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Thalamus provides layer 4 of primary visual cortex with orientation- and direction-tuned inputs

Abstract

Understanding the functions of a brain region requires knowing the neural representations of its myriad inputs, local neurons and outputs. Primary visual cortex (V1) has long been thought to compute visual orientation from untuned thalamic inputs, but very few thalamic inputs have been measured in any mammal. We determined the response properties of 28,000 thalamic boutons and 4,000 cortical neurons in layers 1–5 of awake mouse V1. Using adaptive optics that allows accurate measurement of bouton activity deep in cortex, we found that around half of the boutons in the main thalamorecipient L4 carried orientation-tuned information and that their orientation and direction biases were also dominant in the L4 neuron population, suggesting that these neurons may inherit their selectivity from tuned thalamic inputs. Cortical neurons in all layers exhibited sharper tuning than thalamic boutons and a greater diversity of preferred orientations. Our results provide data-rich constraints for refining mechanistic models of cortical computation.

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Figure 1: In vivo calcium imaging of thalamic axons in V1.
Figure 2: Adaptive optics is essential for tuning curve characterization.
Figure 3: Characterization of tuning properties of L4, L2/3 and L5 neurons in V1.
Figure 4: Orientation tuning of thalamic boutons and neurons in V1.
Figure 5: Direction tuning of thalamic boutons and neurons in V1.

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Acknowledgements

We thank M. Cembrowski, Y. Dan, R. Egnor, A. Kerlin, J. Magee, G. Murphy, S. Sternson, M. Stryker and K. Svoboda for comments on the manuscript. A. Hu for help with histology. This work was supported by Howard Hughes Medical Institute.

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Authors

Contributions

N.J. initiated and oversaw the project, W.S. and N.J. designed the experiments, Z.T. collected calcium imaging data on L4 neurons, and W.S. collected all other data. All of the authors contributed to data analysis and presentation. W.S., B.D.M. and N.J. wrote the paper.

Corresponding author

Correspondence to Na Ji.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 dLGN provides the main thalamic inputs to mouse primary visual cortex (V1).

(a) Neurons in V1 labeled with injected Fluoro-Gold (FG). Scale bar: 100 μm. (b) FG Injection site. Scale bar: 2 mm. (c) Visual thalamus with retrograde-label FG (black) injected in V1 and anterograde-label Cholera Toxin Subunit B/Alexa 594 (red) introduced to the contralateral eye. dLGN and vLGN: dorsal and ventral lateral geniculate nucleus; LP: lateral posterior thalamic nucleus. Scale bar: 100 μm. Representative images from 4 mice.

Supplementary Figure 2 Thalamic axons project to L1 through L4 of mouse V1.

(a) GFP labels visual thalamic neurons and tdTomato labels L4 neurons in Scnn1a-Tg3-Cre mice. (b) GFP+ neurons in dLGN. d, dorsal; m, medial. Scale bar: 100 μm. (c) GFP+ thalamic axons and tdTomato+ L4 neurons in a V1 section from pia (top) to white matter (bottom). Scale bar: 50 μm. (d) Enlarged views of GFP+ axons of areas i–iv in c. Scale bar: 10 μm. (e) In widetype mice with GCaMP6s+ thalamic axons, the same projection pattern was observed from pia (top) to white matter (bottom). Scale bars: 50 μm. (f) Enlarged views of GCaMP6s+ axons of areas i–iv in e. Scale bar: 10 μm. We found no GFP+ or GCaMP6s+ cell bodies in V1. Representative images from 3 mice (GFP and tdTomato) and 21 mice (GCaMP6s).

Supplementary Figure 3 Image registration and calcium transient calculation.

(a) Example frames of a calcium imaging stack of thalamic boutons at 350 μm below pia. Scale bar: 10 μm. (b) Average of 3,120 frames before and after iterations of registration. (c) Area within the green square in b. Red numbers: six regions of interest (ROIs) that are putative thalamic boutons. Scale bar: 10 μm. (d) Average fluorescent signal intensity of each ROI for each frame of the registered stack (upper panels, only part of the data shown); The tallest bin of the fluorescent signal histogram (right panels) is set as the baseline fluorescence F0 (magenta dots in left panels). Calcium transient ΔF/F (lower panel) is then calculated as ΔF/F=(F – F0)/F0. ROI6: a representative fluorescent signal trace across 3,000 frames (~45-minute acquisition for orientation, spatial, and temporal frequency tuning) measured with 156 mW post-objective power.

Supplementary Figure 4 Estimation of the number of independent boutons.

(a) Boutons on the same axon have (b) highly correlated calcium transients. Scale bar: 5 μm. (c) Distribution of correlation coefficient R between boutons visually identified as being on the same axon (76 boutons, 24 axons). Mean: 0.81; s.d.: 0.08. Red dashed line at 0.57: 3 s.d. below the mean; Blue dashed line at 0.65: 2 s.d. below the mean. Correlation coefficients were computed over the entire imaging session. (d) Boutons in one field of view and (e) the matrix of their correlation coefficients. Scale bar: 10 μm. (f) Distribution of correlation coefficients for all fields of view (34,120 boutons, 87 fields of view). (g) Assuming boutons with R less than (upper panel) 0.65 or (lower panel) 0.57 to be from different cells, the percentage of independent boutons for each image section. Superficial image sections have more boutons from the same axons, because of the higher occurrence of horizontally elaborating axon terminals in L1.

Supplementary Figure 5 Population averaged responses of thalamic boutons and neurons in V1.

(a–i) For thalamic boutons: averaged ΔF/F responses of all (a–c) visually responsive, (d–f) AS, and (g–i) DS boutons 0–100 μm, 150–250 μm, and 300–400 μm below pia, respectively. (j–r) For V1 neurons: averaged responses of all (j,m,p) visually responsive, (k,n,q) AS, and (l,o,r) DS L4, L2/3, and L5 neurons, respectively. Green lines: mean vectors of population responses. Boutons: 21 wild-type mice; L4: 3 Scnn1a-Tg3-Cre mice; L2/3: 6 Thy1-GCaMP6 GP4.3 mice; L5: 5 Rbp4-Cre mice.

Supplementary Figure 6 Distributions of preferred motion axis and direction for thalamic boutons, L4, L2/3, and L5 neurons and their statistical distances.

(a) From top to bottom: distributions of preferred motion axis for AS thalamic boutons in L1, L2/3, L4, as well as AS L4, L2/3, and L5 neurons. (b) From top to bottom: distributions of preferred motion direction for DS thalamic boutons in L1, L2/3, L4, as well as DS L4, L2/3, and L5 neurons. a and b: same data as in Fig. 3, plotted in linear histogram. (c) Cumulative distributions of the preferred axis for the AS units. (d) Statistical distances between pairs of distributions in c quantified via Kolmogorov-Smirnov distance. 0 indicates identical distributions. (e) Cumulative distributions of the preferred direction for the DS units. (f) Kolmogorov-Smirnov distances between pairs of distributions in e, with smaller distance indicating more similar distributions.

Supplementary Figure 7 Boutons with different preferred motion axis and direction have similar brightness.

(a) From left to right: Preferred axis distributions of AS boutons in L1, L2/3, and L4. (b) Scatter plots of maximal brightness of AS boutons versus their preferred motion axis. (c) Box-and-whisker plots of brightness for AS boutons within the two shaded windows in b. (d) From left to right: Preferred direction distributions of DS boutons in L1, L2/3, and L4. (e) Scatter plots of maximal brightness of DS boutons versus their preferred motion direction. (f) Box-and-whisker plots of brightness for DS boutons within the three shaded windows in e. +’s are outliers. Brightness differences in c and f are not statistically significant (P>0.06 for all pairs, Kruskal-Wallis test).

Supplementary Figure 8 Orientation tuning of thalamic boutons from mice with different GCaMP6s expression levels in lateral posterior thalamic nucleus.

(a–c) GCaMP6s expression in visual thalamus of three mice: left to right, decreasing levels of expression in lateral posterior thalamic nucleus. (d–f) Orientation tuning of L1 thalamic boutons measured in vivo from Mouse 1, 2, and 3, respectively: upper left, in vivo image; upper right, boutons color-coded by their preferred orientation; lower left, distribution of preferred orientation; lower right, distribution of tuning width. (g–i) Orientation tuning of L4 thalamic boutons characterized in vivo from Mouse 1, 2, and 3, respectively. Scale bar: 500 μm (a–c), 10 μm (d–i).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Table 1 (PDF 3021 kb)

Supplementary Methods Checklist (PDF 385 kb)

In vivo two-photon calcium images of thalamic boutons 70 μm below pia.

Left panel: two-photon images of GCaMP6s+ thalamic boutons 70 μm below pia in V1 of a mouse that was presented with drifting-grating stimuli (top-right inset). Right panel: ΔF/F calcium transients of the axonal varicosities (i.e., putative boutons) labeled in the left panel; scale bar: 100%. Images and transients are the averages of ten trials. (AVI 18041 kb)

In vivo two-photon calcium imaging of thalamic boutons 350 μm below pia.

Left panel: two-photon images of GCaMP6s+ thalamic boutons 350 μm below pia in V1 of a mouse that was presented with drifting-grating stimuli (top-right inset). Right panel: ΔF/F calcium transients of the axonal varicosities (i.e., putative boutons) labeled in the left panel; scale bar: 50%. Images and transients are the averages of ten trials. (AVI 20577 kb)

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Sun, W., Tan, Z., Mensh, B. et al. Thalamus provides layer 4 of primary visual cortex with orientation- and direction-tuned inputs. Nat Neurosci 19, 308–315 (2016). https://doi.org/10.1038/nn.4196

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