Journal: bioRxiv

Article Title: TRACR: an anterograde transneuronal tracing system for genetic access across synapses and longitudinal circuit analysis

doi: 10.64898/2026.02.08.704659

Figure Lengend Snippet: (A) Schematic of TRACR. A presynaptic Sender neuron (green) displays an NRX–GFP ligand enriched at synaptic terminals. A postsynaptic Receiver neuron (magenta) expresses synNotch receptors comprising an extracellular GFP nanobody (LaG17), the Notch core regulatory and transmembrane domains (grey), and the intracellular transcriptional activator tTA. Ligand binding across the synapse [1] triggers intramembrane proteolysis of synNotch [2] and release of tTA, which translocates to the nucleus to activate a TRE-responsive Reporter [3], labeling the postsynaptic neuron with RFP. (B) TRACR components. Sender AAVs encode NRX-GFP, a 2A peptide, and a marker or effector transgene. Sender expression is driven by either a cell-type-specific promoter (Pro) or a pan-neuronal promoter (hSyn), and can be restricted by Cre-dependent DIO. Receiver AAVs encode two synNotch-tTA variants with an N-terminal Myc. TRE-driven reporters used here include AAV-TRE-mRuby2 and Ai63 (TIT-tdTomato) mice. (C) Sender-GFP localization in cultured DIV14 cortical neurons co-infected with AAV-hSyn-Cre and AAV-hSyn-DIO-NRXGFP-T2A-Flpo. Surface staining (green; no Triton X-100 (-TX) shows NRX–GFP at the plasma membrane and at presynaptic sites overlapping with Synapsin1 (red, with sequential permeabilization; inset enlarged at right). (D) synNotch localization in cultured neurons infected with Receiver AAV-hSyn-myc-LaG17-synNotch-tTA. Immunostaining for Myc (white) shows synNotch along neurites and at the cell surface (right, no Triton X-100). (E) Strategy to visualize Sender-GFP within presynaptic terminals of retinal ganglion cells (RGCs) in dorsal lateral geniculate nuclei (dLGN). AAVs encoding Sender (hSyn-DIO-NRXGFP-T2A-Flpo) and synaptic vesicle marker (hSyn-DIO-HA-SV-2A-tdTomato) were delivered to retinas of vGluT2-Cre mice. (F) Confocal image of dLGN (dashed outline) shows NRX-GFP (green) overlap with RGC terminal axons (TdTomato, magenta). Inverted images of tdTomato (middle) and GFP channels (right) show lack GFP along TdTomato+ axon tracts (black arrows). (G) Localization of NRX-GFP ligand (green) within RGC terminal boutons, and overlapping with HA-tagged synaptic vesicle (SV) marker (magenta). (H) AAV strategy to express Sender (hSyn-DIO-NRXGFP-T2A-ChR2-YFP) in the retina, and Receiver (hSyn-synNotch) and Reporter (TRE-mRuby2) in the thalamus of vGluT2-Cre mice. Mice were injected at 3-4 postnatal weeks and harvested at 2 months of age. (I) Section of thalamus showing TRACR labeled retinothalamic connections. Receiver neurons (Myc, orange) are broadly distributed across thalamic nuclei, whereas Reporter expression (RFP, magenta) is restricted to Myc+ Receiver cells within the target region (dLGN) and is absent from non-target nuclei, including laterodorsal (LD) and ventral posteromedial (VPM). Right: enlarged insets. Scale bars: 50 µm (C,D); 200 µm (F, top) and 20µm (F, bottom); 200 µm (H, left) and 10 µm (H, right).

Article Snippet: To create a Sender with a channelrhodopsin marker (pAAV.hSyn-DIO-Nrx3b-GFP-T2A-hChR2(H134R)-eYFP-WPRE-pA), the smFP HA insert was replaced with hChR2(H134R)-eYFP (Addgene plasmid #26973, a gift from Karl Deisseroth; http://n2t.net/addgene:26973 ; RRID:Addgene_26973).

Techniques: Ligand Binding Assay, Labeling, Marker, Expressing, Cell Culture, Infection, Staining, Clinical Proteomics, Membrane, Immunostaining, Injection

Journal: bioRxiv

Article Title: TRACR: an anterograde transneuronal tracing system for genetic access across synapses and longitudinal circuit analysis

doi: 10.64898/2026.02.08.704659

Figure Lengend Snippet: (A) Schematic of the TRACR strategy and predicted activation pattern at the photoreceptor–bipolar cell synapse. At postnatal day (P) 5, mice were co-infected with a Sender AAV driving NRX–GFP expression in photoreceptors (subretinal delivery of AAV-ProC1-NRXGFP-T2A-JawsHA), and an Receiver-AAV (intravitreal delivery of AAV-4xGrm6-MycLaG17-synNotchRAM7-tTA) driving synNotch expression in bipolar cells of Ai63 reporter mice. Synaptic contact between Sender- and Receiver-expressing cells induces TdTomato (RFP) reporter expression in Receiver-positive cells, analyzed at P20. (B) Localization of the NRX–GFP ligand in photoreceptor terminals. Retinal cross-section showing NRX–GFP fluorescence (without GFP amplification) enrichment in photoreceptor terminals, in proximity to presynaptic markers Ribeye (magenta) and PSD95 (white). (C) TRACR activation requires Sender and Receiver expression. Representative P20 retinal sections from Ai63 mice infected with the indicated AAVs and stained for Sender (GFP, green), Receiver (Myc, white), Reporter (TdTomato, magenta), and nuclei (Hoechst, blue). Left: Receiver (Rec)-only controls show minimal ligand-independent reporter activation). Middle: Co-injection of Sender (S) and Receiver results in robust reporter activation confined to Receiver-expressing bipolar cells (middle). Right: Percentage of Reporter-positive Receiver-expressing cells. Each data point represents the mean of three sections from a single retina, 6 retinas per condition. Data are shown as mean ± SD. ****p < 0.0001, Welch’s t-test. (D) TRACR detects connections to rod and cone bipolar cells. Left: Sender- and Receiver-infected retinas from P20 Ai63 mice show rod bipolar cells (RBC; PKC, yellow), and cone bipolar cells (CBC, SCGN, cyan) express RFP (magenta). Right: Percentage of marker-positive Reporter-positive cells. N= 6 retinas. Data are shown as mean ± SD. (E) Ligand proximity alone is insufficient to activate TRACR. Left: Schematic and representative retinal sections showing photoreceptor (PhR)-driven Sender (GFP, green; AAV-ProC1-NRXGFP) and Receiver expression (Myc, white; AAV-ProC1-synNotch). Middle: Schematic and representative sections showing Müller glia-driven Sender (AAV-ProB2-NRXGFP) and Receiver expression (AAV-Grm6-synNotch) in bipolar cells. In both conditions, reporter activation is minimal or absent. Right: Percentage of Reporter-positive Receiver-expressing cells. N = 3 retinas (PhR, Photoreceptor Sender/Receiver), 6 retinas (MG, Müller glia Sender). Data are shown as mean ± SD. Scale bars: 5 μm (B), 20 μm (C, D, E).

Article Snippet: To create a Sender with a channelrhodopsin marker (pAAV.hSyn-DIO-Nrx3b-GFP-T2A-hChR2(H134R)-eYFP-WPRE-pA), the smFP HA insert was replaced with hChR2(H134R)-eYFP (Addgene plasmid #26973, a gift from Karl Deisseroth; http://n2t.net/addgene:26973 ; RRID:Addgene_26973).

Techniques: Activation Assay, Infection, Expressing, Fluorescence, Amplification, Staining, Injection, Marker