Cortico-cortical projections in mouse visual cortex are functionally target-specific

Running title: Target-specific projections in mouse visual cortex

Lindsey L. Glickfeld, Mark L. Andermann, Vincent Bonin and R. Clay Reid*

Department of NeurobiologyHarvard Medical School

Boston, MA, USA

*Corresponding authorEmail: clay_reid@hms.harvard.edu

Supplementary information:Containing 5 supplementary figures and legends

Nature Neuroscience: doi:10.1038/nn.3300

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Supplementary Figure 1: Expression of GCaMP3 in the axonal arborizations of V1 neurons. (a) To target injections and find boundaries, we first obtained images of changes in the intrinsic autofluorescence signal in response to visual stimuli placed at three different positions within the visual field (same mouse as in Figure 1c-e). Decreases in fluorescence represent increases in activity; colored circles mark the center of the activated region in each area. Insets: relative positions of three visual stimuli (40° diameter, square-wave drifting grating). Note that as the visual stimulus moves more lateral, the regions of activation become further apart. (b) Raw fluorescence image from the experiment

st rd ndin a. Circles are from positions marked in a: 1 and 3 positions are closed circles, 2 position is an open circle. Arrows indicate direction of retinotopic organization from nasal to temporal. (c) Top, raw fluorescence image from same mouse as in a, after expression of GCaMP3 in V1. Locations of each area were marked using vasculature landmarks from b. Bottom, pixel-based map of the stimulus location that evoked the best response (see inset for position color code; 25° diameter, square-wave drifting grating). Note the lack of reversals of retinotopy suggesting that the injection was contained entirely within V1. (d) Coronal section of visual cortex after injection with GCaMP3. (e) Raw fluorescence image from same mouse as in c. Fluorescence from V1 is masked with a piece of black tape and the illumination was increased. Locations of each area were marked based on vasculature from b. (f) Schematic of the 25 different visual stimuli presented; note that the stimuli on the diagonal are all at the same speed. Scale in all panels: 500 µm.

stimulus

Glickfeld et al., Nature Neuroscience

SUPPLEMENTARY FIGURES

Nature Neuroscience: doi:10.1038/nn.3300

Supplementary Figure 2: Activation of multiple different axonal components within the same field of view. (a) Average change in fluorescence (dF/F; 24 trials) in response to presentation of visual stimuli at seven different speeds (some speeds were presented as multiple combinations of SF and TF, we chose the following 7 stimuli (from left to right): 1 Hz at 0.32 cpd; 2 Hz at 0.16 cpd; 4 Hz at 0.16 cpd; 4 Hz at 0.08 cpd; 8 Hz at 0.08 cpd; 8 Hz at 0.04 cpd; and 15 Hz at 0.02 cpd) from the same experiment as in Figure 1c-e. Scale: 50 µm. (b) In order to estimate the number of axons in the field of view, we generated a trial-to-trial correlation matrix for all boutons in the field of view, ordered to optimize neighbor relationships. We assumed that boutons on the same axon would be more correlated than boutons on different axons. No corrections were made for stimulus correlations or behavioral state that may lead to increased correlations between different axons; however, note the relatively low correlations between distant boutons. (c) Correlation of each bouton with its neighbor in the ordering from b. Dotted red line at 0.75 represents cutoff used to find peaks (arrowheads) which may represent clusters of boutons from the same axon. This cutoff was chosen to be conservative and err on the side of grouping boutons together to provide a lower bound for the number of axons in the field of view. (d) Normalized response profiles from two boutons chosen from each peak in c. Same axes as in Figure 1e. (e) dF/F time course of four pairs of boutons from d (colors matched to peaks from c and pairs from d). Gray arrows represent stimulus onset times. (f) Location of identified boutons overlaid on the dF/F image (top, same image as in Figure 1d) and F image (bottom). Scale: 15 µm.

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Glickfeld et al., Nature Neuroscience

Nature Neuroscience: doi:10.1038/nn.3300

Supplementary Figure 3: Target specificity of projections does not depend on identification of boutons. (a) Top, maximum projection of dF/F for all visual stimuli from three different fields of view within LM (left, black), PM (middle, blue) and AL (right, red) of an example mouse (mouse A from Figure 2a-b). Bottom, response profile within each area was measured by averaging the entire field of view. Numbers represent peak dF/F. (b) Image (top) and average tuning (bottom) of all significantly visually driven pixels within each field of view (from a). (c) Image (top) and average tuning (bottom) of data (as opposed to fits) from all significantly driven boutons within each field of view. (d) Image (top) and average tuning (bottom) of data from all significantly driven and well-fit boutons within each field of view. (e-h) Average response profile (top) and speed tuning in AL and PM (bottom) for all fields of view across all mice determined by (e) averaging the entire field, (f) all significant pixels, (g) the data from all responsive boutons, (h) and the data (left) or fits (right; from Figure 2c) from all well-fit boutons. All error bars: SEM.

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Glickfeld et al., Nature Neuroscience

Nature Neuroscience: doi:10.1038/nn.3300

Supplementary Figure 4: Lack of a relationship between bouton fluorescence, breadth of fit, or trial-to-trial variability and the increased target specificity of strongly responding boutons. (a) Classification accuracy for discriminating between distributions of boutons in AL and PM by preferred spatial frequency (purple), preferred temporal frequency (green) and peak speed (black). 50% accuracy represents chance. (b) Average bouton fluorescence (F) binned by peak dF/F for boutons in LM (thin black), PM (thin blue), AL (thin red), and in all areas (thick black). (c) Average tuning width (mean of ó and ó ; see Methods) binned by peak dF/F. (d) Average coefficient of SF TF

variation in the peak response amplitude (across trials) binned by peak dF/F. (e-h) Average speed tuning binned by dF/F for all (e) pixels, (f) significantly responsive pixels, (g) significantly responsive boutons, (h) and for the data (left) and fits (right) from all well-fit boutons in AL (red) and PM (blue). All error bars: SEM.

Glickfeld et al., Nature Neuroscience

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Nature Neuroscience: doi:10.1038/nn.3300

Supplementary Figure 5: Expression of GCaMP3 in the axonal arborizations of LM neurons. (a) Changes in the intrinsic autofluorescence signal in response to visual stimuli placed at two different positions within the visual field. Colored circles mark the center of the activated region in each area. Insets: relative positions of the visual stimuli (40° diameter, square-wave drifting grating). (b) Raw

stfluorescence image from the experiment in a. Circles are from positions marked in a: 1 position is a ndclosed circle, 2 position is an open circle. Arrows indicate direction of retinotopic organization from

nasal to temporal. (c) Top, raw fluorescence image from same mouse as in a, after expression of GCaMP3 in LM. Locations of each area were marked based on vasculature from b. Bottom, pixel-based map of the stimulus position that evoked the best response (see inset for position color code; 25° diameter, square-wave drifting grating). Note the lack of reversals of retinotopy suggesting that injection was contained entirely within LM. (d) Raw fluorescence image from same mouse as in c. Fluorescence from LM is masked with a piece of black tape and the illumination was increased. Locations of each area were marked based on vasculature from b. Scale bar in all panels: 500 µm.

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Glickfeld et al., Nature Neuroscience

Nature Neuroscience: doi:10.1038/nn.3300