Laser Scanning Photostimulation Mapping of Intracortical Projections to L2/3 neurons

<div><p>(A) Across-barrel slice showing barrels corresponding to rows A–E (from left to right) under brightfield illumination. In this experiment, the map pattern (red grid) was centered on the septum between the barrels C and D.</p> <p>(B) Examples of synaptic input maps for a L2^Barrel cell (top) and a L3^Barrel cell (bottom). Black pixels are sites where glutamate uncaging evoked direct responses in the recorded cells, polluting the synaptic responses. Dashed lines indicate the barrel positions. Solid white circles show the cell body positions of the recorded neurons.</p> <p>(C) Examples of dendritic morphologies of L2^Barrel and L3^Barrel cells (same cells as in [B]).</p></div

Laser Scanning Photostimulation Mapping of Thalamocortical Projections

<div><p>(A) Montage of a thalamocortical slice.</p> <p>(B) Layout of the experiment. Excitatory neurons were recorded in the barrel cortex while thalamic neurons were photostimulated by glutamate uncaging. The map pattern (red grid) was centered on the POm/VPM boundary. Dashed lines indicate a barrel-column.</p> <p>(C–E) Examples of synaptic input maps for individual L4 (C), L5B (D), and L5A (E) neurons. The pixel values encode the mean amplitudes of EPSCs measured within 100 ms after the stimulus (see [G]). The dashed lines indicate the borders between POm, VPM, and the ventral posterolateral nucleus (VPL) (see [B]). Letters mark a pair of pixels corresponding to the traces shown in (G).</p> <p>(F) Map of standard deviations across trials for an individual cell (same cell as in [E]).</p> <p>(G) Examples of individual EPSCs. The responses were evoked by photostimulation at the sites indicated by letters in (C–E) (two sites per arrow). Arrowheads indicate the timing of the stimulus; dashed lines indicate the averaging window used for analysis.</p> <p>(H) Percentage of cortical cells with thalamic input. The percentage was computed for cells that were within a lateral distance of 300 μm of cells that showed thalamic input in the brain slice.</p></div

Interdigitated Intracortical Relays

<div><p>(A) Average synaptic input maps for positionally defined L2/3 pyramidal cells.</p> <p>(B) Regions of interest used for the analysis in (C–E).</p> <p>(C) The ratio of input from L5A/L4 as a function of the depth of the soma of barrel-related neurons. Dashed black line (top) indicates the pia.</p> <p>(D) The ratio of input from L2/3/L4.</p> <p>(E) Average synaptic input for projections defined by the presynaptic layer, postsynaptic layer, and postsynaptic column. For L3 neurons in septum and barrels, L4 input is significantly stronger than L5A input; for L2 neurons in barrels, L5A input is significantly stronger than L4 input (<i>p</i> < 0.005, Wilcoxon). Note that this pattern differs from that measured in the rat using essentially identical techniques (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040382#pbio-0040382-g003" target="_blank">Figure 3</a>e in [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040382#pbio-0040382-b013" target="_blank">13</a>]). L3 input is significantly stronger for L2/3^Septum cells than for L2/3^Barrel cells (<i>p</i> < 0.05, Wilcoxon).</p> <p>(F) Horizontal profiles of L4 input to L3 cells.</p> <p>(G) Horizontal profiles of L5A input to L2 cells.</p></div

Spatial Resolution of LSPS in the Somatosensory Thalamus

<div><p>(A) Photostimulation-evoked APs recorded in loose-patch mode in a POm cell (arrowheads indicate the stimulus). The traces correspond to the 12 pixels in the boxed region shown in (B).</p> <p>(B) Excitation profile of a single POm cell. The grid was centered on the soma. Pixel values encode the number of APs in a 100-ms window after photostimulation.</p> <p>(C) Average excitation profiles (VPM, <i>n =</i> 7; POm, <i>n =</i> 7).</p> <p>(D) Excitation as a function of lateral distance from the soma (at 0). Responses elicited in each column of the 8 × 8 grid were summed.</p></div

Circuit Diagram of the Thalamocortical and Ascending Intracortical Projections in the Barrel Cortex

<p>Lemniscal projections, green; paralemniscal projections, blue. Thick, thin, and dashed lines denote decreasing density of the projection.</p

Laminar Organization of Thalamocortical Projections

<p>Overlay of input domains for L4, L5A, L5B, and L6A cells. The contours enclose regions where the inputs exceed 50% of the largest responses (VPM, green; POm, blue). Dashed contours correspond to pyramidal cells with apical dendrites that were cut below L1. Shaded area denotes the range of POm/VPM boundaries for all experiments.</p

Thalamic Input Domains Projecting to Individual Cortical Columns

<div><p>(A–D) Schematic of the locations of recorded neurons (left), input maps (middle), and overlaid input domains (right) Circles in the input domains indicate the largest responses from a pair of L4 cells in the same column (A), a pair of L4 cells in neighboring columns (B), a pair of L5A cells in the same column (C) and a pair of L5A cells in neighboring columns (D).</p> <p>(E) Distance between the largest input in the synaptic input maps (green, VPM; blue, POm) from pairs recorded in the same column or in neighboring columns. The average (thick line), and the minimum and maximum distances between largest input across cells (rectangle) are shown. Numbers in parenthesis are the number of pairs. The pairs of cells located in the same column were three L4/L4, five L4/L5B, four L4/L6A, two L5B/L6A, one L6A/L6A, and six L5A/L5A. Pairs of cells located in neighboring columns were four L4/L4, one L4/L5B, and six L5A/L5A cells.</p> <p>(F) Areas of the input domains in VPM (green) and POm (blue) for pairs of L4/L5B/L6A, and L5A cells located in the same column.</p></div

Thalamocortical Topographic Transformations

<div><p>(A) Schematic of the coordinate system. <i>X</i>_ID and <i>Y</i>_ID are the horizontal and vertical coordinates of the input domain, respectively. <i>X</i>_Soma is the horizontal coordinate of the cell position in cortex. <i>X</i>_ID and <i>X</i>_Soma are measured from the VPM/POm boundary (center of the uncaging grid; light red). <i>Y</i>_ID is measured from the bottom of VPM (bottom of the uncaging grid).</p> <p>(B) and (C) Positions of the input domains (<i>Y</i>_ID in B_;<i>X</i>_ID in C) in VPM (green) and POm (blue) as a function of <i>X</i>_Soma. Cells were in L4 (<i>n =</i> 12); L5B (<i>n =</i> 7); L6A (<i>n =</i> 5); and L5A (<i>n =</i> 21). Dashed line shows the linear correlation between <i>X</i>_Soma and <i>Y</i>_ID for VPM and POm domains (<i>r²</i> = 0.72).</p></div

The Magnitude of the PSD-95 Retention Time Is Determined by Its Interactions with the PSD

<div><p>(A) Examples of the time course of paGFP* (black), paGFP*-actin (red), and PSD-95-paGFP* (blue) fluorescence after photoactivation in single spines (age, P10).</p> <p>(B) Retention times for paGFP*, paGFP*-actin, and PSD-95-paGFP* (age, P10) (circles indicate individual spines; bars indicate medians) (median <i>τ</i>_paGFP = 0.47 s, range, 0.28–1.28 s, <i>n</i> = 2 animals, 11 spines; median <i>τ</i>_paGFP-actin = 0.98 min, range, 0.45–6.42 min, <i>n</i> = 3 animals, 26 spines; median <i>τ</i>_PSD-95-paGFP = 22 min, range, 4–59 min, <i>n</i> = 2 animals, 15 spines).</p> <p>(C) Escape time for paGFP* from individual spines (circles) as a function of age. (P10–P30: <i>τ</i>_median = 0.72 s, range 0.28–2.38 s, <i>n</i> = 4 animals, 35 spines; > P60: <i>τ</i>_median = 0.58 s, range 0.24–1.78 s, <i>n</i> = 1 animal, 105 spines).</p></div

Larger Spines Retain PSD-95 Longer

<div><p>(A) Image of two photoactivated spines, a and b, on two branches of the same dendrite. <i>G_a</i>_,max and <i>G_b</i>_,max are the fluorescence intensities of PSD-95-paGFP^* immediately after photoactivation, a measure of PSD size. <i>τ</i>_a<i>,</i> and <i>τ</i>_b are the corresponding retention times.</p> <p>(B) Comparison of retention times and PSD sizes for pairs of spines. Each line corresponds to a pair of spines, a and b (black, positive slope, <i>n</i> = 64; gray, negative slope, <i>n</i> = 18, 6 animals; orange, example from [A]). Spines with larger PSDs have longer retention times.</p></div