ATLAS: a rationally designed anterograde transsynaptic tracer

Rivera, Jacqueline F.; Huang, Haoyang; Weng, Weiguang; Sohn, Heesung; Girasole, Allison E.; Li, Shun; Albanese, Madeline A.; Qin, Melissa; Tao, Can; Klug, Molly E.; Rao, Sadhna; Paletzki, Ronald; Herring, Bruce E.; Kanoski, Scott E.; Zhang, Li I.; Gerfen, Charles R.; Sabatini, Bernardo L.; Arnold, Don B.

doi:10.1038/s41592-025-02670-x

Download PDF

Article
Open access
Published: 01 May 2025

ATLAS: a rationally designed anterograde transsynaptic tracer

Nature Methods volume 22, pages 1101–1111 (2025)Cite this article

15k Accesses
1 Citations
78 Altmetric
Metrics details

Subjects

Abstract

Genetically modified rabies virus can map neural circuits retrogradely from genetically determined cells. However, similar tools for anterograde tracing are not available. Here, we describe a method for anterograde transsynaptic tracing from genetically determined neurons based on a rationally designed protein, ATLAS. Expression of ATLAS in neurons causes presynaptic release of a payload composed of an antibody-like protein, AMPA.FingR, which binds to the N terminus of GluA1, and a recombinase. In the synaptic cleft, AMPA.FingR binds to GluA1, causing the payload to be endocytosed into postsynaptic cells and delivered to the nucleus, where it triggers expression of a recombinase-dependent reporter. In mice, ATLAS mediates monosynaptic transneuronal tracing from random or genetically determined cells that is strictly anterograde, synaptic and nontoxic. Moreover, ATLAS-mediated tracing shows activity dependence, suggesting that it can label active circuits underlying specific behaviors. Finally, ATLAS is composed of modular components that can be independently replaced or modified.

Multiple Transcriptomic Analyses Explore Potential Synaptic Biomarker Rabphilin-3A for Alzheimer's Disease

Article Open access 12 August 2024

The molecular cytoarchitecture of the adult mouse brain

Article Open access 13 December 2023

Non-synaptic function of the autism spectrum disorder-associated gene SYNGAP1 in cortical neurogenesis

Article 09 November 2023

Main

Neural circuits underlying sleep¹, interoception², tactile sensory processing³, sexual behavior⁴, digestion⁵ and parenting⁶ have been studied by identifying critical cellular components using activity-dependent reporters and activating those cells optogenetically or through sensory stimulation. However, fully defining the anatomy and function of these circuits requires the identification of cells upstream and downstream of starter cells. This task is difficult due to the heterogeneity of cell types present in relatively small volumes of neural tissue⁷. Thus, classical tracing methods using proteins such as wheat germ agglutinin and horse radish peroxidase⁸, which are taken up by most neurons, cannot be used to define most neuronal circuits precisely.

A genetically modified form of rabies virus was developed that specifically infects neurons expressing the Avian sarcoma leucosis virus receptor, TVA, enabling tracing from specific cell types⁹. Neural circuits carry information from presynaptic to postsynaptic cells, or anterogradely, but modified rabies virus infects neurons that are presynaptic to starter neurons, traveling retrogradely¹⁰. In addition, it is engineered to jump a single synapse, allowing for precise mapping^11,12. Although modified rabies virus has been broadly used for tracing circuits retrogradely¹³, there is currently no comparable method for tracing circuits from genetically determined cells anterogradely. In particular, although two viruses have recently been used for anterograde tracing from nonspecific cells, an adeno-associated virus (AAV1) and a genetically modified yellow fever virus (YFV), neither can mediate tracing from genetically determined cells^14,15,16.

Here, we introduce ATLAS (anterograde transsynaptic label based on antibody-like sensors), a rationally designed protein that mediates anterograde transsynaptic labeling from genetically determined cells. ATLAS can be expressed specifically in Cre-expressing neurons in transgenic animals and label neurons to which they project. Labeling is strictly anterograde, monosynaptic and activity-dependent in vivo. Furthermore, we provide direct evidence that transneuronal labeling mediated by ATLAS is exclusively transsynaptic. Finally, we show that components of ATLAS can be independently replaced, demonstrating that it is a versatile platform that can be upgraded or modified to perform specific tasks.

Results

Generating AF, a binder to the N terminus of GluA1

We used messenger RNA (mRNA) display, an in vitro selection procedure¹⁷, to identify binders to amino acids 1–394 of Rat GluA1 from a library of mRNA–protein fusions encoding the 10FNIII domain of human FN1, with 17 random residues engineered into the BC and FG loops^18,19. We expressed the selected fibronectin clones in COS7 cells with GluA1 fused to a hemagglutinin tag (GluA1-HA) and Stargazin-V5. Nonpermeabilized labeling of MYC showed that four fibronectin (Fn) clones labeled the surface of COS7 cells colabeled for HA (Extended Data Fig. 1). Following expression in dissociated cultures of cortical neurons, clone Fn9.3 gave the most robust labeling (Extended Data Fig. 1). We refer to this clone as AMPA.FingR (AF; FingR stands for fibronectin intrabody generated with mRNA display¹⁸).

We generated AF-HA protein and found that it bound specifically to a band colabeled with a GluA1 antibody on a western blot of neuronal lysate (Fig. 1a,b). To characterize AF further, we coexpressed a secreted version of it, ss-AF-YFP (ss refers to the GluA1 signal sequence), in dissociated cortical neurons with PSD-95.FingR-tagRFP, which labels postsynaptic sites¹⁸. We found that AF labeling partially overlapped with PSD-95.FingR-tagRFP, suggesting a perisynaptic localization (Fig. 1c and Extended Data Fig. 1). AF also colocalized with endogenous clathrin, consistent with localization to endocytic zones (Fig. 1d). We then tested whether AF-HA protein is endocytosed by adding it to the medium of cortical neurons in culture. We found that virtually all neurons displayed HA labeling (37 of 38, n = 5 independent cultures, Fig. 1e) within 1 h. Furthermore, the addition of 10 mg ml⁻¹ chlorpromazine, a clathrin inhibitor, significantly reduced HA labeling (4.2 ± 0.8 versus 2.1 ± 0.5, P = 0.012, n = 5 cultures, Mann–Whitney U-test, Fig. 1f), confirming that internalization was mediated by endocytosis. All values are expressed as mean ± s.e.m.

**Fig. 1: Mechanisms of release and uptake by ATLAS.**

Since AF binds to GluA1 receptors and is endocytosed postsynaptically, we reasoned that we could make a transsynaptic tracer if we targeted AF to synaptic vesicles by fusing it to the luminal domain of the presynaptic protein, synaptobrevin 2 (VAMP2) (ref. ²⁰). We inserted a BACE1 (β-secretase) cleavage site (BACEcs)²¹ between AF and VAMP2 (to give VAMP2-BACEcs-AF), enabling endogenous BACE to cleave AF from VAMP2, leaving it free to exit the lumen of the synaptic vesicle on exocytosis. In addition, an ALFA-tag (At) was added to label VAMP2 (ref. ²²). To test our strategy, we substituted super ecliptic pHluorin (SEP)²⁰ for AF and coexpressed the resulting plasmid (VAMP2-At-BACEcs-SEP, Fig. 1b) with PSD-95.FingR-tagRFP in cortical neurons in culture. The release from acidic synaptic vesicles into the neutral pH of the synaptic cleft is expected to cause a sudden increase in the fluorescence of SEP, allowing it to be visualized²⁰. Following overnight expression of VAMP2-At-BACEcs-SEP in neuronal cultures, we imaged SEP using widefield epifluorescence microscopy. We saw flashes of fluorescence lasting approximately 2–8 s (images taken at 1 Hz) appearing at points opposite postsynaptic sites (Fig. 1g). This result is consistent with VAMP2 delivering SEP to synaptic vesicles, BACE cleaving off SEP, and SEP being released into the synaptic cleft. We also fused AF to SEP and expressed the resulting protein (VAMP2-At-BACEcs-AF-SEP, Fig. 1b) in culture and observed flashes that were brighter and decayed with a longer time constant (6 ± 2 versus 405 ± 60 s, n = 5 independent cultures, Fig. 1h,i), a significant difference (P < 0.0001, t-test). This result is consistent with AF binding to the postsynaptic membrane following release and remaining until it is endocytosed. To further test whether AF-SEP is released from synaptic vesicles, we examined whether depolarization increases the number of release events. We imaged the same cultures before and after adding 100 mM KCl and counted the number of bright flashes corresponding to release events. We found a roughly 15-fold increase in release events with KCl (50 ± 6 versus 3 ± 1 for control, P = 0.02, t-test, n = 5 independent cultures; Fig. 1j–l). Together, these results are consistent with AF-SEP being released from synaptic vesicles, moving across the synaptic cleft, binding to the adjacent postsynaptic neuron perisynaptically and then being endocytosed.

Given that AF likely binds to the extracellular N-terminal domain of GluA1, we tested whether the expression of ss-AF-YFP affects synaptic transmission to hippocampal CA1 neurons. We found that in cells in hippocampal slices transfected with ss-AF-YFP using biolistics, the amplitudes of evoked excitatory postsynaptic currents (eEPSCs) of AMPA (n = 10, P = 0.31, t-test), NMDA receptors (n = 8, P = 0.70, t-test) and paired-pulse facilitation (n = 6, P = 0.19, t-test) were not significantly different from control cells, consistent with the expression of AF not affecting synaptic transmission (Fig. 1m–p). All pairs of recordings were made from distinct cultured slices and, thus, each is a biological replicate.

Transsynaptic tracing in vitro and in vivo

To generate a protein capable of mediating transsynaptic tracing, we replaced SEP with Cre in VAMP2-At-BACEcs-AF-SEP to give VAMP2-At-BACEcs-AF-Cre (ATLAS_Cre, Fig. 1b). Following expression of ATLAS_Cre, AF-Cre should be released into the synaptic cleft and then bind to AMPA receptors at the postsynaptic membrane. Subsequently, it needs to be endocytosed and carried to the nucleus to provide genetic access to the postsynaptic cell (Fig. 2a). To facilitate this final step, we added the DNA binding domain from CCR5 (refs. ^18,23) to the ATLAS protein, as it contains a robust nuclear localization signal. To test our strategy, we transfected ATLAS_Cre into dissociated cultures infected with AAV encoding a floxed green fluorescent protein (GFP) reporter (AAV8-DIO-GFP). We observed cells with GFP and Cre that were not labeled with the starter cell marker, At, consistent with the transneuronal transport of Cre and recombination of the floxed allele, implying transport of Cre to the nucleus (Fig. 2b–e and Extended Data Fig. 2). Furthermore, At-labeled presynaptic structures were found to be overlapping or immediately adjacent to dendrites belonging to each of the GFP-labeled neurons (Extended Data Fig. 2). Thus, our results are consistent with ATLAS_Cre mediating transsynaptic tracing and providing genetic access to postsynaptic cells.

**Fig. 2: Schematic of ATLAS transsynaptic transport and testing in vitro.**

To test our strategy in vivo, we injected AAV8-ATLAS_cre into the medial prefrontal cortex (mPFC) and AAV8-DIO-mCherry into the striatum (Str) of an adult mouse (Fig. 3a). After 1 week, we found At staining in mPFC and abundant mCherry staining in Str (Fig. 3b,c). In contrast, there was negligible mCherry staining in a control experiment without AAV8-ATLAS_cre (Fig. 3d). Because the projection from mPFC to Str is strictly anterograde²⁴, this result is consistent with AAV8-ATLAS_cre mediating the transsynaptic transport of Cre into the nucleus of the postsynaptic cell, causing mCherry labeling.

**Fig. 3: ATLAS mediates transsynaptic tracing in vivo.**

Tracing from genetically determined cells

To mediate genetically determined tracing, we generated AAV8-DIO-ATLAS_FLP (Fig. 1b), where expression of ATLAS is Cre-dependent and the payload is FLP (Flip recombinase). To test this construct, we traced the projection from the primary visual cortex (V1) to the superior colliculus (SC), which is strictly anterograde²⁵. We coinjected Lenti-CamkII-Cre (a lentivirus expressing Cre specifically in excitatory neurons) and AAV8-DIO-ATLAS_FLP into V1 and AAV8-fDIO-mCherry into SC of wild-type mice. In addition to At staining in V1, we saw abundant staining for mCherry in SC (391 ± 37 cells, n = 7 samples from three mice, Fig. 3e–h and Supplementary Table 1) and a small number of faint cells on the control (35 ± 6, n = 6 samples from three mice), which was subtracted from the previous count. All cell counts are expressed as the number of cells per 0.1 mm unless otherwise specified to allow for comparisons. An advantage of this strategy is that lentivirus has no intrinsic retrograde or anterograde transneuronal transport that could cloud the interpretation of the results, unlike virtually every pseudotype of AAV¹⁵.

To test ATLAS tracing in transgenic Cre mouse lines, we examined the projection from the dorsal lateral geniculate nucleus (dLGN) to V1 in a VIPR2-Cre line expressing Cre in neurons in the dLGN²⁶. We injected AAV8-DIO-ATLAS_FLP into dLGN and AAV8-fDIO-mCherry into V1. Staining of At was found specifically in the dLGN, and mCherry staining in V1 (267 ± 54 cells, n = 5 samples from three mice) was dense in layer IV and less dense in layers II/III and V (Fig. 3i–l), consistent with known anatomy²⁷. These results are consistent with ATLAS tracing anterogradely from Cre-expressing neurons in wild-type and transgenic mice.

Increasing efficiency of transsynaptic labeling with BACE

To measure the efficiency of ATLAS tracing, we injected AAV8-ATLAS_Cre in mPFC and AAV8-DIO-mCherry in Str and counted the number of labeled cells as before after 1 week of incubation. Initially, we observed 144 ± 17 labeled cells (n = 6 samples from three mice). One possible inefficiency in our system is that there may be insufficient endogenous BACE in presynaptic cells to cleave all the AF-Cre from VAMP2. To test this hypothesis, we coexpressed exogenous BACE with ATLAS_Cre and measured the resulting efficiency. When we coinjected AAV8-BACE-HA and AAV8-ATLAS_Cre into mPFC in equal amounts, it did not significantly affect the number of postsynaptic cells labeled with mCherry (180 ± 20 cells, n = 6 samples from three mice, P = 0.38, Tukey’s multiple comparisons, Extended Data Fig. 3). However, when we injected AAV8-BACE-HA and AAV8-ATLAS_Cre in a 5:1 ratio (viral particles), we saw a significant increase in the number of cells in Str labeled with mCherry, compared to both AAV8-ATLAS + BACE 1:1 (P = 0.0027) and AAV-ATLAS alone (286 ± 17, P = 0.0002), n = 6 samples from three mice; Tukey’s multiple comparisons test, Extended Data Fig. 3 and Supplementary Table 1). With this ratio of BACE to ATLAS, we saw approximately 10% of postsynaptic neurons labeled (Extended Data Fig. 3). Thus, when supplemented with exogenous BACE, ATLAS labels cells in the Str at a similar density to YFV¹⁴.

Labeling is transsynaptic

Given its design, it is likely that transneuronal labeling mediated by ATLAS occurs exclusively through synapses. To test this hypothesis in vivo, we asked whether transneuronal labeling could happen from inhibitory neurons. Since the uptake of ATLAS is mediated by GluA1 receptors, which are not found at inhibitory synapses, if we expressed ATLAS in inhibitory neurons, any transneuronal labeling would have to be nonsynaptic (Fig. 4a). Accordingly, we injected AAV8-DIO-ATLAS_FLP and AAV8-fDIO-mCherry into V1 of an SST-Cre mouse, where somatostatin-positive inhibitory interneurons express Cre²⁸. When we examined labeling in the cortex after 2 weeks, we saw 483 cells labeled with mCherry and, of those, only 20 cells were not colabeled with At (Fig. 4b–d and Supplementary Table 1, n = 3 mice). This labeling rate was less than the background rate measured over an equivalent number of sections for control experiments where only AAV8-fDIO-mCherry was injected into V1 (100 cells, n = 3 mice), consistent with no transneuronal labeling above background. This result leads us to conclude that ATLAS labels postsynaptic neurons exclusively through the transsynaptic transfer of proteins.

**Fig. 4: ATLAS does not mediate nonsynaptic transneuronal labeling and transsynaptic labeling is specific.**

ATLAS tracing does not block synaptic transmission

To test whether ATLAS disrupts synaptic transmission, we coinfected AAV8-ATLASsn_Cre (Fig. 1b), AAV8-BACE-HA and AAV8-DIO-Channelrhodopsin 2 (ChR2)-GFP into the mPFC and AAV8-DIO-mCherry in the Str of wild-type mice. Since we found that ATLAS labels ~10% of target neurons in this circuit (Extended Data Fig. 3), we reasoned that it should be possible to find both labeled and nonlabeled cells that are synaptically connected to ChR2-expressing neurons to compare whether ATLAS labeling affects the cell’s physiological properties. Four weeks after the virus injection, we cut coronal slices of Str approximately 350 μm thick and incubated them in artificial cerebral spinal fluid for in vitro slice recording. Whole-cell patch clamp recording was performed to measure 470-nm optically evoked EPSC (eEPSC) from striatal cells (Extended Data Fig. 4). To evaluate the potential effect of ATLAS tracing on synaptic transmission, we compared the striatal cells with and without mCherry expression. Robust evoked EPSCs were recorded in mCherry-expressing cells with an onset latency of <5 ms following the light flash in eight out of eight cells (from three mice), indicating that the presynaptic ChR2-expressing cells were synaptically connected with mCherry-labeled cells (Extended Data Fig. 4). Furthermore, when labeled postsynaptic cells were recorded from, the amplitude of ChR2-evoked EPSCs exhibited a nonsignificant increase compared with those of unlabeled cells (Extended Data Fig. 4, n = 8 labeled cells, n = 7 unlabeled cells from three mice for both, P = 0.28, t-test). Thus, ATLAS did not block synaptic transmission. In addition, there were no significant changes in onset latency (P = 0.22, t-test) and time constant (P = 0.78, t-test, Extended Data Fig. 4). This result is consistent with transsynaptic labeling with ATLAS not causing detectable toxicity. Moreover, we verified that evoked currents are monosynaptic through the sequential application of Tetrodotoxin, which blocks action potentials and polysynaptic transmission, and 4-aminopyridine, which blocks K⁺ channels and facilitates direct activation of presynaptic terminals.

Electrophysiology confirms specificity of ATLAS tracing

To test for the specificity of ATLAS tracing, we measured optogenetically evoked currents in the circuit from the primary visual cortex to the Str²⁹. We coinfected AAV8-ATLASsn_Cre (Fig. 1b) and AAV8-BACE-HA in V1, and AAV8-DIO-mCherry/tdTomato in the dorsomedial Str (DMS). Two weeks later, we injected AAV8-DIO-ChR2-GFP in the same area in V1 as the first injection to manipulate ATLAS-labeled presynaptic inputs while minimizing expression competition between ATLAS and ChR2. Histology 2 weeks after the second injection revealed efficient expression of At, HA, ChR2 in V1 and Cre-dependent mCherry/tdTomato in DMS, consistent with the projection pattern of DMS-projecting V1 neurons (Extended Data Fig. 4). To compare V1 innervation between ATLAS-labeled versus unlabeled postsynaptic neurons, we measured optically evoked EPSCs (oEPSCs) following a 2-ms flash of 473-nm blue light in acute brain slices prepared from mice injected with virus 4 weeks earlier (Fig. 4e). We performed sequential, paired whole-cell recordings where we measured oEPSCs of an ATLAS-labeled (mCherry/tdTomato⁺) and an unlabeled (mCherry/tdTomato⁻) neuron in the same field of view (100 × 100 μm; Fig. 4e–h). Ten pairs of cells (from four mice) were recorded, in which ten out of ten labeled cells carried a current, compared with seven out of ten unlabeled cells (Fig. 4f). To assess the baseline connection probability of the V1-DMS circuit, we randomly patched onto DMS neurons in striatal areas where we saw prominent fiber labeling with GFP. Of the 17 random cells we patched (n = 3 mice), 41% of cells were connected (7 out of 17, Fig. 4f). oEPSCs recorded from labeled cells were significantly larger in amplitude and total charge compared with those of paired unlabeled cells (amplitude −134 ± 38 versus −66 ± 21 pA, P = 0.0020, Wilcoxon signed-rank test, total charge −0.121 ± 0.03 versus −0.038 ± 0.01 pC, P = 0.0020, Wilcoxon signed-rank test, n = 10 mice, plotted as absolute values in Fig. 4g,h). Furthermore, in ten out of ten pairs, the current recorded from the labeled cell had a larger amplitude than that from the unlabeled cell (Fig. 4h). Together, these results indicate that when ATLAS_Cre and a Cre-dependent reporter are simultaneously expressed in different brain regions, cells exhibiting Cre-dependent labeling are likely to receive synaptic inputs from cells expressing ATLASsn_Cre. Furthermore, they demonstrate that cells with stronger synaptic connections are more likely to be labeled by ATLAS.

ATLAS is exclusively anterograde and monosynaptic

A limitation of many virally based anterograde transneuronal labeling methods is that they also mediate labeling in the retrograde direction^14,15. To determine whether ATLAS mediates retrograde labeling in vivo, we injected AAV8-DIO-ATLAS_FLP and Lenti-hsyn-Cre into Str and AAV8-fDIO-mCherry into mPFC (Fig. 5a). Because the mPFC to Str projection is unidirectional and lentivirus does not spread retrogradely, any labeling in mPFC would be due to retrograde transneuronal labeling mediated by ATLAS. However, despite abundant At labeling in Str, indicating that ATLAS was expressed at a high level, we found no labeling above background in mPFC (20 ± 4 cells for experiment, n = 6 samples from three mice, versus 45 ± 14 for control, n = 6 samples from three mice, P = 0.11, t-test, Fig. 5b–e and Supplementary Table 1). These data are consistent with ATLAS not mediating retrograde labeling.

To test whether ATLAS can mediate transsynaptic labeling that is monosynaptic, a critical property for transsynaptic tracing results to be interpretable, we tried tracing the polysynaptic pathway from V1 to SC to the parabigeminal nucleus (PBG)¹⁴. We injected AAV8-ATLAS_Cre into V1 and AAV8-DIO-mCherry into SC and PBG (Fig. 5f). We found AT labeling in V1 and abundant transsynaptic mCherry labeling in SC (Fig. 5g,h) but no staining significantly above background in PBG (3 ± 1 cells for experiment, n = 8 samples from three mice, versus 19 ± 3 for control, n = 6 samples from three mice, P = 0.35, one-way analysis of variance (ANOVA) (Tukey’s test, Fig. 5i,n). Furthermore, when we injected AAV8-ATLAS_Cre into SC, we saw abundant labeling in PBG (181 ± 30 cells, n = 3 samples from three mice) that was significantly above both background and labeling from V1 to PBG (P < 0.0001 for both, one-way ANOVA, Tukey’s multiple comparisons, Fig. 5j–n), demonstrating that ATLAS is capable of tracing the SC to PBG pathway. Thus, transsynaptic labeling mediated by ATLAS is exclusively anterograde and monosynaptic.

Activity dependence of ATLAS

Because ATLAS mediates transsynaptic labeling by releasing AF-recombinase from synaptic vesicles, transsynaptic labeling is likely activity-dependent. To test for activity dependence of transsynaptic labeling in vivo, we injected AAV8-ATLASsn_Cre + AAV8-hHM3Dq-mCherry, an excitatory Dreadd³⁰, into mPFC and AAV8-DIO-GFP into Str (Extended Data Fig. 5) in two mice. We then injected one mouse daily with clozapine N-oxide (CNO) intraperitoneally but not the other. After 1 week, we perfused the mice and performed immunocytochemisty. We found abundant c-FOS labeling in the mPFC of the CNO+ mouse, which overlapped with At labeling (Extended Data Fig. 5 and Supplementary Table 1). In contrast, there was negligible c-FOS labeling in the mPFC of the CNO⁻ mouse (Extended Data Fig. 5 and Supplementary Table 1). In addition, GFP labeling appeared to increase in terms of total fluorescence and number of labeled cells in the CNO⁺ mouse (Extended Data Fig. 5). We then pooled these data with those from similar experiments using AAV8-ATLAS_Cre and found that CNO⁺ mice had significantly higher postsynaptic GFP labeling in the Str versus CNO- mice both in terms of the number of cells labeled (88 ± 12 versus 52 ± 7, n = 5 mice, P = 0.0357, t-test, Extended Data Fig. 5) and total fluorescence (38 ± 4 versus 19 ± 2 a.u., P = 0.0025, t-test, Extended Data Fig. 5). In contrast, the number of presynaptic At stained cells was not significantly different between CNO⁺ and CNO⁻ mice (1,200 ± 100 versus 1,200 ± 100, P = 0.7492, t-test, Extended Data Fig. 5). Thus, our results indicate that the efficiency of transsynaptic labeling was significantly higher in CNO⁺ mice versus CNO⁻ mice, consistent with ATLAS mediating activity-dependent transsynaptic labeling.

An alternate synaptic vesicle targeting domain

ATLAS is composed of multiple independent components, each of which can, in principle, be modified or upgraded to improve efficiency or alter function. We took advantage of this property to optimize the VAMP2 component of ATLAS, which is responsible for presynaptic targeting. Although VAMP2 is widely used as a presynaptic marker, it can produce toxicity when expressed at a high level. Indeed, we saw evidence of toxicity when ATLAS was expressed in the mPFC for 2 and 4 weeks (Extended Data Fig. 6). Accordingly, we exchanged the cytoplasmic domain of VAMP2 for a nanobody against Synaptotagmin 1 (SYTnb)³¹ to give SYTnb-VAMP2Δ-At-BACEcs-AF-Cre (ATLASsn_Cre, Figs. 1b and 6a), where VAMP2Δ consists of the lumenal and transmembrane domains of VAMP2. When we expressed ATLASsn_Cre in the mPFC for 2 or 4 weeks, we saw no evidence of toxicity (Extended Data Fig. 6). To test ATLASsn_Cre, we transfected it into dissociated cultures of cortical neurons while infecting them with AAV8-DIO-mCherry. We saw abundant cells labeled with mCherry but not with At, indicating they were labeled transsynaptically (Fig. 6b). A closeup image of such a postsynaptic cell revealed At labeling opposite its dendritic spines, consistent with presynaptic labeling (Fig. 6b, inset).

**Fig. 6: Transsynaptic tracing with ATLASsn.**

To test AAV8-ATLASsn_Cre in vivo, we injected it and AAV8-BACE-HA into the mPFC and AAV8-DIO-mCherry into the Str. After 2 weeks of incubation, we saw expression of At in the mPFC and abundant mCherry labeling in Str (Fig. 6c,d and Extended Data Fig. 7). The dorsal region of postsynaptic mCherry labeling colocalized almost perfectly with At labeling representing presynaptic termini (Fig. 6d and Extended Data Fig. 7), consistent with ATLASsn mediating transsynaptic labeling. To further test ATLASsn in vivo, we coinfected AAV8-ATLASsn_Cre and AAV8-BACE-GFP in M1 cortex and AAV8-DIO-mCherry in the Str and thalamus (Thal, Fig. 6e). After 2 weeks of incubation, staining of GFP and mCherry revealed labeled presynaptic cells in M1 (Fig. 6f) and postsynaptic cells in Str (Fig. 6g) and Thal (Fig. 6h). Overlap of GFP and mCherry labeling in Str and Thal (Extended Data Fig. 7) is consistent with transsynaptic labeling. Furthermore, minimal background was observed in the Str when AAV8-DIO-mCherry alone was injected (Extended Data Fig. 7).

Labeling of cortical to subcortical circuits

To further test ATLASsn, we used it to trace three additional pathways: from the auditory cortex (A1) to the inferior colliculus, the mPFC to the claustrum and the entorhinal cortex to the hippocampus, respectively. In each case, we injected AAV8-ATLASsn_Cre and AAV8-BACE-HA into the presynaptic area and AAV8-DIO-GFP into the corresponding postsynaptic area. After 2 weeks, we stained presynaptic cells for At and postsynaptic cells for GFP. In each experiment, we found cells with At and GFP labeling, consistent with efficient transsynaptic labeling (inferior colliculus, 173 ± 13 cells, n = 8 samples from three mice; claustrum, 71 ± 14 cells, n = 7 samples from three mice; hippocampus, 132 ± 30 cells, n = 4 samples from two mice, Extended Data Fig. 8 and Supplementary Table 1). Thus, ATLAS is capable of tracing multiple different pathways.

In addition, to understand the time course over which transsynaptic labeling happens, we coexpressed AAV8-ATLASsn_Cre and AAV8-BACE-HA in the mPFC and AAV8-DIO-GFP in the Str and waited 4, 7, 14 and 21 days (n = 8 samples from four mice in each case) before perfusing animals and staining for presynaptic and postsynaptic labels. After 4 days, we found faint labeling of presynaptic cells with At, although labeled cells were relatively abundant (534 ± 114 cells, Extended Data Fig. 9). In contrast, GFP-labeled postsynaptic cells were very sparse at this time point (2 ± 1 cells), and total GFP labeling was minimal (3 ± 1 a.u.). After 7 days, the number of presynaptic cells labeled with At increased (1,150 ± 300, Extended Data Fig. 9), but not significantly (P = 0.3279, one-way ANOVA). However, both the number of labeled postsynaptic cells (52 ± 7 cells) and the total GFP label (35 ± 3 a.u.) were significantly higher (P < 0.0001, ANOVA; Extended Data Fig. 9). Thus, postsynaptically labeled cells become visible between 4 and 7 days postinjection. After 14 and 21 days, the number of labeled presynaptic cells (1,270 ± 260 and 1,070 ± 270 cells) was not significantly different from 7 days (P = 0.9874, P = 9943, ANOVA; Extended Data Fig. 9). The number of postsynaptic cells labeled with GFP at 14 days (63 ± 5 cells) was not significantly different from the number at 7 days (P = 0.4514, ANOVA; Extended Data Fig. 9). However, the cell number at 21 days (86 ± 5 cells) was significantly higher than at 14 days (P = 0.0116, ANOVA). The labeling intensity increased significantly at 14 and 21 days versus 7 days (65 ± 2 and 82 ± 6 a.u., P < 0.0001, ANOVA; Extended Data Fig. 9). Thus, ATLAS transsynaptic labeling increases notably in the first 7 days and at a slower rate thereafter in terms of cell number, but increases in a relatively linear manner in total intensity. In contrast, labeling of presynaptic cells is constant after the first week.

ATLAS mediates transsynaptic labeling in the rat

It is highly desirable for transsynaptic labeling techniques to work in systems other than the mouse. Accordingly, we tested whether ATLAS would work in the rat by injecting AAV8-ATLASsn_Cre and AAV8-BACE-HA into the CA1 region of the ventral hippocampus and AAV8-DIO-tdTomato into the nucleus accumbens (ACB, Fig. 6i), to which the CA1 neurons project. We observed At staining in CA1 (Fig. 6j) and abundant tdTomato-labeled cells in ACB (115 ± 11, n = 3 rats, Fig. 6k), consistent with transsynaptic labeling. Finally, tdTomato labeling of axons within the lateral hypothalamic area (LHA) (Fig. 6l), which receives axonal projections from ACB³², confirms the postsynaptic labeling of neurons within ACB.

Discussion

ATLAS is a rationally designed protein that mediates transsynaptic tracing from genetically determined neurons via a precisely defined mechanism. In contrast, viruses such as rabies, YFV, AAVs and the protein used in the nonviral method Trans-Seq have modes of egress from starter cells and entry into recipient cells that remain mysterious^10,14,15,33. In addition, AAV1, YFV, vesicular stomatitis virus (VSV) and herpes simplex virus-1 (HSV1) undergo a measurable component of retrograde transport in addition to anterograde and have not been shown to mediate tracing that is exclusively transsynaptic^{14,15,16,34,35}.

Because ATLAS protein does not have an intrinsic amplification mechanism like rabies and similar viruses⁹, tracing using ATLAS requires the expression of a recombinase-dependent reporter in the postsynaptic cell. While this could be interpreted as a drawback, it is also a benefit because amplification that occurs in rabies or VSV infection is associated with toxicity^36,37. In the case of tracing using AAV1, which also requires amplification, a transgenic mouse with a floxed reporter transgene can be used instead of postsynaptic injections^15,16. Efficient and consistent transsynaptic labeling occurred in some but not all cases when we expressed ATLAS_Cre in the ai75 floxed reporter mice³⁸. Thus, broad regions containing postsynaptic target areas must be identified a priori and injected with a floxed reporter, which is not required for many virus-based transneuronal labeling techniques. However, this should not be a difficult problem to surmount as regions postsynaptic to specific cell types are easily identified by exogenously expressing a presynaptic marker such as VAMP2 (ref. ³⁹).

Our finding that 100% of postsynaptic striatal cells labeled following expression of ATLAS in V1 were synaptically connected to cells expressing ChR2-GFP, which was coinfected with ATLAS, indicates that postsynaptic cells with Cre-dependent labeling are likely to be synaptically connected with presynaptic starter cells in which ATLAS_Cre is expressed. Also, 70% of neurons not labeled with ATLAS but immediately adjacent to labeled cells were found to be connected to presynaptic cells expressing ChR2-GFP. This result could be due to many factors, including the fact that transsynaptic labeling with ATLAS is not 100% efficient. In addition, Cre-dependent expression of ChR2-GFP could occur in neurons in V1 that are connected with starter cells through local connections, leading to more widespread expression of ChR2-GFP than of ATLAS. In contrast to paired unlabeled neurons, ~40% of random unlabeled cells chosen near labeled presynaptic terminals from V1 were found to be connected to those terminals. These data are consistent with postsynaptic neurons labeled with ATLAS being connected to labeled presynaptic neurons at a rate considerably higher than chance.

Because ATLAS is a protein, it can be evolved to change its properties and improve performance similar to GCaMP⁴⁰ or GRAB sensors⁴¹. For instance, ATLAS, as described in this paper, works only for anterograde tracing from excitatory neurons. However, AF can, in principle, be replaced by a recombinant binder to a neurotransmitter receptor, such as a GABA (γ-aminobutyric acid) or acetylcholine receptor, or even to a receptor for a neuromodulator, such as dopamine or serotonin. The resulting modified ATLAS could be used for tracing virtually any neuronal circuit mediated by the corresponding neurotransmitter–receptor combination. Also, because of its activity dependence, ATLAS could be used to mark circuits that respond to specific stimuli, especially if ATLAS tracing could be gated to occur during a particular time interval. Finally, it may be possible to modify ATLAS to label circuit changes during events such as memory formation⁴² or specific developmental processes such as synaptic pruning⁴³.

Methods

AF production

Amino acids 1–394 of GluA1 grown in Sf9 (Spodoptera frugiperda) cells was used as a target for an mRNA display selection, carried out as described in ref. ⁴⁴. FingRs amplified from the ninth round of selection were fused to a Myc tag and coexpressed in Cos7 cells (ATCC, CRL-1651) with ss-GluA1-HA (ss, signal sequence of GluA1) and Stargazin-V5. Cos7 cells were stained live with anti-MYC antibody, then fixed, permeabilized and stained for HA and V5. Fibronectins labeling the surface of cells expressing GluA1 and Stargazin were then expressed in dissociated cultures. Fibronectin 9.3-labeled endogenous GluA1 and was designated AF.

Plasmid constructs

The ss-AF-YFP consists of DNA encoding amino acids 1–18 of GluA1 inserted upstream of AF and yellow fluorescent protein (YFP). For ATLAS constructs, VAMP2 complementary DNA (cDNA) was inserted upstream of ALFA-tag (SRLEEELRRRLTE) followed by a BACEcs (β-site Amyloid Precursor Protein-cleaving enzyme 1) cut site (EISEVNLDAEFR). VAMP2-BACEcs-SEP has SEP downstream of BACEcs. VAMP2-BACEcs-AF-SEP has AF and SEP downstream of BACEcs. ATLAS_Cre has AF and Cre cDNA downstream of BACEcs. DIO-ATLAS_Flp has a Myc tag (EQKLISEEDL) followed by FLP1 cDNA downstream of BACEcs and nested LoxP sites on either side of the coding sequence. GFP-HA-BACE1 plasmid was purchased (Addgene, no. 165032), and GFP was removed to make BACE-HA. The above sequences were ligated into a pAAV plasmid containing the human synapsin (hsyn) promoter and a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Lenti-hsyn-Cre was purchased from Addgene (no. 86641), and the hSYN1 promoter was replaced with the CamKIIα promoter to generate Lenti-CamKII-Cre.

Dissociated cultures

Pups from timed pregnant Sprague Dawley rats (E19) were used for cultured cortical neurons, prepared as in ref. ⁴⁵. All procedures were approved by the Institutional Animal Care and Use Committee of the University of Southern California.

Protein purification

Human embryonic kidney 293T cells were transfected with the plasmid pCAG-ss-AF-3XHA-TEVcs-HALOTag and the secreted protein AF-HA-TEVcs-HALOTag was collected and incubated with HaloLink resin (Promega, cat. no. G1914) overnight at 4 °C. and then with tobacco etch virus (TEV) protease (New England Biolabs, cat. no. P8112S) overnight at 4 °C. Supernatant was collected and incubated with NEBExpress Ni Resin (New England Biolabs, cat. no. S1428S) overnight at 4 °C to remove the TEV protease. Purified protein was dialyzed into 20 mM HEPES, 250 mM NaCl pH 7.4 and concentrated using the Amicon Ultra centrifugal filters (cat. no. UFC501096).

Endocytosis

Neuronal cultures were incubated with 1 mM purified AF-HA protein for 1 h at 37 °C. Next, cultures were washed three times with 1× Hanks Balanced Salt Solution containing 10 mM HEPES, fixed with 4% paraformaldehyde and stained for anti-HA (Cell Signaling, cat. no. 3724S) and anti-Map2 (Abcam, cat. no. ab5392).

Presynaptic release

Plasmids encoding VAMP2-BACEcs-SEP (1 μg) or VAMP2-BACEcs-AF-SEP (1 μg) with BACE-HA (2 μg) were transfected into 15–18 day in vitro (DIV) cultured neurons. Two days before transfection, cultures were infected with AAV8-Cre and AAV8-DIO-PSD-95.FingR-TdTomato to label postsynaptic sites. Neurons were imaged 3 to 5 days later with or without 100 mM KCl. Time-lapse imaging was performed for either 100 or 200 frames at an interval of 1,000 ms and an exposure of 500 ms. Time constants were measured using curve fitting functions in GraphPad Prism. Videos of SEP release events were generated using ImageJ by taking the difference of adjacent image frames to eliminate background and SEP fluorescence beyond the initial frame after release.

Viruses

The following viruses were purchased from Addgene: AAV8-hSyn-DIO-mCherry no. 50459, AAV8-EF1a-fDIO-mCherry no. 114471, AAV8-hsyn-DIO-eGFP no. 50457 and AAV8.hSyn.hM3D.mCherry no. 50474. All other viruses were generated in the Arnold laboratory using standard methods.

Transsynaptic tracing in culture

Neuronal cultures (14 DIV) were transfected with plasmids for ATLAS (1 μg) and BACE-HA (2 μg) using the calcium phosphate method. Transfection was followed immediately by infection with AAV8-hsyn-DIO-GFP and incubated for 7 days. Neurons were fixed and probed as described.

Immunocytochemistry for dissociated cultures

Neurons were stained using: anti-GFP, 1:10,000, Aves GFP-1020; anti-MYC, 1:1,000, Novus Biologicals NB600-334; anti-Map2, 1:1,000, Abcam ab5392; anti-clathrin, 1:1,000, GeneTex GTX80218; anti-mCherry, 1:1,000, Rockland M11217; anti-RFP, 1:1,000, Invitrogen MA5-15257; anti-GluA1, 1:1,000, EMD Millipore ABN241; anti-PSD-95, 1:3,000, NeuroMab 75-348; anti-HA, 1:1,000, Cell Signaling 3724S; anti-Cre, 1:1,000, EMD Millipore MAB3120; Alexa Fluor 488 goat anti-chicken, 1:1,000, Invitrogen A-11006; Alexa Fluor 594 goat anti-rabbit, 1:1,000, Invitrogen A-11012; Alexa Fluor 594 goat anti-mouse, 1:1,000, Invitrogen A-11005; Alexa Fluor 647 goat anti-mouse, 1:1,000, Invitrogen A-21235; Alexa Fluor 647 goat anti-rabbit, 1:1,000, Invitrogen A-21244; JF549 halotag ligand, 1:1,000, Janelia Farm; JF647 halotag ligand, 1:1,000, Janelia Farm; FluoTag-X2 anti-alfa 647, 1:1,000, NanoTag biotechnologies N1502 AF647. Neurons were mounted using Dapi-fluoromount-G (Electron Microscopy Services, 17984-24).

Electrophysiology (testing of AF)

All experiments were performed in accordance with National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals, and all procedures were approved by the Institutional Animal Care and Use Committee of the University of Southern California. As previously described, 400-μm-thick rat organotypic hippocampal slice cultures were prepared from male and female P6 to P8 Sprague Dawley rats⁴⁶. Sparse biolistic transfections were performed on DIV1 as previously described^47,48. Next, 50 μg of mixed plasmid DNA encoding ss-AF-YFP was coated on 1-μm diameter gold particles. Construct expression was confirmed by YFP epifluorescence. Dual whole-cell recordings of CA1 pyramidal neurons were performed on DIV7–8 through simultaneous recordings from a transfected neuron and a neighboring, nontransfected control neuron. Synaptic responses were evoked by stimulating with a monopolar glass electrode filled with artificial cerebrospinal fluid (ACSF) in the stratum radiatum. AMPAR-evoked EPSCS (-eEPSCs) were measured at −70 mV. NMDAR-eEPSCs were measured at +40 mV and were temporally isolated by measuring amplitudes 150 ms after the stimulus. For paired-pulse facilitation, paired pulses at 40-ms interpulse interval were delivered and the ratio of peak amplitudes was analyzed to generate barplots depicting mean ± s.e.m. Data analysis was performed using Igor Pro v.9.

Animal preparation and stereotaxic surgery (mouse)

All procedures in Figs. 3, 4b–d, 5 and 6c,d and Extended Data Figs. 3, 5, 6, 7a,b, 8 and 9 were performed in accordance with NIH Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee at the University of Southern California. Male and female wild-type mice (C57BL/6J, Jackson Laboratories, strain no. 000664), Vipr2-Cre (Cre expression in dLGN, Jackson Laboratories, strain no. 031332) and Sst-IRES-Cre (Cre expression in somatostatin interneurons, Jackson Laboratories strain no. 013044) mice aged 2–6 months were used. For stereotaxic viral injection, mice were anesthetized in an induction chamber containing 4% isoflurane mixed with oxygen and transferred to a stereotaxic frame with a heating pad. Then, 1.5% isoflurane was delivered throughout the procedure through a nose cone at a rate of 0.5 l min⁻¹. Injection coordinates based on ref. ⁴⁹ were used to mark the location where a small hole (0.5 mm diameter) was drilled. Viruses were delivered through pulled glass micropipettes using pressure injection via a micropump. Total injection volumes ranged from 50 to 800 nl, at 20–60 nl min⁻¹. Following injection, the micropipette was left in place for 15 min to minimize virus diffusion into the pipette track. Animals were administered Ketoprofen (5 mg kg⁻¹) at the beginning of the surgical procedure and again every 24 h for 2 d following surgery.

Injections of viruses (mouse)

For experiments in Figs. 3, 4b–d, 5, 6c,d and Extended Data Figs. 3, 5, 6, 7a,b, 8 and 9, anterograde tracing experiments using ATLAS_cre, 400–600 nl of either ATLAS_Cre or ATLASsn_Cre virus was injected into presynaptic cells in the PFC, Primary auditory cortex (A1) or entorhinal cortex. AAV8-DIO reporter virus (400–800 nl), diluted to minimize the background signal, was injected into postsynaptic cells in the Str, ipsilateral claustrum, bilateral inferior colliculus or ipsilateral dentate gyrus. The animals were euthanized 2 weeks following the injections.

In experiments with DIO-ATLAS_Flp, transgenic VIPR2-Cre mice were injected with AAV8-DIO-ATLAS_Flp (600 nl, 1.0 × 10¹³) at the ipsilateral LGN. A second injection of AAV8-EF1a-fDIO-mCherry (600 nl, 5.0 × 10¹²) was administered at the primary visual cortex (V1). The animals were euthanized after 2 weeks. To test AAV-DIO-ATLAS_Flp, C57BL/6J mice were injected with a viral mixture of AAV8-DIO-ATLAS_Flp and Lenti-hsyn-Cre at V1 and AAV8-EF1a-fDIO-mCherry (600 nl, 5.0 × 12) in the ipsilateral SC and euthanized 2 weeks later. Transgenic SST-Cre mice were injected with a mixture of AAV8-DIO-ATLAS_Flp and AAV8-ef1a-fDIO-mCherry (at 1:1 ratio based on viral particles) at V1 and euthanized 1 week later.

For testing the potential retrograde spread of ATLAS, AAV8-DIO-ATLAS_Flp and Lenti-hsyn-Cre were injected into Str of C57BL/6J mice, and AAV8-EF1a-fDIO-mCherry (800 μl, 5.0 × 10¹²) was injected into mPFC. As the negative control, a group of C57BL/6J mice was injected with AAV8-ef1a-fDIO-mCherry (800 μl, 5.0 × 10¹²) in mPFC. All animals were then euthanized 2 weeks following the injections.

To test for monosynaptic tracing, C57BL/6J mice were injected with AAV8-ATLAS_Cre (600 nl, 1.0 × 10¹³) in V1 and AAV8-hsyn-DIO-mCherry (400 nl, 1.0 × 10¹³) in the PBG. As the positive control, C57BL/6J mice were injected with AAV8-ATLAS_Cre (600 nl, 1.0 × 10¹³) in SC and AAV8-hsyn-DIO-mCherry (400 nl, 1.0 × 10¹³) in PBG. As a negative control, wild-type mice were injected with AAV8-hsyn-DIO-mCherry (400 nl, 1.0 × 10¹³) at PBG. Animals were euthanized 2 weeks following injections.

To test supplementing ATLAS with BACE, mixtures with the following ratios of AAV8-ATLAS_Cre and AAV8-hsyn-BACE-HA: 1:1 ATLAS: BACE, 1:5 ATLAS: BACE, or no BACE based on number of viral particles. These were injected into the C57BL/6J mice in mPFC. AAV8-hsyn-DIO-GFP (800 nl, 1.0 × 10¹³) in ipsilateral Str to visualize postsynaptic cells. Animals were euthanized 2 weeks following injections. To examine the effect of incubation time on efficiency of ATLAS, C57BL/6J mice were injected with a viral mixture of AAV8-ATLASsn_Cre and AAV8-hsyn-BACE-HA (600 nl, 1:3 based on viral particles) in mPFC and AAV8-hsyn-DIO-GFP (800 nl, 1.0 × 10¹³) in ipsilateral Str. Animals were euthanized 4, 7, 14 or 21 days following injections.

For testing the effect of neuronal activity on ATLAS tracing, C57BL/6J mice were injected with AAV8-ATLASsn_Cre or AAV8-ATLAS_Cre, AAV8-hsyn-BACE and AAV8-hHM3Dq-mCherry (600 nl, 1:3:1 based on viral particles) in mPFC and AAV8-hsyn-DIO-GFP (800 nl, 1.0 × 10¹³) in ipsilateral Str. CNO (200 μl, 1.1 mM) was administered to experiment animals through intraperitoneal injection daily following viral injections. Animals were euthanized 7 days after viral injections. A final dose of CNO was administered 45 min before the perfusion.

To determine whether ATLAS shows toxicity compared to ATLASsn, one group of C57BL/6J mice were injected with 600 nl of AAV8-ATLASCre and AAV8-hsyn-BACE (1:3 based on viral particles), and the other group of mice were injected with 600 nl of viral mixture of AAV8-ATLASsn_Cre and AAV8-hsyn-BACE (at a 1:3 ratio based on viral particles) at mPFC. AAV8-hsyn-DIO-GFP (800 nl) was injected into the ipsilateral Str of both groups. Animals were euthanized 14 and 28 days following injections.

In all experiments listed above, presynaptic and postsynaptic injections were performed during the same surgery. Bregma and Lambda coordinates are in millimeters as follows: anteroposterior is the distance from Lambda, mediolateral is the distance from midline and dorsoventral is the distance from Lambda except for V1, A1 and SC, which use the distance from the surface of the cortex. mPFC (6.3, 0.3, 1.7); Str (5.1, 1.8, 3.4); dLGN (2.2, 2.2, 2.9); primary visual cortex (V1) (0.8, 2.4, 0.6); primary auditory cortex (A1) (1.5, 4.2, 0.6); SC (0.6, 0.8, 1.2); inferior colliculus (−0.7, 0.8, 0.6); PBG (0.1, 1.8, 3.3); claustrum (5.4, 2.5, 4.0); entorhinal cortex (0.0, 3.6, 3.6) and dentate gyrus (0.6, 2.8, 2.5).

Histology (mouse)

Mouse brains were perfused with 40 ml of phosphate-buffered saline (PBS), then 40 ml of 4% paraformaldehyde in PBS (Figs. 3, 4b–d, 5, 6c,d and Extended Data Figs. 3, 5, 6, 7a,b, 8 and 9). The brains were fixed overnight in 4% paraformaldehyde at 4^oC and dehydrated in 30% sucrose solution for at least 48 h. Brains were embedded in tissue freezing medium at −45 °C and sectioned to a thickness of 25–35 µm using a cryostat. For immunohistochemistry, antibodies used were: rabbit anti-RFP, Rockland (cat. no. 600-401-379), 1:1,000; chicken anti-GFP antibody, AVES (gfp-1020, cat. no. NC9510598), 1:1,000; Fluotag-X2 anti-ALFA, NanoTag (cat. no. N1502-ALFA647-L), 1:1,000; mouse anti-NeuN antibody, EMD Millipore (cat. no. MAB377), 1:1,000, c-Fos (cat. no. 9F6) Rabbit mAb, Cell Signaling Technology (cat. no. 2250T), 1:1,000; Alexa Flour 488-conjugated goat anti-chicken (A-11039) or anti-mouse IgG (cat. no. A-28175), Thermo Fisher, 1:1,000; Alexa Flour 594-conjugated anti-rabbit IgG, Thermo Fisher (cat. no. A-11012), 1:1,000; Alexa Flour 647-conjugated goat anti-mouse IgG, Thermo Fisher, 1:1,000 (cat. no. A-21235).

Quantification of transsynaptic labeling

Labeled presynaptic cells (At) and postsynaptic cells (GFP or mCherry) were counted within an identical 1-mm² region of 25-μm-thick stained sections of target regions. All sections examined were roughly equal distance from the site of AAV injection. We quantified cells in two representative sections, each 25 µm apart. Then, the data from the selected sections was pooled and multiplied by two to estimate the number of labeled cells within 100 µm. To account for the background expression from spontaneous recombination by the DIO reporter construct, we subtracted the cell counts from control experiments from those in corresponding ATLAS experiments. To quantify activity-dependent transsynaptic labeling, total GFP fluorescence minus background was measured identically in Str in sections at equivalent distances from injection sites using ImageJ. For the SST experiment, At-labeled cells and mCherry-labeled cells were counted within an identical 1 mm² region of 25-μm-thick stained sections. The number of mCherry⁺ cells over the mCherry and At colabeled cells was then calculated.

Stereotaxic surgery and histology (M1 to Str and Thal)

All experiments in Fig. 6e–h were performed in accordance with NIH Guidelines for the Care and Use of Laboratory Animals, and all procedures were approved by the Institutional Animal Care and Use Committee of NIH. The ATLASsn_Cre viral vector AAV8-SYTnb-VAMP2Δ-At-BACEcs-AF-Cre and AAV8-BACE-GFP (1:1, 200 nl) were injected stereotaxically into the motor cortex of wild-type mice at (1.0, 2.0, 1.8) (Fig. 6e–h). They also received an injection of a Cre-dependent reporter vector: AAV8-Ef1a-FLEX-tdTomato (Addgene, 28306) into the Str (0.5, 2.0, 4.0) and thalamus (−1.55, 1.5, 3.75). Following a survival time of 2 weeks, animals were perfused and 50-μm sections were cut with a freezing microtome. The following antibodies were used for immunocytochemistry: rabbit anti-RFP (cat. no. 600-401-379, Rockland Immunochemicals), chicken anti-GFP (cat. no. A10262, Invitrogen), goat anti-chicken 488 (cat. no. A32931, Invitrogen) and goat anti-rabbit 555 (cat. no. A32732, Invitrogen). Sections were counterstained with Neurotrace Blue 435 (cat. no. N21479, Invitrogen) and coverslipped with Aqua-polymount (cat. no. 18606, Polysciences). Slides with 12–15 coronal brain sections were imaged on a Zeiss EpiFluorescence Microscope equipped with Xylis broad spectrum LED illumination (Excelitas Technologies), a Hammamatsu Orca Flash4.0 camera and Ludl stage drive controlled by Neurolucida 360 Imaging software (MBF Bioscience). Images were registered using NeuroInfo v.2022.1.1.

Animal preparation and stereotaxic surgery (rat)

All experiments were performed in accordance with NIH Guidelines for the Care and Use of Laboratory Animals, and all procedures were approved by the Institutional Animal Care and Use Committee of the University of Southern California. Male Sprague Dawley rats (Envigo) weighing 300–400 g were injected with viruses delivered using a micro-infusion pump (Harvard Apparatus) connected to a 33-gauge microsyringe injector attached to a PE20 catheter and Hamilton syringe. Flow rate was 5 μl min⁻¹. Injectors were left in place for 3 min postinjection. Viruses were incubated for 21 days after injection. AAV8-ATLASsn_Cre was unilaterally injected into the CA1v at: −4.9 mm anteroposterior, ±4.8 mm mediolateral, −7.8 mm dorsoventral (zeroing at skull site). AAV-pCAG-FLEX-tdTomato-WPRE (Addgene; cat. no. 51503) was unilaterally injected into the ACB: +1.2 mm anteroposterior, ±1.0 mm mediolateral, −6.75 mm dorsoventral (zeroing at dura). Injection volume was 100 nl per site. For control experiments animals were injected with AAV-pCAG-FLEX-tdTomato-WPRE unilaterally in the ACB, but no injection was made in the ventral CA1. Photomicrographs were acquired using a Nikon 80i (Nikon DS-QI1, 1,280 × 1,024 resolution, 1.45 megapixel) microscope under epifluorescence controlled by NIS-Elements v.C6.10.01.

Histology (rat)

Following perfusion and fixation, Brains were sectioned into 30-μm sections on a microtome cooled with dry ice, collected in an antifreeze solution and stored in a −20 °C freezer. The following antibodies were used for immunocytochemistry: Rabbit anti-RFP (1:2,000, Rockland Inc.; cat. no. 600-401-379; Clonality: Polyclonal) and FluoTag-X2 anti-ALFA (1:1,000, NanoTag Biotechnologies; cat. no. N1502).

Quantification of activity-dependent transsynaptic labeling

For experiments to quantify transsynaptic labeling with and without BACE, mCherry-expressing cells were counted within an identical 1-mm² region over four consecutive 25-μm stained sections of Str. For experiments to quantify activity-dependent transsynaptic labeling, total GFP fluorescence minus background was measured identically in Str sections using ImageJ v.1.49b, Slidebook v.8 and Zen Microscopy v.3.11.

Electrophysiology (PFC to Str)

For experiments in Extended Data Fig. 4, wild-type C57BL/6J mice coinfected with AAV8-ATLASsn_FLP, AAV8-BACE-HA and AAV8-DIO-ChR2-GFP into the mPFC, and DIO-mCherry in Str were used for slice recording. Details of the stereotaxic surgery are the same as in Figs. 3a–d and 6c,d. Four weeks following injections, animals were decapitated following anesthesia, and the brain was rapidly removed and immersed in an ice-cold dissection buffer (composition 60 mM NaCl, 3 mM KCl, 1.25 mM NaH₂PO₄, 25 mM NaHCO₃, 115 mM sucrose, 10 mM glucose, 7 mM MgCl₂, 0.5 mM CaCl₂; saturated with 95% O₂ and 5% CO₂; pH 7.4). Coronal slices of 350 µm thickness were sectioned by a vibrating microtome (Leica VT1000s) and recovered for 30 min in a submersion chamber filled with warmed (35 °C) ACSF (composition 119 mM NaCl, 26.2 mM NaHCO₃, 11 mM glucose, 2.5 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, and 1.2 NaH₂PO₄, 2 mM sodium pyruvate, 0.5 mM VC). Str neurons surrounded by GFP⁺ fibers were visualized under a fluorescence microscope (Olympus BX51 WI). Patch pipettes (~4–5 MΩ resistance) filled with a cesium-based internal solution (composition 125 mM cesium gluconate, 5 mM TEA-Cl, 2 mM NaCl, 2 mM CsCl, 10 mM HEPES, 10 mM EGTA, 4 mM ATP, 0.3 mM GTP and 10 mM phosphocreatine; pH 7.25; 290 mOsm) were used for whole-cell recordings. Signals were recorded under voltage-clamp mode at a holding voltage of −70 mV for excitatory currents, filtered at 2 kHz and sampled at 10 kHz. Tetrodotoxin (1 μM) and 4-aminopyridine (1 mM) were added to the external solution for recording monosynaptic responses to blue light stimulation (5-ms pulse, 3-mW power, 5–10 trials).

Surgery (V1 to Str)

Experiments in Fig. 4e–h were performed in accordance with NIH Guidelines for the Care and Use of Laboratory Animals, and all procedures were approved by the Institutional Animal Care and Use Committee of Harvard Medical School. Injections were performed through a pulled glass at a rate of 30 nl min⁻¹. After injections, pipettes were slowly withdrawn (less than 100 µm s⁻¹) at least 5 min after the end of the infusion. Viruses were injected in two rounds of surgeries to minimize expression competition between ATLAS viruses (that is ATLASsn_Cre, BACE-HA) and Cre-dependent ChR2. Wild-type animals undergo the first round of surgery between 4 and 8 weeks of age (Fig. 4e–h). Before the start of surgery, presynaptic virus cocktail was prepared by mixing AAV8-hSyn-ATLASsn_Cre and AAV8-hSyn-BACE-HA in 3:1 ratio (titer is 2 × 10¹³ gc ml⁻¹ for both viruses). During surgery, presynaptic virus cocktail was injected at three separate sites in V1. In addition, AAV8-hSyn-DIO-mCherry or AAV9-CAG-DIO-tdTomato was injected into two separate sites in Str. Then 14 days after the first surgery, AAV8-hSyn-DIO-ChR2-GFP was injected at the same three V1 sites as the first surgery.

Coordinates of V1 injection sites were as follows (coordinates are relative to bregma and dorsoventral is relative to the brain surface, below). Here, 300 nl of presynaptic virus cocktail (AAV8-ATLASsn_Cre and AAV8-BACE-HA) was injected per site for the first surgery. Then 200 nl of AAV8-hSyn-DIO-ChR2 was injected per site for the second surgery.

V1 site 1: −2.75 mm anteroposterior, ±2.5 mm mediolateral, −0.65 mm dorsoventral
V1 site 2: −3.80 mm anteroposterior, ±2.9 mm mediolateral, −0.65 mm dorsoventral
V1 site 3: −3.80 mm anteroposterior, ±2.0 mm mediolateral, −0.65 mm dorsoventral

Next, 500 nl of postsynaptic virus (Cre-dependent mCherry or tdTomato) was injected at Str site 1 (anterior) while 400 nl of virus was injected at Str site 2 (posterior).

Str site 1: 0.70 mm anteroposterior, ±1.60 mm mediolateral, −2.22 mm dorsoventral
Str site 2: −0.58 mm anteroposterior, ±2.75 mm mediolateral, −2.75 mm dorsoventral

Electrophysiology (V1 to Str)

For experiments in Fig. 4e–h, brain slices were obtained from 2- to 3-month-old mice (both male and female) using standard techniques. The experiments closely followed the procedures outlined in previous studies^50,51. Mice were anesthetized using isoflurane inhalation and subsequently subjected to transcardial perfusion with ice-cold ACSF composed of the following (in mM): 125 NaCl, 2.5 KCl, 25 NaHCO₃, 2 CaCl₂, 1 MgCl₂, 1.25 NaH₂PO₄ and 11 glucose (final osmolarity of 300–305 mOsm kg⁻¹). The brain was removed from the skull, placed in ice-cold ACSF and 250–300-μm coronal brain slices were cut and initially placed in a holding chamber for 10 min at 34 °C in a choline-based solution composed of (in mM): 110 choline chloride, 25 NaHCO₃, 2.5 KCl, 7 MgCl₂, 0.5 CaCl₂, 1.25 NaH₂PO₄, 25 glucose, 11.6 ascorbic acid and 3.1 pyruvic acid. Following choline incubation, slices were transferred to a second chamber with ACSF at 34 °C for a minimum of 30 min. The chamber was subsequently shifted to room temperature for the duration of the experiment. All recordings were made within 4–5 h of slicing. The temperature was maintained at 32 °C and carbogen-bubbled ACSF was perfused at a rate of 2–3 ml min⁻¹ for all experiments.

Monosynaptically connected striatal neurons were identified by their mCherry-positive or tdTomato-positive somata. Fluorescence-negative neurons with GABAergic interneuron physiological properties (membrane tau decay <0.8 ms for both fast-spiking and persistent low-threshold spiking subtypes; input resistance >500 MΩ in persistent low-threshold spiking subtype) were excluded from the analysis.

Striatal neurons were patched in whole-cell voltage-clamp configurations using borosilicate glass electrodes (3–5 MΩ) filled with cesium-based (voltage clamp) internal solution to measure EPSCs. The solution contained (in mM): 135 CsMeSO₃, 10 HEPES, 1 EGTA, 3.3 QX-314 (Cl− salt), 4 Mg-ATP, 0.3 Na-GTP, 8 Na₂-phosphocreatine, with pH adjusted to 7.3 using CsOH, resulting in an osmolarity of 295 mOsm kg⁻¹. All recorded neurons exhibited electrophysiological characteristics of spiny projection neurons. All synaptic currents were recorded with a cesium-based internal and monitored at a holding potential of −70 mV. Fluorescent positive and negative neurons were patched as randomly ordered sequential pairs, strictly within one field of view of each other. ChR2-mediated synaptic currents from V1 were optically evoked using 2-ms pulses of 473-nm light at light powers of 2 mW controlled through an acousto-optic modulator.

To assess the baseline connection probability of V1-DMS circuit viruses were injected in V1 at identical locations, titer and volume and at identical times as described above (see Surgery section for Fig. 4e–h). After 4 weeks, brain slices were obtained and patched in whole-cell voltage-clamp configurations as described above. We first found the field of view with maximal ChR2 fiber expression in DMS and randomly patched cells within it. ChR2-mediated synaptic currents from V1 were optically evoked using as above.

Quality control criteria were a stable baseline membrane resistance (variation in R_m < 10%) and a stable baseline resting potential with median standard deviation of ≤0.5 mV across recordings. Optically evoked EPSCs were analyzed post hoc using MATLAB v.2024a.

Statistics and reproducibility

Experiments in Fig. 1c were performed in three independent cultures; in Fig. 1d, two cultures; in Fig. 1e–g, five cultures; in Fig. 1h, four cultures; in Fig. 1n, ten slices; in Fig. 1o, eight slices; in Fig. 1p, six slices; in Fig. 2b–e, in three independent cultures. Experiments in Figs. 3l and 4b–d were performed in one mouse bilaterally and another mouse unilaterally. Experiments in Fig. 6b were performed in three cultures, in Fig. 6c–h, in three mice and in Fig. 6j–l, in three rats.

Experiments in Extended Data Figs. 1a–h and 2a–g were performed in two cultures; in Extended Data Fig. 6, in two mice; in Extended Data Fig. 7, in three mice. All values are expressed as mean ± s.e.m.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

Images used in quantifying transsynaptic experiments are available on the G-Node database at https://gin.g-node.org/Haoyangh/ATLAS_nature_method_2024.git. All original microscopic images are available on request to the corresponding author, as these images require additional annotation and context to be accurately interpreted. AAV8-ATLASsn_Cre, AAV8-DIO-ATLASsn_Flp and AAV-DIO-BACE-HA sequences and plasmids are available at Addgene, IDs 232351, 232352 and 232353. Source data are provided with this paper.

References

Weber, F. & Dan, Y. Circuit-based interrogation of sleep control. Nature 538, 51–59 (2016).
Article CAS PubMed Google Scholar
Zhao, Q. et al. A multidimensional coding architecture of the vagal interoceptive system. Nature 603, 878–884 (2022).
Article CAS PubMed PubMed Central Google Scholar
Condylis, C. et al. Dense functional and molecular readout of a circuit hub in sensory cortex. Science 375, eabl5981 (2022).
Article CAS PubMed PubMed Central Google Scholar
Bayless, D. W. et al. A neural circuit for male sexual behavior and reward. Cell 186, 3862–3881 e3828 (2023).
Article CAS PubMed PubMed Central Google Scholar
Jarvie, B. C. & Knight, Z. A. Breaking down a gut-to-brain circuit that prevents malabsorption. Cell 185, 2393–2395 (2022).
Article CAS PubMed Google Scholar
Kohl, J. et al. Functional circuit architecture underlying parental behaviour. Nature 556, 326–331 (2018).
Article CAS PubMed PubMed Central Google Scholar
Network, B. I. C. C. A multimodal cell census and atlas of the mammalian primary motor cortex. Nature 598, 86–102 (2021).
Article Google Scholar
van der Want, J. J., Klooster, J., Cardozo, B. N., de Weerd, H. & Liem, R. S. Tract-tracing in the nervous system of vertebrates using horseradish peroxidase and its conjugates: tracers, chromogens and stabilization for light and electron microscopy. Brain Res. Protoc. 1, 269–279 (1997).
Article Google Scholar
Callaway, E. M. & Luo, L. Monosynaptic circuit tracing with glycoprotein-deleted rabies viruses. J. Neurosci. 35, 8979–8985 (2015).
Article CAS PubMed PubMed Central Google Scholar
Wickersham, I. R., Finke, S., Conzelmann, K. K. & Callaway, E. M. Retrograde neuronal tracing with a deletion-mutant rabies virus. Nat. Methods 4, 47–49 (2007).
Article CAS PubMed Google Scholar
Weible, A. P. et al. Transgenic targeting of recombinant rabies virus reveals monosynaptic connectivity of specific neurons. J. Neurosci. 30, 16509–16513 (2010).
Article CAS PubMed PubMed Central Google Scholar
Wickersham, I. R. et al. Monosynaptic restriction of transsynaptic tracing from single, genetically targeted neurons. Neuron 53, 639–647 (2007).
Article CAS PubMed PubMed Central Google Scholar
Luo, L., Callaway, E. M. & Svoboda, K. Genetic dissection of neural circuits: a decade of progress. Neuron 98, 865 (2018).
Article CAS PubMed PubMed Central Google Scholar
Li, E. et al. Anterograde transneuronal tracing and genetic control with engineered yellow fever vaccine YFV-17D. Nat. Methods 18, 1542–1551 (2021).
Article CAS PubMed PubMed Central Google Scholar
Zingg, B. et al. AAV-mediated anterograde transsynaptic tagging: mapping corticocollicular input-defined neural pathways for defense behaviors. Neuron 93, 33–47 (2017).
Article CAS PubMed Google Scholar
Zingg, B., Peng, B., Huang, J., Tao, H. W. & Zhang, L. I. Synaptic specificity and application of anterograde transsynaptic AAV for probing neural circuitry. J. Neurosci. 40, 3250–3267 (2020).
Article CAS PubMed PubMed Central Google Scholar
Roberts, R. W. & Szostak, J. W. RNA-peptide fusions for the in vitro selection of peptides and proteins. Proc. Natl Acad. Sci. USA 94, 12297–12302 (1997).
Article CAS PubMed PubMed Central Google Scholar
Gross, G. G. et al. Recombinant probes for visualizing endogenous synaptic proteins in living neurons. Neuron 78, 971–985 (2013).
Article CAS PubMed PubMed Central Google Scholar
Batori, V., Koide, A. & Koide, S. Exploring the potential of the monobody scaffold: effects of loop elongation on the stability of a fibronectin type III domain. Protein Eng. 15, 1015–1020 (2002).
Article CAS PubMed Google Scholar
Miesenbock, G., De Angelis, D. A. & Rothman, J. E. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394, 192–195 (1998).
Article CAS PubMed Google Scholar
Vassar, R. et al. Beta-secretase cleavage of Alzheimer’s amyloid precursor protein by the transmembrane aspartic protease BACE. Science 286, 735–741 (1999).
Article CAS PubMed Google Scholar
Gotzke, H. et al. The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications. Nat. Commun. 10, 4403 (2019).
Article PubMed PubMed Central Google Scholar
Perez, E. E. et al. Establishment of HIV-1 resistance in CD4⁺ T cells by genome editing using zinc-finger nucleases. Nat. Biotechnol. 26, 808–816 (2008).
Article CAS PubMed PubMed Central Google Scholar
Anastasiades, P. G. & Carter, A. G. Circuit organization of the rodent medial prefrontal cortex. Trends Neurosci. 44, 550–563 (2021).
Article CAS PubMed PubMed Central Google Scholar
Benavidez, N. L. et al. Organization of the inputs and outputs of the mouse superior colliculus. Nat. Commun. 12, 4004 (2021).
Article CAS PubMed PubMed Central Google Scholar
Zhuang, J. et al. Laminar distribution and arbor density of two functional classes of thalamic inputs to primary visual cortex. Cell Rep. 37, 109826 (2021).
Article CAS PubMed PubMed Central Google Scholar
Cruz-Martin, A. et al. A dedicated circuit links direction-selective retinal ganglion cells to the primary visual cortex. Nature 507, 358–361 (2014).
Article CAS PubMed PubMed Central Google Scholar
Gerfen, C. R. The neostriatal mosaic: multiple levels of compartmental organization. Trends Neurosci. 15, 133–139 (1992).
Article CAS PubMed Google Scholar
Khibnik, L. A., Tritsch, N. X. & Sabatini, B. L. A direct projection from mouse primary visual cortex to dorsomedial striatum. PLoS ONE 9, e104501 (2014).
Article PubMed PubMed Central Google Scholar
Roth, B. L. DREADDs for neuroscientists. Neuron 89, 683–694 (2016).
Article CAS PubMed PubMed Central Google Scholar
Queiroz Zetune Villa Real, K. et al. A versatile synaptotagmin-1 nanobody provides perturbation-free live synaptic imaging and low linkage-error in super-resolution microscopy. Small Methods 7, e2300218 (2023).
Article PubMed Google Scholar
O’Connor, E. C. et al. Accumbal D1R neurons projecting to lateral hypothalamus authorize feeding. Neuron 88, 553–564 (2015).
Article PubMed Google Scholar
Tsai, N. Y. et al. Trans-Seq maps a selective mammalian retinotectal synapse instructed by Nephronectin. Nat. Neurosci. 25, 659–674 (2022).
Article CAS PubMed PubMed Central Google Scholar
Beier, K. T. et al. Anterograde or retrograde transsynaptic labeling of CNS neurons with vesicular stomatitis virus vectors. Proc. Natl Acad. Sci. USA 108, 15414–15419 (2011).
Article CAS PubMed PubMed Central Google Scholar
Lo, L. & Anderson, D. J. A Cre-dependent, anterograde transsynaptic viral tracer for mapping output pathways of genetically marked neurons. Neuron 72, 938–950 (2011).
Article CAS PubMed PubMed Central Google Scholar
Lin, K. et al. A mutant vesicular stomatitis virus with reduced cytotoxicity and enhanced anterograde trans-synaptic efficiency. Mol. Brain 13, 45 (2020).
Article PubMed PubMed Central Google Scholar
Sun, L. et al. Differences in neurotropism and neurotoxicity among retrograde viral tracers. Mol. Neurodegener. 14, 8 (2019).
Article PubMed PubMed Central Google Scholar
Daigle, T. L. et al. A suite of transgenic driver and reporter mouse lines with enhanced brain-cell-type targeting and functionality. Cell 174, 465–480 e422 (2018).
Article CAS PubMed PubMed Central Google Scholar
Xu, W. & Sudhof, T. C. A neural circuit for memory specificity and generalization. Science 339, 1290–1295 (2013).
Article CAS PubMed PubMed Central Google Scholar
Zhang, Y. et al. Fast and sensitive GCaMP calcium indicators for imaging neural populations. Nature 615, 884–891 (2023).
Article CAS PubMed PubMed Central Google Scholar
Sun, F. et al. Next-generation GRAB sensors for monitoring dopaminergic activity in vivo. Nat. Methods 17, 1156–1166 (2020).
Article CAS PubMed PubMed Central Google Scholar
Liu, X. et al. Optogenetic stimulation of a hippocampal engram activates fear memory recall. Nature 484, 381–385 (2012).
Article CAS PubMed PubMed Central Google Scholar
Faust, T. E., Gunner, G. & Schafer, D. P. Mechanisms governing activity-dependent synaptic pruning in the developing mammalian CNS. Nat. Rev. Neurosci. 22, 657–673 (2021).
Article CAS PubMed PubMed Central Google Scholar
Olson, C. A., Liao, H.-I., Sun, R. & Roberts, R. W. mRNA display selection of a high-affinity, modification-specific phospho-IkappaBalpha-binding fibronectin. ACS Chem. Biol. 3, 480–485 (2008).
Article CAS PubMed PubMed Central Google Scholar
Lewis, T. L. Jr., Mao, T., Svoboda, K. & Arnold, D. B. Myosin-dependent targeting of transmembrane proteins to neuronal dendrites. Nat. Neurosci. 12, 568–576 (2009).
Article CAS PubMed PubMed Central Google Scholar
Stoppini, L., Buchs, P. A. & Muller, D. A simple method for organotypic cultures of nervous tissue. J. Neurosci. Methods 37, 173–182 (1991).
Article CAS PubMed Google Scholar
Schnell, E. et al. Direct interactions between PSD-95 and stargazin control synaptic AMPA receptor number. Proc. Natl Acad. Sci. USA 99, 13902–13907 (2002).
Article CAS PubMed PubMed Central Google Scholar
Lu, W. et al. Subunit composition of synaptic AMPA receptors revealed by a single-cell genetic approach. Neuron 62, 254–268 (2009).
Article CAS PubMed PubMed Central Google Scholar
George Paxinos, K. F. Paxinos and Franklin’s the Mouse Brain in Stereotaxic Coordinates 4th edn, Vol. 1 (Elsevier/Academic, 2013).
Google Scholar
Saunders, A. et al. A direct GABAergic output from the basal ganglia to frontal cortex. Nature 521, 85–89 (2015).
Article CAS PubMed PubMed Central Google Scholar
Wallace, M. L. et al. Genetically distinct parallel pathways in the entopeduncular nucleus for limbic and sensorimotor output of the basal ganglia. Neuron 94, 138–152 e135 (2017).
Article CAS PubMed PubMed Central Google Scholar

Download references

Acknowledgements

We thank all Arnold laboratory members for helpful discussions. We also thank A. Delgadillo, A. Bareghamyan, R. Yan, X. Xi and C. McWilliam for technical assistance, and F. Wang for crucial input at the beginning stages of this project. We thank E. Liman for her insightful comments on the manuscript. From the Sabatini laboratory, we thank W. Wang, S. Tang and K. Reinhold for excellent experimental assistance and intellectual input. This work was supported by a grant from the National Institute of Mental Health (NIMH) and the Brain Initiative (grant no. MH116989) to D.B.A., NIMH IRP award (grant no. ZIA MH002497-34) to C.R.G., a grant (no. DK104897) to S.E.K. from NIDDK, by NINDS grant (no. R37NS046579) to B.L.S. and by Brain Initiative NRSA (grant no. 5F32MH125596) to A.E.G.

Author information

These authors contributed equally: Jacqueline F. Rivera, Haoyang Huang.

Authors and Affiliations

Department of Biological Sciences, Dornsife College, University of Southern California, Los Angeles, CA, USA
Jacqueline F. Rivera, Haoyang Huang, Weiguang Weng, Heesung Sohn, Melissa Qin, Molly E. Klug, Sadhna Rao, Bruce E. Herring, Scott E. Kanoski & Don B. Arnold
Neuroscience Graduate Program, University of Southern California, Los Angeles, CA, USA
Haoyang Huang, Heesung Sohn, Sadhna Rao, Bruce E. Herring, Scott E. Kanoski & Don B. Arnold
Howard Hughes Medical Institute, Department of Neurobiology, Harvard Medical School, Boston, MA, USA
Allison E. Girasole, Shun Li, Madeline A. Albanese & Bernardo L. Sabatini
Center for Neural Circuits and Sensory Processing Disorders, Zilkha Neurogenetic Institute, Department of Physiology and Neuroscience, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
Can Tao & Li I. Zhang
Section on Neuroanatomy, National Institute of Mental Health, Bethesda, MD, USA
Ronald Paletzki & Charles R. Gerfen
Department of Biomedical Engineering, Viterbi School of Engineering, University of Southern California, Los Angeles, CA, USA
Don B. Arnold

Authors

Jacqueline F. Rivera
View author publications
Search author on:PubMed Google Scholar
Haoyang Huang
View author publications
Search author on:PubMed Google Scholar
Weiguang Weng
View author publications
Search author on:PubMed Google Scholar
Heesung Sohn
View author publications
Search author on:PubMed Google Scholar
Allison E. Girasole
View author publications
Search author on:PubMed Google Scholar
Shun Li
View author publications
Search author on:PubMed Google Scholar
Madeline A. Albanese
View author publications
Search author on:PubMed Google Scholar
Melissa Qin
View author publications
Search author on:PubMed Google Scholar
Can Tao
View author publications
Search author on:PubMed Google Scholar
Molly E. Klug
View author publications
Search author on:PubMed Google Scholar
Sadhna Rao
View author publications
Search author on:PubMed Google Scholar
Ronald Paletzki
View author publications
Search author on:PubMed Google Scholar
Bruce E. Herring
View author publications
Search author on:PubMed Google Scholar
Scott E. Kanoski
View author publications
Search author on:PubMed Google Scholar
Li I. Zhang
View author publications
Search author on:PubMed Google Scholar
Charles R. Gerfen
View author publications
Search author on:PubMed Google Scholar
Bernardo L. Sabatini
View author publications
Search author on:PubMed Google Scholar
Don B. Arnold
View author publications
Search author on:PubMed Google Scholar

Contributions

D.B.A. conceived and designed the study, assisted with data analysis and wrote the paper with input from all authors. J.F.R. generated the AF and performed the biochemistry and neuronal culture-related experiments. J.F.R. and M.Q. designed and generated the plasmid constructs. H.H., W.W. and H.S. performed and analyzed all in vivo tracing experiments in mice except for those mentioned below. J.F.R., W.W., H.S., H.H. and M.Q. generated the AAVs and lentiviruses. L.I.Z. and C.T. designed, and C.T. performed, the electrophysiology experiments in Extended Data Fig. 4a–f. A.E.G., S.L. and B.L.S. designed, and A.E.G., S.L. and M.A.A. performed, the electrophysiology experiments in Fig. 4e–h and the related experiments in Extended Data Fig. 4g,h. S.R. and B.E,H. designed, and S.R. performed, the electrophysiology experiments in Fig. 1. C.R.G. designed, and R.P. performed, the experiments in Fig. 6e–h and Extended Data Fig. 7c–h. M.E.K. and S.E.K. designed, and M.E.K. performed, the experiments in Fig. 6i–l.

Corresponding author

Correspondence to Don B. Arnold.

Ethics declarations

Competing interests

An international patent application (no. PCT/US2024/044732) has been filed. Inventors: D.B.A., J.F.R., H.H. and W.W. Status of application: submitted but not reviewed. The entire manuscript is covered in the patent application. The remaining authors declare no competing interests.

Peer review

Peer review information

Nature Methods thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editor: Nina Vogt, in collaboration with the Nature Methods team.

Additional information

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

Extended data

Extended Data Fig. 1 Testing of the AMPA.FingR.

a. COS7 cells cotransfected with ss-Fn9.3-MYC, Stargazin-V5, and GluA1-HA. b. Permeabilized labeling of HA in same cells as in a. c. COS7 cells cotransfected with Fn6.5-MYC, Stargazin-V5, and GluA1-HA. d. Permeabilized labeling of HA in same cells as in c. e., f. Labeling following co-expression of ss-Fn9.3-MYC with GFP in cultured cortical neurons. g., h. Lack of MYC surface labeling following co-expression of ss-Fn9.11-MYC with GFP in cultured cortical neurons. i. Live cortical neuron in culture expressing ss-AF-YFP (green) and PSD-95.FingR-tagRFP (magenta). Figure 1c is a closeup of the boxed region. j. DIC image of cell in i. k. Closeup of boxed area in j. Arrowheads point to the same synapses in k., l., and m. l. Closeup of boxed area in i. showing ss-AF-YFP. m. Closeup of boxed area in i showing PSD-95.FingR-tagRFP. Scale bars 20 μm in a.-h., 10 μm in i.-m.

Extended Data Fig. 2 Transsynaptic labeling in culture with ATLAS.

a. Presynaptic neuron labeled with At. b. Postsynaptic neurons labeled with GFP in same experiment as in a. c. Merge of a. and b. showing presynaptic neuron (At) labeled in magenta and postsynaptic neurons (GFP) labeled in green. d. Closeup of boxed region d from panel c. showing adjacent/overlapping presynaptic termini (magenta) and dendrites or spines (green) indicated by arrowhead, consistent with synaptic contacts between the presynaptic neuron in a. and postsynaptic neuron 1 in c. e. Closeup of boxed region e from panel c. showing adjacent/overlapping presynaptic termini (magenta) and dendrites or spines (green) indicated by arrowhead, consistent with synaptic contacts between the presynaptic neuron in a. and postsynaptic neuron 2 in c. f. Closeup of boxed region f from panel c. showing adjacent/overlapping presynaptic termini (magenta) and dendrites or spines (green) indicated by arrowhead consistent with synaptic contacts between the presynaptic neuron in a. and postsynaptic neuron 3 in c. g. Closeup of boxed region g from panel c. showing adjacent/overlapping presynaptic termini (magenta) and dendrites or spines (green) indicated by arrowhead. Scale bars 100 μm in c, 10 μm in d-g.

Extended Data Fig. 3 Improving the efficiency of ATLAS by co-expressing BACE.

a. mCherry (magenta) and endogenous NeuN (green) labeling in Str in mouse brain infected with AAV8-ATLAS_Cre in mPFC and AAV8-DIO-mCherry in Str. b. Same as in a. with a 1:1 ratio (viral particles) of AAV8-ATLAS_Cre:BACE-HA injected into mPFC and AAV8-DIO-mCherry in Str. c. Same as in a. with 1:5 ratio of AAV8-ATLAS_Cre:BACE-HA injected into mPFC and AAV8-DIO-mCherry in the Str. d. Closeup of Str from same experiment as in c. e. Control staining from an experiment with an injection of AAV8-DIO-mCherry only in Str. f. Expressing ATLAS + BACE-HA 1:5 in mPFC leads to significantly higher labeling density in Str than ATLAS alone or ATLAS + BACE 1:1. (Tukey’s multiple comparisons test). Scale bars 100 μm.

Source data

Extended Data Fig. 4 ATLAS tracing does not disrupt synaptic transmission.

a. Schematic of configuration for whole cell patch clamp recording from Str neurons following ATLAS-mediated tracing from mPFC. b. Example traces of light-evoked EPSCs from mCherry- and mCherry+ striatal neurons following optical activation of inputs from mPFC. c. Statistics comparing onset latency of eEPSC between mCherry- (n = 7 cells from 3 animals) and mCherry+ (n = 8 cells from 3 animals, mean +/− SEM) striatal neurons. Two-tailed unpaired t-test, N.S., P = 0.2251. d. Statistics comparing amplitudes of eEPSCs measured in mCherry- (n = 7 cells from 3 animals) and mCherry+ (n = 8 cells from 3 animals, mean +/− SEM) striatal neurons. Two-tailed unpaired t test, N.S., P = 0.2766. e. Statistics comparing time constants of eEPSCs measured in mCherry- (n = 7 cells from 3 animals, mean +/− SEM) and mCherry+ (n = 8 cells from 3 animals) striatal neurons. Two-tailed unpaired t-test, N.S., P = 0.2342. f. Traces illustrate the monosynaptic nature of the excitatory input to mCherry-labeled striatal neurons. eEPSC was suppressed by TTX application and rescued by 4AP. g. High magnification histological sections showing presynaptic expression patterns in V1 of BACE (upper left), Cre-dependent ChR2 (upper right), ATLASsn_Cre (At, lower left), and merged (lower right), as in experiments in Fig. 4e–h. h. High magnification histological sections showing postsynaptic expression patterns in the dorsomedial striatum of mCherry/tdTomato (upper left), Cre-dependent ChR2 (upper right), and ATLASsn_Cre (At, lower left), and merged (lower right), as in experiments in Fig. 4e–h. Experiments in g. and h. were performed in 3 mice. Scale bars 100 μm.

Source data

Extended Data Fig. 5 In vivo transsynaptic labeling with ATLAS is activity-dependent.

a. Coronal section from mPFC of mouse infected with AAV8-ATLAS_Cre + AAV8-hHM3Dq-mCherry in mPFC and AAV8-DIO-GFP in Str, stained after one week. b. Same section as in a. labeled for endogenous c-FOS (green). c. Merge of a. and b. d. Closeup of marked region in c. e. GFP labeling of postsynaptic cells in Str. f. Closeup of marked region in e. g.Str from same mouse as a.-e. showing c-FOS labeling (red). h. Same section as g. showing GFP labeling. i. Merge of g. and h. j. Coronal section from mouse as in a. subjected to intraperitoneal injections of CNO. k. Same section as j. labeled for endogenous c-FOS. l. Merge of j. and k. m. Closeup of marked region in l. showing colocalization of c-FOS and At (arrowheads). n. GFP labeling of postsynaptic cells in Str. o.Closeup of marked region in n. p. Str from same mouse as in j-r labeled with c-FOS (red). q. Same section as p showing GFP labeling. r. Merge of p. and q. s. Significantly more cells in Str were labeled with GFP in mice injected with CNO vs. those without (n = 5, unpaired t-test, two-tailed). Each data point represents a biological replicate in a different mouse. t. Intensity of GFP label in Str was significantly greater in mice injected with CNO vs. mice without CNO (n = 5, unpaired t-test, two-tailed). Each data point represents a biological replicate in a different mouse. u. The number of cells labeled with At in mPFC was not significantly different in mice injected with CNO vs. in mice without CNO (n = 5, unpaired t-test, two-tailed). Each data point represents a biological replicate in a different mouse. Scale bar 100 μm. Error bars depict mean +/− SEM.

Source data

Extended Data Fig. 6 Toxicity associated with ATLAS, but not ATLASsn.

a. Coronal section of mPFC following expression of ATLASsn_Cre for 2 weeks. Toxicity is evidenced by degenerating processes (arrowhead) and swollen cell bodies (arrow). Experiments were performed in two mice. b. Coronal section of mPFC following expression of ATLASsn_Cre for 2 weeks. Experiments were performed in two mice. c. Same as a. following expression for 4 weeks. Degenerating processes (arrowhead) and swollen cell bodies (arrows) are apparent. Experiments were performed in two mice. d. Same as b. following expression for 4 weeks.

Extended Data Fig. 7 Transsynaptic labeling of ATLASsn_Cre from the cortex to the striatum and thalamus.

a. Coronal section from striatum of mouse in Fig. 6c, d injected with AAV8-ATLASsn_Cre in medial prefrontal cortex and AAV8-DIO-mCherry in the striatum and incubated for two weeks showing At labeling of presynaptic termini. b. Coronal section from striatum of same mouse as in a. showing mCherry-labeled neurons. c. Coronal section at the level of the striatum of mouse brain injected with AAV8-ATLASsn_Cre + AAV8-BACE-GFP in the M1 cortex and AAV8-DIO-mCherry in the striatum and thalamus. Sections were stained for GFP (green), mCherry (red), and fluorescent Nissl stain (blue). d. Closeup of area marked in c. showing overlap of projections from neurons expressing AAV8-BACE-GFP (green) and neurons expressing mCherry. e. Coronal section at the level of the thalamus of the same mouse brain as in c. and d. showing projections of neurons expressing BACE-GFP (green), neurons expressing mCherry (red) and Nissl stain (blue). f. Closeup of area marked in e. showing overlap of projections from neurons expressing AAV8- BACE-GFP (green) and neurons expressing mCherry. g. Coronal section at the level of the striatum of a control mouse injected with AAV8-DIO-mCherry in the striatum in an identical manner to the mice in c.-f. but with no injections in the cortex. Negligible mCherry labeling (red) is visible in the striatum. Sections were also stained with Nissl stain (blue). h. Closeup of area marked in g. showing negligible mCherry labeling. Scale bars in a, b, d, f, and h 100 μm; in c, e, and g 1 mm.

Extended Data Fig. 8 Transsynaptic labeling of ATLASsn_Cre from cortical to subcortical areas.

a. Coronal section from auditory cortex (A1) of a mouse injected with AAV8-ATLASsn_Cre and AAV8-BACE-HA in A1, and AAV8-DIO-GFP in bilateral inferior colliculi (IC) and incubated for two weeks showing At labeling of presynaptic neurons. b. Coronal section from the same mouse as in a. showing GFP labeling in bilateral IC consistent with bilateral postsynaptic staining. c. Closeup of area marked in b. d. Coronal section from prefrontal cortex of a mouse injected with AAV8-ATLASsn_Cre and AAV8-BACE-HA in the prefrontal cortex, and AAV8-DIO-GFP in the claustrum (Cl) and incubated for two weeks showing At labeling of presynaptic neurons. e. Coronal section from the same mouse as in d. showing GFP labeling in Cl. f. Closeup of area marked in e. g. Horizontal section from entorhinal cortex of a mouse injected with AAV8-ATLASsn_Cre and AAV8-BACE-HA in the entorhinal cortex (EC), and AAV8-DIO-GFP in the hippocampus, and incubated for two weeks showing At labeling of presynaptic neurons. h. Horizontal section from the same mouse as in g. showing GFP labeling of dentate granule cells in the dentate gyrus (DG) and cells in the subiculum (Sub). i. Closeup of area marked in h. j. Quantitation of labeling of anterograde pathways. PFC-STR, Extended Data Fig. 3; PFC-CL, Extended Data Fig. 8d–f.; A1-IC, Extended Data Fig. 8a–c.; EC-Hp, Extended Data Fig. 8g–i.; SC-PBG, Fig. 5j–l.; LGN-V1, Fig. 3i–l.; V1-SC, Fig. 3e–h. Each data point represents a biological replicate in a different mouse. Error bars depict mean +/− SEM. Scale bars 100 μm.

Source data

Extended Data Fig. 9 Time course of transsynaptic tracing from mPFC to striatum.

a. Coronal section from PFC of a mouse injected with AAV8-ATLASsn_Cre and AAV8-BACE-HA in PFC, and AAV8-DIO-GFP in Str, and incubated for four days. b. Coronal section from the same mouse as in a. c. Coronal section from PFC of an experiment similar to that in a. incubated for 7 days. d. Coronal section of Str from the same mouse as in c. e. Coronal section from PFC of an experiment similar to that in a. and c. incubated for 14 days. f. Coronal section of Str from the same mouse as in e. g. Coronal section from PFC of an experiment similar to that in a., c., and e. incubated for 21 days. h. Coronal section of striatum from the same mouse as in g. i. The number of cells labeled in the PFC between 4 days and 21 days. j. The total intensity of GFP labeling in Str between 4 and 21 days. k. The number of cells labeled with GFP in Str between 4 and 21 days. Each data point represents a biological replicate in a different mouse. Scale bars 100 μm.

Source data

Supplementary information

Reporting Summary

Supplementary Table 1

Quantitation for Figs. 3–6 and Extended Data Figs. 3–5, 8 and 9.

Source data

Source Data Fig. 1

Statistical source data for Fig. 1i–p. Entire lane of western blot in 1a for AF. Entire lane of western blot in 1a for GluA1. The same lane is labeled with both AF and GluA1 with different secondary antibodies and fluorophores.

Source Data Fig. 4

Statistical source data for Fig. 4b–d,g,h.

Source Data Fig. 5

Statistical source data for Fig. 5e,n.

Source Data Extended Data Fig. 3

Statistical source data for Extended Data Fig. 3f.

Source Data Extended Data Fig. 4

Statistical source data for Extended Data Fig. 4c–e.

Source Data Extended Data Fig. 5

Statistical source data for Extended Data Fig. 5s–u.

Source Data Extended Data Fig. 8

Statistical source data for Extended Data Fig. 8j.

Source Data Extended Data Fig. 9

Statistical source data for Extended Data Fig. 9i–k.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, 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 you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. 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 http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Cite this article

Rivera, J.F., Huang, H., Weng, W. et al. ATLAS: a rationally designed anterograde transsynaptic tracer. Nat Methods 22, 1101–1111 (2025). https://doi.org/10.1038/s41592-025-02670-x

Download citation

Received: 16 February 2024
Accepted: 11 March 2025
Published: 01 May 2025
Issue Date: May 2025
DOI: https://doi.org/10.1038/s41592-025-02670-x

Subjects

Abstract

Similar content being viewed by others

Main

Results

Generating AF, a binder to the N terminus of GluA1

Transsynaptic tracing in vitro and in vivo

Tracing from genetically determined cells

Increasing efficiency of transsynaptic labeling with BACE

Labeling is transsynaptic

ATLAS tracing does not block synaptic transmission

Electrophysiology confirms specificity of ATLAS tracing

ATLAS is exclusively anterograde and monosynaptic

Activity dependence of ATLAS

An alternate synaptic vesicle targeting domain

Labeling of cortical to subcortical circuits

ATLAS mediates transsynaptic labeling in the rat

Discussion

Methods

AF production

Plasmid constructs

Dissociated cultures

Protein purification

Endocytosis

Presynaptic release

Viruses

Transsynaptic tracing in culture

Immunocytochemistry for dissociated cultures

Electrophysiology (testing of AF)

Animal preparation and stereotaxic surgery (mouse)

Injections of viruses (mouse)

Histology (mouse)

Quantification of transsynaptic labeling

Stereotaxic surgery and histology (M1 to Str and Thal)

Animal preparation and stereotaxic surgery (rat)

Histology (rat)

Quantification of activity-dependent transsynaptic labeling

Electrophysiology (PFC to Str)

Surgery (V1 to Str)

Electrophysiology (V1 to Str)

Statistics and reproducibility

Reporting summary

Data availability

References

Acknowledgements

Author information

Authors and Affiliations

Contributions

Corresponding author

Ethics declarations

Competing interests

Peer review

Peer review information

Additional information

Extended data

Supplementary information

Source data

Rights and permissions

About this article

Cite this article

Share this article

Search

Quick links