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. 2016 Sep 26;5:e19332. doi: 10.7554/eLife.19332

Figure 2. Subcellular ChR2-assisted mapping of V1→PM connections to L2/3 PV and Pyr cells.

(a) Coronal slices showing injection (left) and target (right) sites two weeks after the injection of AAV2/1.CAG.ChR2-Venus.WPRE.SV40 into V1. Scale bar, 500 µm. Select target areas indicated in right panel. SC, superior colliculus. (b) Schematic of laser-scanning photostimulation of ChR2-expressing axon terminals during whole-cell recording of a biocytin-filled neuron. TTX and 4-AP are added to the bath solution, and the blue laser is delivered successively one spot at a time in a grid pattern separated by 75 µm. (c) EPSCssCRACM in a PV (left) and a neighboring Pyr (right) cell upon photostimulation. Grey shapes denote the location of the cell body of the recorded neuron. (d) Heat map of mean EPSCs within 75 µs after photostimulation for the EPSCs in 3c. Reconstructions of respective biocytin-filled neurons are superimposed on heat map. (e) Average heat map of 14 neighboring PV-Pyr cell pairs in L2/3 receiving V1→PM input, normalized to largest pixel value between a pair. PV cells receive substantially stronger input. (f) Scatter plot denoting the relative input strengths to 14 PV-Pyr cell pairs. Each data point represents a pair with the respective EPSCs in the PV (vertical axis) and the Pyr (horizontal axis) cell. The total EPSC in PV cells is significantly larger than that in neighboring Pyr cells (p<0.001, Wilcoxon signed-rank test). Solid black line: mean slope, blue line: mean slope after normalizing currents to mean cell conductance. (g) The mean time to peak of EPSCs after photostimulation is larger in Pyr cells than in PV cells (*p<0.05, paired t-test).

DOI: http://dx.doi.org/10.7554/eLife.19332.007

Figure 2.

Figure 2—figure supplement 1. V1→PM pathway in a PV-tdT mouse.

Figure 2—figure supplement 1.

(a−e) Image of coronal section two weeks after the injection of AAV2/1.CAG.ChR2-Venus.WPRE.SV40 into V1. Slice includes areas PM, AL, and V1. PV cells in red (a,c), ChR2-Venus-expressing axons in green (b,c). Merged image in (c). Dotted lines demarcate AL/V1 and V1/PM boundaries indicated by the sharp decline of M2 expression between V1 and surrounding areas (d). V1 is characterized by a thick band of M2 expression (purple) in L4, showing that the axonal terminations (green) lie outside V1 (d,e). (f) Higher magnification view of PM from (c) with layers indicated.
DOI: http://dx.doi.org/10.7554/eLife.19332.008
Figure 2—figure supplement 2. Paired recordings of excitation-dependent, PV cell-mediated inhibition of Pyr cells.

Figure 2—figure supplement 2.

(a) Coronal section through V1 and PM of a PV-Cre × Ai9 mouse in which PV cells express tdT (red). Scale bar, 200 µm. Inset: Higher magnification of the boxed region shows a high density of tdT-expressing dendrites in L1 even though PV cell bodies are not found in this layer. (b) Left, PV cell identified by tdT expression targeted for whole-cell recordings. Scale bar, 20 µm. Middle, same cell as in left panel, imaged under DIC-IR optics. PV cell shows a non-adapting, fast-spiking firing pattern (inset, red trace) upon current injection. Right, A Pyr cell under infrared optics exhibits an adapting spiking physiology (blue trace) upon current injection. (c) Schematic of paired recordings of a PV (red) and a Pyr (blue) cell. Successively increasing current steps (100, 200, 300 and 400 pA) were injected into the PV cell under current clamp, and inhibitory currents (IPSCs) were recorded in the Pyr cell held at 0 mV under voltage clamp. (d) Example trace in a L2/3 PV-Pyr connected pair in PM. Increasing current injections into the PV cell results in stronger inhibitory drive to the Pyr cell. (e) Example traces of a connected PV-Pyr cell pair in L5 of PM. (f) Pooled data from connected PV-Pyr pairs in L2/3 (left) and L5 (right) show that increasing the excitation of PV cells results in stronger inhibition of synaptically connected Pyr cells (p<0.001 for both sets of data; ANOVA). Mean IPSCs measured over 75 ms after start of current step. (g) Probability of a PV cell connected to a neighboring Pyr cell (<100 µm) in L2/3 and L5 of PM.
DOI: http://dx.doi.org/10.7554/eLife.19332.009