Article Figures & Data
Figures
- Figure 1.
aPC principal cells are organized in a continuous gradient. A, Plot of input resistance versus depth in layer II of the aPC. The color code refers to the burst index: red, low spike frequency adaptation; blue, high spike frequency adaptation. Within layer II, a functional and morphological diversity of different principal cells can be identified. Gray lines indicate populations defined for statistical comparisons between superficial and deep cells using a k-means cluster algorithm. In layer II, we distinguish two clusters for analysis: L2S containing more semilunar-like cells and L2P with more pyramidal-like cells. B, Comparison of the two clusters for the parameters underlying the analysis yields significant differences. C–G, Biocytin reconstructions and input–output curves of cells demonstrating the vertical transition. C, “Canonical” semilunar cell (cluster L2S); D, semilunar–pyramidal transition cell (cluster L2S); E, superficial pyramidal cell (cluster L2P); F, superficial to deep pyramidal transition cell (cluster L2P); G, deep pyramidal cell (L3P). H, Biocytin reconstruction and input–output curve of a multipolar cell used for calibration (L3M).
- Figure 2.
Resolution of photoactivation by glutamate uncaging. A–C, Sketches on top mark the position of three example principal cells within the aPC coronal slice. Below, biocytin reconstructions are overlayed with direct inputs evoked by LSPS measured in current clamp. Perisomatic suprathreshold activation is demonstrated by red APs. The stimulation pattern consisted of points with 30 μm spacing. Spatial profiles of excitability of the main aPC excitatory cells were performed at different laser intensities. A, Layer II pyramidal cell (L2P, blue) to represent layer II cells (L2C, see D); B, layer III pyramidal cell (L3P, green); C, multipolar cell (L3M, orange). D–F, Distribution histograms of suprathreshold activation as a function of the distance from the calibrated cells' somata. APs were counted in 10 μm spatial bins. Shaded boxes correspond to d*, where 75% of all inputs were observed. L2Ps and L2Ss grouped as L2Cs (n = 26), 83.5 μm; L3 pyramidal cell (L3P; n = 6), 97.2 μm; multipolar cell (L3M; n = 9), 170.7 μm.
- Figure 3.
Mapping local recurrent feedback excitation. A, Representative traces displaying direct activation of neurons measured in current clamp. The UV flash is indicated by the dark blue line. The light blue shaded area indicates the 100 ms critical time interval analyzed for indirect inputs by the detection algorithm. B, Representative traces displaying the different classes of events in voltage clamp detected by the analysis algorithm. E, Indirect excitatory response; D, direct excitatory response; I, indirect inhibitory response. C, Averaged PSC incidence rates for L2S (red), L2P (blue), and L3P (green) plotted over time. The arrows indicate the time point of photostimulation. A significant photostimulation-related increase can be observed in all cell types. D, Frequency distribution histogram of the latency of the first and last AP after the UV flash in 10 ms bins (370 traces with APs from 27 cells). E, Example map of an L2S (red triangle, target cell). F, Example map of an L2P (blue triangle, target cell). Left, Low-magnification DIC images of the aPC coronal slice. Right, Corresponding scanning rasters and target cells are projected on the schematic drawings of the DIC images. Colored points indicate positive synaptic points detected as inputs from source cells. L I, layer I (not mapped); L II, layer II; L III <300 μm, the first 300 μm of layer III parallel to the layer II/layer III border; L III >300 μm, the part of layer III below 300 μm. The raster consisted of stimulation points separated by 30 μm. G, Example traces from the stimulation raster (red in A2): 200 ms around the UV flash (indicated by dark blue line) are plotted. The light blue shaded area indicates the 100 ms critical time interval analyzed for indirect inputs by the detection algorithm. Traces positive for intracortical inputs are plotted in red, and negative traces are plotted in black.
- Figure 4.
Intracortical connectivity increases from superficial to deep. A, Statistical comparison of the connectivity coefficient of L2S, L2P (as distinguished by the cluster analysis), and L3P. From layer II, L2Ps (7.3 ± 2.2, n = 13) and L3Ps (7.7 ± 2.5, n = 15) display a significantly higher connectivity coefficient than L2Ss (1.0 ± 0.3, n = 19; L2P vs L2S, p < 0.001; L3P vs L2S, p < 0.05). Compared with L2Ss, L2Ps do not display significantly larger intracortical connectivity from superficial layer III (L2P: 5.2 ± 1.4, n = 13; L2S: 1.2 ± 0.5, n = 15; L2P vs L2S) and from deep layer III (L2P: 6.7 ± 3.1, n = 14; L2S: 1.8 ± 0.8, n = 15; L2P vs L2S). From both superficial and deep layer III, L3Ps were significantly higher connected than L2Ss (superficial layer III, L3Ps: 14.4 ± 4.3, n = 14; deep layer III, L2Ps: 15.6 ± 2.8, n = 17; superficial layer III, L3P vs L2S: p < 0.01; deep layer III, L3P vs L2S: p < 0.01). From layer II and superficial layer III, the average intracortical connectivity of L2Ps was not significantly different compared with L3Ps (layer II: L2P, 7.3 ± 2.2; L3P, 7.7 ± 2.5; p > 0.05; layer III: L2P, 5.2 ± 1.4; L3P, 14.4 ± 4.3; L2Ps vs L3Ps: p > 0.05). From deep layer III, we observed a significantly higher connectivity index for L3Ps than for L2Ps (L2P, 6.7 ± 3; L3P, 15.6 ± 2.8; p < 0.01, Kruskal–Wallis test used for all comparisons). B, Overall connectivity from all layers correlates with the target cell's input resistance (r = −0.49; p < 0.001; n = 53). C, Overall connectivity from all layers correlates with the target cell's burst index (r = 0.66; p < 0.001; n = 53).
- Figure 5.
L3Ps receive asymmetric inputs on the ventrodorsal axis. A, Left, DIC image of the aPC superimposed with the layering used for analysis. Right, Scanning raster and layering in the same magnification; positive synaptic points indicative of source-cell activation are colored in green, and the target cell is depicted by a green triangle. B, Median (black lines) and 75% percentiles of the position coefficient Δ are plotted for layer II, superficial III, and deep III for L2Ps (blue) and L3Ps (green). L2Ps show symmetrically distributed intracortical inputs [layer II median Δ, 0.12 (n = 14); superficial layer III Δ, 0 (n = 13); deep layer III Δ, 0 (n = 12); p > 0.05 for deep layer III vs layer II inputs onto L2Ps, Mann–Whitney U test]. L3Ps have a clearly asymmetric position shift of their deep inputs toward the dorsal orientation [layer II median Δ, −0.12 (n = 14); superficial layer III Δ, 0 (n = 16); deep layer III median Δ, −0.34 (n = 18); p < 0.05 for deep layer III vs layer II inputs onto L3Ps, Mann–Whitney U test].
- Figure 6.
Single-cell excitability probing of sensory layer Ia inputs to deep and superficial layer II neurons. A, Top, aPC bolus loaded with OG-BAPTA1-AM. The overlay of green fluorescence and transmitted infrared to visualize a bolus-loaded cell and patch pipette are shown. The black box indicates position of cell used for simultaneous cell-attached recording and Ca2+ imaging. Middle, Five subsequent synaptically induced single APs measured in a cell-attached patch underlying the averaged Ca2+ transient at the bottom. B, Inset, Overlay of fluorescence and transmitted infrared (scale bars, 50 μm). The high-magnification image of layer II neurons is from the inset. Numbers 1–8 label example cells corresponding to the traces in C. Blue boxes correspond to superficial layer II cells, white boxes correspond to cells neither attributable to superficial and deep half of rows (middle), and red boxes correspond to cells in deep half of layer II cell rows (scale bar, 50 μm). C, Averaged Ca2+ traces evoked by extracellular electrical stimulation of layer Ia (layer Ib blocked by baclofen) inputs to superficial (red) and deep (blue) layer II cells as labeled in B. The vertical gray lines correspond to the time points each of the five pulses were delivered with an interpulse interval of 50 ms. Black arrows are placed in the stimulation interval in which the onset could be detected. D, Top, On average, superficial cells in layer II display the onset of averaged Ca2+ transients indicating AP firing after significantly less stimuli than deep cells in layer II [mean stimulus number (Stim. number), 2.1 ± 0.1 vs 2.5 ± 0.16 a.u., respectively; p < 0.05, Mann–Whitney U test]. Bottom, Cumulative frequency of data displayed at the top.
- Figure 7.
Circuit diagram of aPC sensory and intracortical excitatory microcircuits. Organization of an excitatory microcircuit in the aPC is shown. A, Sensory input via the LOT (gray dashed line) is projected to the L2S (red downward triangle), L2P (blue upward triangle), and L3P (green upward triangle) via synapses (gray discs) in layer Ia. The radius of the discs indicates the input strength. The colored vertical bars indicate the dendrites of the respective cell types. B–D, The strength of the intracortical connections (colored discs) is arranged in a superficial to deep gradient complementing sensory input strength. Colored dashed lines thereby indicate the axons. Note that LSPS does not allow us to distinguish between semilunar and layer II pyramidal cell inputs (B, red and blue circles). The intracortical projection strength from deep layer III multipolar cells (D, orange) follows the same gradient as the L2S/L2P projections (blue, red) and the L3P projections (C, green). The strong synaptic pathway from those multipolar cells to the L3P cells exhibits a spatial asymmetry (D, orange semicircle) in that most of these projections arise from multipolar cells located more dorsally than the L3P.
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