Spatial Clustering of Inhibition in Mouse Primary Visual Cortex

Abstract

Whether mouse visual cortex contains orderly feature maps is debated. The overlapping pattern of geniculocortical inputs with M2 muscarinic acetylcholine receptor-rich patches in layer 1 (L1) suggests a non-random architecture. Here, we found that L1 inputs from the lateral posterior thalamus (LP) avoid patches and target interpatches. Channelrhodopsin-2-assisted mapping of excitatory postsynaptic currents (EPSCs) in L2/3 shows that the relative excitation of parvalbumin-expressing interneurons (PVs) and pyramidal neurons (PNs) by dLGN, LP, and cortical feedback is distinct and depends on whether the neurons reside in clusters aligned with patches or interpatches. Paired recordings from PVs and PNs show that unitary inhibitory postsynaptic currents (uIPSCs) are larger in interpatches than in patches. The spatial clustering of inhibition is matched by dense clustering of PV terminals in interpatches. The results show that the excitation/inhibition balance across V1 is organized into patch and interpatch subnetworks, which receive distinct long-range inputs and are specialized for the processing of distinct spatiotemporal features.

Keywords: inhibition; intracortical feedback; parvalbumin interneurons; thalamocortical connections; visual cortex.

Figure 1. Patchy dLGN→V1 and LP→V1 projections to L1.

Tangential sections through L1 of V1 of Chrm2tdT (A-C, E-G) and C57BL/6J mice (I-K). (A-C) Spatially clustered dLGN→V1 projections anterogradely traced with AAV2/1.hSyn.EGFP (green) overlap with M2tdT+ patches (purple). (D) Normalized fluorescence intensity of dLGN→V1 input to M2tdT+ patches (for contour maps, Figure S1) is higher than to M2tdT-interpatches. (E-G) Patchy LP→V1 projections traced with AAV2/1.hSyn.EGFP (green) overlap with M2tdT- interpatches. (H) Normalized fluorescence intensity of LP inputs to M2tdT-interpatches is higher than to M2tdT+ patches. (I-K) Interdigitating patchy LP→V1 and dLGN→V1 projections traced in the same mouse with AAV2/1hSyn.tdTomato (purple) and AAV2/1.hSyn.EGFP (green), respectively. (L) LP→V1 input is weaker in dLGN+ patches (proxies of M2tdT+ patches; A-C) than in dLGN- interpatches. Mean ± SD, KS (Kolmogorov-Smirnov test), N = number of mice.

Figure 2. Patchy LM→V1, AL→V1 and PM→V1 projections to L1.

Tangential sections through L1 of V1 of C57BL/6J mice (A, C-H). (A) Overlapping clusters of dLGN→V1 (green, AAV2/1.hSyn.EGFP) and LM→V1 (purple, AAV2/1hSyn.tdTomato injection [*]) projections in patches (dLGN+ patches are proxies of M2+ patches) of L1. (B) Frequency distribution of fluorescence intensity of LM→V1 projections (normalized to mean in patches) shows stronger inputs to dLGN+ patches. (C-H) Interdigitating patterns of AL→V1 (D, purple, tracing with tdTomato) and PM→V1 (E, green, tracing with EGFP) projections. Immunostaining for M2 (C) showing that AL→V1 overlap with M2+ patches (F). PM→V1 input to M2-interpatches alternates with AL→V1 to M2+ patches (G, H). (I) Frequency distribution of fluorescence intensity of AL→V1 projections indicates stronger input to M2+ patches. (J) Distribution of PM→V1 intensity indicates stronger input to interpatches where AL→V1 is weak. Same conventions as in Figure 1.

Figure 3. Tangential slices: sCRACM of dLGN→V1 input to L1 onto L2/3 PNs and PVs in patches and interpatches.

(A) Confocal Z-stack showing ChR2-Venus labeled dLGN→V1 projections in L1 and Alexa 594 hydrazide-filled pairs of L2/3 PNs and PVs in patch and interpatch. (B, D) Whole cell patch clamp recordings from PN (black triangle) and PV (red circle) in patches and interpatches in the same slice. Each trace represents average of EPSCs (3 to 5 per neuron) of PNs and PVs in patches (Bi, Biii) and interpatches (Di, Diii) upon laser stimulation (blue dots, 75 × 75 μm grid) of ChR2-expressing dLGN→V1 terminals. Heatmaps of responses evoked at different locations of the dendritic arbor (white profiles) of PN and PV in patches (Bii, Biv) and interpatches (Dii, Div). (C, E) Each dot represents relative strength of dLGN→V1 input (summed pixels of significant EPSCs) of a pair of L2/3 PNs and PVs in patch (C) and interpatch (E). Red line denotes mean slope from zero, blue line shows mean slope after normalizing currents to mean conductance. (F) Distribution of dLGN→V1 input strength across dendritic tree. Grey bars represent input area as number of pixels with non-zero EPSCs. Red bars EPSC density. (G) Box plots of dLGN→V1 EPSCs from PVs and PNs in patches and interpatches. (C, E, F) Wilcoxon signed-rank test (Wt) (***p < 0.001, **p < 0.01).

Figure 4. Coronal slices: sCRACM of dLGN→V1 and LP→V1 input to L2/3 PNs and PVs in patches and interpatches.

(A) dLGN→V1 projections (green) traced with AAV2/1.hSyn.ChR2(H134R).EYFP, showing dense terminations in patches (P) and sparse input to interpatches (IP) in L1. Biocytin-filled L2/3 PN (magenta) recorded in patch, showing apical dendrites projecting to patch and basal dendrites descending to uniformly labeled dLGN-recipient deep L2/3. (B) Box plots of recordings from L2/3 PNs showing that EPSCs in L1 (normalized to total EPSCs) in IP (N = 20) are significantly smaller than in P (N = 23). (C) Box plots of recordings from L2/3 PNs showing that the proportion of EPSCs (normalized to total EPSC) in L1 of interpatches is smaller (N = 20, p = 0.0025, paired t-test) than in L2/3. No significant (ns) laminar differences in patches (N = 23). Blue dot denotes mean L1 or L2/3 EPSC. (D, E) Relative strengths of EPSCs from neighboring pairs of L2/3 PNs and PVs evoked by dLGN→V1 input. Recordings from patches (D) and interpatches (E) obtained in different slices. Mean slope of currents from zero (red), slope after normalization to conductance (blue). (F) Similar EPSC_PV/EPSC_PN ratio in patches and interpatches. (G) EPSCs evoked by LP→V1 input to pairs of neighboring PNs (dots) in patches and interpatches recorded in the same slice, showing stronger inputs to interpatches. (H, I) EPSCs evoked by LP→V1 inputs to pairs of PNs and PVs in patches and interpatches recorded in different slices, showing similar inputs to PNs and PVs. (J) EPSC_PV/EPSC_PN ratio in patches and interpatches. (K) EPSC_PV/EPSC_PN ratio evoked by stimulation of dLGN→V1 and LP→V1 axons. Pooled responses from patches and interpatches.

Figure 5.. Tangential slices: sCRACM of LM→V1 input to L1 onto L2/3 PNs and PVs in patches and interpatches.

(A) Venus labeled LM→V1 projections in L1 and Alexa 594 hydrazide-filled pairs of L2/3 PNs and PVs in patch and interpatch. (B, D) Whole cell patch clamp recordings in the same slice. Each trace represents average of EPSCs evoked by laser stimulation of ChR2-expressing LM→V1 terminals recorded from PNs and PVs in patches (Bi, Biii) and interpatches (Di, Diii). Heatmaps of responses from PN and PV in patches (Bii, Biv) and interpatches (Dii, Div). (C, E) Dots represent relative strength of LM→V1 input to a pair of L2/3 PNs and PV in patches (C) and interpatches (E). (F) Distribution of LM→V1 input strength across dendritic tree of PNs and PVs in patches and interpatches. Grey bars denote input areas, red bars represent current densities. (G) Box plots of strength of LM→V1-evoked EPSCs from PVs and PNs in patches and interpatches. (C, E, F, G). Wt (***p < 0.001, ns = not significant). Same conventions as in Figure 3.

Figure 6.. Clustering of PV neurons in V1.

(A-C) Tangential 40 μm section through V1 at 40 μm below pial surface (see, S1 in M) showing overlapping pattern of immunolabeled M2+ patches (cyan, arrows) with PVtdT processes (magenta). (D-F) Section ~80 μm below surface (see, S2 in M; section aligned to A) showing overlapping pattern (arrows) of PV processes with M2− interpatches. (G-I) Section ~120 μm below surface (see, S3 in M; aligned to B) showing overlapping pattern (arrows) of PV cell bodies, dendrites and boutons with M2− interpatches. (J) Normalized PVtdT intensity (to mean intensity in patches) in L1A showing higher PV in patches. (K) Normalized PVtdT intensity (to mean intensity in patches) in L2 showing higher PV in interpatches. (J, K) KS, mean ± SD. (L) Biocytin-filled L2/3 PV+ BCs (coronal plane) showing that dendrites (blue) branch in L1B-2. Axons (red) of cells in patches (P, left panel) and interpatches (IP, right panel) branch near the cell body with little spread to neighboring IPs or Ps, respectively. Left panel shows patch-cell with asymmetrical axon arbor, largely contained within P. Elongated shape of axonal arbor is due to oblique section angle across P. PVs are less frequent in P. (M) Diagram of coronal section. Strong (solid lines) dLGN→V1, LM→V1, AL→V1 inputs to M2+ Ps (blue). Strong LP→V1, PM→V1 inputs to M2− IPs. High density of PV+ BC cell bodies, dendrites, axo-somatic and axo-dendritic boutons in L1B-2 of IPs. Putative PV+ ChC dendrites are denser in Ps and extend to L1A. Dense axo-axonic connections in L2/3 of Ps.

Figure 7.. Stronger inhibition in interpatches than patches.

(A, B) Diagram of recordings in coronal slices of unitary uIPSCs and uEPSCs in synaptically reciprocally connected pairs of L2/3 PNs and PVs (PN↔PV) aligned with ChR2.Venus-expressing dLGN→V1 patches (solid green) and interpatches (green outline) in L1. Recordings of inward uIPSCs from PNs with high [Cl⁻] internal solution at −70 mV holding potential. Recordings from PVs with K-gluconate internal solution. (C, D) uEPSCs (inward red trace) from PVs and uIPSCs (inward black trace) from PNs, after presynaptic spike from PNs or PVs, respectively, in patch (C) and interpatch (D). uEPSC in patches and interpatches have similar amplitudes and are blocked by DNQX (green C, D). In the reverse connection uIPSCs (black trace) in interpatches (D) were larger than in patches (C), and were blocked by Picrotoxin (blue C, D). The insets in (C) and (D) show that in the PV→PN and PV←PN direction postsynaptic responses exhibit monosynaptic delays. (E, F) uEPSCs and uIPSCs recorded in pairs of PNs and PV in patches and interpatches. In most pairs, uIPSCs in interpatches are larger than in patches. (G) Average uEPSCs from reciprocally connected PV↔PN pairs are similar, whereas uIPSCs in interpatches are larger (***p < 0.001). (H) Average I/E ratios of PV↔PN pairs in patches and interpatches. I/E balance in interpatches is tilted toward inhibition (***p < 0.001, paired t-test). (I) Average charge of uEPSCs and uIPSCs in patches and interpatches, showing that excitatory charge transfer at PN→PV contacts in patches and interpatches is similar. In interpatches the inhibitory charge transfer at PV→PN contacts is larger (*** p < 0.001, one-way ANOVA, Bonferroni correction). (J) I/E ratio showing that in reciprocally connected PV↔PN pairs in interpatches, uEPSCs are more strongly (** p < 0.01, two-sample t-test) opposed by uIPSCs. (K) The percentages of the total number of reciprocally connected PV↔PN pairs in patches (Ps) is similar (ns, Chi square test) in patches and interpatches (IPs). (L) The onset latency of uIPSC in Ps and IPs is similar (ns, Chi square test). (M) The rise time of uIPSCs in IPs is faster than in Ps (***p<0.001, paired t-test).