Local Order within Global Disorder: Synaptic Architecture of Visual Space  Benjamin Scholl, Daniel E. Wilson, David Fitzpatrick  Neuron  Volume 96, Issue 5, Pages 1127-1138.e4 (December 2017) DOI: 10.1016/j.neuron.2017.10.017 Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Measuring 1D Spatial Receptive Fields of Individual Somata and Spines (A) Two-photon projection of a cortical cell soma and an example ΔF/F fluorescence trace during visual stimulation (left). Stimulus-averaged responses for each bar location in visual space revealed spatial and polarity selectivity (right). Stimulus onset is shown at the beginning of each response. Black lines and gray shading indicate mean and SE, respectively. Temporal specificity of responses to individual bars allows separation of ON (e.g., increase in luminance) or OFF (e.g., decrease in luminance) responses, shown as red and blue, respectively. Peak ΔF/F responses extracted from stimulus-triggered cycles were used to generate ON and OFF spatial RFs (right). Data were fit to a Gaussian curve. (B) The same as (A) for an individual spine with strong OFF responses to somatically-distal spatial locations. Spine residual ΔF/F fluorescence trace (green) shows large, independent, isolated calcium events. (C) The same as (B) for a spine (from the same cortical cell) with strong ON responses. (D) The same as (B) for a spine exhibiting ON-OFF responses. (E) The same as (B) for a spine with segregated ON and OFF responses. See also Figures S1–S3. Neuron 2017 96, 1127-1138.e4DOI: (10.1016/j.neuron.2017.10.017) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Lack of Global Dendritic Organization for Spine Spatial Preferences (A and B) Example neuron with all serially imaged visually responsive spines and corresponding soma colored by spatial preference (scale bar, 50 μm). Spatial preferences are shown for dominant polarity for each spine. (B) The same as (A) for another cortical cell. (C) Distribution of normalized spine responses for a single bar location is shown with a dendritic center of mass (gray ellipse). Ellipse axes are two SDs of spatial patterns of synaptic responses. Inset: center of mass ellipses for each stimulus spatial location. (D) An example of the overlap of the center of mass ellipses calculated for two different spatial locations. (E) The relationship between bar position distance and center of mass overlap. (F) The relationship between bar position distance relative to the soma preference and center of mass overlap. (G) The relationship between bar position distance and center of mass overlap for ON (red) and OFF (blue) spatial patterns is shown separately. (H) The distribution of spine ON spatial preferences, relative to the soma, is shown at distal (>75 μm) and proximal (<75 μm) dendritic locations. (I) The same as (H) for OFF spatial preferences. (J) Distribution of spine ON-OFF ratios at distal and proximal dendritic locations. See also Figure S3. Neuron 2017 96, 1127-1138.e4DOI: (10.1016/j.neuron.2017.10.017) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Local Clustering of Spine Spatial RF Properties (A) Example dendritic branch with spines sharing similar spatial preference and RF properties. Spines are colored based on spatial preference for ON and OFF. Spines with multiple colors indicate strong ON and OFF responses with different spatial preference. Spatial ON and OFF ΔF/F responses (mean and SE) are shown with a fitted Gaussian curve. (B) Tuning correlation between spines is dependent on dendritic distance. Data shown are mean and SE with an exponential curve fit (black line). A bootstrapped shuffled correlation is also shown (gray). (C) The distribution of the mean correlation of clustered spine pairs (correlation > 0.5, distance < 10 μm) with soma RF (black outline). Also shown is the distribution of RF correlation between all individual spines and somata (gray). See also Figure S4. Neuron 2017 96, 1127-1138.e4DOI: (10.1016/j.neuron.2017.10.017) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Synaptic Clusters Exhibit Spatiotemporal Correlation (A) Example dendritic branch with responsive dendritic spines, colored by spatial preference. Co-active synaptic events occurred across repeated presentations of a bar at a single spatial location. (B) The trial-to-trial correlation between spines was dependent on dendritic distance. Data shown are mean and SE with an exponential curve fit (black line). A bootstrapped shuffled correlation is also shown (gray). See also Figure S5. (C) The probability of co-active synaptic events is plotted relative to spine dendritic distance. Data shown are mean and SE, and a bootstrapped shuffled correlation is shown in gray. (D) The relationship between spine tuning correlation and trial-to-trial correlation. Individual points (gray squares) are shown with mean and SE (black circles). (E) The distribution of spatial length constants was measured across cells. Neuron 2017 96, 1127-1138.e4DOI: (10.1016/j.neuron.2017.10.017) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 Local Clustering of Spontaneous Activity and Drifting Grating Evoked Co-activity (A) Two-photon projection of an example dendritic branch with dendritic spines. (B) Time series traces of ΔF/F activity from a subset of dendritic spines (black) and corresponding global dendritic activity (gray). Note that spine time series traces are back propagating action potential (bAP)-subtracted, and an exponential filter has been applied (STAR Methods). (C) The relationship between spine correlation of spontaneous activity and distance along the dendritic shaft. Data are shown as mean and SE with an exponential curve fit (black line). A bootstrapped shuffled correlation is shown in gray. (D) Two-photon projection of an example dendritic segment. (E) Individual dendritic spine responses (black) to the presentation of a single oriented grating (0°) as well as the corresponding dendritic response (gray). (F) The trial-to-trial correlation between spines during drifting grating presentation is dependent on dendritic distance. Data are shown as mean and SE with an exponential curve fit (black line). A bootstrapped shuffled correlation is shown in gray. Neuron 2017 96, 1127-1138.e4DOI: (10.1016/j.neuron.2017.10.017) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 Synaptic Clusters Convey Functionally Distinct Spatial RF Properties (A) A population of spines from an example cell and Z-scored spatial RF responses for both polarities. (B) Left: 2D visualization of the principal-component coefficients for the synaptic population in (A) (computed using t-SNE; Maaten and Hinton, 2008). Each point represents one spine. Center: the k-means cluster evaluation score for these coefficients. Right: 2D visualization of principal-component coefficients for the synaptic population in (A). Colors indicate group labels from k-means clustering. (C) Dendritic locations of a subset of spines from (A). Each spine is colored to indicate the group label from k-means clustering; the group label from k-means clustering is also indicated next to each spine. (D) Neighboring spines less than 5 μm distant are more likely to found in the same functional cluster compared with a bootstrapped shuffle (left). Neighboring spines within a 5- to 10-μm or 10- to 15-μm distance are no more likely to be from the same functional cluster than chance (center and right). Shown are points from individual cells (white) and the population (black) in mean and SE. (E) Spatial RFs were generated by averaging spine spatial RFs within the same defined cluster. Data are shown as mean and SE for ON (red) and OFF (blue) responses, normalized within each functional cluster. Also shown is a soma RF (far right). (F) The relationship between the correlation of functional clusters and soma RFs and the proportion of spines in a corresponding functional cluster. Neuron 2017 96, 1127-1138.e4DOI: (10.1016/j.neuron.2017.10.017) Copyright © 2017 Elsevier Inc. Terms and Conditions