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In vivo measurement of afferent activity with axon-specific calcium imaging

Nature Neuroscience volume 21, pages 1272–1280 (2018)Cite this article

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Abstract

In vivo calcium imaging from axons provides direct interrogation of afferent neural activity, informing the neural representations that a local circuit receives. Unlike in somata and dendrites, axonal recording of neural activity—both electrically and optically—has been difficult to achieve, thus preventing comprehensive understanding of neuronal circuit function. Here we developed an active transportation strategy to enrich GCaMP6, a genetically encoded calcium indicator, uniformly in axons with sufficient brightness, signal-to-noise ratio, and photostability to allow robust, structure-specific imaging of presynaptic activity in awake mice. Axon-targeted GCaMP6 enables frame-to-frame correlation for motion correction in axons and permits subcellular-resolution recording of axonal activity in previously inaccessible deep-brain areas. We used axon-targeted GCaMP6 to record layer-specific local afferents without contamination from somata or from intermingled dendrites in the cortex. We expect that axon-targeted GCaMP6 will facilitate new applications in investigating afferent signals relayed by genetically defined neuronal populations within and across specific brain regions.

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Fig. 1: Axonal enrichment of GCaMP sensor driven by GAP43 targeting motif in dissociated cells and in vivo.
Fig. 2: Axon-GCaMP maintains photostability and displays enhanced SNR.
Fig. 3: Axon-GCaMP6s improves SNR and image-wise correlations in long-distance axons.
Fig. 4: Imaging thalamocortical afferents deep in cortical tissue with axon-GCaMP6s.
Fig. 5: Axon-GCaMP6s permits layer-specific recording of axons projected from local neurons, without contamination from somatodendritic signals.

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Acknowledgements

We thank H. Chen at the vector core of vision center at UCD for virus production and L. Looger and B. Mensh for insightful comments on the manuscript. This work was supported by funding to L.T. (DP2MH107056, U01NS090604, and U01NS099714 from the National Institutes of Health and Rita Allen Foundation), to L.P. (Marie Curie PCIG12-GA-2012-334353, the European Union’s Seventh Framework Program FP7/2007-2013 under grant agreement No. 600925, and the Champalimaud Foundation), to N.J. (Howard Hughes Medical Institute and grants U01 NS103573 and U01 NS103489 from the National Institutes of Health), and a Human Frontiers Research Grant to L.T. and L.P. (RGY0085/2013).

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Medicine, University of California Davis, Davis, CA, USA

    Gerard Joey Broussard, Elizabeth K. Unger, Xian Xiao & Lin Tian

  2. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA

    Yajie Liang, Guanghan Meng & Na Ji

  3. Champalimaud Center for the Unknown, Lisbon, Portugal

    Marina Fridman & Leopoldo Petreanu

  4. Department of Physics, Department of Molecular & Cellular Biology, University of California Berkeley, Berkeley, CA, USA

    Guanghan Meng & Na Ji

  5. Westlake Institute for Advanced Study, Hangzhou, China

    Xian Xiao

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Contributions

L.P. and L.T. initiated the project. G.J.B. and L.T. designed the projects; developed axon-targeted GCaMP6; performed characterization of ADR, photostability, and SNR in vitro in dissociated neuronal culture; and performed histological characterization of ADR and expression in vivo. Y.L. G.M., and N.J. performed in vivo characterization of dLGN axons in V1, deep-brain imaging, and layer-specific cortical axonal recording in V1. M.F. and L.P. performed in vivo characterization of L1 axons in V1 projecting from LP and area LM and examined frame-to-frame correlations. G.J.B. analyzed data from in vivo imaging with help from Y.L., M.F., and G.M. E.K.U. performed viral injections. X.X. performed pilot experiments for sensor characterization. G.J.B. and L.T. wrote the manuscript with critical input from all authors.

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Correspondence to Leopoldo Petreanu or Lin Tian.

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Integrated supplementary information

Supplementary Figure 1 Axon-GCaMP6 efficiently transports to the axonal arbor of dissociated hippocampal neurons.

(a) Schematic representation of all GCaMP targeting variants. (b) Normalized axon to dendrite ratio (nADR) for all variants. Data plotted as mean ± s.e.m. F(13,77) = 14.61, P = 0.000082 by one way ANOVA; for individual comparisons to u-GCaMP6m, *P<0.05, ****P<0.0001, by Tukey-Kramer test for multiple comparisons. (c) nADR as a function of time post-transfection for all GCaMP6m constructs. (d) Representative neuron with nADR binned by distance from cell soma. Similar results were obtained for 8-10 cells from three independent cultures. (e) Axon-GCaMP6 is enriched across the entire axonal arbor of dissociated hippocampal neurons. For (b),(c), and (e), data plotted as mean ± s.e.m. n = 4-10 cells from at least 2 independent cultures for each construct.

Supplementary Figure 2 Bicistronic constructs reveal that similar expression levels result in superior labeling of axon termini by axon-GCaMP6s.

(a) Tissue sections from an animal injected with rAAV-hSynapsin-axon-GCaMP6s-P2A-mRuby3-WPRE-SV40 at dLGN demonstrating the extent of labeling in the GCaMP (top images), mRuby3 (center images), and the merge of the two channels (bottom images). Large injections drove transduction in dLGN and off-target in vLGN and CA3 regions. Of these structures, only dLGN forms the projection shown in V1. Note the bright GCaMP labeling in axons of all transduced structures even in off-target regions. (b) Tissue sections from an animal injected with rAAV-hSynapsin-GCaMP6s-P2A-mRuby3-WPRE-SV40 at dLGN demonstrating the extent of labeling in the GCaMP (top images), mRuby3 (center images), and the merge of the two channels (bottom images). The similarity of staining in the mRuby3 channel indicates that similar patterns of transduction and levels of protein expression and were driven in each animal. dLGN axons in the thalamorecipient layer of V1 are much more weakly labeled by u-GCaMP6s. All images taken with identical settings and presented with same look up table. Note that brighter labeling of dLGN in the green channel for the axon-GCaMP6s animal is due to vLGN-derived fibers of passage as seen in Fig. 1e. Similar results were obtained 4 animals per construct. All scale bars: 1mm.

Supplementary Figure 3 Retrograde labeling was similar between bicistronic constructs expressing axon-GCaMP6s and u-GCaMP6s.

Representative images of V1 section from an animal injected with rAAV-hSynapsin-axon-GCaMP6s-P2A-mRuby3-WPRE-SV40 (a) and rAAV-hSynapsin-GCaMP6s-P2A-mRuby3-WPRE-SV40 (b). Middle and bottom panels show zoomed in images (medial and lateral region of V1) in both green and red fluorescence showing a higher degree of retrograde transduction, particularly in deep L6. Note the similarity of retrograde transduction including that layer 6 cells and more medial regions are more frequently transduced by retrograde transport of virus. Similar or lower levels of retrograde transduction were observed in 6 of a total of 8 injected animals. Scale bars: 100 μm or 10 μm as indicated in example panels.

Supplementary Figure 4 Axon-GCaMP6m has diffusion characteristics superior to syGCaMP6m and comparable to u-GCaMP6m.

(a-b) Schematic representation of the FLIP and the FRAP experiments. Scale bar 5 μm. (c) FLIP data from the indicated constructs. Constructs such as GAP43-GCaMP6m (i.e., axon-GCaMP6m) fused to only palmitoylation domains showed higher photostability than those fused to myristoylation domains or peptides with additional binding partners. Similar results were obtained in data from at least two independent cultures per construct. (d) Demonstration of the procedure used for quantifying diffusion coefficients on a laser-scanning confocal. A Gaussian was fit to the ratio of the fluorescence profile of an axon from directly after the bleaching pulse and during the baseline period to extract parameter Re. A double-exponential was then fit to the recovery curve to extract the time to the half-maximal recovery, t1/2 (see methods). (e) Quantification of diffusion coefficients extracted from individual experiments for u-GCaMP6m and axon-GCaMP6m. Because the post-bleach profile of syGCaMP6m was fit poorly by a Gaussian, we did not attempt to quantify a diffusion coefficient for this probe. Data presented as mean ± s.e.m. *P<0.029. Wilcoxon’s rank-sum test; n = 4 cells from 2 plates per construct. (f) Time course of fluorescence intensity normalized to the first 50 seconds of acquisition during imaging of dLGN projection termini of data presented as averages in Fig. 2g. Rows correspond to individual analyzed ROIs while columns represent time bins. Note that a majority of syGCaMP6s ROIs exhibit substantial bleaching during the imaging epoch while both u-GCaMP6s and axon-GCaMP6s maintain much of their signal output.

Supplementary Figure 5 Axon-GCaMP6s enriches in axons, which led to enhanced SNR. Axon-GCaMP6s displayed similar kinetics and expression level compared to u-GCaMP6s.

(a) Quantification of green to red fluorescent intensity in each analyzed ROI. Green fluorescence was quantified during the pre-stimulus baseline period. (b) Scatterplots of individual ROIs with marginal distribution of ΔF/F0 vs. F0 (top) and SNR vs. F0 (bottom). (c) Averaged SNR of time-lapse traces across all ROIs normalized to the maximum values in response to 10FP (left) and 80FP (right). Data plotted as mean ± s.e.m. (d) Half-decay time from all analyzed ROIs in response to 10, 20, 40, and 80FPs. Boxplot partitions represent data quartile edges. Two-way ANOVA revealed that while FP number had a significant effect on decay times, the construct used to transfect cells did not. FP number: F(6,84221) = 4389.35, P < 4.94E-324; Construct: F(2,84221) = 1.99, P = 0.14, Two-way ANOVA. (e) Representative field of axons in the red channel. Scale bar 5 μm. (f) Quantification of ROI sizes in all analyzed ROIs. There was no difference in the sizes of selected ROIs. P = 0.94, Student’s t-test. (g) Representative field of axons shown in (e) overlayed with selected ROIs in cyan. (h) Quantification of red fluoresence in all selected ROIs. The fluorescent intensity of mRuby was slightly lower in the axon-GCaMP6s experiments, which indicates that increased brightness in axons is unlikely due to relative overexpression of axon-GCaMP6s. P = 1.44E-33, Student’s t-test. All analyses in (a-h) based on n = 9646 ROIs from u-GCaMP6s and n = 8687 ROIs from axon-GCaMP6s with data taken from 9 experiments performed on 3 independent transfections per construct.

Supplementary Figure 6 Thalamocortical axons labeled with axon-GCaMP6s exhibit ΔF/F similar to those of u-GCaMP6s.

(a) Schematic representation of viral injection in dLGN followed by imaging V1. (b-c) Trial-averaged traces of ΔF/F0 (b) and cumulative distribution (c) of all ROIs of dLGN axons in V1. ∆F/F of axon-GCaMP6s was significantly higher compared to u-GCaMP6s. P = 5.41E-73, Kolmogorov-Smirnov test. n=712 ROIs from 4 animals for axon-GCaMP6s and n=14478 ROIs for u-GCaMP6s from 19 animals. (d) Schematic representation of viral injection in LP followed by imaging in V1. (e-f) Trial-averaged traces of ΔF/F0 (e) and cumulative distribution (f) of all ROIs of LP axons in V1. P = 0.44 by Kolmogorov-Smirnov test. n=1393 ROIs from 3 animals for axon-GCaMP6s and n=917 ROIs from 4 animals for u-GCaMP6s.

Supplementary Figure 7 Axon-GCaMP6s and u-GCaMP6s report calcium signals similarly in putative presynaptic and adjacent axonal compartments.

(a) Schematic of imaging experiments of dissociated neurons virally transduced to express axon-GCaMP6s or u-GCaMP6s and synaptophysin-mRuby2 (sy-mRuby2) presented with representative imaged axonal field. Scale bar 10 μm. (b) Images of axonal segments expressing sy-mRuby2 taken before (magenta, top panel) and after (cyan, middle panel) an ~8 minute functional imaging session. Stable puncta indicated by channel overlap (orange arrows, bottom panel) were putatively identified as pre-synaptic compartments. Sy-mRuby2-labeled unstable puncta that transited axon segments (aquamarine arrows, bottom panel) were putatively identified as adjacent axonal compartments. Scale bar: 3 μm. (c) ΔF/F traces for response of individual analyzed ROIs. Point at which field stimulus was applied indicated by vertical dotted black line. (d) Response of all analyzed ROIs classified as indicated in figure legend. Note that both u-GCaMP6s and axon-GCaMP6s show a higher response in the ROIs classified as putative pre-synapses. (d) (top) Cumulative distribution of basal fluorescence in analyzed ROIs (stable puncta, 12.97; unstable puncta, 7.33; P = 1.23E-30 by the Kolmogorov-Smirnov test). (bottom) Cumulative distribution of ΔF/F of analyzed ROIs in reponse to application of 5 field stimuli (stable puncta, 2.76; unstable puncta, 1.93; P = 2.57E-4 by the Kolmogorov-Smirnov test). These experimental data were derived from n = 222 (stable) and 199 (unstable) ROIS from two independent cultures per construct. (e) Schematic representation of viral injection in LP, followed by in vivo imaging in V1 of projected axons. (f) Representative images of L1 axons projected from LP with ROIs covering putative presynaptic compartments (bouton-like sturctures, red) or nearby putative axonal shaft (non-varicose segments, pink). Scale bar: 10 μm. (g) Trial-averaged traces of ΔF/F time course from all responsive ROIs to preferred stimulus direction. Point at which moving stimulus presented indicated by vertical dotted black line. (h) (top) Cumulative distribution of normalized fluorescence intensity of LP axons in V1 in either bouton-like or shaft-like compartments (bouton, 20.34; shaft, 17.45; P= 0.11 by the Kolmogorov-Smirnov test). (bottom) Cumulative distribution of ΔF/F responses of individual ROIs with median values indicated by vertical lines as well as color-coded numeric values (bouton, 0.25; shaft, 0.12; P = 1.10E-5 by the Kolmogorov-Smirnov test). Data were analyzed from n = 120 (bouton) and 114 (shaft) ROIs taken from 3 imaging experiments from two animals.

Supplementary Figure 8 In vivo photostability of axon-GCaMP6s results in better SNR than syGCaMP6s.

(a) Schematic representation of viral injection in dLGN followed by in vivo imaging in V1 of projected axons. (b) Averaged fluorescence of representative ROIs in axons transduced by axon-GCaMP6s (top) or syGCaMP6s (bottom). The syGCaMP6s ROI showed a marked decrease in fluorescence output over the course of the imaging session. (c) The same data as presented in (b) with traces aligned based on the stimulus which was presented and color-coded to indicate trial order. The syGCaMP6s data shows low responses in later trials. (d) Representative images of L1 axons projected from dLGN of axon-GCaMP6s or syGCaMP6s expressing axons. Scale bars:10 μm. (e) Normalized fluorescent intensity averaged over the first 50 frames of axon-GCaMP6s (5.57) and syGCaMP6s (3.45) in dLGN axons projecting to V1 (P = 3.13E-19 by the Kolmogorov-Smirnov test). (f-h) Trial averaged traces of ΔF/F (f) with cumulative distribution from the first and tenth trials alone (g) or averaged over all trials (h). For the single trial analysis of ΔF/F, one-way ANOVA revealed a significant difference across group means (F(3,1723) = 38.32; P = 2.86E-24) with significant pairwise differences between all groups as assessed by Tukey-Kramer posthoc test. ∆F/F averaged across trials for axon-GCaMP6s was significantly higher compared to syGCaMP6s (axon-GCaMP6s: 0.72; syGCaMP6s: 0.65, P = 0.016, Kolmogorov-Smirnov test). (i-k) Trial averaged traces of SNR (i) with cumulative distribution from the first and tenth trials alone (j) or averaged over all trials (k). For the single trial analysis of SNR, one-way ANOVA revealed a significant difference across group means (F(3,1723) = 6.76; P = 0.0002) with significant pairwise differences between the first trial distributions and the tenth trial of syGCaMP6s by the Tukey-Kramer posthoc test. SNR averaged across trials for axon-GCaMP6s was significantly higher compared to syGCaMP6s (axon-GCaMP6s: 2.73; syGCaMP6s: 1.63, P = 1.14E-25, Kolmogorov-Smirnov test). All analyses based on n = 712 ROIs from 3 animals (axon-GCaMP6s) and 150 ROIs from 2 animals (syGCaMP6s). Note that the trial averaged traces for axon-GCaMP6s data have been omitted as these data are presented in Supplementary Fig. 6 b and Fig. 3 d. Because single trial data was noisier than trial-averaged data, outliers were omitted from CDFs, but not median or statistical calculations in (g) and (j) to emphasize separation of the distributions.

Supplementary Figure 9 Axon-GCaMP6f improves SNR in long-distance axons.

(a) Schematic representation of viral injection in dLGN followed by in vivo imaging in V1 of projected axons. (b) Representative images of L1 axons projected from dLGN demonstrating enhanced brightness of axon-GCaMP6f expressing axons. Scale bars: 10 μm. (c) Normalized average fluorescent intensity of axon-GCaMP6f (5.32) and u-GCaMP6f (0.58) in dLGN axons projecting to V1 (P = 9.87E-98 by the Kolmogorov-Smirnov test). (d-e) Trial-averaged traces of ΔF/F0 (d) and cumulative distribution (e) of all ROIs of dLGN axons in V1. ∆F/F of axon-GCaMP6f was significantly higher compared to u-GCaMP6f (axon-GCaMP6f: 63.58; u-GCaMP6f: 39.28, P = 1.77E-10, Kolmogorov-Smirnov test). (f-g) Trial-averaged traces of SNR (f) and cumulative distribution (g) of all ROIs of dLGN axons in V1. SNR of axon-GCaMP6f was significantly higher compared to u-GCaMP6f (axon-GCaMP6s: 1.23; u-GCaMP6s: 0.77, P = 1.20E-09, Kolmogorov-Smirnov test). For u-GCaMP6f we analyzed n = 262 ROIs from 2 animals, for axon-GCaMP6s we analyzed n = 190 ROIs from 2 animals.

Supplementary Figure 10 Axon-GCaMP6s displays similar direction and orientation preference, as well as kinetics, compared with u-GCaMP6s.

Schematic of viral injection into dLGN (a) or LP (d) followed by imaging in V1. Mean preference of PDFs for orientation of axis-selective axons (b) and direction preference of direction-selective axons (c) of all analyzed ROIs from dLGN axons in V1 were averaged across animals. There was a highly significant difference in orientation preference when data from both constructs were pooled, however, no difference in orientation tuning due to which construct was expressed was detected as assessed by two-way ANOVA. Orientation: F(3,275) = 140.44, P < 4.94E-324; Construct: F(1,275) = 0.001, P = 0.97. n=228 binned observations of ROI orientation preference from 4 animals for u-GCaMP6s and n=48 binned observations of ROI orientation preference from 3 animals for axon-GCaMP6s. Mean preference of PDFs for orientation (e) and direction preference (f) of all analyzed ROIs from LP axons in V1 were averaged across animals. There was no significant difference in orientation preference across data from u-GCaMP6s and axon-GCaMP6s as assessed by two-way ANOVA. Orientation: F(7,151) = 1.07, P = 0.39; Construct: F(1,151) = 0.02, P = 0.89. n=72 binned observations of ROI orientation preference from 4 animals for u-GCaMP6s and n=80 binned observations of ROI orientation preference from 3 animals for axon-GCaMP6s. Data shown as mean ± s.e.m. Too few boutons from LM u-GCaMP6s imaging sessions were included to establish reliable orientation and direction preferences. (g-i) Kinetics of calcium responses in vivo is correlated with probe concentration in axonal termini. (g) Schematic representation of viral injection in dLGN followed by in vivo imaging in V1 of projected axons. (h) Representative traces of axonal responses averaged across 10 trials of preferred direction for the indicated sensor with a double exponential fit for time to half-decay of signal (tauoff). (i) Scatter plot of the decay times against the baseline fluorescence for ROIs with SNR value above 1. Marginal distributions are shown as kernel densities on the appropriate axes with median values indicated by orthogonal line and color-coded text. When controlling for basal fluorescence as a readout of protein production level, no significant affect was observed of construct on response kinetics. F(1,1541) = 2.76, P = 0.10, ANCOVA. For u-GCaMP6s we analyzed n=502 ROIs from 4 animals, for axon-GCaMP6s n=1043 ROIs from 3 animals.

Supplementary Figure 11 LM axons labeled with axon-GCaMP6s are brighter and exhibit higher ΔF/F than u-GCaMP6s 2 weeks post-transduction.

(a) Schematic representation of viral injection in LM followed by imaging in V1 2 weeks post-injection. (b) Representative images of L1 axons projecting to V1 from all transduced structures demonstrating enhanced brightness of axon-GCaMP6s axons. Scale bars:10 μm. (c) Trial-averaged traces of SNR time course from all ROIs responsive to preferred stimulus direction. Point at which moving stimulus presented indicated by vertical dotted black line. (d) Cumulative distribution of SNR of individual ROIs with median values indicated by vertical lines as well as color-coded numeric values. Axon-GCaMP6s, 1.09; u-GCaMP6s, 0.35; P = 5.62E-12 by the Kolmogorov-Smirnov test. n=42 ROIs from 2 animals for u-GCaMP6s and n=1269 ROIs from 3 animals for axon-GCaMP6s. (e) Time course of image-wise correlation (Pearson’s rho) values for LM boutons. Data plotted as mean (solid line) ± s.e.m. (shaded regions); n = 6 imaging sessions from two animals for each construct. (f) Normalized average fluorescent intensity of axon-GCaMP6s and u-GCaMP6s. P = 9.72E-27 by Kolmogorov-Smirnov test. For u-GCaMP6s we analyzed 42 ROIs from 2 animals, for axon-GCaMP6s we analyzed 1269 ROIs from 3 animals. (g-h) Trial-averaged traces of ΔF/F0 in response to preferred stimulus direction (g) and cumulative distribution (h) of all ROIs of LM axons in V1. P = 1.06E-07 by Kolmogorov-Smirnov test. (i-j) Far more visually responsive ROIs were found in axon-GCaMP6s than u-GCaMP6s imaging sessions. (i) Number of responsive ROIs found per imaging session. **P = 0.0028 by Wilcoxon’s rank sum test. (j) Percentage of analyzed ROIs that were visually responsive per imaging session. Data presented as mean ± s.e.m. **P = 0.0028 by Wilcoxon’s rank sum test. A total of n = 9 imaging sessions were taken from axon-GCaMP6s animals, while n = 4 were taken from u-GCaMP6s animals.

Supplementary Figure 12 Axon-GCaMP6s improves contrast across imaging depths in cortical tissue in vivo.

(a) Images of dLGN axons labled with axon-GCaMP6s or u-GCaMP6s at the indicated depth. All images are on an inverted look up table (LUT) to enhance contrast for the human eye. LUT-matched images were produced by normalizing images to the square of the excitation power and then matching the maximum and minimum values of the image histograms. Contrast-optimized images were produced by histogram streching with saturation of the top 0.4% of pixels. Contrast-optimized images are overlayed by analyzed line ROIs. This experiment was repeated in 2 animlas per construct with similar results. Scale bars (yellow) are 10μm. (b) Averaged fluorescence profiles of the ROIs shown in a. Profiles were normalized to emphasize the increase in contrast depth achieved with axon-GCaMP6s relative to u-GCaMP6s at both analyzed depths. Data plotted as mean normalized profile (dark line) ± s.d. (shaded region). (c) Distributions of minimum profile depths indicating contrast for all analyzed profiles. At both 50 and 350 μm depth axon-GCaMP6s had a lower minimum indicating a higher contrast relative to u-GCaMP6s. 50 μm: P = 4.94E-16, Wilcoxon’s rank sum test; n = 60 profiles from 2 animals for each construct. 350 μm: P = 1.91E-11, Wilcoxon’s rank sum test; n = 60 profiles from 2 animals for each construct.

Supplementary Figure 13 Imaging thalamocortical afferents deep in cortical tissue with axon-GCaMP6f.

(a) Experimental schematic demonstrating that brighter axon-GCaMP6f axons can be imaged at deeper cortical layers than u-GCaMP6f axons. (b) Comparison of axon-GCaMP6f and u-GCaMP6f fluorescence normalized to the square of the excitation laser power as a function of imaging depth. Data plotted as individual ROIs with binned values (light color) and binned means ± s.e.m (dark colors), and log-linear fit across the depth. When correcting for fluorescence loss due to depth, axon-GCaMP6f was found to produce significantly higher fluorescence output. F(1,156) = 495.11, P < 4.94E-324, ANCOVA. For u-GCaMP6f, n = 60 analyed ROIs, for axon-GCaMP6f, n = 100 analyzed ROIs. Data were taken from two imaging sessions per depth bin across two animals per construct. (c-e) Responses of dLGN axons in V1 in deep layers. (c) Representative images of dLGN axons projecting to V1 with overlays indicating analyzed ROI location at 600μm below pia. Scale bars 3 μm. (d) Average ΔF/F traces aligned to stimulus direction of ROIs indicated in (c) with baseline indicated by dotted black line. Data plotted as mean (black line) ± s.e.m. (gray shading). n = 10 trials per direction. (e) Tuning curves for individual ROIs. (f) Representative images from dLGN axons expressing u-GCaMP6f at 600μm depth with look-up table matched to axon-GCaMP6f images (left) or optimized for contrast (right) demonstrating the complete lack of detected expression and signal generated by u-GCaMP6f.

Supplementary Figure 14 Axon-GCaMP6s allows imaging of local afferents without contamination from dendritic signal.

(a) Representative image of axon-GCaMP6s expressing processes in Scnn1a-Cre-Tg3 animal projected from layer 4 into the supergranular layers in V1 in vivo. No dendritic spines were found in any of the images taken from these animals. (b) Representative image of u-GCaMP6s expressing processes in Scnn1a-Cre-Tg3 animal projected from layer 4 into the supergranular layers in V1 in vivo. In contrast to the axon-GCaMP6s images, there were multiple frames that had clear morphological evidence of spines, indicative of dendritic contamination. Dendritic structures are indicated by arrows in this image. Sacle bars 10 μm. Axon-GCaMP6s enables determination of layer-specific tuning properties of L5 (c-f) and L6 (g-j) axons across cortical layers in V1. Representative images of axons at indicated depth with overlays indicating analyzed ROIs (c, g) and color-coded tuning map demonstrating pixel-wise tuning across analyzed images (d, h). Scale bar 10 μm. (e, i) Average ΔF/F traces aligned to stimulus direction of individual ROIs. Data plotted as mean (black line) ± s.e.m. (gray shading). (f, j) Tuning curves of individual ROIs. n = 10 trials per direction of visual stimulus. Data were taken from 2 animals per genetic line.

Supplementary Figure 15 Local axons labeled by axon-GCaMP6s can be imaged in deep cortical regions like thalamocortical pathways.

(a) Schematic representation of the viral injection of FLEX-hSynapsin1-GCaMP6s to transduce layer 4 cells of Scnn1a-Tg3-Cre animals. (b) Expected pattern of transduction of layer 4 pyramidal cells in V1: axons well-labeled by axon-GCaMP6s with sub-threshold expression in the somatodendritic compartment. (c) axon-GCaMP6s fluorescence normalized to the square of laser intensity plotted as a function of imaging depth. Data presented as individual axons (light red), means per bin with s.e.m. (dark red), and log-linear fit to all datapoints; Two imaging sessions per depth bin across two animals. (d) Representative images of axons at indicated depth (518 and 600 μm) with overlays indicating analyzed ROIs. (e) Color-coded tuning map demonstrating pixel-wise tuning across analyzed images. Scale bar 10 μm. (f) Average ΔF/F traces aligned to stimulus direction of individual ROIs labeled in (d) with baseline indicated by dotted black line. Data plotted as mean (black line) ± s.e.m. (gray shading); n = 10 trials per direction. (g) Tuning curves for individual ROIs. Data were taken from two Scnn1a-Tg3-cre animals.

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Supplementary Video 1 - Axon-GCaMP6 provides brighter labelling for thalamocortical axons.

Left panel shows a representative recording session from axons in superficial V1 of an animal injected with virus axon-GCaMP6s in dLGN during presentation of visual stimuli. Right panel is the same, but the virus used encoded u-GCaMP6s.

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Broussard, G.J., Liang, Y., Fridman, M. et al. In vivo measurement of afferent activity with axon-specific calcium imaging. Nat Neurosci 21, 1272–1280 (2018). https://doi.org/10.1038/s41593-018-0211-4

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