Author response: Postsynaptic cell type and synaptic distance do not determine efficiency of monosynaptic rabies virus spread measured at synaptic resolution

Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Abstract Retrograde monosynaptic tracing using glycoprotein-deleted rabies virus is an important component of the toolkit for investigation of neural circuit structure and connectivity. It allows for the identification of first-order presynaptic connections to cell populations of interest across both the central and peripheral nervous system, helping to decipher the complex connectivity patterns of neural networks that give rise to brain function. Despite its utility, the factors that influence the probability of transsynaptic rabies spread are not well understood. While it is well established that expression levels of rabies glycoprotein used to trans-complement G-deleted rabies can result in large changes in numbers of inputs labeled per starter cell (convergence index [CI]), it is not known how typical values of CI relate to the proportions of synaptic contacts or input neurons labeled. And it is not known whether inputs to different cell types, or synaptic contacts that are more proximal or distal to the cell body, are labeled with different probabilities. Here, we use a new rabies virus construct that allows for the simultaneous labeling of pre- and postsynaptic specializations to quantify the proportion of synaptic contacts labeled in mouse primary visual cortex. We demonstrate that with typical conditions about 40% of first-order presynaptic excitatory synapses to cortical excitatory and inhibitory neurons are labeled. We show that using matched tracing conditions there are similar proportions of labeled contacts onto L4 excitatory pyramidal, somatostatin (Sst) inhibitory, and vasoactive intestinal peptide (Vip) starter cell types. Furthermore, we find no difference in the proportions of labeled excitatory contacts onto postsynaptic sites at different subcellular locations. Editor's evaluation This study provides valuable new information on using monosynaptic rabies virus for trans-synaptic tracing, a widely used tool to map synaptic inputs to specific types of neurons in the mammalian brain. The authors provide convincing quantitative information about the efficiency for the rabies virus to cross synapses of visual cortical neurons in the mouse. They further demonstrate that the cortical cell type and location of synapse in the postsynaptic cell do not appear to affect the efficiency for the rabies virus to cross synapses. https://doi.org/10.7554/eLife.89297.sa0 Decision letter eLife's review process Introduction Monosynaptic rabies tracing using glycoprotein (G)-deleted rabies virus (RVdG) is a powerful tool for the study of neural circuit connectivity. This method enables scientists to label, genetically manipulate, or monitor the activity of brain-wide monosynaptic inputs to cell populations of interest. Since its introduction (Wickersham et al., 2007a; Wickersham et al., 2007b) it has been widely used for the identification of presynaptic inputs to single neurons (Marshel et al., 2010; Rancz et al., 2011; Rossi et al., 2020; Wertz et al., 2015), projection-defined neurons (Cruz-Martín et al., 2014; Levine et al., 2014), adult-born neurons (Deshpande et al., 2013; Garcia et al., 2014), transplanted neurons (Doerr et al., 2017; Grealish et al., 2015), hPSC-derived organoid neurons (Andersen et al., 2020; Miura et al., 2020), and genetically defined excitatory neurons (DeNardo et al., 2015; Kim et al., 2015), inhibitory neurons (Miyamichi et al., 2013; Wall et al., 2016), and non-neuronal cell types (Clark et al., 2021; Mount et al., 2019). In addition to being used for the identification of inputs, the incorporation of Ca2+ indicators and light-activated opsins into rabies reagents (Osakada et al., 2011) allows rabies tracing experiments to probe the relationship between function and connectivity (Rossi et al., 2020; Tian et al., 2016; Wertz et al., 2015; Wester et al., 2019). The ability of genetically modified RVdG to selectively spread retrogradely between synaptically connected cells allows for the identification of presynaptic partners regardless of their distance from one another and has led to novel insights throughout the nervous system. Despite the utility and widespread use of monosynaptic rabies tracing to study neural connectivity, there is uncertainty about the efficiency of transsynaptic spread from starter cells to input neurons and what factors influence the probability of spread. Although studies quantifying inputs to single neurons have provided some insight into the efficiency of spread (Marshel et al., 2010; Miyamichi et al., 2011; Rancz et al., 2011; Wertz et al., 2015) results vary widely across experimental conditions and no direct measurements of spread efficiency are available. Furthermore, using the convergence index (CI), defined as the number of rabies-labeled input neurons divided by the number of starter cells, recent studies have shown that the number of input neurons labeled per starter cell can be changed by as much as 10-fold by modifying the rabies glycoprotein (Kim et al., 2016), replacing the rabies virus strain (Reardon et al., 2016), or adjusting the level of glycoprotein expression (Lavin et al., 2020). Despite allowing quantitative comparisons between different reagents and conditions, these studies relied on methods that result in large animal to animal variability, require tedious counting across many animals, and can display high variability depending on the number of starter neurons (Tran-Van-Minh et al., 2022). Most importantly, CI measurements fail to quantify what proportion of all inputs are labeled. Additionally, the use of CI fails to address questions of the influence of biological factors on rabies spread efficiency, such as distance of synapses to the starter cell soma or differences of spread from different starter cell types. In this study we examine the efficiency of RVdG retrograde spread from starter cell to input neurons at the synaptic level. We designed a new genetically modified rabies virus that labels presynaptic terminals with synaptophysin-RFP (SynPhRFP) and excitatory postsynaptic densities with postsynaptic density-95-GFP (PSD95GFP). Because more than 99% of PSD95 postsynaptic puncta colocalize with a presynaptic terminal (Micheva et al., 2010), this construct allows us to quantify the proportion of excitatory synapses on a starter cell that have their corresponding input neuron labeled with rabies virus (defined here as synaptic fraction [SF]). We find that with the particular reagents and conditions that we used, rabies retrogradely labels about 35–40% of excitatory synaptic contacts onto each of the excitatory and inhibitory cell populations we tested. Furthermore, we found that SF does not vary by the proximity or distance of synapses to the starter cell soma or by the type of neuronal dendrite. Finally, we model the factors that influence the relationship between SF and the fraction of input neurons labeled (input fraction [IF]) and show that under plausible conditions for our results in mouse visual cortex the IF/SF ratio is about 0.75, so an SF of 40% corresponds to an IF of 30% of input neurons labeled. This analysis also allows an estimate of the probability of rabies spread across a single synaptic contact (unitary synaptic efficiency, U) which we estimate to be about 0.28 (28%) when SF = 0.4. Overall, this study provides insight into some long-standing questions about the efficiency of monosynaptic rabies tracing. We further discuss that desired tracing efficiency varies depending on experimental aims, the likely relationships between SF and proportion of input neurons that are labeled, and that efficiency was not maximized with the experimental design used here. Results RVdG-PSD95GFP-SynPhRFP viral construct allows simultaneous fluorescent labeling of pre- and postsynaptic specializations To investigate the transsynaptic spread of rabies virus from starter cells to input neurons at synaptic resolution, we developed a high-throughput and precise method to directly identify synapses labeled by transsynaptic spread. We created a new deletion-mutant rabies virus construct that expresses two synaptic fusion protein transgenes from the rabies G locus (Figure 1A), modeled after existing rabies constructs known to label multiple subcellular compartments of neurons (Wickersham et al., 2013). One gene encodes for a fusion protein of the excitatory postsynaptic marker, postsynaptic density 95 (PSD-95), and enhanced green fluorescent protein (eGFP). The other encodes for a fusion protein of the presynaptic marker synaptophysin (SynPh) and the bright and photostable red fluorescent protein TagRFP-T (Shaner et al., 2008). We chose the synaptophysin-TagRFP-T (SynPhRFP) fusion protein as it has previously been shown to result in a punctate red fluorescent pattern that colocalizes with varicosities on axons when expressed from the rabies genome (Wickersham et al., 2013). Injection of SAD-B19 EnvA+ RVdG-PSD95GFP-SynPhRFP in primary visual cortex (V1) of Sim1Cre mice expressing TVA and oG in Cre+ layer 5 neurons (derived from adeno-associated viruses [AAV] helper viruses, see Materials and methods) resulted in strong punctate green and red fluorescent labeling (Figure 1B), with green labeling prominent on dendrites and dendritic spines (Figure 2E and F). Super-resolution Airyscan imaging (see Materials and methods) revealed that GFP labeling colocalized with a subset of the puncta labeled with antibody staining against endogenous PSD-95 (Figure 1C). In addition to synaptic labeling there were typically nuclear aggregates of both red and green fluorescent protein in infected neurons (Figure 1B). Figure 1 Download asset Open asset 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. 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-v1.zip Download elife-89297-fig1-data1-v1.zip Figure 2 Download asset Open asset 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 mm. (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 mm (top) and scale bar = 2 mm (bottom). Efficiency of rabies transsynaptic spread across excitatory synapses onto excitatory starter neurons To quantify the proportion of synaptic contacts labeled with rabies virus, we injected a mixture of three Cre-dependent helper AAVs into the primary visual cortex (V1) of Nr5a1-Cre mice to target initial rabies infection to a sparse population of L4 excitatory neurons (Harris et al., 2014; Figure 3A, B). (1) AAV8-nef-AO-66/71-TVA950 (AAV-CIAO-TVA) expresses the TVA receptor for the avian sarcoma leukosis virus envelope protein, EnvA, which is necessary for entry of pseudotyped EnvA+ RVdG into Cre+ cells. However, recombinant-independent off-target leak expression of transgenes is common in recombinase-dependent DIO and FLEX AAV constructs. Because even miniscule quantities of TVA leak expression is sufficient for pseudotyped rabies to enter off-target cells and confound results (Callaway and Luo, 2015), it was necessary to minimize leak expression of TVA. We therefore used the novel cross-over insensitive ATG-out (CIAO) AAV construct, which has been shown to nearly eliminate leak expression and provide reliable and targeted transgene expression (Fischer et al., 2019), to express TVA in our population of interest. CIAO constructs have the ATG codon placed outside of the loxp mutant pairs loxp66/71 sites to ensure the gene coding region is out of frame with the ATG start signal in the absence of Cre-mediated recombination. Experiments in which AAV-CIAO-TVA and EnvA+ RVdG were injected into V1 of Cre-negative mice resulted in 15.83±11.33 rabies-infected neurons (mean ± SEM, n=3 mice) indicating low levels of Cre-independent TVA expression. (2) AAV8-hSyn-FLEX-H2BBFP-oG expresses optimized rabies glycoprotein (oG) (Kim et al., 2016), which allows for trans-complementation in EnvA+ RVdG-infected neurons, also termed starter cells, allowing the rabies to spread retrogradely into presynaptically connected inputs. Co-expression of nuclear mTagBFP2 (BFP), a brighter and more photostable blue fluorescent protein (Subach et al., 2008), and oG allows for the unambiguous identification of starter cells (Figure 3C and E), defined as any cell that expresses both oG and rabies transgenes. (3) Because H2BBFP only labels the nucleus of starter cells, we also used AAV-CAG-FLEX-smFP_myc to generate cytoplasmic labeling, allowing dendrites of starter cells to be traced. This AAV expresses spaghetti monster fluorescent protein (smFP), which consists of dark non-fluorescent GFP fused to 10 Myc epitope tags that can be combined with anti-Myc antibody staining to attain bright fluorescent labeling of subcellular structures (Viswanathan et al., 2015). Infection of L4 excitatory neurons with AAV-CAG-FLEX-smFP_myc resulted in strong dendritic labeling that improved the ability to accurately trace apical and basal dendrites of starter cells (Figure 3C, E, and F). Three weeks after injection of a mixture of the three AAV helper viruses, EnvA+ RVdG-PSD95GFP-SynPhRFP was injected into the same location in V1 and allowed to express for 7 days. As expected from transsynaptic spread, we observed that many thalamic dLGN long-distance inputs to L4 excitatory neurons were reliably labeled with RVdG-PSD95GFP-SynPhRFP (Figure 3D). Figure 3 Download asset Open asset 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 mm 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 mm and middle and bottom scale bar = 0.5 mm. (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). To quantify the proportion of excitatory synaptic contacts labeled on starter neurons (defined as SF), we first identified starter neurons that also expressed smFP using widefield fluorescence microscopy. Select areas of starter neuron dendrites that could be traced back to their parent cell bodies were imaged using Airyscan super-resolution microscopy (Huff et al., 2017) to increase the resolution and signal-to-noise and allow visualization of single synaptic puncta (Figure 3F). To assess the transsynaptic spreading efficiency, we quantified the proportion of starter cell excitatory postsynaptic specializations, labeled with PSD95GFP, that were directly apposed with rabies-labeled presynaptic terminals, labeled with SynPhRFP (Figure 3F). Because L4 excitatory neurons connect to one another, the direct connections between starter cells can generate co-labeling of pre- and postsynaptic specializations independent from transsynaptic spread. Co-labeling independent of transynaptic spread might also result from direct rabies infection of the small numbers of non-starter neurons infected with rabies due to leak expression of TVA (see above). It was therefore important to begin by quantifying how much of this 'background label' is present under conditions in which similar numbers of L4 neurons are directly infected with EnvA+ RVdG-PSD95GFP-SynPhRFP, but there is no transsynaptic spread. To quantify this we calculated the proportion of PSD95GFP puncta apposed to SynPhRFP in experiments that omitted oG (no AAV8-hSyn-FLEX-H2BBFP-oG, Figure 2A, B, and C). The omission of oG prevents retrograde spread of rabies virus and labeling of input neurons (Figure 3D), therefore observed SynPhRFP colocalization with PSD95GFP must be a result of L4 to L4 excitatory starter neuron connections and/or infection via TVA leak expression. We found that neurons in which oG was used for trans-complementation displayed significantly higher proportions of PSD95GFP puncta colocalized with SynPhRFP compared to neurons in which oG was omitted (50.61 ± 3.92% vs 8.86 ± 1.79% mean ± SEM respectively, Wilcoxon rank-sum test, p=0.001, n=9 neurons across 3 mice and n=5 neurons across 2 mice; Figure 3G). Within experimental condition groups, puncta colocalization did not vary significantly across neurons from distinct experimental animals. The difference between these values (50.61–8.86%) yields an estimate of 42% of postsynaptic densities whose presynaptic partners are transsynaptically labeled (SF = 0.42). To determine how SF is related to distance from the cell body we sampled from different portions of the dendritic arbors of L4 starter cells. Sampled areas within 75 µm of the soma were classified as proximal dendritic domains and areas within 75 µm of the pial surface were distal distal domains are to be about µm from the We found no significant differences between SF at proximal of apical dendrites compared to the distal as by PSD95GFP and SynPhRFP puncta colocalization ± vs ± respectively, Wilcoxon rank-sum test, n=9 neurons across 3 Figure 3G). We also compared SF at basal dendrites apical dendrites and observed no significant difference ± vs ± respectively, Wilcoxon rank-sum test, n=9 neurons across 3 mice and neurons across 3 Figure 3G). that a fraction of the observed synaptic labeling might than direct spread of rabies virus at the observed synapse (see This fraction is small to the variability in our measured SF at distal proximal or apical basal dendrites. Efficiency of rabies transsynaptic spread across excitatory synapses onto inhibitory starter neurons To assess differences in efficiency of rabies retrograde spread for different starter cell types, we monosynaptic rabies tracing using RVdG-PSD95GFP-SynPhRFP as but using mouse that express in two distinct of inhibitory We used the and to target initial infection to somatostatin or vasoactive intestinal peptide inhibitory neurons (Figure We and as both low levels of connections compared to neurons, which connect to each other et al., et al., 2013). We found no significant difference between the of PSD95GFP puncta apposed to SynPhRFP on dendrites compared to dendrites ± vs respectively, Wilcoxon rank-sum test, n=9 neurons across 3 mice per Figure values are to the SF for labeling inputs to L4 starter cells see above). We also whether SF based on distance from the cell starter cells, colocalization of PSD95GFP with SynPhRFP on proximal dendritic domains 75 µm of the cell did not significantly from distal dendritic domains 75 µm of the pial ± vs ± respectively, Wilcoxon rank-sum test, Figure We observed similar results for starter cells, with no difference between SF at proximal distal domains ± vs ± respectively, Wilcoxon rank-sum test, Figure Figure Download asset Open asset Efficiency of transsynaptic spread from inhibitory starter cells to excitatory inputs. (A) Representative example images of V1 injection site for mouse (top) and mouse obtained using widefield fluorescence microscopy at 10×. Scale bar = 200 µm. Max intensity projection reconstruction of images obtained using Airyscan super-resolution imaging at 63×. image of (B) and (D) starter neurons and SynPhRFP+) labeled with smFP_myc. Scale bar = 20 µm. and Zoomed in images of boxed in and 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, of postsynaptic densities (PSD95GFP) on and starter cells apposed with rabies-labeled presynaptic terminals (SynPhRFP). Middle, colocalization on the distal vs proximal domains of dendrites. Right, colocalization on the distal vs proximal domains of 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) = p-value > 0.05 = not significant (ns). between and Because studies have found large variability in the number of rabies-labeled input cells per starter cell (CI), there is interest in what fraction of all input neurons are labeled for the of the numbers of inputs to each neuron is so it is not to IF from with in Materials and we provide a analysis and quantitative of the relationships between SF and IF for cortical starter Overall, these indicate that the SF data we have here provide a estimate and important new insight into IF under the tracing conditions that we have it is important to factors that SF likely to be than and more in Materials and methods) indicate that for the cortical we our observed SF of 40% likely corresponds to an IF of about We further estimate that when SF is 40% for a cortical the probability of rabies spread across a single synaptic contact (unitary synaptic efficiency, U) is likely to be about this spreading efficiency is likely to be it can allow for of strong input (see but there is for spread the experimental or for labeling to all input Figure 5 Download asset Open asset from (A) inputs between layer excitatory neurons and (B) the number of synapses per excitatory neuron to excitatory neuron (A) The of inputs to a layer excitatory neuron that provide common input to another layer excitatory neuron of neurons at between layer on identification of presynaptic inputs to layer cells. (B) of the numbers of synapses per for excitatory neuron to excitatory neuron connections identified in of a from mouse visual cortex. is on a number of synapses per = ± The of connections only one synaptic Figure Download asset Open asset between synaptic fraction input fraction and synaptic efficiency on synapses per input neuron and proportions of neurons with differences in and SF against the ratio of neurons with values of 1 3 (A) or 2 (C) for different values of and of IF SF against the of neurons with values of 1 3 (B) or 2 (D) for different values of is the expected relationship between SF and IF and what are the factors that SF likely to be than We have defined SF as the proportion of synaptic contacts that are labeled on a starter neuron. It is important to that SF typically not be the same as the proportion of input neurons that are labeled In Materials and methods with Figures we