Skip to main content

Recombinase-independent AAV for anterograde transsynaptic tracing


Viral transsynaptic labeling has become indispensable for investigating the functional connectivity of neural circuits in the mammalian brain. Adeno-associated virus serotype 1 (AAV1) allows for anterograde transneuronal labeling and manipulation of postsynaptic neurons. However, it is limited to delivering an AAV1 expressing a recombinase which relies on using transgenic animals or genetic access to postsynaptic neurons. We reasoned that a strong expression level could overcome this limitation. To this end, we used a self-complementary AAV of serotype 1 (scAAV1) under a strong promoter (CAG). We demonstrated the anterograde transneuronal efficiency of scAAV1 by delivering a fluorescent marker in mouse retina-superior colliculus and thalamic-amygdala pathways in a recombinase-independent manner in the mouse brain. In addition to investigating neuronal connectivity, anterograde transsynaptic AAVs with a strong promoter may be suitable for functional mapping and imaging.


Significant progress has been made in the fields of neurophysiology and neuroanatomy since the discovery of viral approaches to map out brain connectivity. The viral approach is a powerful tool to label interconnected brain regions, allowing cell-type-specific identification and manipulation. Retrograde mapping of the presynaptic inputs to a specific brain region is achievable via different viral tools that vary in their degree of specificity (axonal vs. transsynaptic transport) and efficiency [1, 2]. Anterograde tracing has remained largely limited to labeling axonal input to the downstream postsynaptic region. However, the recent discovery of the adeno-associated virus serotype 1 (AAV1) property to label postsynaptic neurons transsynaptically advanced the field. AAV1 expressing a recombinase has been used in various studies to label and manipulate postsynaptic neurons. The major limitation is that a recombinase-dependent transgene should be expressed in downstream targets, which is doable only via transgenic animals or, alternatively, via viral expression postsynaptically, which requires prior knowledge of the target [1, 3, 4]. This study aims to optimize a method allowing recombinase-independent anterograde transsynaptic labeling of postsynaptic neurons in the mammalian brain.


We optimized the expression parameters to achieve stronger expression of an AAV1 vector that carries a green fluorescent protein (GFP). The vector is self-complementary, I.e., carrying double-stranded DNA and under short-CAG as a promoter at a high titer (>1.0 × 10E13 vg/ml) [5]. To test the efficiency of the vector, we conducted two separate experiments in well-characterized long-range monosynaptic and non-reciprocal visual and auditory pathways. In the first experiment, we injected the vector into the mouse retina and imaged the downstream target in the superior colliculus (SC) [6, 7]. In the second experiment, the virus was injected into the medial geniculate nucleus (MGn), and we imaged the basolateral amygdala (BLA) [8,9,10,11]. We also evaluated whether an AAV1 vector with an alternative (and possibly weaker) promoter, human Synapsin (hSyn), would yield a comparable level of transsynaptic anterograde labeling.

We delivered the scAAV1 GFP under either short.CAG or hSyn to the retina, and 3 weeks later, we harvested the brains and retinae to examine the GFP labeling postsynaptically (Fig. 1A–H). The two vectors were delivered separately at an equal titer and volume. We observed that scAAV1.short.CAG.GFP resulted in a significantly higher number of transsynaptically labeled GFP neurons in the SC, in comparison to scAAV1.hSyn.GFP (Fig. 1C–E). In the mice injected with scAAV1.short.CAG.GFP in the retina, we observed that the contralateral SC had robust neuronal GFP labeling with clear neuronal morphology. In contrast, the ipsilateral had very sparse labeling, which is consistent with basic retinal-collicular anatomy (Fig. 1F, G). Unlike the efficient transsynaptic anterograde labeling we observed in the SC from the retina, we did not observe any somatic labeling in the lateral geniculate nucleus (LGN). This lower labeling efficiency in the LGN compared to the SC is consistent with the original study where AAV1 has been reported to show anterograde transsynaptic labeling properties [3]. Next, we tested the intracerebral efficiency by injecting the scAAV1.short.CAG.GFP in the MGn. This resulted in robust GFP labeling only in the ipsilateral BLA with clear neuronal morphology. However, there was no labeling in the contralateral BLA, which is consistent with the known neuroanatomy (Fig. 1I–L) [8, 9]. To further confirm the directionality, from the MGn to the BLA, we injected a retro AAV2 vector expressing mRuby in the MGn and imaged downstream and upstream regions of the MGn. We did not observe any neural labelling in the BLA, as a downstream target. However, we observed numerous labelling in an MGn-upstream region cortical, the auditory cortex, consistent with the reported corticothalamic circuitry [11, 12] (Additional file 1: Fig. S1A–D). To confirm the neuronal specificity, we co-stained for the astrocytic marker, glial fibrillary acidic protein, GFAP, in the BLA. We did not observe any co-labeling of the transneuronally labelled GFP cells and the GFAP positive cells (Additional file 1: Fig. S1E).

Fig. 1
figure 1

A Diagram showing the experimental timeline for the retinal injection of either scAAV1.hSyn.GFP (n = 3) or scAAV1.short.CAG.GFP (n = 4). B Image of a retina injected with scAAV1.short.CAG.GFP. C, D Representative image showing GFP-labeled neurons in the SC after scAAV1.hSyn.GFP (C) and scAAV.1 short.CAG.GFP (D) retinal injection; arrows point to labelled neurons. E The number of GFP-positive neurons in the SC is significantly higher in mice with scAAV1.short.CAG.GFP retinal injection (n = 4) compared to mice with scAAV1.hSyn.GFP retinal injection (n = 3; Unpareid t-test, p = 0.0027). F–H Representative images from the ipsilateral SC (F), contralateral SC (G), and LGN (H). The insets below are high-magnification images of the boxed regions. I Diagram showing the injection of AAV.1 short.CAG.GFP in the MGn and the anterograde transsynaptically labeled neurons in the BLA (n = 4). J Representative image of the injection site in the MGn. K, L Representative images showing that the ipsilateral BLA has GFP-positive neurons; arrows point to labelled neurons (L) after AAV1.short.CAG.GFP in the MGn, while the contralateral BLA (K) does not. The boxes below are high magnification of the respective regions. Results are reported as mean ± SEM. **p < 0.01


Here we investigated the possibility of achieving anterograde transsynaptic labeling in a recombinase independent manner using an AAV1 vector. We demonstrated that scAAV1.short.CAG. GFP could transneuronally label downstream postsynaptic targets with a single virus injection in wild-type mice. After injecting the vector into the retina and the MGn, we observed reliable labeling in the SC and the BLA, respectively. Single-stranded AAV1 CAG GFP was reported to lack transneuronal anterograde labeling capacity, which might be due to the low expression level [3]. Consistent with this hypothesis, we observed that scAAv1.short.CAG.GFP yields more robust transsynaptic labeling in comparison to a possibly weaker promoter like scAAV1.hSyn.GFP. Our results indicate that the transsynaptic transfer of transgenes that express optogenetic/chemogenetic actuators might be achievable by using the AAV1 expressing the transgene of interest in the mammalian brain. Similarly, in an avian model, avian-adenoassociated virus (an AAV variant) shows recombinase-independent transneuronal labeling, putatively with a similar underlying mechanism to the AAV1 [13]. These viral tools will expand the potential utility of viral approaches for circuit interrogation in a pathway and cell-type specific manner. Future studies will be needed to optimize AAV-mediated transsynaptic tracing to allow for strict anterograde labeling and, thereby, make it applicable in reciprocally connected brain regions. Similarly, optimizations that allow for using AAV1 at a lower titer while maintaining the anterograde transsynaptic property might reduce the toxicity at the injection site.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request.


  1. Luo L, Callaway EM, Svoboda K. Genetic dissection of neural circuits: a decade of progress. Neuron. 2018;98(2):256–81.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Tervo DGR, Hwang BY, Viswanathan S, Gaj T, Lavzin M, Ritola KD, et al. A designer AAV variant permits efficient retrograde access to projection neurons. Neuron. 2016;92(2):372–82.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Zingg B, Chou XL, Zhang ZG, Mesik L, Liang F, Tao HW, Zhang LI. AAV-mediated anterograde transsynaptic tagging: mapping corticocollicular input-defined neural pathways for defense behaviors. Neuron. 2017;93(1):33–47.

    Article  CAS  PubMed  Google Scholar 

  4. Zingg B, Peng B, Huang J, Tao HW, Zhang LI. Synaptic specificity and application of anterograde transsynaptic AAV for probing neural circuitry. J Neurosci. 2020;40(16):3250–67.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. McCarty DM, Monahan PE, Samulski RJ. Self-complementary recombinant adenoassociated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis. Gene Ther. 2001;8(16):1248–54.

    Article  CAS  PubMed  Google Scholar 

  6. Huerta MF, Harting JK. Connectional organization of the superior colliculus. Trends Neurosci. 1984;7(8):286–9.

    Article  Google Scholar 

  7. Kitanishi T, Tashiro M, Kitanishi N, Mizuseki K. Intersectional, anterograde transsynaptic targeting of neurons receiving monosynaptic inputs from two upstream regions. Commun Biol. 2022;5(1):149.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Barsy B, Kocsis K, Magyar A, Babiczky Á, Szabó M, Veres JM, et al. Associative and plastic thalamic signaling to the lateral amygdala controls fear behavior. Nat Neurosci. 2020;23(5):625637.

    Article  Google Scholar 

  9. Hintiryan H, Bowman I, Johnson DL, Korobkova L, Zhu M, Khanjani N, et al. Connectivity characterization of the mouse basolateral amygdalar complex. Nat Commun. 2021;12(1):2859.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Khalil V, Faress I, Mermet-Joret N, Kerwin P, Yonehara K, Nabavi S. Subcortico-amygdala pathway processes innate and learned threats. Elife. 2023;12:e85459.

    Article  PubMed  PubMed Central  Google Scholar 

  11. LeDoux JE, Ruggiero DA, Reis DJ. Projections to the subcortical forebrain from anatomically defined regions of the medial geniculate body in the rat. J Comparat Neurol. 1985;242(2):182–213.

    Article  CAS  Google Scholar 

  12. Bartlett EL, Stark JM, Guillery RW, Smith PH. Comparison of the fine structure of cortical and collicular terminals in the rat medial geniculate body. Neuroscience. 2000;100(4):811–28.

    Article  CAS  PubMed  Google Scholar 

  13. Ito T, Ono M, Matsui R, Watanabe D, Ohmori H. Avian adeno-associated virus as an anterograde transsynaptic vector. J Neurosci Methods. 2021;359: 109221.

    Article  PubMed  Google Scholar 

Download references


We thank members of the Nabavi laboratory for their suggestions. We thank Jean-Charles Paterna and Viral Vector Facility (VVF) of the Neuroscience Center Zurich (ZNZ) for their support. Figures were Created with


This study was supported by ERC starting Grant (22736), by Independent Research (DFF), Novo Nordisk. Foundation (NNF16OC0023368), and AUFF NOVA grants to SN. Additionally, SN was supported by the Danish Research Institute of Translational Neuroscience (19958), and by PROMEMO (Center of Excellence for Proteins in Memory funded by the Danish National Research Foundation) (DNRF133). HY was supported by The Lundbeck Foundation LF Postdocs (R347-2020-2299), Daiichi Sankyo Foundation of Life Science, and The Uehara Memorial Foundation research fellowship.

Author information

Authors and Affiliations



IF and SN conceived and designed the study. IF wrote the original draft with inputs from VK, HY, KY, and SN. IF, VK, KY, and SN designed the experiments. IF, VK, HY, and SS performed the experiments. VK made the figure.

Corresponding author

Correspondence to Islam Faress.

Ethics declarations

Ethics approval and consent to participate

All the procedures were performed on C57BL/6JRJ wildtype (Janvier, France). Mice were naïve and acclimated to the vivarium for at least a week before the beginning of the experiment. The mice were 6–8 weeks old at the beginning of the experimental procedures. Animals were group-housed (3–4 per cage) with enriched conditions in a 12 h light/dark cycle (the light switches on at 6 A.M.) with constant levels of humidity and temperature (22 ± 1 °C). Food and water were provided ad libitum. Experiments were conducted at Aarhus University in the Biomedicine department, Ole Worms Allé 8, Aarhus 8000. All the experimental procedures were conducted according to the Danish Animal Experiment Inspectorate.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1

: Figure S1 A Diagram showing the injection of Retro/AAV.2hSyn.mRuby in the MGn. Representative image of the injection site in the MGn. Representative images showing DAPI nuclear staining (left), and the lack of retrograde labeling and dense axonal labeling (middle) in the BLA. The right panel shows the merge between the nuclear staining and the mRuby axonal labeling. The zoomed-in image shows the lack of overlap between the nuclear staining and the mRuby labeling. Representative images of DAPI nuclear labeling (left) and axonal and retrograd neuronal mRuby expression in the auditory cortex (Actx) from the same mouse shown in panels B and C. The right panel shows a zoomed-in image of the merge of the nuclear staining and the mRuby labeling. Representative images of DAPI nuclear labeling (left) and anterograde transsynaptically GFP labeled neurons (middle). Glial fibrillary acidic protein (GFAP) expressing cells in magenta and a merge between the previous panels on the three images of an ipsilateral amygdala from a mouse injected with scAAV1.short.CAG.GFP in the MGn (n=4). The images show the lack of colocalization between the transsynaptically labeled GFP+ neurons and the GFAP+ cells in the amygdala.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Faress, I., Khalil, V., Yamamoto, H. et al. Recombinase-independent AAV for anterograde transsynaptic tracing. Mol Brain 16, 66 (2023).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI:


  • AAV
  • Transsynaptic
  • Neural-circuit
  • Viral-tracing
  • Neuroanatomy