The columnar and laminar organization of inhibitory connections to neocortical excitatory cells

Abstract

The cytoarchitectonic similarities of different neocortical regions have given rise to the idea of 'canonical' connectivity between excitatory neurons of different layers within a column. It is unclear whether similarly general organizational principles also exist for inhibitory neocortical circuits. Here we delineate and compare local inhibitory-to-excitatory wiring patterns in all principal layers of primary motor (M1), somatosensory (S1) and visual (V1) cortex, using genetically targeted photostimulation in a mouse knock-in line that conditionally expresses channelrhodopsin-2 in GABAergic neurons. Inhibitory inputs to excitatory neurons derived largely from the same cortical layer within a three-column diameter. However, subsets of pyramidal cells in layers 2/3 and 5B received extensive translaminar inhibition. These neurons were prominent in V1, where they might correspond to complex cells, less numerous in barrel cortex and absent in M1. Although inhibitory connection patterns were stereotypical, the abundance of individual motifs varied between regions and cells, potentially reflecting functional specializations.

Figure 1. Targeted ChR2 expression in GABAergic interneurons

(a, b) Targeting constructs. Homology sequences are indicated in dark grey, promoters in yellow, open reading frames in red, and selection markers in light grey. (a) Construct used to generate the R26::ChR2-EGFP allele. Cre-mediated excision of a triple-polyA transcriptional STOP cassette (3x PA, black) flanked by loxP sites enables ChR2-EGFP expression from the CAG promoter. (b) Construct used to generate the Gad2::Cre-ER^T2 allele. An internal ribosome entry sequence (IRES, light red) separates the Gad65 and Cre-ER^T2 reading frames. (c) Gad2::Cre^T2 R26::ChR2-EGFP mice after tamoxifen induction express ChR2-GFP in Cre-positive cells (top), which comprise both Gad65- and Gad67-positive interneurons (middle), but not CamKIIα–positive pyramidal cells (bottom). See Supplementary Fig. 1 for an analysis of interneuron subtypes and Supplementary Table 1 for statistics. The left and center columns show raw confocal images; the right column displays the corresponding colocalization maps, which were produced by multiplying the two fluorescence channels on a pixel-by-pixel basis and normalizing the resulting product image to 8 bits.

Figure 2. Genetically targeted photostimulation of GABAergic interneurons

(a, b) Responses to photostimulation in cell-attached (a) and whole-cell current-clamp recordings (b); native resting potentials are indicated in parentheses. Interneurons follow trains of 20-ms optical pulses at 10 Hz with action potentials; pyramidal neurons are unresponsive to light. (c) ChR2-mediated photocurrents desensitize during repeated optical stimulation at frequencies >1 Hz (left) but remain stable at stimulation frequencies ≤0.2 Hz (right). (d) Spiking probabilities as a function of time after stimulus onset were estimated by analyzing 29–198 light-evoked action potentials per cell (n=4 interneurons in cell-attached recordings; colored traces). Spike times are defined as the times at which the upstroke of an action potential reaches half-maximal amplitude. Average spike latencies (± s.d.) are indicated in matching colors. (e) Wide-field optical stimulation at 5 Hz (grey bars) evokes IPSCs in pyramidal cells voltage-clamped at 0 mV (grey traces). IPSCs are abolished after bath application of 10 μM gabazine (red trace, top) or 0.5 μM TTX (red trace, bottom).

Figure 3. Optogenetic mapping of inhibitory connectivity

(a) Contour plots depict spiking probabilities (left) and depolarization amplitudes (right) of three interneurons as functions of stimulus location. Blue dots indicate stimulation points; arrowheaded scale bars of 100 μm point to the pial surface. Perisomatic illumination reliably elicits action potentials (left). Positioning the focus of the stimulating beam near dendritic branches does not cause higher spiking probabilities or larger depolarizations than illumination of dendrite-free neuropil equidistant from the soma (right). (b) Spiking probabilities of 9 interneurons as functions of the distance of the stimulation spot from the soma. Cells were recorded in the cell-attached (n=4 cells, green traces) or whole-cell configuration (n=5 cells, blue traces). (c) Sequential illumination of 10 different locations at 2.5 Hz (20 ms, 2 mW, grey tick marks). Illumination of sites marked by blue arrows gives rise to reproducible IPSCs in the recorded pyramidal cell. (d) Maps of inhibitory inputs to pyramidal neurons in V1 (top row) or S1 (bottom row), located in L2/3 (left column) or L5B (right column) at comparable depths (± 7 μm) from the surface of the slice. Two neurons were recorded simultaneously to test whether cells within the same local network could exhibit different connectivity patterns. Cell positions are indicated by triangles; a filled triangle denotes the postsynaptic target for each map. Color on a heat scale symbolizes the average amount of charge flowing during 100 ms following the onset of the IPSC, at a holding voltage of 0 mV.

Figure 4. Horizontal (columnar) organization of inhibitory connections

(a, b) Overlay maps of inhibitory inputs to pairs of simultaneously recorded pyramidal neurons in layer 2/3 of S1. The maps depict the locations of inhibitory inputs but not their strength and have been scaled to the size of a standard somatosensory barrel (yellow outlines). Cell positions are marked by triangles. Data from the left cell in each pair are coded in blue, data from the right cell in red. (a) Pairs of pyramidal neurons in the same barrel-related column. (b) Pairs of pyramidal neurons in adjacent barrel-related columns. (c) Horizontal profiles of input distributions show the interpolated number of inhibitory inputs as a function of horizontal distance from the center of the input distribution for each layer, ignoring the laminar (vertical) location of these inputs. Horizontal distances were scaled to the size of a standard somatosensory barrel (yellow outlines) and center-aligned; the number of inhibitory inputs to an excitatory neuron in a given layer at a given distance from the map center was then averaged. The intensity of blue color symbolizes the density of input sources. See Methods and Supplementary Table 3 for statistical detail.

Figure 5. Vertical (laminar) organization of inhibitory connections

(a–e) Average strength of inhibitory input from the indicated source layers (rows) to excitatory neurons located in L2/3 (a), L4 (b), L5A (c), L5B (d) and L6 (e). An example of a neurobiotin-filled excitatory neuron, recovered after recording, is shown to the left of each panel. The figure summarizes data from 30 neurons in M1, 54 neurons in S1, and 53 neurons in V1. The strength of a connection is expressed as the average percentage of inhibitory charge flow arising from identified inputs in a layer. L5 represents the sum of L5A and L5B. Values are represented numerically (s.d. in parentheses) and by the intensity of grey shading. Colored boxes indicate significant differences (P < 0.05), either between two cortical areas (red) or between one area and the other two (blue), as determined by one-way ANOVA and Tukey-HSD post-hoc tests (Supplementary Tables 2 and 4).

Figure 6. Area-specific differences in the laminar organization of inhibitory connections

(a) Discriminant analysis of the laminar source distributions of inhibitory inputs to excitatory neurons in different target layers of M1, S1, and V1 (Supplementary Table 8). Neurons are represented as points in the coordinate system spanned by the discriminant functions Y1 and Y2. Borders between colored areas indicate decision boundaries for assigning neurons to M1 (blue), S1 (yellow), and V1 (red). Data points whose fill color matches the background color are classified correctly. Black squares indicate the centroid positions for all cells in each cortical area. (b) Abundance of interneuron subtypes expressing parvalbumin (P), somatostatin (S), and VIP (V) in in the respective cortical layers and areas; pie chart diameters represent overall interneuron densities (Supplementary Table 9). (c) Percentages and absolute numbers of inputs, charge flow, and failure rates (means+s.d.) of the four translaminar motifs exhibiting area-specific differences. Colored bars symbolize data for M1 (blue), S1 (yellow), and V1 (red). Asterisks denote statistically significant differences between cortical areas (P < 0.05), as determined by ANOVA and Tukey-HSD post-hoc tests (Supplementary Tables 5, 6, 7 and 10).

Figure 7. Cell-specific differences in the laminar organization of inhibitory connections

(a, b) Hierarchical clustering of pyramidal cells in layers 2/3 (a) and 5B (b) of M1, S1, and V1. Classification variables were the strengths of intra- and translaminar inhibitory inputs, quantified as normalized inhibitory charge flow. Neurons in both layers fall into two well-separated clusters: a minor population of neurons receiving strong translaminar inhibition (left cluster), and a major population of predominantly home-layer inhibited neurons (right cluster). Bootstrap estimates of cluster distances at the first bifurcation level are 0.64±0.11 and 0.71±0.09 (means±s.d.) for pyramidal cells in layers 2/3 (a) and 5B (b), respectively. Pie charts indicate the average strengths of inhibitory input from the dominant translaminar layer in black (layer 6 for neurons in layer 5B, and layer 5 for neurons in layers 2/3), the home layer in grey, and other layers in white. Colored letters denote the cortical area from which each observation is derived. Note the high frequency of V1 neurons and the absence of M1 neurons in the clusters receiving dominant translaminar inhibition.