Spinal Inhibitory Interneuron Diversity Delineates Variant Motor Microcircuits

Abstract

Animals generate movement by engaging spinal circuits that direct precise sequences of muscle contraction, but the identity and organizational logic of local interneurons that lie at the core of these circuits remain unresolved. Here, we show that V1 interneurons, a major inhibitory population that controls motor output, fractionate into highly diverse subsets on the basis of the expression of 19 transcription factors. Transcriptionally defined V1 subsets exhibit distinct physiological signatures and highly structured spatial distributions with mediolateral and dorsoventral positional biases. These positional distinctions constrain patterns of input from sensory and motor neurons and, as such, suggest that interneuron position is a determinant of microcircuit organization. Moreover, V1 diversity indicates that different inhibitory microcircuits exist for motor pools controlling hip, ankle, and foot muscles, revealing a variable circuit architecture for interneurons that control limb movement.

Figure 1. Transcription Factors Enriched in V1 Interneurons

(A) Isolation of V1 and dI4/dIL^A interneurons. Left, interneuron populations. Middle, En1::Cre (V1) and Ptf1a::Cre (dI4/dIL^A) lineage-traced interneurons in p0 lumbar spinal cord. Right, FACS-isolated eYFP⁺ interneurons for microarray analysis. (B) Scatter plot of expression levels of transcription factors (TFs, red) enriched in V1 interneurons from p0 mice. (C) TFs with > 3-fold enrichment (p ≤ 0.02, one-way ANOVA) at one or more developmental ages. See also Figure S1.

Figure 2. V1 Transcriptional Diversity and Cladistic Analysis

(A) TFs (red) label subsets of V1 interneurons (green) in L3-L5 spinal segments from p0 En1.nLacZ mice. Scale bars = 100 μm or 10 μm (inset). (B) V1 interneurons expressing TFs at p0 L3-L5 spinal segments (n ≥ 3 animals, mean ± SEM). See Figure S2B,C for characterization of TFs in dI4/dIL^A and V2a interneurons. (C) Coverage of V1 interneurons in p0 L3-L5 spinal cord. Anti-FoxP2, MafA, Pou6f2, and Sp8 antibodies label 64.2 ± 0.6% of V1 interneurons (n = 3). Application of 14 antibodies - Bhlhb5, FoxP1, FoxP2, MafB, Nr3b2, Nr4a2, Nr5a2, Oc1, Oc2, Otp, Pou6f2, Prdm8, Prox1, and Sp8 - labels 90.4 ± 0.8% of V1 interneurons (n = 3). Mean ± SEM. (D) Matrix of pairwise overlap for 148 out of 171 comparisons. N.M. = not measured, due to antibody incompatibility. (E) V1 interneurons segregate into four clades defined by mutually exclusive expression of FoxP2, MafA, Pou6f2, and Sp8 (< 1% overlap in each pairwise comparison). Clades are further subdivided by distinct TFs (black). Dotted line represents additional V1 cell types. The number of V1 cell types is indicated in parenthesis. See also Figures S2D and S3C.

Figure 3. Spatial Segregation of V1 Interneuron Subpopulations

(A) V1 interneurons in p0 L3-L5 segments of En1.nLacZ mice. D/V axis range: 132 to −265 μm; M/L axis range: 127 to 487 μm, 5th-95th percentiles from central canal. Contours represent density at the 30^th-90^th percentiles. (B) Spatial clustering of V1^Pou6f2/Nr5a2 interneurons (blue, F_a = 0.236) (p < 0.00001, one-tailed Monte Carlo test compared to parental V1). (C) M/L biases in distributions of V1^Sp8 (X_epicenter = 162 μm) and V1^Pou6f2 (X_epi = 403 μm) interneurons. p < 1 × 10⁻²⁰, Wilcoxon Rank Sum test in x-axis, V1^Sp8 or V1^Pou6f2 vs V1^Parental, and V1^Sp8 vs V1^Pou6f2. (D) D/V biases in distributions of V1^Pou6f2 (Y_epi = 66 μm), V1^FoxP4 (Y_epi = −158 μm), and V1^MafA (Y_epi = −277 μm) interneurons. V1^Sp8 interneurons (Y_epi = 72 μm) also occupy a dorsal position. p < 1 × 10⁻²⁰, Wilcoxon Rank Sum test in y-axis for V1^Pou6f2, V1^FoxP4, V1^MafA, or V1^Sp8 vs V1^Parental. E) Subdivision of V1^Pou6f2 interneurons into medial (Nr5a2⁺, blue) and lateral (Lmo3⁺, red) subsets in p0 L3-L4 spinal segments. (F) V1^Prdm8 interneurons fractionate into dorsal Sp8⁺ (blue) and ventral FoxP4⁺ (red) composite groups. (G) V1, V1^Pou6f2, and V1^Sp8 settling position at L3 (blue) or L5 (red) in p0 mice. p < 0.0001 for L3 vs L5, 2D KS test. (H) Constancy of x,y position (mean ± SD) for V1 interneurons expressing Sp8 (n = 7), Pou6f2/Nr5a2 (n = 8), Pou6f2/Lmo3 (n = 4), FoxP4 (n = 7), Nr3b2/Nr5a2 (n = 8), Nr3b3/Prox1 (n = 6), and MafA (n = 7 animals). (I) Spatial distributions of seven V1 subsets. Contours represent 60^th-90^th percentile densities. See also Figures S3 and S4, and Table S1.

Figure 4. Electrophysiological Characterization of V1 Clades

(A-C) Physiology of V1^FoxP2 interneurons. (A) V1^FoxP2 interneurons (n = 5) targeted for recording and filled with Cascade Blue in En1::Cre; FoxP2::Flpo; RCE.dual.GFP mice. Scale bar = 20 μm. (B) Firing properties of V1^FoxP2 cells show a prominent after-hyperpolarization (AHP, arrow), a non-bursting phenotype, and an absence of spike frequency adaptation (SFA). (C) Instantaneous firing (IF) frequency for each action potential (dot) through pulses of increasing current amplitudes (20 pA steps). Little or no SFA is observed below 460 pA. (D-F) Physiology of V1^{Pou6f2/lateral} interneurons. (D) Position of V1^{Pou6f2/lateral} interneurons (n = 7) in MafB::GFP; En1::Cre; Rosa.lsl.tdT mice. (E) Transient low-threshold depolarization (arrow), with an initial burst (asterisks), and the presence of SFA throughout the pulse. (F) SFA, indicated by the decreasing instantaneous frequency of successive action potentials. (G-I) Physiology of V1^{Pou6f2/medial} interneurons. (G) Position of V1^{Pou6f2/medial} interneurons (n = 7). (H) Neurons exhibit a non-burst phenotype and a weak low-threshold depolarization (arrow, H). (I) IF plot showing SFA. (J-L) Physiology of V1^R interneurons, representing the V1^MafA clade. (J) Position of V1^R interneurons (n = 6) in En1::Cre; Rosa.lsl.tdT; MafB::GFP mice. (K) Neurons show prominent low-threshold depolarization (arrow), and burst firing (asterisks). (L) IF plot shows absence of SFA. See also Figure S5.

Figure 5. Relative Position of V1^R and V1^Sp8 Interneurons to Motor Pools

(A) V1^Sp8 interneurons (green), in p12 lumbar spinal cord of En1::Cre; Sp8::FlpoER^T2; RCE.dual.GFP mice. (B) V1^R interneurons (yellow, colocalization mask of eGFP and calbindin immunoreactivity) in ~p21 En1::Cre; RCE.lsl.GFP lumbar spinal cord. (C) V1^R and V1^Sp8 position with respect to GL, TA, and IF motor pools in ~p21 mice. Motor pool D/V positions: GL: 84 ± 3 μm, TA: 291 ± 6 μm, IF: 321 ± 15 μm, from dorsal border of ventral funiculus. (D) D/V position of V1^R interneurons (yellow) with respect to CTB-backfilled GL, TA, and IF motor pools (MN, red) in ~p21 lumbar spinal cord. D/V distances: V1^R ventral to GL, TA, and IF motor neurons by 8 ± 3 μm, 242 ± 14 μm, and 264 ± 13 μm, respectively. p < 0.0001, one-way ANOVA; Bonferroni post-hoc test: p < 0.001, TA or IF vs GL. M/L distances were not significantly different (p = 0.99, one-way ANOVA). (E) D/V position of V1^Sp8 interneurons (green) with respect to CTB-backfilled GL, TA, and IF motor pools (red) in ~p21 lumbar spinal cord. V1^Sp8 dorsal to GL, TA, and IF by 332 ± 8 μm, 139 ± 23 μm, and 50 ± 8 μm, respectively (p < 0.0001, one-way ANOVA; Bonferroni post-hoc test: p < 0.001, TA or IF vs GL; p < 0.05, TA vs IF). In the M/L axis, V1^Sp8 interneurons were significantly closer to IF than to GL or TA (192 ± 11 μm versus 406 ± 26 μm or 382 ± 33 μm, respectively; p < 0.01, one-way ANOVA; Bonferroni post-hoc test; p < 0.01, IF vs GL or TA). Values are mean ± SEM, n ≥ 3 animals per condition. Scale bars = 100 μm or 50 μm (inset). See also Figure S6.

Figure 6. Specificity of Sensory-Interneuron Connectivity at Individual Joints

(A) Assay of proprioceptive input to V1^R interneurons. (B-C) CTB⁺; vGluT1⁺ proprioceptive input to V1^R interneurons. (C) Percent of V1^R interneurons with pool-specific input. p = 0.0007, one-way ANOVA; Bonferroni post-hoc test: p < 0.01, GL vs TA or IF, n = 3 animals. Innervation density (inputs/100 μm dendrite) for neurons receiving sensory input: GL, 5.1 ± 1.0; TA, 2.8 ± 1.9. All data are mean ± SEM. Scale bar = 2 μm. (D) Assay of monosynaptic input from pool-specific Ia afferents onto V1^R interneurons. (E) Left, V1^R interneurons (red, labeled in En1::Cre; Rosa.lsl.tdT mice) targeted for intracellular recording, filled with Alexa-488 hydrazide (green). Right, stimulation of L5 ventral root (vr-L5) at different intensities evoked graded short-latency synaptic potentials. Scale bar = 10 μm. (F) V1^R interneurons (GL: 6/11 cells; TA: 1/11 cells; IF: 0/11 cells) receiving monosynaptically-evoked EPSPs from peripheral muscles. (G) Monosynaptic EPSPs in a V1^R interneuron (black) or GL motor neuron (gray) following GL muscle stimulation. (H) Short-latency EPSPs evoked in V1^R interneurons after GL stimulation (3 superimposed responses evoked at 0.1 Hz stimulation frequency, black). Stimulation from TA (gray) or IF (brown) muscle sensory fibers resulted in long (>20 msec), variable latencies, indicative of polysynaptic activation. Arrow, stimulation artifact. (I) Left, latencies from synaptically evoked responses in V1^R interneurons after stimulation of GL, TA, and IF muscles in p4 to p5 mice. The latency from GL muscle stimulation was significantly shorter than from TA or IF (p < 0.05, one-way ANOVA). Right, coefficient of variation (CV_onset) of synaptic response latency. Red line = mean. (J) Assay of proprioceptive sensory input onto V1^Sp8 interneurons. (K-L) CTB⁺; vGluT1⁺ proprioceptive input to V1^Sp8 interneurons. (L) V1^Sp8 interneurons with pool-specific input. p = 0.004, one-way ANOVA; Bonferroni post hoc test: p < 0.01, GL vs TA; p < 0.001, TA vs IF; n.s., GL vs IF, n ≥ 3 animals. Average CTB⁺; vGluT1⁺ input density/100 μm of V1^Sp8 dendrite: GL: 0.92 ± 0.23; TA: 2.20 ± 0.14; IF: 0 ± 0; p < 0.0001, oneway ANOVA; Bonferroni post hoc test: p < 0.01, GL vs TA; p < 0.05, GL vs IF; p < 0.001, TA vs IF. Similar labeling efficiency was observed for GL, TA, and IF sensory afferents (Figure S7C). Scale bar = 2 μm. See also Figure S7.

Figure 7. Specificity of Interneuron-Motor Neuron Interconnectivity at Individual Joints

(A) Assay of pool-specific motor input to interneurons. (B-C) V1^R interneurons receive CTB⁺; vAChT⁺ input from GL and TA (arrows) but not IF motor neurons (MN). Scale bar = 2 μm. (C) Left, V1^R interneurons with input from GL, TA, or IF MNs. p = 0.02, one-way ANOVA; Bonferroni post-hoc test: p < 0.05, GL or TA vs IF. Right, CTB⁺ MN inputs/100 μm of V1^R dendrite length. p = 0.002, one-way ANOVA; Bonferroni post-hoc test: p < 0.01, GL or TA vs IF; p > 0.5, GL vs TA, n ≥ 3 animals, and 23 (GL), 24 (TA), or 15 (IF) cells. (D-E) Absence of MN input to V1^Sp8 interneurons. GL, n = 4 animals, 43 cells; TA, n = 2 animals, 52 cells; IF, n = 3 animals, 43 cells. (F) Assay of interneuron input onto motor pools. (G-H) V1^R interneurons preferentially innervate GL and TA relative to IF motor pools, on proximal MN dendrites (H, left) or soma (H, right). p < 0.0001, one-way ANOVA; Bonferroni post hoc test: p < 0.001, GL or TA vs IF, n = 4 animals, and 31 (GL), 21 (TA), or 27 (IF) cells. (I-J) V1^Sp8 interneurons sparsely and uniformly innervate motor pools acting on different joints. Number of V1^Sp8 inputs/100 μm MN dendrite or 100 μm² of soma area, normalized to V1^Sp8 interneuron number. p = 0.53 or 0.65 for dendrites and soma, respectively, one-way ANOVA, n ≥ 3 animals, 35 (GL), 42 (TA), or 59 (IF) cells. Scale bars = 2 μm. All data are mean ± SEM. (K) V1^R and V1^Sp8 microcircuits operating on hip, ankle, and foot motor neurons. Solid and dotted lines represent prevalent and sparse synaptic connectivity. See also Figure S7.