Journal: The Journal of Neuroscience

Article Title: Synaptic Specificity and Application of Anterograde Transsynaptic AAV for Probing Neural Circuitry

doi: 10.1523/JNEUROSCI.2158-19.2020

Figure Lengend Snippet: Application in descending pathways to the spinal cord. A, Retrograde labeling of spinal-projecting cortical and subcortical neuronal populations following injections of AAVretro-hSyn-Cre (red) and AAVretro-hSyn-GFP (green) into the left side of the cervical and lumbar spinal cord, respectively, in Ai14 mice. Scale bars: A–C, 500 µm. B, Lack of axonal projections from spinal cord to selected brain regions following cervical injection of AAV1-hSyn-GFP (green) and lumbar injection AAV1-CAG-tdTomato (red). C, Corresponding injections of scAAV1-hSyn-Cre (red; 100 nl injection volume) into different spinal-projecting brain regions (A, top) in Ai14 mice. D, Different patterns of transsynaptic labeling at cervical, thoracic, and lumbar segments of the spinal cord for each injection following a 2 week postinjection survival time. Each column corresponds to the injection site shown in C. Scale bars, 250 µm. E, Quantification of the average number of anterograde transsynaptically labeled cells per coronal section of cervical (C) thoracic (T), or lumbar (L) spinal cord for each injection (n = 1 mouse each). Error bar = SD.

Article Snippet: In addition, we observed that AAV1-hSyn-Flp was capable of labeling comparable numbers of cells in SC compared with Cre-expressing AAV1 constructs ( C , D ), suggesting this approach may be applied interchangeably with AAV1 viruses expressing different forms of recombinase. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Virus Titer Source Addgene Plasmid ID AAV1 hSyn-Cre-WPRE 2.5 × 10 13 GC/ml Addgene 105553 scAAV1 hSyn-Cre 2.8 × 10 13 GC/ml Vigene Biosciences AAV1 hSyn-Flp 5.5 × 10 13 GC/ml Vigene Biosciences 51669 AAVretro-hSyn-Cre 1.5 × 10 14 GC/ml Vigene Biosciences 105537 AAV1-CMV-Cre 2.7 × 10 13 GC/ml Addgene 105537 AAV5-CMV-Cre 2.8 × 10 13 GC/ml Addgene 105537 AAV6-CMV-Cre 3.5 × 10 13 GC/ml Addgene 105537 AAV8-CMV-Cre 4.4 × 10 13 GC/ml Addgene 105537 AAV9-CMV-Cre 1.6 × 10 14 GC/ml Addgene 105537 AAVPHP.B-CMV-Cre 2.3 × 10 13 GC/ml SignaGen CAV2-CMV-Cre 1.3 × 10 12 GC/ml Montpellier Viral Core Adenovirus (Ad5-CMV-Cre) 3.0 × 10 12 GC/ml Kerafast Lentivirus (LV-CMV-Cre) 1.0 × 10 8 GC/ml Cellomics Tech Baculovirus (BAC-CMV-Cre) 3.7 × 10 10 GC/ml Uni. of Iowa G-deleted Rabies virus (RV-Cre-GFP) 8.6 × 10 8 GC/ml Salk Institute G-deleted Rabies virus (RV-GFP) 5.5 × 10 8 GC/ml Salk Institute AAV1-EF1a-DIO-Flp-WPRE 1.5 × 10 14 GC/ml Vigene Biosciences 87306 AAV d J-EF1a-fDIO-YFP-WPRE 2.5 × 10 13 GC/ml UNC Viral Core 55641 AAV1-CAG-FLEX-GFP-WPRE 1.7 × 10 13 GC/ml Addgene 51502 AAVretro-hSyn-GFP-WPRE 1.7 × 10 14 GC/ml Vigene Biosciences 105539 AAV1 hSyn-GFP-WPRE 3.2 × 10 13 GC/ml Addgene 105539 AAV1-EF1a-DIO-hChR2-eYFP-WPRE 1.6 × 10 13 GC/ml Addgene 20298 AAV d J-CMV-TeNT-P2A-GFP 5.7 × 10 12 GC/ml Stanford Viral Core AAVretro-EF1a-Cre-WPRE 2.3 × 10 13 GC/ml Salk Institute 55636 Open in a separate window List of viruses used in this study Moreover, among the four AAV1 viruses tested, we observed a nearly twofold increase in the number of anterograde transsynaptically labeled cells when using AAV1-hSyn-Cre-WPRE compared with scAAV1-hSyn-Cre, AAV1-CMV-Cre, or AAV1-hSyn-Flp, which each lack the WPRE ( D ; ).

Techniques: Labeling, Injection

Journal: The Journal of Neuroscience

Article Title: Synaptic Specificity and Application of Anterograde Transsynaptic AAV for Probing Neural Circuitry

doi: 10.1523/JNEUROSCI.2158-19.2020

Figure Lengend Snippet: Comparison of anterograde transsynaptic spread, toxicity, and retrograde transport potential. A, Injection of AAV1-hSyn-Cre-WPRE in V1 of Ai14 mice (top). Overexpression of Cre may result in cell death at the injection site, as seen by a reduction in Nissl stain intensity and irregular cell morphology (middle). Bottom, Anterograde transsynaptically labeled neurons in SC. B, Injection of scAAV1-hSyn-Cre in Ai14 mice (top). This virus lacks the WPRE enhancer and shows no apparent toxicity in neurons at the injection site (middle). Bottom, Anterograde transsynaptically labeled neurons in SC. C, AAV1-hSyn-Flp injection in Frt-GFP mice (top). This virus also lacks the WPRE enhancer and shows no apparent toxicity (middle). Bottom, Anterograde transsynaptically labeled neurons in SC. All injections 60 nl total volume, 4 week postinjection survival time. Scale bars, 500 µm. D, Quantification of total number of cells labeled in SC for different Cre or Flp-expressing viruses injected into V1 (4 week postinjection survival, 60 nl injection, n = 4 mice each). Error bar = SD. E, Injection of AAVretro-hSyn-Cre in IC of Ai14 mice (left). Retrograde labeling in A1 (middle), and close-up of dashed region (right). Scale bars: left and middle, 500 µm; right, 100 µm. F, Injection of scAAV1-hSyn-Cre in IC of Ai14 mice(left). Retrograde labeling in A1 (middle), and close-up of dashed region (right). Scale bars: left and middle, 500 µm; right, 100 µm. G, Injection of scAAV1-hSyn-Cre in A1 of Ai14 mice (left). Anterograde transsynaptic labeling of cells in IC (middle), and close up of dashed region (right). Scale bars: left and middle, 500 µm; right, 100 µm. H, Quantification of retrograde labeling in A1 following injection of scAAV1-hSyn-Cre or AAVretro-hSyn-Cre in IC (n = 3 mice for each group). All injections 80 nl total volume, 2 week postinjection survival time. Error bar = SD. ***p < 0.001, t test. I, Quantification of anterograde transsynaptic labeling in IC following injection of scAAV1-hSyn-Cre or AAVretro-hSyn-Cre in A1 (n = 3 mice for each group). All injections 80 nl total volume, 2 week postinjection survival time. Error bar = SD. *p < 0.05, t test.

Article Snippet: In addition, we observed that AAV1-hSyn-Flp was capable of labeling comparable numbers of cells in SC compared with Cre-expressing AAV1 constructs ( C , D ), suggesting this approach may be applied interchangeably with AAV1 viruses expressing different forms of recombinase. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Virus Titer Source Addgene Plasmid ID AAV1 hSyn-Cre-WPRE 2.5 × 10 13 GC/ml Addgene 105553 scAAV1 hSyn-Cre 2.8 × 10 13 GC/ml Vigene Biosciences AAV1 hSyn-Flp 5.5 × 10 13 GC/ml Vigene Biosciences 51669 AAVretro-hSyn-Cre 1.5 × 10 14 GC/ml Vigene Biosciences 105537 AAV1-CMV-Cre 2.7 × 10 13 GC/ml Addgene 105537 AAV5-CMV-Cre 2.8 × 10 13 GC/ml Addgene 105537 AAV6-CMV-Cre 3.5 × 10 13 GC/ml Addgene 105537 AAV8-CMV-Cre 4.4 × 10 13 GC/ml Addgene 105537 AAV9-CMV-Cre 1.6 × 10 14 GC/ml Addgene 105537 AAVPHP.B-CMV-Cre 2.3 × 10 13 GC/ml SignaGen CAV2-CMV-Cre 1.3 × 10 12 GC/ml Montpellier Viral Core Adenovirus (Ad5-CMV-Cre) 3.0 × 10 12 GC/ml Kerafast Lentivirus (LV-CMV-Cre) 1.0 × 10 8 GC/ml Cellomics Tech Baculovirus (BAC-CMV-Cre) 3.7 × 10 10 GC/ml Uni. of Iowa G-deleted Rabies virus (RV-Cre-GFP) 8.6 × 10 8 GC/ml Salk Institute G-deleted Rabies virus (RV-GFP) 5.5 × 10 8 GC/ml Salk Institute AAV1-EF1a-DIO-Flp-WPRE 1.5 × 10 14 GC/ml Vigene Biosciences 87306 AAV d J-EF1a-fDIO-YFP-WPRE 2.5 × 10 13 GC/ml UNC Viral Core 55641 AAV1-CAG-FLEX-GFP-WPRE 1.7 × 10 13 GC/ml Addgene 51502 AAVretro-hSyn-GFP-WPRE 1.7 × 10 14 GC/ml Vigene Biosciences 105539 AAV1 hSyn-GFP-WPRE 3.2 × 10 13 GC/ml Addgene 105539 AAV1-EF1a-DIO-hChR2-eYFP-WPRE 1.6 × 10 13 GC/ml Addgene 20298 AAV d J-CMV-TeNT-P2A-GFP 5.7 × 10 12 GC/ml Stanford Viral Core AAVretro-EF1a-Cre-WPRE 2.3 × 10 13 GC/ml Salk Institute 55636 Open in a separate window List of viruses used in this study Moreover, among the four AAV1 viruses tested, we observed a nearly twofold increase in the number of anterograde transsynaptically labeled cells when using AAV1-hSyn-Cre-WPRE compared with scAAV1-hSyn-Cre, AAV1-CMV-Cre, or AAV1-hSyn-Flp, which each lack the WPRE ( D ; ).

Techniques: Comparison, Injection, Over Expression, Staining, Labeling, Virus, Expressing

Journal: The Journal of Neuroscience

Article Title: Synaptic Specificity and Application of Anterograde Transsynaptic AAV for Probing Neural Circuitry

doi: 10.1523/JNEUROSCI.2158-19.2020

Figure Lengend Snippet: Anatomical evidence for the synaptic specificity of viral spread. A, Illustration shows that following injection in an upstream brain region, AAV1-Cre is transported down axons and may be released through the synapse to transduce postsynaptically connected neurons (red cells, in Cre-dependent tdTomato background). The extent to which extra-synaptic release of virus may contribute to the local transduction of unconnected cell types (gray cells) remains unclear. B, Strategy for testing the synaptic specificity of viral spread in an anatomically defined circuit. Postsynaptic labeling was examined in the simple lobule of the CB following injections in the inferior olive or PN (left). Mossy fiber afferents from the PN are known to innervate GCs but not PCs (right). On the other hand, climbing fiber afferents from the IO pass through the granule layer and selectively innervate PCs, but not GCs. Locations of cell bodies in different layers of the CB are indicated by dashed lines (molecular layer, Purkinje layer, or granule layer). C, Approach for labeling neurons postsynaptic to mossy fibers in the CB. The scAAV1-hSyn-Cre was injected into the PN of Ai14 x GAD67-GFP transgenic mice. Bottom, Example injection site (red). Blue, fluorescent Nissl stain. Scale bar, 500 µm. D, A coronal section through simple lobule showing pontine afferents and postsynaptic neurons labeled in the granule layer of the cerebellum (red). GAD67-GFP+ PCs and molecular layer interneurons are labeled in green. Blue, Fluorescent Nissl stain. Scale bar, 250 µm. E, Higher-magnification view of the dashed region shown in D. TdTomato+/GFP− GCs (red; costained with Nissl, blue, arrowheads) and mossy fiber terminals (red) were observed in the granule layer, along with large GAD67-GFP+ neurons (yellow, asterisk). Scale bar, 25 µm. F, Approach for labeling neurons postsynaptic to climbing fibers in CB. The scAAV1-hSyn-Cre was injected into the IO of Ai14 x GAD67-GFP mice. Bottom, Example injection site (red). Scale bar, 500 µm. G, Climbing fiber afferents and postsynaptic neurons (red) labeled in the granule, Purkinje, and molecular layers of the simple lobule. Most labeled neurons in the granule cell layer colocalized with GAD67-GFP+ expression. Scale bar, 250 µm. Solid box shows close up of a MLI. Scale bar, 25 µm. H, Higher-magnification view of the dashed region shown in G. Cre+/tdTomato+/GFP+ neurons were observed in the granule layer (yellow, asterisks), Purkinje cell layer (yellow, arrowhead), and molecular layer (cells shown in G). I, Quantification of the total number of GCs (top) and PCs (bottom) counted across four sections of the simple lobule for each animal injected in PN or IO (mean plotted for n = 4 animals for each pathway). Error bar = SD. ***p < 0.001, t test.

Article Snippet: In addition, we observed that AAV1-hSyn-Flp was capable of labeling comparable numbers of cells in SC compared with Cre-expressing AAV1 constructs ( C , D ), suggesting this approach may be applied interchangeably with AAV1 viruses expressing different forms of recombinase. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Virus Titer Source Addgene Plasmid ID AAV1 hSyn-Cre-WPRE 2.5 × 10 13 GC/ml Addgene 105553 scAAV1 hSyn-Cre 2.8 × 10 13 GC/ml Vigene Biosciences AAV1 hSyn-Flp 5.5 × 10 13 GC/ml Vigene Biosciences 51669 AAVretro-hSyn-Cre 1.5 × 10 14 GC/ml Vigene Biosciences 105537 AAV1-CMV-Cre 2.7 × 10 13 GC/ml Addgene 105537 AAV5-CMV-Cre 2.8 × 10 13 GC/ml Addgene 105537 AAV6-CMV-Cre 3.5 × 10 13 GC/ml Addgene 105537 AAV8-CMV-Cre 4.4 × 10 13 GC/ml Addgene 105537 AAV9-CMV-Cre 1.6 × 10 14 GC/ml Addgene 105537 AAVPHP.B-CMV-Cre 2.3 × 10 13 GC/ml SignaGen CAV2-CMV-Cre 1.3 × 10 12 GC/ml Montpellier Viral Core Adenovirus (Ad5-CMV-Cre) 3.0 × 10 12 GC/ml Kerafast Lentivirus (LV-CMV-Cre) 1.0 × 10 8 GC/ml Cellomics Tech Baculovirus (BAC-CMV-Cre) 3.7 × 10 10 GC/ml Uni. of Iowa G-deleted Rabies virus (RV-Cre-GFP) 8.6 × 10 8 GC/ml Salk Institute G-deleted Rabies virus (RV-GFP) 5.5 × 10 8 GC/ml Salk Institute AAV1-EF1a-DIO-Flp-WPRE 1.5 × 10 14 GC/ml Vigene Biosciences 87306 AAV d J-EF1a-fDIO-YFP-WPRE 2.5 × 10 13 GC/ml UNC Viral Core 55641 AAV1-CAG-FLEX-GFP-WPRE 1.7 × 10 13 GC/ml Addgene 51502 AAVretro-hSyn-GFP-WPRE 1.7 × 10 14 GC/ml Vigene Biosciences 105539 AAV1 hSyn-GFP-WPRE 3.2 × 10 13 GC/ml Addgene 105539 AAV1-EF1a-DIO-hChR2-eYFP-WPRE 1.6 × 10 13 GC/ml Addgene 20298 AAV d J-CMV-TeNT-P2A-GFP 5.7 × 10 12 GC/ml Stanford Viral Core AAVretro-EF1a-Cre-WPRE 2.3 × 10 13 GC/ml Salk Institute 55636 Open in a separate window List of viruses used in this study Moreover, among the four AAV1 viruses tested, we observed a nearly twofold increase in the number of anterograde transsynaptically labeled cells when using AAV1-hSyn-Cre-WPRE compared with scAAV1-hSyn-Cre, AAV1-CMV-Cre, or AAV1-hSyn-Flp, which each lack the WPRE ( D ; ).

Techniques: Injection, Transduction, Virus, Labeling, Transgenic Assay, Staining, Expressing

Journal: The Journal of Neuroscience

Article Title: Synaptic Specificity and Application of Anterograde Transsynaptic AAV for Probing Neural Circuitry

doi: 10.1523/JNEUROSCI.2158-19.2020

Figure Lengend Snippet: Tetanus toxin inhibition of viral spread. A, Experimental design and timeline of virus injections. AAV trafficked to the synapse may be released through synaptic vesicles in a VAMP2-dependent manner (top). Tetanus toxin cleaves VAMP2, preventing synaptic vesicle fusion and potential release of AAV. B, Control experiment. AAV1-hSyn-GFP injection in V1 followed by scAAV1-hSyn-Cre injection 2 weeks later in Ai14 mice (top). 2 weeks after the second injection, GFP+ axons (green) and anterograde transsynaptically labeled neurons (red cells) were observed in SC (middle, 10×; bottom, 40×). Blue, fluorescent Nissl stain. C, AAVDJ-CMV-TeNT-P2A-GFP injection in V1 followed by scAAV1-hSyn-Cre injection 2 weeks later in Ai14 mice (top). 2 weeks after the second injection, GFP+/TeNT+ axons (green) were found in SC, however, very few transsynaptically labeled neurons (red cells) were observed. Scale bars: B, C, top, 500 µm; middle, 250 µm; bottom, 25 µm. D, Quantification of number of anterograde transsynaptically labeled cells in SC for each injection time point (n = 8 mice for control and 14 d groups, n = 4 mice for the remaining groups). Error bar = SD. ***p < 0.001; n.s., no significance, t test.

Article Snippet: In addition, we observed that AAV1-hSyn-Flp was capable of labeling comparable numbers of cells in SC compared with Cre-expressing AAV1 constructs ( C , D ), suggesting this approach may be applied interchangeably with AAV1 viruses expressing different forms of recombinase. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Virus Titer Source Addgene Plasmid ID AAV1 hSyn-Cre-WPRE 2.5 × 10 13 GC/ml Addgene 105553 scAAV1 hSyn-Cre 2.8 × 10 13 GC/ml Vigene Biosciences AAV1 hSyn-Flp 5.5 × 10 13 GC/ml Vigene Biosciences 51669 AAVretro-hSyn-Cre 1.5 × 10 14 GC/ml Vigene Biosciences 105537 AAV1-CMV-Cre 2.7 × 10 13 GC/ml Addgene 105537 AAV5-CMV-Cre 2.8 × 10 13 GC/ml Addgene 105537 AAV6-CMV-Cre 3.5 × 10 13 GC/ml Addgene 105537 AAV8-CMV-Cre 4.4 × 10 13 GC/ml Addgene 105537 AAV9-CMV-Cre 1.6 × 10 14 GC/ml Addgene 105537 AAVPHP.B-CMV-Cre 2.3 × 10 13 GC/ml SignaGen CAV2-CMV-Cre 1.3 × 10 12 GC/ml Montpellier Viral Core Adenovirus (Ad5-CMV-Cre) 3.0 × 10 12 GC/ml Kerafast Lentivirus (LV-CMV-Cre) 1.0 × 10 8 GC/ml Cellomics Tech Baculovirus (BAC-CMV-Cre) 3.7 × 10 10 GC/ml Uni. of Iowa G-deleted Rabies virus (RV-Cre-GFP) 8.6 × 10 8 GC/ml Salk Institute G-deleted Rabies virus (RV-GFP) 5.5 × 10 8 GC/ml Salk Institute AAV1-EF1a-DIO-Flp-WPRE 1.5 × 10 14 GC/ml Vigene Biosciences 87306 AAV d J-EF1a-fDIO-YFP-WPRE 2.5 × 10 13 GC/ml UNC Viral Core 55641 AAV1-CAG-FLEX-GFP-WPRE 1.7 × 10 13 GC/ml Addgene 51502 AAVretro-hSyn-GFP-WPRE 1.7 × 10 14 GC/ml Vigene Biosciences 105539 AAV1 hSyn-GFP-WPRE 3.2 × 10 13 GC/ml Addgene 105539 AAV1-EF1a-DIO-hChR2-eYFP-WPRE 1.6 × 10 13 GC/ml Addgene 20298 AAV d J-CMV-TeNT-P2A-GFP 5.7 × 10 12 GC/ml Stanford Viral Core AAVretro-EF1a-Cre-WPRE 2.3 × 10 13 GC/ml Salk Institute 55636 Open in a separate window List of viruses used in this study Moreover, among the four AAV1 viruses tested, we observed a nearly twofold increase in the number of anterograde transsynaptically labeled cells when using AAV1-hSyn-Cre-WPRE compared with scAAV1-hSyn-Cre, AAV1-CMV-Cre, or AAV1-hSyn-Flp, which each lack the WPRE ( D ; ).

Techniques: Inhibition, Virus, Control, Injection, Labeling, Staining

Journal: The Journal of Neuroscience

Article Title: Synaptic Specificity and Application of Anterograde Transsynaptic AAV for Probing Neural Circuitry

doi: 10.1523/JNEUROSCI.2158-19.2020

Figure Lengend Snippet: Forward mapping of topographically organized brain regions. A, Schematic diagram depicting the parcellation of brain regions (in Y) based on their topographical input (from X) using an anterograde transsynaptic approach. Secondary injections of AAV1-Flex-GFP enable further mapping of axonal outputs (to Z) for any given input-defined subregion. A retrograde approach may not provide access to the same population because of collateralization of divergent outputs. B, Top, tdTomato expression at the injection site, MOp-ul, following first injection of scAAV1-hSyn-Cre. Bottom, Cre+/GFP+ neurons are shown in PN following secondary injection of AAV1-hSyn-Flex-GFP in PN. Scale bar, 250 µm. C, Axonal projections to the cerebellum from PN neurons defined by their input from MOp-ul. Axons primarily target the contralateral parafloccular sulcus (PflS; top left) and the ventral PRM. Scale bar, 500 µm. D, Top, first injection in MOp-ll. Bottom, Cre+/GFP+ neurons in PN following secondary injection of AAV1-hSyn-Flex-GFP in PN. Scale bar, 250 µm. E, Axonal targeting in CB. Output was primarily restricted to the (COP (top right and bottom), but also collateralize more sparsely to lobules III, IV, and V (top left). Scale bars, 500 µm. F, Quantification of the density of axon terminals in PRM or COP for PN neurons defined by their input from MOp-ul (blue) or MOp-ll (green; n = 3 mice for each group). Values represent the fraction of fluorescent axon signal divided by the total area of the target lobule. Error bar = SD. ***p < 0.001, t test. G, Schematic summary of axonal projections to cerebellum from MOp-ul and MOp-ll-recipient PN neurons (green and blue, respectively). Posterior view of the cerebellum is shown.

Article Snippet: In addition, we observed that AAV1-hSyn-Flp was capable of labeling comparable numbers of cells in SC compared with Cre-expressing AAV1 constructs ( C , D ), suggesting this approach may be applied interchangeably with AAV1 viruses expressing different forms of recombinase. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Virus Titer Source Addgene Plasmid ID AAV1 hSyn-Cre-WPRE 2.5 × 10 13 GC/ml Addgene 105553 scAAV1 hSyn-Cre 2.8 × 10 13 GC/ml Vigene Biosciences AAV1 hSyn-Flp 5.5 × 10 13 GC/ml Vigene Biosciences 51669 AAVretro-hSyn-Cre 1.5 × 10 14 GC/ml Vigene Biosciences 105537 AAV1-CMV-Cre 2.7 × 10 13 GC/ml Addgene 105537 AAV5-CMV-Cre 2.8 × 10 13 GC/ml Addgene 105537 AAV6-CMV-Cre 3.5 × 10 13 GC/ml Addgene 105537 AAV8-CMV-Cre 4.4 × 10 13 GC/ml Addgene 105537 AAV9-CMV-Cre 1.6 × 10 14 GC/ml Addgene 105537 AAVPHP.B-CMV-Cre 2.3 × 10 13 GC/ml SignaGen CAV2-CMV-Cre 1.3 × 10 12 GC/ml Montpellier Viral Core Adenovirus (Ad5-CMV-Cre) 3.0 × 10 12 GC/ml Kerafast Lentivirus (LV-CMV-Cre) 1.0 × 10 8 GC/ml Cellomics Tech Baculovirus (BAC-CMV-Cre) 3.7 × 10 10 GC/ml Uni. of Iowa G-deleted Rabies virus (RV-Cre-GFP) 8.6 × 10 8 GC/ml Salk Institute G-deleted Rabies virus (RV-GFP) 5.5 × 10 8 GC/ml Salk Institute AAV1-EF1a-DIO-Flp-WPRE 1.5 × 10 14 GC/ml Vigene Biosciences 87306 AAV d J-EF1a-fDIO-YFP-WPRE 2.5 × 10 13 GC/ml UNC Viral Core 55641 AAV1-CAG-FLEX-GFP-WPRE 1.7 × 10 13 GC/ml Addgene 51502 AAVretro-hSyn-GFP-WPRE 1.7 × 10 14 GC/ml Vigene Biosciences 105539 AAV1 hSyn-GFP-WPRE 3.2 × 10 13 GC/ml Addgene 105539 AAV1-EF1a-DIO-hChR2-eYFP-WPRE 1.6 × 10 13 GC/ml Addgene 20298 AAV d J-CMV-TeNT-P2A-GFP 5.7 × 10 12 GC/ml Stanford Viral Core AAVretro-EF1a-Cre-WPRE 2.3 × 10 13 GC/ml Salk Institute 55636 Open in a separate window List of viruses used in this study Moreover, among the four AAV1 viruses tested, we observed a nearly twofold increase in the number of anterograde transsynaptically labeled cells when using AAV1-hSyn-Cre-WPRE compared with scAAV1-hSyn-Cre, AAV1-CMV-Cre, or AAV1-hSyn-Flp, which each lack the WPRE ( D ; ).

Techniques: Expressing, Injection

Journal: The Journal of Neuroscience

Article Title: Synaptic Specificity and Application of Anterograde Transsynaptic AAV for Probing Neural Circuitry

doi: 10.1523/JNEUROSCI.2158-19.2020

Figure Lengend Snippet: Application with sparse labeling approaches for reconstructing single neuron morphology. A, For a given Cre+ cell population (red), sparse labeling (green) may be achieved by coinjecting AAV1-DIO-Flp at increasingly lower titers along with high titer AAVDJ-fDIO-YFP. To establish a titering curve, PV neurons in V1 were targeted with coinjections of AAVDJ-fDIO-YFP (final titer: 1.2 × 1013 GC/ml) and AAV1-DIO-Flp (final titers: 7.5 × 1010, 109, or 108 GC/ml) in PV-Cre x Ai14 mice. B, Examples of YFP cell labeling (green) achieved at each titer step. Red, PV-Cre+/tdTomato+ cells. Blue, Fluorescent Nissl. Scale bar, 250 µm. C, 40× magnification of a YFP+ PV neuron labeled in (B, middle). Scale bar, 25 µm. D, Quantification of the number of YFP+/PV+ cells labeled at each titer step (n = 4 mice each). Error bar = SD. E, Injection of scAAV1-hSyn-Cre in the ACA of GAD67-GFP x Ai14 mice transsynaptically labels neurons in PAG. Labeled cell density is greatest in the PAGdl (middle), and both inhibitory (GFP+/tdTomato+) and presumed excitatory (GFP-/tdTomato+) cell types are labeled (bottom; 40× magnification). Scale bars: middle, 250 µm; bottom, 50 µm. F, Strategy for sparse labeling of input- and genetically-defined cell populations. AAV1-DIO-Flp titer for injection in ACA is reduced to 1.5 × 1012 GC/ml to achieve sparse anterograde transsynaptic labeling in PAGdl. Individual Vglut2-Cre+/Flp+ neurons may then be targeted with an injection of high titer AAVDJ-fDIO-YFP to specifically label glutamatergic neurons in PAGdl that receive input from ACA. G) Example of a single Vglut2-Cre+/Flp+ neuron labeled in PAGdl (green). An additional neuron was found in the superior colliculus (arrowhead). Right, 40× magnification of YFP+ (green, top) and Vglut2-Cre+/Tom+ (red, bottom) neuron in PAGdl. Blue, Fluorescent Nissl. Scale bars: left, 250 µm; right, 25 µm. H, 40× magnification of local dendrites and axonal projection of the YFP+ PAGdl neuron shown in G. Scale bar, 50 µm.

Article Snippet: In addition, we observed that AAV1-hSyn-Flp was capable of labeling comparable numbers of cells in SC compared with Cre-expressing AAV1 constructs ( C , D ), suggesting this approach may be applied interchangeably with AAV1 viruses expressing different forms of recombinase. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Virus Titer Source Addgene Plasmid ID AAV1 hSyn-Cre-WPRE 2.5 × 10 13 GC/ml Addgene 105553 scAAV1 hSyn-Cre 2.8 × 10 13 GC/ml Vigene Biosciences AAV1 hSyn-Flp 5.5 × 10 13 GC/ml Vigene Biosciences 51669 AAVretro-hSyn-Cre 1.5 × 10 14 GC/ml Vigene Biosciences 105537 AAV1-CMV-Cre 2.7 × 10 13 GC/ml Addgene 105537 AAV5-CMV-Cre 2.8 × 10 13 GC/ml Addgene 105537 AAV6-CMV-Cre 3.5 × 10 13 GC/ml Addgene 105537 AAV8-CMV-Cre 4.4 × 10 13 GC/ml Addgene 105537 AAV9-CMV-Cre 1.6 × 10 14 GC/ml Addgene 105537 AAVPHP.B-CMV-Cre 2.3 × 10 13 GC/ml SignaGen CAV2-CMV-Cre 1.3 × 10 12 GC/ml Montpellier Viral Core Adenovirus (Ad5-CMV-Cre) 3.0 × 10 12 GC/ml Kerafast Lentivirus (LV-CMV-Cre) 1.0 × 10 8 GC/ml Cellomics Tech Baculovirus (BAC-CMV-Cre) 3.7 × 10 10 GC/ml Uni. of Iowa G-deleted Rabies virus (RV-Cre-GFP) 8.6 × 10 8 GC/ml Salk Institute G-deleted Rabies virus (RV-GFP) 5.5 × 10 8 GC/ml Salk Institute AAV1-EF1a-DIO-Flp-WPRE 1.5 × 10 14 GC/ml Vigene Biosciences 87306 AAV d J-EF1a-fDIO-YFP-WPRE 2.5 × 10 13 GC/ml UNC Viral Core 55641 AAV1-CAG-FLEX-GFP-WPRE 1.7 × 10 13 GC/ml Addgene 51502 AAVretro-hSyn-GFP-WPRE 1.7 × 10 14 GC/ml Vigene Biosciences 105539 AAV1 hSyn-GFP-WPRE 3.2 × 10 13 GC/ml Addgene 105539 AAV1-EF1a-DIO-hChR2-eYFP-WPRE 1.6 × 10 13 GC/ml Addgene 20298 AAV d J-CMV-TeNT-P2A-GFP 5.7 × 10 12 GC/ml Stanford Viral Core AAVretro-EF1a-Cre-WPRE 2.3 × 10 13 GC/ml Salk Institute 55636 Open in a separate window List of viruses used in this study Moreover, among the four AAV1 viruses tested, we observed a nearly twofold increase in the number of anterograde transsynaptically labeled cells when using AAV1-hSyn-Cre-WPRE compared with scAAV1-hSyn-Cre, AAV1-CMV-Cre, or AAV1-hSyn-Flp, which each lack the WPRE ( D ; ).

Techniques: Labeling, Injection

Journal: The Journal of Neuroscience

Article Title: Synaptic Specificity and Application of Anterograde Transsynaptic AAV for Probing Neural Circuitry

doi: 10.1523/JNEUROSCI.2158-19.2020

Figure Lengend Snippet: Verification of functional synaptic connectivity. A, Strategy for slice recording from transsynaptically labeled neurons in the IC (red) following coinjection of scAAV1-hSyn-Cre and AAV1-DIO-ChR2-YFP into A1. B, ChR2-expressing axon terminals (green) surrounding a tdTomato-labeled neuron and neighboring nonlabeled neurons (blue, Nissl stain). Scale bar, 25 µm. C, Average LED-evoked excitatory (recorded at −70 mV) and inhibitory (0 mV) currents in an example tdTomato+ IC neuron before (left) and after (right) perfusing in TTX and 4-AP. LED stimulation is marked by a blue bar. D, Fraction of transsynaptically labeled (red) and neighboring nonlabeled (gray) neurons showing monosynaptic excitatory currents in response to LED stimulation. ***p < 0.001, χ2 test, 28 cells in each group. E, Summary of amplitudes of average monosynaptic excitatory currents evoked by LED for all labeled (red) and nonlabeled (gray) recorded neurons (neurons showing zero currents were excluded). Error bar = SD. There is n.s., no significance, unpaired t test.

Article Snippet: In addition, we observed that AAV1-hSyn-Flp was capable of labeling comparable numbers of cells in SC compared with Cre-expressing AAV1 constructs ( C , D ), suggesting this approach may be applied interchangeably with AAV1 viruses expressing different forms of recombinase. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Virus Titer Source Addgene Plasmid ID AAV1 hSyn-Cre-WPRE 2.5 × 10 13 GC/ml Addgene 105553 scAAV1 hSyn-Cre 2.8 × 10 13 GC/ml Vigene Biosciences AAV1 hSyn-Flp 5.5 × 10 13 GC/ml Vigene Biosciences 51669 AAVretro-hSyn-Cre 1.5 × 10 14 GC/ml Vigene Biosciences 105537 AAV1-CMV-Cre 2.7 × 10 13 GC/ml Addgene 105537 AAV5-CMV-Cre 2.8 × 10 13 GC/ml Addgene 105537 AAV6-CMV-Cre 3.5 × 10 13 GC/ml Addgene 105537 AAV8-CMV-Cre 4.4 × 10 13 GC/ml Addgene 105537 AAV9-CMV-Cre 1.6 × 10 14 GC/ml Addgene 105537 AAVPHP.B-CMV-Cre 2.3 × 10 13 GC/ml SignaGen CAV2-CMV-Cre 1.3 × 10 12 GC/ml Montpellier Viral Core Adenovirus (Ad5-CMV-Cre) 3.0 × 10 12 GC/ml Kerafast Lentivirus (LV-CMV-Cre) 1.0 × 10 8 GC/ml Cellomics Tech Baculovirus (BAC-CMV-Cre) 3.7 × 10 10 GC/ml Uni. of Iowa G-deleted Rabies virus (RV-Cre-GFP) 8.6 × 10 8 GC/ml Salk Institute G-deleted Rabies virus (RV-GFP) 5.5 × 10 8 GC/ml Salk Institute AAV1-EF1a-DIO-Flp-WPRE 1.5 × 10 14 GC/ml Vigene Biosciences 87306 AAV d J-EF1a-fDIO-YFP-WPRE 2.5 × 10 13 GC/ml UNC Viral Core 55641 AAV1-CAG-FLEX-GFP-WPRE 1.7 × 10 13 GC/ml Addgene 51502 AAVretro-hSyn-GFP-WPRE 1.7 × 10 14 GC/ml Vigene Biosciences 105539 AAV1 hSyn-GFP-WPRE 3.2 × 10 13 GC/ml Addgene 105539 AAV1-EF1a-DIO-hChR2-eYFP-WPRE 1.6 × 10 13 GC/ml Addgene 20298 AAV d J-CMV-TeNT-P2A-GFP 5.7 × 10 12 GC/ml Stanford Viral Core AAVretro-EF1a-Cre-WPRE 2.3 × 10 13 GC/ml Salk Institute 55636 Open in a separate window List of viruses used in this study Moreover, among the four AAV1 viruses tested, we observed a nearly twofold increase in the number of anterograde transsynaptically labeled cells when using AAV1-hSyn-Cre-WPRE compared with scAAV1-hSyn-Cre, AAV1-CMV-Cre, or AAV1-hSyn-Flp, which each lack the WPRE ( D ; ).

Techniques: Functional Assay, Labeling, Expressing, Staining

Journal: The Journal of Neuroscience

Article Title: Synaptic Specificity and Application of Anterograde Transsynaptic AAV for Probing Neural Circuitry

doi: 10.1523/JNEUROSCI.2158-19.2020

Figure Lengend Snippet: List of viruses used in this study

Article Snippet: In addition, we observed that AAV1-hSyn-Flp was capable of labeling comparable numbers of cells in SC compared with Cre-expressing AAV1 constructs ( C , D ), suggesting this approach may be applied interchangeably with AAV1 viruses expressing different forms of recombinase. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Virus Titer Source Addgene Plasmid ID AAV1 hSyn-Cre-WPRE 2.5 × 10 13 GC/ml Addgene 105553 scAAV1 hSyn-Cre 2.8 × 10 13 GC/ml Vigene Biosciences AAV1 hSyn-Flp 5.5 × 10 13 GC/ml Vigene Biosciences 51669 AAVretro-hSyn-Cre 1.5 × 10 14 GC/ml Vigene Biosciences 105537 AAV1-CMV-Cre 2.7 × 10 13 GC/ml Addgene 105537 AAV5-CMV-Cre 2.8 × 10 13 GC/ml Addgene 105537 AAV6-CMV-Cre 3.5 × 10 13 GC/ml Addgene 105537 AAV8-CMV-Cre 4.4 × 10 13 GC/ml Addgene 105537 AAV9-CMV-Cre 1.6 × 10 14 GC/ml Addgene 105537 AAVPHP.B-CMV-Cre 2.3 × 10 13 GC/ml SignaGen CAV2-CMV-Cre 1.3 × 10 12 GC/ml Montpellier Viral Core Adenovirus (Ad5-CMV-Cre) 3.0 × 10 12 GC/ml Kerafast Lentivirus (LV-CMV-Cre) 1.0 × 10 8 GC/ml Cellomics Tech Baculovirus (BAC-CMV-Cre) 3.7 × 10 10 GC/ml Uni. of Iowa G-deleted Rabies virus (RV-Cre-GFP) 8.6 × 10 8 GC/ml Salk Institute G-deleted Rabies virus (RV-GFP) 5.5 × 10 8 GC/ml Salk Institute AAV1-EF1a-DIO-Flp-WPRE 1.5 × 10 14 GC/ml Vigene Biosciences 87306 AAV d J-EF1a-fDIO-YFP-WPRE 2.5 × 10 13 GC/ml UNC Viral Core 55641 AAV1-CAG-FLEX-GFP-WPRE 1.7 × 10 13 GC/ml Addgene 51502 AAVretro-hSyn-GFP-WPRE 1.7 × 10 14 GC/ml Vigene Biosciences 105539 AAV1 hSyn-GFP-WPRE 3.2 × 10 13 GC/ml Addgene 105539 AAV1-EF1a-DIO-hChR2-eYFP-WPRE 1.6 × 10 13 GC/ml Addgene 20298 AAV d J-CMV-TeNT-P2A-GFP 5.7 × 10 12 GC/ml Stanford Viral Core AAVretro-EF1a-Cre-WPRE 2.3 × 10 13 GC/ml Salk Institute 55636 Open in a separate window List of viruses used in this study Moreover, among the four AAV1 viruses tested, we observed a nearly twofold increase in the number of anterograde transsynaptically labeled cells when using AAV1-hSyn-Cre-WPRE compared with scAAV1-hSyn-Cre, AAV1-CMV-Cre, or AAV1-hSyn-Flp, which each lack the WPRE ( D ; ).

Techniques: Plasmid Preparation, Virus

Journal: The Journal of Neuroscience

Article Title: Synaptic Specificity and Application of Anterograde Transsynaptic AAV for Probing Neural Circuitry

doi: 10.1523/JNEUROSCI.2158-19.2020

Figure Lengend Snippet: Accessing topographically precise, input-defined cell populations through corticofugal pathways. A, Strategy for labeling cell populations that receive input from upper limb- (green) and lower limb- (red) related primary motor cortex in Ai14 x Frt-GFP (Cre and Flp reporter) mice. B, Injection sites for AAV1-hSyn-Flp in MOp-ul (top, green) and scAAV1-hSyn-Cre in MOp-ll (bottom, red) in an Ai14 x Frt-GFP mouse. Scale bar, 500 µm. C, Anterograde transsynaptic labeling of cells that receive input from upper limb- (green) or lower limb- (red) related primary motor cortex. Many closely apposed, non-overlapping cell populations were observed in mid- and hindbrain structures, including the PN (second row, right). None of the structures shown project back to motor cortex, with the exception of the thalamus (top row, third from left), which may contain both retrograde and anterograde transsynaptic labeling of cell bodies. Scale bars, 250 µm. GR, Gracile nucleus; MA3, medial accessory oculomotor nucleus; MARN, magnocellular reticular nucleus; PRN, pontine reticular nucleus.

Article Snippet: In addition, we observed that AAV1-hSyn-Flp was capable of labeling comparable numbers of cells in SC compared with Cre-expressing AAV1 constructs ( C , D ), suggesting this approach may be applied interchangeably with AAV1 viruses expressing different forms of recombinase. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Virus Titer Source Addgene Plasmid ID AAV1 hSyn-Cre-WPRE 2.5 × 10 13 GC/ml Addgene 105553 scAAV1 hSyn-Cre 2.8 × 10 13 GC/ml Vigene Biosciences AAV1 hSyn-Flp 5.5 × 10 13 GC/ml Vigene Biosciences 51669 AAVretro-hSyn-Cre 1.5 × 10 14 GC/ml Vigene Biosciences 105537 AAV1-CMV-Cre 2.7 × 10 13 GC/ml Addgene 105537 AAV5-CMV-Cre 2.8 × 10 13 GC/ml Addgene 105537 AAV6-CMV-Cre 3.5 × 10 13 GC/ml Addgene 105537 AAV8-CMV-Cre 4.4 × 10 13 GC/ml Addgene 105537 AAV9-CMV-Cre 1.6 × 10 14 GC/ml Addgene 105537 AAVPHP.B-CMV-Cre 2.3 × 10 13 GC/ml SignaGen CAV2-CMV-Cre 1.3 × 10 12 GC/ml Montpellier Viral Core Adenovirus (Ad5-CMV-Cre) 3.0 × 10 12 GC/ml Kerafast Lentivirus (LV-CMV-Cre) 1.0 × 10 8 GC/ml Cellomics Tech Baculovirus (BAC-CMV-Cre) 3.7 × 10 10 GC/ml Uni. of Iowa G-deleted Rabies virus (RV-Cre-GFP) 8.6 × 10 8 GC/ml Salk Institute G-deleted Rabies virus (RV-GFP) 5.5 × 10 8 GC/ml Salk Institute AAV1-EF1a-DIO-Flp-WPRE 1.5 × 10 14 GC/ml Vigene Biosciences 87306 AAV d J-EF1a-fDIO-YFP-WPRE 2.5 × 10 13 GC/ml UNC Viral Core 55641 AAV1-CAG-FLEX-GFP-WPRE 1.7 × 10 13 GC/ml Addgene 51502 AAVretro-hSyn-GFP-WPRE 1.7 × 10 14 GC/ml Vigene Biosciences 105539 AAV1 hSyn-GFP-WPRE 3.2 × 10 13 GC/ml Addgene 105539 AAV1-EF1a-DIO-hChR2-eYFP-WPRE 1.6 × 10 13 GC/ml Addgene 20298 AAV d J-CMV-TeNT-P2A-GFP 5.7 × 10 12 GC/ml Stanford Viral Core AAVretro-EF1a-Cre-WPRE 2.3 × 10 13 GC/ml Salk Institute 55636 Open in a separate window List of viruses used in this study Moreover, among the four AAV1 viruses tested, we observed a nearly twofold increase in the number of anterograde transsynaptically labeled cells when using AAV1-hSyn-Cre-WPRE compared with scAAV1-hSyn-Cre, AAV1-CMV-Cre, or AAV1-hSyn-Flp, which each lack the WPRE ( D ; ).

Techniques: Labeling, Injection