Postsynaptic cell type and synaptic distance do not determine efficiency of monosynaptic rabies virus spread measured at synaptic resolution
- Systems Neurobiology Laboratories, The Salk Institute for Biological Studies, United States
- Neuroscience Graduate Program, University of California, San Diego, United States
- Medical Scientist Training Program, University of California, San Diego, United States
- Allen Institute for Brain Science, United States
Figures
Figure 1
RVdG-PSD95GFP-SynPhRFP allows simultaneous fluorescent labeling of pre- and postsynaptic densities.
(A) Schematic of RVdG-PSD95GFP-SynPhRFP viral construct design. Two transgenes were inserted into the G locus of the rabies genome. One encodes a presynaptically targeted fluorescent fusion protein, synaptophysin TagRFP-T (SynPhRFP), and the other a postsynaptically targeted fluorescent fusion protein, PSD-95 eGFP (PSD95GFP). (B) Coronal sections of Sim1Cre mouse expressing TVA and oG in V1 infected with EnvA+ RVdG-PSD95GFP-SynPhRFP imaged at 20× with confocal microscopy. Top row shows neurons expressing both PSD95GFP and SynPhRFP fusion proteins. Bottom row shows a zoomed in max intensity projection reconstructed image of the region enclosed by the dashed square in the top row. White arrows point to PSD-95 puncta, blue arrows to synaptophysin puncta, and gray arrows to large non-specific nuclear fluorescent aggregates. Scale bars represent 100 µm (top row) or 10 µm (bottom row). (C) Airyscan super-resolution max intensity projection reconstructed images taken at 63× showing colocalization of PSD95GFP fusion protein expressed from the rabies genome with anti-PSD95 antibody staining in magenta. Scale bar = 1 µm.
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Figure 1—source data 1
Plasmid sequence for RVdG-PSD95GFP-SynPhRFP construct.
Plasmid sequence for a new genetically modified rabies virus that labels presynaptic terminals with synaptophysin-RFP (SynPhRFP) and excitatory postsynaptic densities with postsynaptic density-95-GFP (PSD95GFP).
- https://cdn.elifesciences.org/articles/89297/elife-89297-fig1-data1-v2.zip
Figure 2
Experimental design for control experiments omitting oG.
(A) Schematic illustration of experimental design and timeline for control experiments without glycoprotein. Nr5a1Cre mice were injected in V1 with a mixture of AAV-CIAO-TVA and AAV-FLEX-smFP-myc. Three weeks later EnvA+ RVdG-PSD95GFP-SynPhRFP was injected into the same site and allowed to express for 7 days. (B) Representative image of V1 injection site, obtained using widefield fluorescence microscopy at 10×. Scale bar = 200 µm. (C) Max intensity projection reconstruction of images obtained using Airyscan super-resolution imaging at 63×. Top, example image of rabies-infected neuron labeled with smFP_myc, without glycoprotein. Bottom, zoomed in image of boxed region in top image, illustrating PSD-95 puncta colocalized with cytoplasmic smFP_myc. Scale bar = 20 µm (top) and scale bar = 2 µm (bottom).
Figure 3
Efficiency of transsynaptic spread from excitatory L4 Nr5a1+ starter cells to excitatory inputs.
(A) Schematic illustration of experimental design and timeline for monosynaptic rabies tracing. Nr5a1Cre mice were injected in V1 with a mixture of AAV-CIAO-TVA, AAV-FLEX-H2BBFP-oG, and AAV-FLEX-smFP_myc. Three weeks later EnvA+ RVdG-PSD95GFP-SynPhRFP was injected into the same site and allowed to express for 7 days. (B) Schematic of rabies retrograde spread efficiency quantification paradigm. Starter neurons are distinguished from input neurons based on expression of nuclear BFP from AAV-FLEX-H2BBFP-oG in addition to fusion proteins from EnvA+ RVdG-PSD95GFP-SynPhRFP. Starter neurons expressing smFP_myc are used for synaptic quantification to allow tracing of distal dendrites. Synaptic fraction is measured by quantifying the proportion of postsynaptic densities (PSD95GFP) on the starter neuron apposed with rabies-labeled presynaptic terminals (SynPhRFP). (C) Representative example images of V1 injection site, obtained using widefield fluorescence microscopy at ×10 magnification. Scale bar = 200 µm. (D) Coronal section example images obtained using widefield fluorescence microscopy at 10× showing long-range monosynaptic input neurons in dorsal lateral geniculate nucleus (dLGN) to Nr5a1+ L4 neurons in V1 when using the new RVdG construct (top). No retrograde spread is observed when glycoprotein is omitted, see Figure 2 for additional information. Insets are zoomed in images of dashed box regions. Scale bar represents 1 mm in hemisection image or 200 µm in inset. (E) Max intensity projection reconstruction of images obtained using Airyscan super-resolution imaging at 63×. Left, example image of starter neuron (H2BBFP+,PSD95GFP+, and SynPhRFP+) labeled with smFP_myc. Middle, example image of the distal domain of an apical dendrite of a starter neuron. Right, example image of the proximal domain of a basal dendrite. Scale bar = 20 µm (all three). (F) Spatial resolution using Airyscan imaging is sufficient to quantify rabies transsynaptic spread at the synaptic level. Zoomed in max intensity projection reconstructed image of boxed region in (E) right, illustrating PSD-95 puncta colocalized with cytoplasmic smFP_myc. Top, yellow boxed region highlights a spine with PSD-95 puncta without an apposed rabies-labeled presynaptic density. White boxed region highlights a spine with PSD-95 puncta with an apposed rabies-labeled presynaptic density. Middle row, zoomed in max intensity projection reconstructed images of yellow boxed region and bottom rows are zoomed in max intensity projection reconstructed images of white boxed region. Top, scale bar = 2 µm and middle and bottom scale bar = 0.5 µm. (G) Percent of postsynaptic densities (PSD95GFP) on Nr5a1+ starter cells apposed with rabies-labeled presynaptic terminals (SynPhRFP). Left, quantification of colocalization at baseline (no glycoprotein) due to L4 to L4 connections compared to colocalization from transsynaptic spread (with glycoprotein). Middle, colocalization on the distal vs proximal domains of apical dendrites. Right, colocalization on apical vs basal dendrites. Values are reported as mean ± SEM. Statistics were calculated from the Wilcoxon rank-sum test for non-parametric comparisons. Individual data points (circles) indicate values for each neuron. n (number of neurons) = 5 and 9 and N (number of mice) = 2 and 3 for no oG and oG groups respectively. p-value > 0.05 = not significant (ns).
Figure 4
Efficiency of transsynaptic spread from inhibitory starter cells to excitatory inputs.
(A) Representative example images of V1 injection site for SstCre mouse line (top) and VipCre mouse line (bottom), obtained using widefield fluorescence microscopy at 10×. Scale bar = 200 µm. (B–E) Max intensity projection reconstruction of images obtained using Airyscan super-resolution imaging at 63×. Example image of Vip+ (B) and Sst+ (D) starter neurons (H2BBFP+, PSD95GFP+, and SynPhRFP+) labeled with smFP_myc. Scale bar = 20 µm. (C and E) Zoomed in images of boxed regions in (B and D) respectively, illustrating PSD-95 puncta colocalized with cytoplasmic smFP_myc. Top, yellow boxed region highlights a spine with PSD-95 puncta without an apposed rabies-labeled presynaptic density. White boxed region highlights a spine with PSD-95 puncta with an apposed rabies-labeled presynaptic density. Middle row, zoomed in images of yellow boxed region and bottom rows are zoomed in images of white boxed region. Top, scale bar = 2 µm and middle and bottom scale bar = 0.5 µm. (F) Left, percent of postsynaptic densities (PSD95GFP) on Vip+ and Sst+ starter cells apposed with rabies-labeled presynaptic terminals (SynPhRFP). Middle, colocalization on the distal vs proximal domains of Sst+ dendrites. Right, colocalization on the distal vs proximal domains of Vip+ dendrites. Values are reported as mean ± SEM. Statistics were calculated from Wilcoxon rank-sum test for non-parametric comparisons. Individual data points (circles) indicate values for each neuron. n (number of neurons) = 9 and N (number of mice) = 3. p-value > 0.05 = not significant (ns).
Figure 5
Quantification from electron micrographic reconstructions of: (A) shared inputs between layer 4 excitatory neurons and (B) the number of synapses per excitatory neuron to excitatory neuron connection.
(A) The percentage of inputs to a layer 4 excitatory neuron that provide common input to another layer 4 excitatory neuron (percentage of neurons connected) at increasing distances between layer 4 neurons. Based on identification of 17,883 presynaptic inputs to layer 4 cells. (B) Distribution of the numbers of synapses per connection for 76,678 excitatory neuron to excitatory neuron connections identified in electron micrographic reconstructions of a volume from adult mouse visual cortex. Y-axis is plotted on a log scale. Average number of synapses per connection = 1.0965 ± 0.3726 (STD). The overwhelming majority of connections involve only one synaptic contact.
Figure 6
Relationships between synaptic fraction (SF), input fraction (IF), and unitary synaptic efficiency (U) depend on synapses per input neuron (M) and proportions of neurons with differences in M.
(A and C) SF against the ratio (R) of neurons with M values of 1 versus either 3 (A) or 2 (C) for different values of U. (B and D) Ratio of IF versus SF against the R of neurons with M values of 1 versus either 3 (B) or 2 (D) for different values of U.
Author response image 1
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/89297/elife-89297-mdarchecklist1-v2.pdf
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Source data 1
Raw data of pre-and post-synaptic densities for Figures 2—4.
Raw data includes counts for Nr5a1, Som, and vasoactive intestinal peptide (Vip) starter cells and control experiments omitting oG.
- https://cdn.elifesciences.org/articles/89297/elife-89297-data1-v2.xlsx
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Postsynaptic cell type and synaptic distance do not determine efficiency of monosynaptic rabies virus spread measured at synaptic resolution
eLife 12:e89297.
https://doi.org/10.7554/eLife.89297
