL, VPM, and Po). (D) Many labeled neurons were found in contralateral S1. (E–H) Individual channels from the field shown in panel (A). (E) Anti-parvalbumin staining (not shown in panel A). (F) Anti-EGFP staining, indicating expression from the first, Cre-dependent AAV. Note that, at this dilution, the EGFP signal is quite dim even with immune amplification. (G) mTagBFP2, indicating expression from the second, tTA-dependent AAV. (H) mCherry, indicating the presence of the ΔG RV. (I–L) Injection site after using two-helper combination at 1:200 and 1:20 (see main text): few mCherry-labeled cells are seen. (I) Overlay of (J–L). (J) Anti-EGFP staining: no signal is visible, even with amplification. (K) mTagBFP2 signal. A few blue cells are seen even at these dilutions. (L) mCherry expressed by RV. Scale bar in (A): 200 μm, applies to all panels.

Figure 7

Results using recommended dilutions of the single tricistronic helper. (A–G) Use of the single tricistronic helper AAV at 1:10 dilution (see main text) also labeled many presynaptic neurons (but see Figure 7). (A) Injection site in S1; individual channels from this field are shown in panels (E–G). (B) Labeled neurons in ipsilateral S2. (C) Labeled neurons in ipsilateral thalamus (VPL, VPM, and Po). (D) Labeled neurons in contralateral S1. (E) Anti-parvalbumin staining (not shown in panel A). (F) Anti-EGFP staining, indicating expression from the first, Cre-dependent AAV. (G) mCherry, indicating the presence of the ΔG RV. (H–J) Injection site after using a single tricistronic helper: many mCherry-labeled neurons are present. (H) Overlay of (I,J). (I) Anti-EGFP staining: the significant signal is seen even in these Cre-negative mice. (J) mCherry expressed by RV. Scale bar in (A): 200 μm, applies to all panels.

Figure 8

Inputs to midbrain dopaminergic cells using recommended dilutions of two-helper combination. (A–H) Results in DAT-Cre mice. (A) Injection site in substantial nigra reticulata (SNR): overlay of panels (F–H). (B) RV-labeled cells in the dorsal striatum. (C) RV-labeled cells in ventral striatum. (D) RV-labeled cells in the cortex. (E) Anti-tyrosine hydroxylase (TH) staining (not shown in panel A). (F) Anti-EGFP staining, indicating expression from the first, Cre-dependent AAV. (G) mTagBFP2, indicating expression from the second, tTA-dependent AAV. (H) tdTomato, reporting activity of the FLPo-encoding RV. (I–L) Results in Cre-negative mice (injection site shown). (I) Overlay of (J–L). (J) anti-EGFP staining: no signal is visible, even with immunostaining. (K) mTagBFP2 signal. no signal is visible, even with amplification. (L) tdTomato, reporting activity of the FLPo-encoding RV. Only one labeled cell is visible. Scale bar in (A): 200 μm, applies to all panels.