DOI: 10.5061/dryad.5x69p8dc7
title: Principles of visual cortex excitatory microcircuit organization

description: Synapse-specific connectivity and dynamics determine microcircuit function
 but are challenging to explore with classic paired recordings due to their
 low throughput. We therefore implemented optomapping, a ∼100-fold faster
 two-photon optogenetic method. In mouse primary visual cortex (V1), we
 optomapped 30,454 candidate inputs to reveal 1,790 excitatory inputs to
 pyramidal, basket, and Martinotti cells. Across these cell types,
 log-normal distribution of synaptic efficacies emerged as a principle. For
 pyramidal cells, optomapping reproduced the canonical circuit but
 unexpectedly uncovered that the excitation of basket cells concentrated to
 layer 5 and that of Martinotti cells dominated in layer 2/3. The
 excitation of basket cells was stronger and reached farther than the
 excitation of pyramidal cells, which may promote stability. Short-term
 plasticity surprisingly depended on cortical layer in addition to target
 cell. Finally, optomapping revealed an overrepresentation of shared inputs
 for interconnected layer-6 pyramidal cells. Thus, by resolving the
 throughput problem, optomapping uncovered hitherto unappreciated
 principles of V1 structure.
description: We optogenetically stimulated indivudal excitatory neurons by
 spiral-scanning a 1040-nm femtosecond laser beam over cell expressing
 soma-targetted ChroME opsin while simultaneously recording a postsynaptic
 neuron in whole-cell configuration. This data was acquired using custom
 software called "jScan" (https://github.com/pj-sjostrom/jScan) in combination with MultiPatch (https://github.com/pj-sjostrom/MultiPatch ), running in Igor Pro (Wavemetrics Inc). In offline analysis using custom software called "CMap" (see jScan github link above) running in Igor Pro, time-locked excitatory postsynaptic potentials (EPSPs) consistently evoked across 20 repetitions were taken to denote the existence of a synaptic connection. Initial EPSP magnitude due to a 30-Hz train of three laser pulses was used as a metric of synaptic strength. Short-term plasticity was measured from the EPSP train. The electrophysiology traces were processed with CMap so that EPSP amplitude etc was mapped onto the location of optogenetically stimulated presynaptic neurons. This process was repeated for several nearby fields-of-view (FOV), to create an overall 'optomap'. The data pertaining to each optomap (e.g., connectivity, EPSP amplitude, short-term plasticity...) was exported for each postsynaptic cell into individual data files and collected into folders for the same cell type (e.g., "L23 PCs", "L23 BCs", "L23 MCs", etc across L2/3, L5, and L6). In subsequent offline analysis using custom software called "CMetaMap" (see jScan github link above) running in Igor Pro, these folders with extacted optomap data were compiled across the same cell types (again, "L23 PC", "L23 BC", "L23 MC", etc across L2/3, L5, and L6). For example, to view the data that went into Figure 3 of Chou et al., please use the most recent version of CMetaMap to sequentialy load the data from the folders "L23 PC", "L23 BC", and "L23 MC". In addition, we provide Excel sheets for each figure in the paper. These Excel sheets contain the raw data points that went into the figure. Also see the Notes provided in each Excel sheet for additional information on the data structure.
description: # Principles of visual cortex excitatory microcircuit organization
 [https://doi.org/10.5061/dryad.5x69p8dc7](https://doi.org/10.5061/dryad.5x69p8dc7) # Introduction We optogenetically stimulated individual excitatory neurons by spiral-scanning a 1040-nm femtosecond laser beam over cell expressing soma-targeted ChroME opsin while simultaneously recording a postsynaptic pyramidal cell (PC), basket cell (BC), and/or Martinotti cell (MC) in whole-cell configuration. This data was acquired using custom software called "jScan" ([https://github.com/pj-sjostrom/jScan](https://github.com/pj-sjostrom/jScan)) in combination with MultiPatch ([https://github.com/pj-sjostrom/MultiPatch](https://github.com/pj-sjostrom/MultiPatch) ), running in Igor Pro (Wavemetrics Inc). In offline analysis using custom software called "CMap" (see jScan github link above) running in Igor Pro, time-locked excitatory postsynaptic potentials (EPSPs) consistently evoked across 20 repetitions were taken to denote the existence of a synaptic connection. Initial EPSP magnitude due to a 30-Hz train of three laser pulses was used as a metric of synaptic strength. Short-term plasticity was measured from the EPSP train as the paired pulse ratio (EPSP2/EPSP1). The electrophysiology traces were processed with CMap so that EPSP amplitude etc was mapped onto the spatial location of optogenetically stimulated presynaptic neurons. This process was repeated for several nearby fields-of-view (FOV), to create an overall 'optomap'. # Description of the data and file structure We have submitted our data in two different formats, which are organized into two folders (“CMetaMap” and “Figures”). ## CMetaMap The data pertaining to each optomap (e.g., connectivity, EPSP amplitude, short-term plasticity...) was exported for each postsynaptic cell into individual .itx data files and collected into folders for the same cell type (e.g., "L23 PCs", "L23 BCs", "L23 MCs", “L5 PCs”, “L5 BCs”, “L5 MCs”, “L6 PCs”, “L6 BCs”, “L6 MCs”). In subsequent offline analysis using custom software called "CMetaMap" (see jScan github link above and Code/Software section below) running in Igor Pro, these folders with extracted optomap data were compiled across the same cell types (again, "L23 PC", "L23 BC", "L23 MC", etc). For example, to view the data that went into Figure 3 of Chou et al., please use the most recent version of CMetaMap to sequentially load the data from the folders "L23 PC", "L23 BC", and "L23 MC". ## Figures In addition, we provide Excel sheets for each figure in the paper. These Excel sheets contain the data points that went into the figure, which are meant to enable users to recreate the figures in Chou et al. Descriptions for the data structure in each file are given below. Also see the Notes provided in each Excel sheet for additional information on the data structure. ### Fig 1 and 2.xlsx #### *Sheet: Fig 1G* In this sample experiment, a BC was patched, and surrounding candidate presynaptic PCs were optomapped. Each row represents a spiral-scanned candidate presyn PC. Spatial coordinates are drawn as if looking down on the acute slice. Spatial coordinates are in reference to the postsynaptic (patched) cell (i.e. spatial coordinates of the patched cell are 0,0). *Presyn Layer*: The layer location of the candidate presyn PC. “1” ==Layer 2/3, “2” == Layer 4, “3” == Layer 5, “4” == Layer 6. Remember that all candidate presyn cells are PCs due to Emx1-cre dependent expression of the opsin. *In Column*: Boolean denoting whether the candidate presyn PC is located within 100 µm of the patched cell, on the horizontal (x) axis (parallel to layer boundaries). *X coordinate*: Spatial coordinate along the x-axis in µm. The x-axis runs parallel to layer boundaries, values increase as one moves laterally (away from the brain midline). *Y coordinate*: Spatial coordinate along the y-axis in µm. The y-axis runs perpendicular to layer boundaries, values increase as one moves from Layer 1 to Layer 6. *EPSP Amplitude (V)*: Average excitatory postsynaptic potential (EPSP) amplitude recorded in the patched cell in response to spiral scanning a postsynaptic cell 20 times every ~15 seconds. If a spiral-scanned PC did not elicit an EPSP, the entry was left blank, and the cell was deemed unconnected. These values were converted to mV in the figure. #### *Sheet: Fig 2A\_PC, Fig 2A\_BC, Fig 2A\_MC* In Figure 2, we showed the synaptic connections mapped onto one sample postsynaptic PC, BC, or MC. Panel A shows the spatial location and EPSP amplitude of presynaptic PCs. Data structure is the same as in sheet Fig 1G. The frequency distribution of EPSP amplitudes for each sample neuron in Figure 2A are then plotted on a log scale in Figure 2B. ### Fig 3.xlsx, Fig 4.xlsx, Fig 5.xlsx #### *Sheet: Fig3A\_PCs, Fig3A\_BCs, Fig3A\_MCs* Ensemble optomaps were generated for Layer 2/3 PCs, BCs, and MCs. These sheets contain the data for individual optomaps that make up each ensemble optomap (see CMetaMap section above for more details). As above, each row represents a spiral-scanned candidate presyn PC. Spatial coordinates are drawn as if looking down on the acute slice. Spatial coordinates are in reference to the corresponding postsynaptic (patched) cell (i.e. spatial coordinates of each patched cell are 0,0). *Date*: The experiment date as YYYYMMDD. One mouse was used per experiment day, so this column can be used as an identifier for individual animals during analysis. *Cell number*: Identifier for different postsynaptic cells from each experiment day. Data in this column is nested in the Date column. E.g. Cell 3 from 20211027 is different from Cell 3 from 20220609. *Presyn Layer*: The layer location of the candidate **presyn** PC. “1” ==Layer 2/3, “2” == Layer 4, “3” == Layer 5, “4” == Layer 6. Remember that all postsynaptic cells in this file were located in Layer 2/3. Remember that all candidate presyn cells are PCs due to Emx1-cre dependent expression of the opsin. *EPSP Amplitude (V)*: Average excitatory postsynaptic potential (EPSP) amplitude recorded in the patched cell in response to spiral scanning a presynaptic cell 20 times every ~15 seconds. If a spiral-scanned PC did not elicit an EPSP, the entry was left blank, and the cell was deemed unconnected. These values were converted to mV in the figure. *Sex*: The sex of the animal (“M” == male, “F” == female) *Age (days)*: The age of the animal, in postnatal days. *X coordinate*: Spatial coordinate along the x-axis in µm. The x-axis runs parallel to layer boundaries, values increase as one moves laterally (away from the brain midline). *Y coordinate*: Spatial coordinate along the y-axis in µm. The y-axis runs perpendicular to layer boundaries, values increase as one moves from Layer 1 to Layer 6. *In Column*: Boolean denoting whether the candidate presyn PC is located within 100 µm of the patched cell, on the horizontal (x) axis (parallel to layer boundaries). This data has a nested structure: Animal (variables: Sex, Age) &lt; Postsyn cell &lt; Presyn cell (variables: Presyn Layer, EPSP Amplitude, X coordinate, Y coordinate, In Column) #### *Sheet: Fig3B* Data was grouped into presyn layer and cell type. Mean connectivity (in %) and standard error of the mean (SEM) were plotted for each group. Mean connectivity was averaged across individual postsynaptic neurons (i.e. the average connectivity of all L2/3, L4, L5, or L6 inputs onto each postsyn neuron in a group) for display purposes only. Statistical tests (generalized linear mixed models) were conducted on individual presyn neurons, with postsyn cell and animal included as random factors. Note that only *In Column*connections were included. #### *Sheet: Fig3C* As in the previous sheet, data was grouped into presyn layer and cell type. Mean EPSP amplitude (in mV) and standard error of the mean (SEM) were plotted for each group. Mean EPSP amplitude was averaged across individual postsynaptic neurons (i.e. the average EPSP amplitude of all L2/3, L4, L5, or L6 inputs onto each postsyn neuron in a group) for display purposes only. Statistical tests (linear mixed models) were conducted on individual presyn neurons, with postsyn cell and animal included as random factors. Note that only *In Column* connections were included.   ### Fig 4.xlsx, Fig 5.xlsx Ensemble optomaps were generated for Layer 5 (Figure 4) and Layer 6 (Figure 5) PCs, BCs, and MCs. The data structure is the same as Fig 3.xlsx. ### Fig 6.xlsx For a comprehensive visualization of excitatory connectivity, all optomapped data (From Figure 3, 4, and 5) was grouped by presyn layer, postsyn layer, and cell type (e.g. L2/3 inputs onto L2/3 PCs, L4 inputs onto L2/3 PCs, etc). Connectivity (Fig6A), EPSP amplitude (Fig6B), and path strength (connectivity x amplitude, Fig6C) for each group were displayed as heatmap matrices to enable qualitative comparison. This sheet contains the numeric values for each matrix element. Normalized values within each postsynaptic cell group (all PCs, all BCs, and all MCs) were used for heatmap colors. Instructions for creating these heatmaps in Igor are included in the jScan github repository (see link above), in the “How to use CMetaMap” document. ### Fig 7.xlsx #### *Sheet: Fig7A* In this sample experiment, a PC, BC, and MC (postsynaptic) were simultaneously patched and optomapped. Data in these sheets are structured as in Fig 1 and 2.xlsx. Each row represents a spiral-scanned candidate presyn PC and the resulting response (if any) in the three postsynaptic (patched) neurons. Spatial coordinates are drawn as if looking down on the acute slice. **Spatial coordinates in this sheet are in reference to the postsynaptic (patched) MC** (i.e. spatial coordinates of the patched MC is 0,0). *Presyn Layer*: The layer location of the candidate presyn PC. “1” ==Layer 2/3, “2” == Layer 4, “3” == Layer 5, “4” == Layer 6. Remember that all candidate presyn cells are PCs due to Emx1-cre dependent expression of the opsin. *In Column*: Boolean denoting whether the candidate presyn PC is located within 100 µm of the patched cell, on the horizontal (x) axis (parallel to layer boundaries). *X coordinate*: Spatial coordinate along the x-axis in µm. The x-axis runs parallel to layer boundaries, values increase as one moves laterally (away from the brain midline). *Y coordinate*: Spatial coordinate along the y-axis in µm. The y-axis runs perpendicular to layer boundaries, values increase as one moves from Layer 1 to Layer 6. *PC EPSP Amplitude (V)*: Average excitatory postsynaptic potential (EPSP) amplitude recorded in the postsyn (patched) PC in response to spiral scanning a presyn cell 20 times every ~15 seconds. If a spiral-scanned PC did not elicit an EPSP, the entry was left blank, and the cell was deemed unconnected. These values were converted to mV in the figure. *PC PPR*: Average paired pulse ratio (PPR = EPSP2/EPSP1) of postsynaptic responses from 20 spiral scans. Detection threshold was set to 0.1 mV, therefore, if EPSP1 was &lt; 0.1 mV, PPR was not included in the dataset. *BC EPSP Amplitude (V)*: Average excitatory postsynaptic potential (EPSP) amplitude recorded in the postsyn (patched) BC in response to spiral scanning a presyn cell 20 times every ~15 seconds. If a spiral-scanned PC did not elicit an EPSP, the entry was left blank, and the cell was deemed unconnected. These values were converted to mV in the figure. *BC PPR*: Average paired pulse ratio (PPR = EPSP2/EPSP1) of postsynaptic responses from 20 spiral scans. Detection threshold was set to 0.1 mV, therefore, if EPSP1 was &lt; 0.1 mV, PPR was not included in the dataset. *MC EPSP Amplitude (V)*: Average excitatory postsynaptic potential (EPSP) amplitude recorded in the postsyn (patched) MC in response to spiral scanning a presyn cell 20 times every ~15 seconds. If a spiral-scanned PC did not elicit an EPSP, the entry was left blank, and the cell was deemed unconnected. These values were converted to mV in the figure. *MC PPR*: Average paired pulse ratio (PPR = EPSP2/EPSP1) of postsynaptic responses from 20 spiral scans. Detection threshold was set to 0.1 mV, therefore, if EPSP1 was &lt; 0.1 mV, PPR was not included in the dataset. #### *Sheet: Fig7C* We compared all PPR recorded in each patched cell from Fig7A. Only presyn PCs located within 200 µm (on the x-axis) of the patched cells were included in this dataset. #### *Sheet: Fig7D\_E\_raw* This sheet contains PPR from the entire optomapping dataset. Data has a similar structure as Fig3, Fig4, and Fig5. Only presyn PCs located within 200 µm (on the x-axis) of the and deemed connected to the patched cells were included in this dataset. *Postsyn cell type*: The type of the postsynaptic (patched) cell. Either “PC”, “BC”, or “MC” *Presyn Layer*: The layer location of the candidate presyn PC. “1” ==Layer 2/3, “2” == Layer 4, “3” == Layer 5, “4” == Layer 6. Remember that all candidate presyn cells are PCs due to Emx1-cre dependent expression of the opsin. *Postsyn Layer*: The layer location of the patched postsyn cell. “1” ==Layer 2/3, “2” == Layer 4, “3” == Layer 5, “4” == Layer 6. *Date*: The experiment date as YYYYMMDD. One mouse was used per experiment day, so this column can be used as an identifier for individual animals during analysis. *Cell number*: Identifier for different postsynaptic cells from each experiment day. Data in this column is nested in the Date column. E.g. Cell 3 from 20211027 is different from Cell 3 from 20220609. *EPSP Amplitude (V)*: Average excitatory postsynaptic potential (EPSP) amplitude recorded in the patched cell in response to spiral scanning a presynaptic cell 20 times every ~15 seconds. If a spiral-scanned PC did not elicit an EPSP, the entry was left blank, and the cell was deemed unconnected. These values were converted to mV in the figure. *PPR*: Average paired pulse ratio (PPR = EPSP2/EPSP1) of postsynaptic responses from 20 spiral scans. Detection threshold was set to 0.1 mV, therefore, if EPSP1 was &lt; 0.1 mV, PPR was not included in the dataset. *Sex*: The sex of the animal (“M” == male, “F” == female) *Age (days)*: The age of the animal, in postnatal days. #### *Sheet: Fig7D* For a comprehensive visualization of PPR, all optomapped connections (data from Fig7D_E_raw) was grouped by presyn layer, postsyn layer, and cell type (e.g. L2/3 inputs onto L2/3 PCs, L4 inputs onto L2/3 PCs, etc). Mean PPR for each group was displayed as heatmap matrices to enable qualitative comparison. This sheet contains the numeric values for each matrix element. Mean PPR values were used for heatmap colours. Instructions for creating these heatmaps in Igor are included in the jScan github repository (see link above), in the “How to use CMetaMap” document. #### *Sheet: Fig7E* Quantitative comparison of PPR (using linear mixed models) revealed that PPR depended on postsynaptic cell type and presynaptic layer location. To visualize this, we grouped our data by postsynaptic cell type and presynaptic layer location and plotted the mean and SEM for each group. *Presyn Layer*: The layer location of the candidate presyn PC. “1” ==Layer 2/3, “2” == Layer 4, “3” == Layer 5, “4” == Layer 6. Remember that all candidate presyn cells are PCs due to Emx1-cre dependent expression of the opsin. *Cell Type*: The type of the patched postsyn neuron. Either “PC”, “BC”, or “MC” *Mean PPR*: Mean PPR of this group. *SEM*: Standard error of the mean for the PPR of this group. *n connections*: Number of optomapped connections in this group. ### Fig 8.xlsx #### *Sheet: Fig8A* In this sample experiment, three postsyn PCs were simultaneously patched and optomapped. Data in these sheets are structured as in Fig 7.xlsx. Each row represents a spiral-scanned candidate presyn PC and the resulting response (if any) in the three postsynaptic (patched) neurons. Spatial coordinates are drawn as if looking down on the acute slice. **Spatial coordinates in this sheet are in reference to postsynaptic (patched) PC1** (i.e. spatial coordinates of the patched PC1 is 0,0). Only presyn PCs located within 200 µm (on the x-axis) of the patched cells were included in this dataset. *Presyn Layer*: The layer location of the candidate presyn PC. “1” ==Layer 2/3, “2” == Layer 4, “3” == Layer 5, “4” == Layer 6. Remember that all candidate presyn cells are PCs due to Emx1-cre dependent expression of the opsin. *X coordinate*: Spatial coordinate along the x-axis in µm. The x-axis runs parallel to layer boundaries, values increase as one moves laterally (away from the brain midline). *Y coordinate*: Spatial coordinate along the y-axis in µm. The y-axis runs perpendicular to layer boundaries, values increase as one moves from Layer 1 to Layer 6. *PC1 EPSP amplitude (V)*: Average excitatory postsynaptic potential (EPSP) amplitude recorded in the postsyn (patched) PC1 in response to spiral scanning a presyn cell 20 times every ~15 seconds. If a spiral-scanned PC did not elicit an EPSP, the entry was left blank, and the cell was deemed unconnected. These values were converted to mV in the figure. *PC2 EPSP amplitude (V)*: Average excitatory postsynaptic potential (EPSP) amplitude recorded in the postsyn (patched) PC2 in response to spiral scanning a presyn cell 20 times every ~15 seconds. If a spiral-scanned PC did not elicit an EPSP, the entry was left blank, and the cell was deemed unconnected. These values were converted to mV in the figure. *PC3 EPSP amplitude (V)*: Average excitatory postsynaptic potential (EPSP) amplitude recorded in the postsyn (patched) PC3 in response to spiral scanning a presyn cell 20 times every ~15 seconds. If a spiral-scanned PC did not elicit an EPSP, the entry was left blank, and the cell was deemed unconnected. These values were converted to mV in the figure. ### Fig S1.xlsx In these experiments, we patched and recorded from ChroME-expressing PCs to characterize their response to 1040 nm spiral scans. This validated the precision and reliability of our method. Note that in this Excel file, *Cell ID *columns in one sheet are not related to *Cell ID* columns in another sheet. #### *Sheet: FigS1B* We determined the laser power required for spiral scans to reliably induce action potentials (a.k.a. spikes). We spiral-scanned each cell at varying laser powers and measured the spike probability. *Cell ID*: Unique ID for each patched ChroME-expressing PC *Power (mW)*: Laser power used, in mW, measured at the back aperture of the objective. *Spike Probability (%)*: Calculated as the percentage of 15-25 spiral scans that induced a spike in the patched cell. #### *Sheet: FigS1D* We determined the maximum frequency at which spiral scans can induce spikes. *Cell ID*: Unique ID for each patched ChroME-expressing PC *Frequency (Hz)*: Frequency of a train of 3-5 spiral scans, repeated 3-5 times. *Spike Probability (%)*: Calculated as the percentage of 15-25 spiral scans that induced a spike in the patched cell. #### *Sheet: FigS1F* We determined the probability for a single spiral scan to induce more than one spike. We compared this probability to the probability of a single 5-ms-long 1.3-nA depolarizing current injection to induce more than one spike, in the same cell. This current injection protocol is regularly used to induce spikes in electrophysiology experiments. A paired t-test was used for this comparison. *Cell ID*: Unique ID for each patched ChroME-expressing PC *2P_ExtraSpikeProbability*: Calculated as the percentage of ~10 spiral scans that induced a spike in the patched cell. *CurrInj_ExtraSpikeProbability*:  Calculated as the percentage of ~10 current injection pulses that induced a spike in the patched cell. #### *Sheet: FigS1H\_I* We determined the latency and jitter of spiral-scan-induced spikes. This indicates the temporal precision of our method. *Cell ID*: Unique ID for each patched ChroME-expressing PC *Latency (ms)*: The latency was measured as the time (in ms) between the onset of the spiral scan and the spike inflection point. This was averaged over 9 spiral scans. *Jitter (ms)*: The jitter was quantified as the maximum range of the 9 spike latencies measured in each cell (in ms). #### *Sheet: FigS1J* We determined the lateral spatial resolution of spiral scans by recording from a patched ChroME-expressing PC while spiral scanning in surrounding regions. Spatial coordinates are drawn as if looking down on the acute slice. Spatial coordinates are in reference to the patched cell (i.e. spatial coordinates of the patched cell are 0,0). *Cell ID*: Unique ID for each patched ChroME-expressing PC *Direction*: Either “X” or “Y”. Direction of spatial displacement of the spiral scan. The x-axis runs parallel to layer boundaries. (away from the brain midline). The y-axis runs perpendicular to layer boundaries. *Offset (µm)*:  Magnitude of spatial displacement, in µm, in the corresponding direction. X-coordinates increase as one moves laterally (away from the brain midline). Y-coordinates increase as one moves from Layer 1 to Layer 6. *Spike Probability (%)*: Calculated as the percentage of 15-25 spiral scans that induced a spike in the patched cell. The z-axis runs perpendicular to the cut surface of the slice, in other words, the z-axis represents the depth of the acute slice. Values decrease as one moves deeper into the slice. #### *Sheet: FigS1K* We determined the axial spatial resolution of spiral scans by recording from a patched ChroME-expressing PC while spiral scanning above and below the patched PC. The axial plane (a.k.a z-axis) runs perpendicular to the cut surface of the slice, in other words, the z-axis represents the depth of the acute slice. Spatial coordinates are in reference to the patched cell (i.e. spatial coordinates of the patched cell are 0,0). Values decrease as one moves deeper into the slice. *Cell ID*: Unique ID for each patched ChroME-expressing PC *Z offset (µm)*:  Magnitude of z-displacement, in µm. Z-coordinates are in reference to the patched cell (i.e. Z-coordinate of the patched cell is 0). Values decrease as one moves deeper into the slice. *Spike Probability (%)*: Calculated as the percentage of 15-25 spiral scans that induced a spike in the patched cell. ### Fig S2.xlsx We determined the 2-photon (2P) excitation point spread function, a characteristic of our specific microscope setup. To do this, we took 2P image stacks of fluorescent beads at 1040 nm then quantified the fluorescence intensity profiles of the individual beads in the x-, y-, and z-axes (columns of the data table). X and Y intensity profiles were measured over a randomly selected distance covering the diameter of the bead. Each data point represents 1 pixel, at a scale of 30.88 pixels/ µm. Z intensity profiles were measured over the entire stack. Each data point represents 1 slice of the stack, at a scale of 1 slice/ µm. Intensity profiles were peak aligned and background fluorescence was subtracted post-hoc. ### Fig S3.xlsx We assessed the effect of undesirable artifacts that arise from the preparation of acute slices on our connectivity measurements. For example, neuronal processes (dendrites and axons) might be severed, resulting in an underestimation of connectivity. #### *Sheet: FigureS3D* In this sample experiment, we optomapped candidate presyn PCs onto a patched postsyn BC at various depths in the acute slice to determine whether neurons closer to the cut surface of the slice were less likely to be deemed connected due to severed neurites. Each row represents one candidate presyn PC. Spatial coordinates are drawn as if looking down on the acute slice. Spatial coordinates are in reference to the patched cell (i.e. spatial coordinates of the patched cell are 0,0,0). *X coordinate*: Spatial coordinate along the x-axis in µm. The x-axis runs parallel to layer boundaries, values increase as one moves laterally (away from the brain midline). *Y coordinate*: Spatial coordinate along the y-axis in µm. The y-axis runs perpendicular to layer boundaries, values increase as one moves from Layer 1 to Layer 6. *Z coordinate*:  Spatial coordinate along the z-axis, values decrease as one moves deeper into the slice. *Connected*: Boolean value indicating whether the spiral scanned PC was deemed connected to the patched postsyn cell or not. #### *Sheet: FigureS3E* As in FigS3D, we measured the connectivity at various z-depths for 3 postsyn (patched) cells. *Distance from slice surface (µm)*: The z-distance. Note that this was measured in reference to the **surface of the acute slice,** not the patched cell. *Cell1Connectivity (%)*: Connectivity rate of patched cell 1, calculated as number of connected presyn cells / number of spiral-scanned candidate presyn cells. *Cell2Connectivity (%)*: Connectivity rate of patched cell 2, calculated as number of connected presyn cells / number of spiral-scanned candidate presyn cells. *Cell3Connectivity (%)*: Connectivity rate of patched cell 3, calculated as number of connected presyn cells / number of spiral-scanned candidate presyn cells. #### *Sheet: FigureS3F* In order to verify that our optomapping experiments were not conducted at z depths that are negatively affected by slicing artifacts, we measured the optomapping depth (in µm) of 25 randomly selected experiments. #### *Sheet: FigureS3G* In addition to cell depth, the cutting angle of the acute slice could result in unwanted artifacts (e.g. PC apical dendrites could be severed). We therefore measured the cell depth and the cut angle of optomapping experiments in Layer 5 and Layer 6 PCs. *Date*: The experiment date as YYYYMMDD. One mouse was used per experiment day, so this column can be used as an identifier for individual animals during analysis. *Cell number*: Identifier for different postsynaptic cells from each experiment day. Data in this column is nested in the Date column. E.g. Cell 3 from 20211027 is different from Cell 3 from 20220609. *Postsyn layer*: Layer location of the postsynaptic (patched) cell. “5” == Layer 5, “6” == Layer 6 *Connectivity (%)*: Observed connectivity rate of the patched cell, calculated as number of connected presyn cells / number of spiral-scanned candidate presyn cells. *Actual angle*: Cutting angle of the acute brain slice, in degrees. When values are positive, the apical dendrite is angling towards the surface of the slice. When values are negative, the apical dendrite is angling deeper into the slice. *Depth*: The z-distance of the patched cell from the slice surface. *Angle/depth*: The *Actual angle */ *Depth*. *Abs(ang/depth)*: The absolute value of the *Actual angle */ *Depth*. This metric was used as a proxy for the “goodness” of the acute slice preparation. #### *Sheet: FigureS3H* To determine the optomapping method’s sensitivity to detecting connections, we conducted 2-20 repeats of optomapping sweeps and compared the observed connectivity. We found that 20 sweeps were sufficient for detecting synaptic connections, meaning we did not underestimate the observable connectivity. *FOV_ID*: Each optomapping sweep was conducted in a microscope field of view (FOV). *n repeats*: The number of repeated optomapping sweeps tested. *tested*: The number of spiral-scanned candidate presyn PCs. *connected*: The number of spiral-scanned candidate presyn PCs deemed connected. *connectivity (%)*: Observed connectivity rate, calculated as number of connected presyn cells / number of spiral-scanned candidate presyn cells. *normalized connectivity*: *connectivity (%)* was normalized to the observed connectivity at 20 repeats. ### Fig S4.xlsx We characterized the electrophysiological response resulting from direct optogenetic activation (“direct activation”) and optogenetic-induced synaptic transmission (“synaptic response”). #### *Sheet: FigS4E\_F\_G\_H* Data for direct activation responses. Each row represents a spiral scan location that elicited direct activation. Responses were verified to be direct because these experiments were conducted while the brain slice was bathed in pharmacological compounds that blocked excitatory synaptic transmission (AP5 and NBQX). *Date*: The experiment date as YYYYMMDD. One mouse was used per experiment day, so this column can be used as an identifier for individual animals during analysis. *Cell number*: Identifier for different postsynaptic cells from each experiment day. Data in this column is nested in the Date column. E.g. Cell 3 from 20211027 is different from Cell 3 from 20220609. *X Coordinate*: Spatial coordinate along the x-axis in µm. The x-axis runs parallel to layer boundaries, values increase as one moves laterally (away from the brain midline). *Y Coordinate*: Spatial coordinate along the y-axis in µm. The y-axis runs perpendicular to layer boundaries, values increase as one moves from Layer 1 to Layer 6. *Depol amplitude (mV)*: Average depolarization amplitude recorded in the patched cell in response to spiral scanning a candidate presyn cell 20 times every ~15 seconds. Synaptic responses were blocked, therefore, depolarization was a result of direct optogenetic activation only. *PPR*: Average paired pulse ratio (PPR = depol2/depol1) of responses from 20 spiral scans. Detection threshold was set to 0.1 mV, therefore, if depol1 was &lt; 0.1 mV, PPR was not included in the dataset. #### *Sheet: FigS4I\_J* Data for direct activation and synaptic responses. Each row represents a spiral scan location that elicited depolarization. Spiral scans were repeated 20 times in control conditions then 20 times under AP5 and NBQX blockade. If spiral-scan-induced depolarization persisted with blockers, responses were deemed direct, otherwise, responses were deemed synaptic. *Date*: The experiment date as YYYYMMDD. One mouse was used per experiment day, so this column can be used as an identifier for individual animals during analysis. *cell*: Identifier for different postsynaptic cells from each experiment day. Data in this column is nested in the Date column. E.g. Cell 3 from 20211027 is different from Cell 3 from 20220609. *Direct or synaptic*: “D” or “S”. *Amp (mV)*: Average peak depolarization amplitude recorded in the patched cell in response to spiral scanning a candidate presyn cell 20 times every ~15 seconds, in the absence of synaptic blockers. Response could be direct or synaptic. *CV*: Coefficient of variation of the depolarization trace over 20 repeats. *PPR*: Average paired pulse ratio (PPR = depol2/depol1) of responses from 20 spiral scans. Detection threshold was set to 0.1 mV, therefore, if depol1 was &lt; 0.1 mV, PPR was not included in the dataset. *Blocker_Amp*: Average peak depolarization amplitude recorded in the patched cell in response to spiral scanning a candidate presyn cell 20 times every ~15 seconds, with synaptic blockers washed in. Direct activation responses only. If the response does not have a direct activation component, this field is left blank. *Blocker_CV*: Coefficient of variation of the depolarization trace over 20 repeats, with synaptic blockers washed in. Direct activation responses only. If the response does not have a direct activation component, this field is left blank. *Blocker_PPR*: Average paired pulse ratio (PPR = depol2/depol1) of responses from 20 spiral scans, with synaptic blockers washed in. Detection threshold was set to 0.1 mV, therefore, if depol1 was &lt; 0.1 mV, PPR was not included in the dataset. Direct activation responses only. If the response does not have a direct activation component, this field is left blank. *Blocker_Baseline*: Average depolarization amplitude recorded in the patched cell 1 ms after spiral scanning a candidate presyn cell 20 times every ~15 seconds, with synaptic blockers washed in. Direct activation responses only. If the response does not have a direct activation component, this field is left blank. ### Fig S5 and S6.xlsx We characterized the morphological and electrophysiological properties of the neurons patched in this study. Morphological properties were measured from 3D manual reconstructions using confocal imaging of biocytin filled neurons. Electrophysiological properties were measured in response to 500-ms-long depolarizing current injections. #### *Sheet: FigS5A* The first 2 principal components were obtained from the properties identified in sheet FigS5C. Data from L2/3, L5, and L6 were processed separately *Date*: The experiment date as YYYYMMDD. One mouse was used per experiment day, so this column can be used as an identifier for individual animals during analysis. *Cell ID*: Identifier for different postsynaptic cells from each experiment day. Data in this column is nested in the Date column. E.g. Cell 3 from 20211027 is different from Cell 3 from 20220609. *PC1*: Principal component 1 *PC2*: Principal component 2 *ID*: A combination of electrophysiological and morphological properties was used to segregate cells by agglomerative hierarchical clustering. The resulting clusters IDs are listed in this column. #### *Sheet: FigS5C* Electrophysiological and morphological properties of patched neurons. *Cell Type*: The classification of the patched neuron, as determined in the previous sheet. *Layer*: The layer location of the patched neuron. *Date*: The experiment date as YYYY-MM-DD. One mouse was used per experiment day, so this column can be used as an identifier for individual animals during analysis. *Cell ID*: Identifier for different postsynaptic cells from each experiment day. Data in this column is nested in the Date column. E.g. Cell_03 from 2021-10-27 is different from Cell_03 from 2022-06-09. *spike threshold (mV)*: The membrane potential of the cell at the action potential inflection point. *spike height (mV)*: The amplitude of the action potential, measured from resting membrane potential. *spike half-width (ms)*: The half-width of the action potential. *spike afterhyperpolarization (mV)*: The amplitude of the afterhyperpolarization following the action potential (measured from the inflection point) *rheobase (nA)*: The minimum amount of depolarizing current required to induce an action potential. *frequency (Hz)*: The frequency of action potentials in response to a 500-ms-long rheobase current injection. *instantaneous frequency (Hz)*: The frequency of the first 2 action potentials in response to a 500-ms-long rheobase current injection. *accommodation (%)*: The ratio of the frequency of the first 2 action potentials to the frequency of the last 2 action potentials in response to a 500-ms-long rheobase current injection. *spike CV (%)*: The coefficient of variation of spike amplitude. *spike latency (ms)*: The time between current injection onset and action potential inflection point. *V_m (mV)*: The resting membrane potential. *R_input (MOhm)*: The input resistance. *tau_m (ms)*: The membrane time constant. *PPR*: Average paired pulse ratio (PPR = depol2/depol1) of responses from 20 spiral scans. Detection threshold was set to 0.1 mV, therefore, if depol1 was &lt; 0.1 mV, PPR was not included in the dataset. *desc axon max dist (µm)*: The Euclidean distance to the terminal point of the longest downward-projecting axon branch. *asc axon max dist (µm)*: The Euclidean distance to the terminal point of the longest upward-projecting axon branch. *desc dendr max dist (µm)*: The Euclidean distance to the terminal point of the longest downward-projecting dendrite branch. *asc axon max dist (µm)*: The Euclidean distance to the terminal point of the longest upward-projecting dendrite branch. #### *Sheet: FigS6C* Ensemble Sholl analysis on the morphological reconstructions. We drew concentric circles extending out from the soma and counted the number of axonal and dendritic segments that crossed each concentric circle. Cells were grouped by cell type and layer location. Each row represents one concentric circle in the Sholl analysis. *Cell Type*: The classification of the patched neuron, as determined in the previous sheet. *Layer*: The layer location of the patched neuron. *Radius bin*: The radius of the concentric circle. *Mean Axon Crossings*: The mean number of crossings for axonal segments. *Mean Dendrite Crossings*: The mean number of crossings for dendritic segments. *Axon pos SEM*: The positive standard error of the mean of number of crossings for axonal segments. *Axon neg SEM*: The negative standard error of the mean of number of crossings for axonal segments. *Dendrite pos SEM*: The positive standard error of the mean of number of crossings for dendritic segments. *Dendrite neg SEM*: The negative standard error of the mean of number of crossings for dendritic segments. #### *Sheet: FigS6D* Ensemble analysis of the length of axonal and dendritic segments in each layer. Cells were grouped by cell type and layer location. *Layer*: Neocortical layer. *Axon SEM*: The standard error of the mean of the length of axonal segments in each layer. *Axon Mean*: The mean length of axonal segments in each layer. *Dendr SEM*: The standard error of the mean of the length of dendritic segments in each layer. *Dendr Mean*: The mean length of dendritic segments in each layer. ### Fig S7.xlsx We plotted the frequency distribution of EPSP amplitudes measured by optomapping. Data was grouped by postsynaptic layer and postsynaptic cell type (sheets). In Figure S7 of the publication, frequency distributions were color-coded by postsynaptic cell. *Postsyn Layer*: Layer location of the postsynaptic cell. “1” ==Layer 2/3, “2” == Layer 4, “3” == Layer 5, “4” == Layer 6. *Date*: The experiment date as YYYYMMDD. One mouse was used per experiment day, so this column can be used as an identifier for individual animals during analysis. *Cell number*: Identifier for different postsynaptic cells from each experiment day. Data in this column is nested in the Date column. E.g. Cell 3 from 20211027 is different from Cell 3 from 20220609. *EPSP Amplitude (mV)*: Average excitatory postsynaptic potential (EPSP) amplitude recorded in the patched cell in response to spiral scanning a presynaptic cell 20 times every ~15 seconds. If a spiral-scanned PC did not elicit an EPSP, the entry was left blank, and the cell was deemed unconnected. ### Fig S8.xlsx We measured the distance between the postsynaptic (patched) neuron and presynaptic neurons identified via optomapping. #### *Sheet: PC, BC, and MC* These 3 sheets contain information for 3 sample experiments, where a postsynaptic PC, BC, or MC was optomapped. *Presyn Layer*: The layer location of the candidate presyn PC. “1” ==Layer 2/3, “2” == Layer 4, “3” == Layer 5, “4” == Layer 6. Remember that all candidate presyn cells are PCs due to Emx1-cre dependent expression of the opsin. *EPSP Amplitude*: Average excitatory postsynaptic potential (EPSP) amplitude (in mV) recorded in the patched cell in response to spiral scanning a presynaptic cell 20 times every ~15 seconds. *X coordinate*: Spatial coordinate along the x-axis in µm. The x-axis runs parallel to layer boundaries, values increase as one moves laterally (away from the brain midline). *Y coordinate*: Spatial coordinate along the y-axis in µm. The y-axis runs perpendicular to layer boundaries, values increase as one moves from Layer 1 to Layer 6. #### *FigS8B* Ensemble connectivity for excitatory inputs onto L5 neurons, based on binned lateral distances from the patched postsynaptic neuron. *Cell Type*: Cell type of postsynaptic neuron. *lateral distance*: Binned lateral distances from the patched postsynaptic neuron.  *Mean Connectivity (%)*: Mean connectivity, calculated as number of connected neurons / number of spiral scanned neurons * 100%, within the lateral distance bin. *Connectivity SEM*: Standard error of the mean for connectivity, calculated as number of connected neurons / number of spiral scanned neurons, within the lateral distance bin. Sample Connectivity: Connectivity, calculated as number of connected neurons / number of spiral scanned neurons * 100%, of one sample postsynaptic neuron within the lateral distance bin. #### *FigS8C\_D* Ensemble connectivity for excitatory inputs onto L5 neurons, based on binned lateral distances from the patched postsynaptic neuron. *Cell type*: Cell type of postsynaptic neuron. *Layer*: Layer location of the postsynaptic cell. *Date*: The experiment date as YYYYMMDD. One mouse was used per experiment day, so this column can be used as an identifier for individual animals during analysis. *Cell ID*: Identifier for different postsynaptic cells from each experiment day. Data in this column is nested in the Date column. E.g. Cell 3 from 20211027 is different from Cell 3 from 20220609. *DWM*: Distance-weighted metric for connectivity. The DWM is a non-parametric analytical equivalent to the decay constant of numerically fit exponentials. #### *FigS8E* Ensemble connectivity for excitatory inputs onto L5 neurons, based on binned lateral distances from the patched postsynaptic neuron. *Cell Type*: Cell type of postsynaptic neuron. *lateral distance*: Layer location of the postsynaptic cell. *Mean Connectivity (%)*: Mean connectivity, calculated as number of connected neurons / number of spiral scanned neurons * 100%, within the lateral distance bin. *Connectivity SEM*: Standard error of the mean for connectivity, calculated as number of connected neurons / number of spiral scanned neurons, within the lateral distance bin. *PC exp fit*: Fitted exponential curve for pooled connectivity onto all patched PCs up to 550 µm. *PC normalized exp fit*: Normalized values of the previous column *PC x*: x-coordinates (distance from soma of patched cell) for the exponential fits.B*PC exp fit*: Fitted exponential curve for pooled connectivity onto all patched PCs up to 550 µm. *BC exp fit*: Fitted exponential curve for pooled connectivity onto all patched BCs up to 550 µm. *BC normalized exp fit*: Normalized values of the previous column *BC x*: x-coordinates (distance from soma of patched cell) for the exponential fits. *MC exp fit*: Fitted exponential curve for pooled connectivity onto all patched MCs up to 550 µm. *MC normalized exp fit*: Normalized values of the previous column *MC x*: x-coordinates (distance from soma of patched cell) for the exponential fits. ### Fig S9.xlsx We verified the connectivity detection capabilities of optomapping by using optomapping results to target cells for paired patch recordings. We compared the EPSP amplitudes and PPR measured at the same connection using optomapping and paired recording. *Date*: The experiment date as YYYYMMDD. One mouse was used per experiment day, so this column can be used as an identifier for individual animals during analysis. *preCell*: Identifier for the patched presynaptic cell from each experiment day. Data in this column is nested in the Date column. E.g. Cell 3 from 20211027 is different from Cell 3 from 20220609. *postCell ID*: Identifier for the patched postsynaptic cell from each experiment day. Data in this column is nested in the Date column. E.g. Cell 3 from 20211027 is different from Cell 3 from 20220609. *Opto Tested*: The number of candidate presynaptic cells spiral scanned. *Opto Connected*: The number of connected presynaptic cells identified by optomapping. *Opto Connectivity*: The connectivity rate measured by optomapping, calculated as Opto Tested / Opto Connected. *Opto EPSP amp (mV)*: Average excitatory postsynaptic potential (EPSP) amplitude (in mV) recorded in the patched cell in response to spiral scanning a presynaptic cell 20 times every ~15 seconds. *paired EPSP amp (mV)*: Average excitatory postsynaptic potential (EPSP) amplitude (in mV) recorded in the patched cell in response to suprathreshold depolarizing current injections delivered to the patched presynaptic cell 20 times every ~15 seconds. *Opto PPR*: Average paired pulse ratio (PPR = depol2/depol1) of responses from 20 spiral scans. Detection threshold was set to 0.1 mV, therefore, if depol1 was &lt; 0.1 mV, PPR was not included in the dataset. *paired PPR*: Average paired pulse ratio (PPR = depol2/depol1) of responses from 20 suprathreshold current injection pulses delivered to the patched presynaptic cell. Detection threshold was set to 0.1 mV, therefore, if depol1 was &lt; 0.1 mV, PPR was not included in the dataset. ### Fig S10.xlsx Synaptic short-term dynamics depends on postsynaptic cell type. In PC→BC and PC→PC connections, we observed short-term depression, meaning successive postsynaptic responses elicited by a train of APs decrease in size. On the other hand, PC→MC connections exhibit short-term facilitation, where successive postsynaptic responses increase in size. Thus far, we have used the amplitude of the first EPSP in a train of three as a measure of synaptic connection strength. This method may underestimate the strength of short-term facilitating synapses. We therefore measured the peak postsynaptic depolarization over the period of three successive spiral scans. #### *FigS10A* The data in this sheet correspond to the pooled connectivity heat maps for excitatory connections onto L2/3, L5, and L6 MCs. *Date*: The experiment date as YYYYMMDD. One mouse was used per experiment day, so this column can be used as an identifier for individual animals during analysis. *Cell number*: Identifier for different postsynaptic cells from each experiment day. Data in this column is nested in the Date column. E.g. Cell 3 from 20211027 is different from Cell 3 from 20220609. *Postsyn Layer*: The layer location of the **postsyn** MC. “1” ==Layer 2/3, “2” == Layer 4, “3” == Layer 5, “4” == Layer 6. Remember that all postsynaptic cells in this file were located in Layer 2/3. *Presyn Layer*: The layer location of the candidate **presyn** PC. “1” ==Layer 2/3, “2” == Layer 4, “3” == Layer 5, “4” == Layer 6. Remember that all postsynaptic cells in this file were located in Layer 2/3. Remember that all candidate presyn cells are PCs due to Emx1-cre dependent expression of the opsin. *EPSP Amplitude (mV)*: Average excitatory postsynaptic potential (EPSP) amplitude recorded in the patched cell in response to the first spiral scan in a train of three onto a candidate presyn cell 20 times every ~15 seconds. If a spiral-scanned PC did not elicit an EPSP, the entry was left blank, and the cell was deemed unconnected. These values were converted to mV in the figure. *Max Depol (mV)*: Average peak depolarization amplitude recorded in the patched cell in response to a train of three 30-Hz spiral scans repeated 20 times every ~15 seconds. If a spiral-scanned PC did not elicit an EPSP, the entry was left blank, and the cell was deemed unconnected. *X coordinate*: Spatial coordinate along the x-axis in µm. The x-axis runs parallel to layer boundaries, values increase as one moves laterally (away from the brain midline). *Y coordinate*: Spatial coordinate along the y-axis in µm. The y-axis runs perpendicular to layer boundaries, values increase as one moves from Layer 1 to Layer 6. *In Column*: Boolean denoting whether the candidate presyn PC is located within 100 µm of the patched cell, on the horizontal (x) axis (parallel to layer boundaries). #### *FigS10B* Ensemble EPSP1 amplitude and peak depolarization amplitude of excitatory inputs onto MCs, pooled by pre- and postsynaptic layer location. *Cell Type*: Cell type of the postsynaptic cell. Only patched MCs were included in this dataset. *Presyn Layer*: The layer location of the candidate **presyn** PC. *Postsyn Layer*: The layer location of the **postsyn** MC. *Measurement*: Denotes whether the measurement in this row corresponds to EPSP1 (“EPSP1”) or peak depolarization amplitude (“MaxDepol”). *Mean (mV)*: Average depolarization amplitude of the corresponding measurement (EPSP1 or MaxDepol) for the ensemble group. *SEM*: Standard error of the mean. #### *FigS10C* Heatmap matrices for the connection strength and path strength of excitatory inputs onto MCs based on peak depolarization. This sheet contains the numeric values for each matrix element. Normalized values within each postsynaptic cell group (all PCs, all BCs, and all MCs) were used for heatmap colors. Instructions for creating these heatmaps in Igor are included in the jScan github repository (see link above), in the “How to use CMetaMap” document. #### *FigS10D* To characterize the temporal properties of postsynaptic activation in different neuron types, we plotted the latency (from spiral scan onset) to peak depolarization for postsynaptic PCs, BCs, and MCs. We found no differences by pre- or postsynaptic layer location (data not shown). *Cell Type*: Cell type of the postsynaptic cell. *MaxDepol (mV)*: Average peak depolarization amplitude recorded in the patched cell in response to a train of three 30-Hz spiral scans repeated 20 times every ~15 seconds. *Latency (ms)*: The average latency to peak depolarization, measured from the onset of spiral scans, in response to three 30-Hz spiral scans repeated 20 times every ~15 seconds. # Code/Software CMap and CMetaMap both run in Igor Pro 9 (Wavemetrics Inc.) on either Windows or OSX. For instance, we most recently used Igor Pro 9.05 and OSX Sonoma 14.4.1, but this should not be important — Igor is generally reliably cross-platform. Note that a PDF manual (with instructions for installing Igor Pro, loading the data, using CMetaMap, and statistical analysis in R) is provided at the jScan github repository (([https://github.com/pj-sjostrom/jScan](https://github.com/pj-sjostrom/jScan)), called "How to use CMetaMap". jScan and MultiPatch only run in Igor on a Windows machine, because these scripts rely on National Instruments (NI) boards for data acquisition, and Igor Pro only supports NI boards for Windows machines using the NIDAQmx XOP plugin, which does not exist for OSX. Thus, for viewing this data set, Igor on either platform works. But for acquiring more data of the same kind, Igor on a Windows machine must be used.