Inhibition of inhibition in visual cortex: the logic of connections between molecularly distinct interneurons

Abstract

Cortical inhibitory neurons contact each other to form a network of inhibitory synaptic connections. Our knowledge of the connectivity pattern underlying this inhibitory network is, however, still incomplete. Here we describe a simple and complementary interaction scheme between three large, molecularly distinct interneuron populations in mouse visual cortex: parvalbumin-expressing interneurons strongly inhibit one another but provide little inhibition to other populations. In contrast, somatostatin-expressing interneurons avoid inhibiting one another yet strongly inhibit all other populations. Finally, vasoactive intestinal peptide-expressing interneurons preferentially inhibit somatostatin-expressing interneurons. This scheme occurs in supragranular and infragranular layers, suggesting that inhibitory networks operate similarly at the input and output of the visual cortex. Thus, as the specificity of connections between excitatory neurons forms the basis for the cortical canonical circuit, the scheme described here outlines a standard connectivity pattern among cortical inhibitory neurons.

Figure 1. Three non-overlapping Cre-driver lines

a) Confocal double fluorescence images of coronal sections through visual cortex of three Cre-driver lines (Pvalb-Cre (left); Sst-Cre (center); VIP-Cre (right)). Cre expression pattern (labeled in red, revealed by crossing the Cre-driver lines with the ROSA-tdTomato reporter line; left sub-panels) counterstained with anti Pvalb antibody (labeled in green; center sub-panels) and overlay (right sub-panels). Note labeling of Pvalb-Cre cells but not Sst-Cre or VIP-Cre cells with anti Pvalb antibodies (yellow cells in right sub-panel). Scale bar: 100μm; same for all panels in figure 1. b) Schematic of overlap of Cre lines with respect to Pvalb antibody labeling (top) and quantification of overlap (bottom). The left and right ordinates refer to the left and right data-columns of the same cre-line, respectively. Error bars represent s.e.m. (Pvalb: n=1548 cells, 4 sections, 2 mice; Sst: n=1933 cell, 6 sections, 2 mice; VIP: n=1465 cells, 6 section, 2 mice). c) Confocal double fluorescence images of coronal sections through visual cortex of the three Cre-driver lines crossed with the HTR3a-GFP line. Cre expression pattern (labeled in red, revealed by crossing the Cre-driver lines with the ROSA-tdTomato reporter line; left sub-panels); HTR3a-GFP (labeled green; center sub-panels) and overlay (right sub-panels). Note co-labeling of VIP-Cre cells but not Pvalb-Cre or Sst-Cre cells with GFP (yellow cells in right sub-panel). d) Schematic of overlap of Cre lines with cells labeled in the HTR3a-GFP line (top) and quantification of overlap (bottom). The left and right ordinates refer to the left and right data-columns of the same cre-line, respectively. Error bars represent s.e.m. (Pvalb: n=1373 cells, 4 sections, 2 mice; Sst: n=1666 cells, 4 sections, 2 mice; VIP: n=1243 cells, 4 sections, 2 mice). e) as in (c) but three Cre-driver lines crossed with the GAD67-GFP line. f) Quantification of overlap of Cre-lines with cells labeled in the GAD67-GFP line. (Pvalb: overlap=36.2±0.5 s.e.m, n=1548 cells, 4 sections, 2 mice; Sst: overlap=30.4±1.5 s.e.m, n=3869, 6 sections, 2 mice; VIP: overlap=17.4±1 s.e.m, n=3033 cells, 6 section, 2 mice).

Figure 2. Five molecularly distinct interneuron categories defined by scRT-PCR

a) Six example cells whose genes were amplified by scRT-PCR. Cells are categorized according to their primary (black) and secondary (gray) gene expression: cell1-Pvalb/Tac1, cell2-Sst/Pdyn/Grin3a, cell3-VIP/Tac2, cell4-Tnfaip8l3/Sema3c, cell5-undefined, cell6-Pvalb/Sst/VIP/Tnfaip8l3 (discarded). Full-length gels are presented in Supp. Fig. 2a. b) Histogram of coexpression of primary and secondary markers from the analysis of 474 single cells (n=474 cells; n=415 slices; n=134 mice). Matching primary and secondary markers are illustrated in the same color. c) Dichotomous categorization scheme of postsynaptic interneurons based on primary and secondary marker expression. Numbers in brackets represent the percentage of cells categorized according to the scheme (PM = primary marker, SM = secondary marker, UD = undefined). d) Expression pattern of nine marker genes (four primary and five secondary markers) in 474 cells. Each row is a different cell; each column is a different gene. The color of the primary markers is the same as the color of the co-expressed secondary markers. Cells are sorted and grouped in different categories (labeled on the right) according to their primary and secondary expression pattern. e) Schematic of genetically defined 3 presynaptic interneuron classes and 6 postsynaptic interneuron categories. The L1 interneuron category contains all unidentified (UD) neurons located in layer 1.

Figure 3. Individual neuronal contributions of the three interneuron classes onto pyramidal cells

a) Top: Schematic of paired recording configuration. Bottom: Average unitary IPSCs (uIPSC) recorded in pyramids in response to an action potential evoked in a defined presynaptic interneuron. Each trace represents the average postsynaptic current of a different paired recording. Pvalb cells (left; n=12; 12/12 connected pairs; 5 slices; 3 mice), Sst cells (center; n=12; 12/12 connected pairs; 6 slices; 2 mice) and VIP cells (right; n=32; 4/32 connected pairs; 12 slices; 5 mice;). b) Summary histogram of unitary uIPSQ recorded in pyramids and mediated by the three different presynaptic interneuron classes (Pvalb: n=12; Sst: n= 12; VIP: n=4; error bars=s.e.m). c) Summary histogram of the connectivity between the three presynaptic interneuron classes and postsynaptic pyramidal cells. d) Summary histogram of individual neuronal contribution (uIPSQ × P_con) of the three presynaptic interneuron classes onto pyramidal cells normalized by the individual neuronal contribution of Pvalb cells.

Figure 4. Pvalb cells mainly inhibit one another

a) Schematic of experimental configuration: ChR2 expressing Pvalb-Cre cells are photo-stimulated while recording from a pyramidal cell (Pyr) and a neighboring GAD65/67 positive inhibitory neuron expressing GFP. b) Example IPSCs simultaneously recorded in the reference pyramid (black) and in one of the six different interneuron categories (different colors). The order of the six pyramid IPSCs (top to bottom) matches the order of the IPSC simultaneously recorded in each of the six interneuron categories. For simplicity, all traces were scaled such that the pyramid IPSCs have the same peak amplitude. c–h) The inhibitory postsynaptic charge (IPSQ) evoked by Pvalb cell photostimulation and recorded in individual interneurons (IPSQ_IN; y-axis) is plotted against the IPSQ simultaneously recorded in a pyramidal cell (IPSQ_Pyr; x-axis; see (a) for symbol legend). Dotted line is unity line. Category(n of cells/slices/mice): Pvalb(16/15/10), Sst(9/9/6), VIP(15/12/9), Tnfaip8l3(9/5/5), Undefined-UD(16/12/9), L1(7/5/5). i) Panel showing mean±s.e.m of individual neuronal contributions (INC) of all recorded pairs of the respective category. Note that Pvalb cells receive most inhibition. j) Schematic illustration of the inhibition mediated by Pvalb cells onto each interneuron category (abbreviation as in Fig. 2e).

Figure 5. Sst cells inhibit all other categories but one another

a) Schematic of experimental configuration: ChR2 expressing Sst-Cre cells are photo-stimulated while recording from a pyramidal cell (Pyr) and a neighboring GAD65/67 positive inhibitory neuron expressing GFP. b) Example IPSCs simultaneously recorded in the reference pyramid (black) and in one of the six different interneuron categories (different colors). The order of the six pyramid IPSCs (top to bottom) matches the order of the IPSC simultaneously recorded in each of the six interneuron categories. For simplicity, all traces were scaled such that the pyramid IPSCs have the same peak amplitude. c–h) Left panels: The inhibitory postsynaptic charge (IPSQ) evoked by Sst cell photostimulation and recorded in individual interneurons (IPSQ_IN; y-axis) is plotted against the IPSQ simultaneously recorded in a pyramidal cell (IPSQ_Pyr; x-axis; see (a) for symbol legend). Dotted line is unity line. Note that all inhibitory neuron categories receive inhibition comparable to that simultaneously recorded in pyramids, but for Sst cells that receive none (d). Category(n of cells/slices/mice): Pvalb(13/10/5), Sst(12/6/5), VIP(10/7/6), Tnfaip8l3(13/10/6), Undefined-UD(16/13/6), L1(8/6/5). i) Panel showing mean±s.e.m of individual neuronal contributions (INC) of all recorded pairs of the respective category. j) Schematic illustration of the inhibition mediated by Sst cells onto each interneuron category (abbreviation as in Fig. 2e).

Figure 6. VIP cells preferentially inhibit Sst cells

a) Schematic of experimental configuration: ChR2 expressing VIP-Cre cells are photo-stimulated while recording from a pyramidal cell (Pyr) and a neighboring GAD65/67 positive inhibitory neuron expressing GFP. b) Example IPSCs simultaneously recorded in the reference pyramid (black) and in one of the six different interneuron categories (different colors). The order of the six pyramid IPSCs (top to bottom) matches the order of the IPSC simultaneously recorded in each of the six interneuron categories. For simplicity, all traces were scaled such that the pyramid IPSCs have the same peak amplitude. c–h) The inhibitory postsynaptic charge (IPSQ) evoked by VIP cell photostimulation and recorded in individual interneurons (IPSQ_IN; y-axis) is plotted against the IPSQ simultaneously recorded in a pyramidal cell (IPSQ_Pyr; x-axis; see (a) for symbol legend). Dotted line is unity line. Note that only Sst cells receive substantial inhibition (d). Also, note the x and y-axes are expanded by one order of magnitude as compared to Pvalb-Cre (Fig. 4) and Sst-Cre (Fig. 5). Category(n of cells/slices/mice): Pvalb(29/20/12), Sst(11/8/6), VIP(20/14/8), Tnfaip8l3(18/12/8), Undefined-UD(7/5/4), L1(6/5/4). i) Panel showing mean±s.e.m of individual neuronal contributions (INC) of all recorded pairs of the respective category. j) Schematic illustration of the inhibition mediated by VIP cells onto each interneuron category (abbreviation as in Fig. 2e).

Figure 7. Comparing individual neuronal contributions among cortical interneurons

a) Heat map of the normalized individual neuronal contributions of the three presynaptic interneuron classes onto the six postsynaptic interneuron categories. b) Top: Schematic of paired recording configuration. Bottom: Average unitary IPSCs (uIPSC) recorded in pyramids in response to an action potential evoked in a defined presynaptic interneuron. Each trace represents the average postsynaptic current of a different paired recording. Pvalb onto Pvalb cells (left; n=13; 13/13 connected pairs, 6 slices, 3 mice), Sst onto Pvalb cells (center; n=14; 12/14 connected pairs, 6 slices, 3 mice) and VIP onto Sst cells (right; n=16; 10/16 connected pairs, 7 slices, 3 mice). c) Summary histogram of unitary uIPSQ recorded in interneurons and mediated by the three different presynaptic interneuron classes (Pvalb → Pvalb: n=13; Sst → Pvalb: n=12; VIP → Sst: n=10; error bar=s.e.m). d) Summary histogram of the connectivity probability between the three presynaptic interneuron classes and the respective postsynaptic interneurons. e) Summary histogram of individual neuronal contribution (uIPSQ × Connectivity probability) of the three presynaptic interneuron classes onto interneurons normalized by the individual neuronal contribution of Pvalb onto pyramid cells. f) Schematic illustration of the connectivity pattern between the three presynaptic interneuron classes (Pvalb, Sst, VIP) and 6 postsynaptic interneuron categories (Pvalb, Sst, VIP, Tnfaip8l3, UD, L1) in layer 2/3 and 5 of mouse visual cortex (abbreviation as in Fig. 2e). g) Schematic illustration of the inhibitory connections among the three largest classes of interneurons (Pvalb, Sst, VIP) and pyramidal cells (abbreviation as in Fig. 2e).