Nature Neuroscience: doi: /nn Supplementary Figure 1. Expression of GCaMP6s in LGN and their axons in V1.

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1 Supplementary Figure 1 Expression of GCaMP6s in LGN and their axons in V1. (a, b) Coronal section of LGN and V1 expressing GCaMP6s. a Coronal slice (bregma -2.3 mm) including thalamus confirmed that GCaMP6s expression was restricted within LGN. b The section of V1 was stained with fluorescent Nissl (red) and layers were assigned according to the size and density of cells (Anderson et al., Brain Res. 2009) (top). The same section from the top panel with GCaMP6s signal (green, bottom) is shown with assigned layers. (c) A part of V1 area (a bottom) was expanded. Intensity of GCaMP6s signal (LGN axons, green) was summed horizontally and plotted as normalized intensity on the right along the cortical depth. LGN axons distributing in layer 4 were particullary numerous and much less amount in layers 1 and

2 2/3. Small numbers of axons can be also seen in layers 5 and 6. We repeated the experiments three times. Previous studies showed the LGN projection pattern to V1 in higher mammals. In cat, X and Y cells mainly target layers 4 and 6, while W cells mainly target layer 1 and some amount to layers 3, 4a, 5a (Boyd & Matsubara, J. Comp. Neurol. 1996). In macaque monkey, similar to cat, P and M cells target mainly layer 4 and less amount layer 6, while K cells target mainly layers 1 and 2/3 (Lund, Annu. Rev. Neurosci. 1988). Therefore, projection patterns from LGN to V1 in mouse is similar to cat and macaque in principle. (d) Three-dimensionally reconstructed image of V1 from Scnn1a-cre/Ai14 mouse in vivo. Most of the cell bodies (red) can be seen at the depth of ~400μm from the pia mater. (e) Merged image of LGN axons (green) and layer 4 neurons (red) obtained from Scnn1a-cre/Ai14 mouse. LGN axons (green) can be seen among layer 4 neurons (red). Some imagings from layer 4 were obtained by using Scnn1a-cre/Ai14 mice (11 mice). (f, g, h) An example of cranial window for the in vivo imaging (f). Intrinsic optical imaging was performed and retinotopic map was created to determine the V1/V2 border (11 mice, g). tdtomato (red) was co-expressed with GCaMP6s (green) in LGN axons. Because GCaMP6s signal is low at the base level, we used the co-expressed tdtomato signal to find the area to image. Two-photon calcium imagings of LGN axons were typically obtained several ROIs (eg. white rectangles) within V1 (h). The dotted line indicates V1/V2 border determined from the retinotopic map (f-h).

3 Supplementary Figure 2 Orientation selectivity of LGN axons in layer 1 and 4. Orientation maps of LGN axons recorded in layer 1 (a, n = 123 from 37 mice) and layer 4 (b, n = 98 from 30 mice). Colored pixels indicate the orientation selective [hue: preferred orientation, lightness: response magnitude, and saturation: gosi]. White pixels indicate responsive but orientation non-selective. Orientation selectivity of LGN axons was typically much lower in layer 4 than layer 1.

4 Supplementary Figure 3 Proportion of orientation selective LGN axons and V1 neurons. Histograms of proportion of orientation selective LGN boutons in layer 1 (a, n = 123 images from 37 mice), 2/3 (b, n = 97 images from 36 mice) and 4 (c, n = 98 images from 30 mice) and V1 neurons in layer 2/3 (d, n = 26 volume images from 14 mice) and 4 (e, n = 14 volume images from 10 mice) were shown (see Online Methods for criteria). Proportion of orientation selective LGN axons in layer 4 (c) was significantly lower than those in layers 1 and 2/3 LGN axons (a, b) and V1 neurons (d, e) (p < 0.001, Mann-Whitney U-test with Bonferroni correction).

5 Supplementary Figure 4 Point spread function in layer 1 and 4 after spherical aberration correction. The image sizes of LGN axonal shaft were measured in layer 1 and 4 (10 axonal shafts from each layer). These values

6 were used to estimate the PSF in layers 1 and 4. Full width at the half maximum (FWHM) of axonal shaft for lateral and axial-directions were 0.48 ± 0.03 µm and 2.83 ± 0.12 µm for layer 1 and 0.58 ± 0.05 µm and 3.71 ± 0.15 µm for layer 4 respectively. The image size of lateral direction was not statistically different between layers 1 and 4 (a: p = 0.06 paired t-test). The image size in axial-direction was statistically different between layers 1 and 4 (d: p < 0.01 paired t-test). Despite of statistical difference of image sizes, expansion of image size of shafts in layer 4 was not so big (1.3 times lager than that in layer 1), indicating PSF is mostly maintained in layer 4. Fluorescence profiles of lateral and axial directions and their Gaussian fittings are shown for layer 1 (b, c) and layer 4 (e, f) respectively.

7 Supplementary Figure 5 Relation between gosi (or OSI) and contamination ratio. gosi (a) and OSI (b) were plotted as a function of contamination ratio in layer 1 (n = 2,924 boutons from 37 mice), 2/3 (n = 2,818 boutons from 36 mice) and 4 (n = 1,722 boutons from 30 mice) (see Methods section for the calculation formula of gosi and OSI). In our studies, raw calcium signal data to visual stimulations were corrected to minimize the out-of-focus signal contamination. The correction was done by subtracting the out-of-focus signal multiplied by the contamination ratio from the raw data (see Online Methods). We confirmed that our conclusion was affected neither by the contamination ratio nor by which formula to use to calculate orientation selectivity (gosi or OSI).

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9 Supplementary Figure 6 Relation between density of LGN axons and gosi in layer 1 and 4. (a) To examine the possibility that contamination with untuned neuropil signal from dense layer 4 axons lowered the orientation selectivity of LGN axons in layer 4, we compared the gosi of boutons in images with densely versus sparsely labeled layer 4 and layer 1. We plotted the gosi as a function of density of responsive boutons for layer 1 and layer 4 LGN axons. Regression analysis revealed that the correlations between mean gosi and density of responsive boutons were statistically significant for both layers 1 (n = 123 images from 37 mice, p < 0.01) and 4 (n = 98 images from 30 mice, p < 0.001). The slopes of regression lines for layer 1 (gosi = x density of responsive boutons ) and layer 4 (gosi = x density of responsive boutons ) were not statistically different (p = 0.66). Therefore, we assumed these slopes were parallel and we performed analysis of covariance. We found the distributions of gosis for these layers were statistically different (p < 0.001) after removing the effect of density of responsive boutons. (b) Representative images of LGN boutons were shown from low and high responsive bouton density images of layer 1 and layer 4. These results indicated that our conclusion that the orientation selectivity was lower in layer 4 LGN axons than in layer 1 was not due to the higher axonal density in layer 4 than in layer 1.

10 Supplementary Figure 7 Distributions of OSI of LGN axons and V1 neurons. Distribution of OSI from LGN axons in layer 1 (a, n = 2,924 boutons from 37 mice), 2/3 (b, n = 2,818 boutons from 36 mice) and 4 (c, n = 1,722 boutons from 30 mice) and V1 neurons in layer 2/3 (d, n = 9,629 neurons from14 mice) and 4 (e, n = 5,102 neurons from10 mice) are plotted. Distribution from layer 4 LGN axons was significantly different from layer 1 LGN axons and V1 layers 2/3 and 4 neurons (p < 0.001, Mann-Whitney U-test with Bonferroni correction). We obtained the similar results from OSI as from gosi. Orientation selectivity was overall lower in layer 4 LGN axons than LGN axons in superficial layers and V1 neurons.

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12 Supplementary Figure 8 Relation between the orientation bias and the strength of orientation selectivity. We observed that LGN axons had strong orientation bias (Figure 3f-h). To know if this bias for orientation preferences has any relationship with orientation selectivity, boutons were divided into two groups according to the strength of orientation selectivity (gosi); one group has gosi between 0.33 and 0.67 (a-e, moderate), another has gosi more than 0.67 (f-j, strong). Distribution of preferred orientation in each group was plotted for LGN axons (a-c, f-h) and V1 neurons (d,e, i,j). In layers 1 and 2/3, orientation bias was not related with strength of orientation selectivity. However, in layer 4, cardinal bias toward 90 degree became more evident in the group of strongly orientation selective boutons (gosi > 0.67). Therefore, there was a laminar difference in the relation between orientation selectivity and the orientation bias. These results may suggest that the source of strongly orientation selective inputs to layer 4 and to the superficial layers might be different. n indicates number of boutons.

13 Supplementary Figure 9 Proportion of direction selective LGN axons and V1 neurons. Histograms of proportion of direction selective LGN boutons in layer 1 (a, n = 123 images from 37 mice), 2/3 (b, n = 97 images from 36 mice) and 4 (c, n = 98 images from 30 mice) and V1 neurons in layer 2/3 (d, n = 13 images from 5 mice) and 4 (e, n = 12 images from 6 mice) were shown (see Online Methods for criteria). Proportion of direction selective LGN boutons in layer 4 (c) was significantly lower than those in layer 1 (a, p<0.001, Mann-Whitney U-test with Bonferroni correction) and V1 neurons in layer 4 (e, p<0.01, Mann-Whitney U-test with Bonferroni correction).

14 Supplementary Figure 10 Distribution of preferred directions of LGN axons. Distribution of preferred direction of LGN axons in layer 1 (a, n = 1,954 boutons from 37 mice), 2/3 (b, n = 1,630 boutons from 36 mice) and 4 (c, n = 931 boutons from 30 mice). They show cardinal bias which is less prominent (see Supplementary Fig. 11 for the possible reason) than preferred orientation (see Fig. 3). Red triangles indicate cardinal directions.

15 Supplementary Figure 11 Dissociation between preferred orientations and directions of LGN axons. Dissociation between preferred orientation and direction. (a-c) The distribution of bin difference between preferred orientation and direction from LGN axons in layer 1 (a, n = 1,235 boutons from 37 mice), 2/3 (b, n = 1,019 boutons from 36 mice), and 4 (c, n = 495 boutons from 30 mice). Although most of the boutons (~75 %) showed that preferred orientation and direction fell into the same bin (a - c), the rest of boutons were in the next bin. In such cases, the polar plots of the tuning curves showed that two direction peaks were not in the perfect opposition, which caused the dissociation of preferred orientation and direction by more than 15 degrees (d - f). The dissociation contributed to weaker direction bias (Supplementary Fig. 10) than the orientation bias (Fig. 3f-j).

16 Supplementary Figure 12 Spatial frequency tunings of LGN axons and V1 neurons. (a-e) Distribution of preferred spatial frequency was shown as histograms for LGN axons in layer 1 (a, n = 3,817 boutons from 20 mice), 2/3 (b, n = 2,669 boutons from 18 mice) and 4 (c, n = 5,576 boutons from 20 mice) and V1 neurons in layer 2/3 (d, n = 7,635 neurons from 10 mice) and 4 (e, n = 6,210 neurons from 9 mice). Mean preferred frequency of layer 4 LGN axons (c) was statistically different from other layers axons (a, b) and V1 neurons (d, e) ( p < with layer 1 axons and V1 neurons, p < 0.05 with layer 2/3 axons, Mann-Whitney U-test with Bonferroni correction). (f) Proportion of SPF selective LGN axons and V1 neurons were not statistically different (p > 0.05, Mann-Whitney U-test with Bonferroni correction). Mean preferred SPF was significantly lower in layer 4 LGN axons than layer 4 V1 neurons, suggesting further computation in the cortical circuit.

17 Supplementary Figure 13 Summary. Summary. Percentages of strongly-selective (gosi > 0.67), moderately-selective (0.33 < gosi < 0.67), and weakly-selective or non-selective (gosi < 0.33) boutons among responsive LGN boutons in V1 layers 1, 2/3, and 4, and neurons among responsive neurons in V1 layers 2/3 and 4 are indicated.

18 Supplementary Table 1 N of Images (Animals) N of Analyzed responsive boutons or neurons N of Orientation selective boutons or neurons LGN axons L1 123 (37) ,924 LGN axons L2/3 97 (36) 7,916 2,818 LGN axons L4 98 (30) 9,698 1,722 V1 neurons L2/3 26 (14) 15,310 9,629 V1 neurons L4 14 (10) 8,797 5,102 Kondo & Ohki, Supplementary Table 1

19 Supplementary Table 2 N of Images N of Analyzed responsive boutons or neurons N of Direction selective boutons or neurons (Animals) LGN axons L1 123 (37) ,954 LGN axons L2/3 97 (36) 7,916 1,630 LGN axons L4 98 (30) 9, V1 neurons L2/3 13 (5) V1 neurons L4 12 (6) 1, Kondo & Ohki, Supplementary Table 2

20 Supplementary Table 3 N of Images N of Analyzed responsive boutons or neurons N of SPF selective boutons or neurons (Animals) LGN axons L1 42 (20) LGN axons L2/3 29 (18) LGN axons L4 55 (20) V1 neurons L2/3 15 (10) V1 neurons L4 12 (9) Kondo & Ohki, Supplementary Table 3