Spinal Locomotor Circuits Develop Using Hierarchical Rules Based on Motorneuron Position and Identity

Abstract

The coordination of multi-muscle movements originates in the circuitry that regulates the firing patterns of spinal motorneurons. Sensory neurons rely on the musculotopic organization of motorneurons to establish orderly connections, prompting us to examine whether the intraspinal circuitry that coordinates motor activity likewise uses cell position as an internal wiring reference. We generated a motorneuron-specific GCaMP6f mouse line and employed two-photon imaging to monitor the activity of lumbar motorneurons. We show that the central pattern generator neural network coordinately drives rhythmic columnar-specific motorneuron bursts at distinct phases of the locomotor cycle. Using multiple genetic strategies to perturb the subtype identity and orderly position of motorneurons, we found that neurons retained their rhythmic activity-but cell position was decoupled from the normal phasing pattern underlying flexion and extension. These findings suggest a hierarchical basis of motor circuit formation that relies on increasingly stringent matching of neuronal identity and position.

Figure 1. GCaMP6f reliably reports neural activity in spinal motorneurons

A) Electrical stimulation of the ventral root (black ticks) evoked calcium signals in LMC (cyan) and MMC (green) motorneurons. Increasing numbers of stimuli evoked larger amplitude, longer duration responses. LMC and MMC motorneurons respond with similar kinetics and summation to ventral root stimulation, but signals are larger in the LMC. Inset, diagram of experimental setup. Hb9:GCaMP6f signals were imaged through the ventral surface of the spinal cord. B) Increasing numbers of stimuli evoked a linear increase in the response amplitude of spinal motorneurons. Amplitudes normalized to response amplitude of the single stimulus ΔF/F. C) Single stimuli evoked fast rising, exponentially decaying responses. Trains of 3 stimuli at 2.5 and 5Hz evoked separable fluorescence peaks corresponding to each stimulus with temporal summation. Stimulation at rates faster than the imaging frame rate (10 Hz stimuli, 8.3 Hz imaging) generated larger responses without detectable peaks from individual stimuli. D) Examples of neurochemically (NMA and serotonin) evoked motorneuron electrical and GCaMP6f signals in individual motorneurons Electrical signals (black) and raw imaging signals (green) are superimposed. Motorneuron activity related GCaMP6f fluorescence signals are evident for isolated single spikes (top), spike bursts (middle) and tonic firing (bottom). E) Phase contrast (Dodt) and fluorescence image of visually targeted cell attached recording from a GCaMP6f expressing motorneuron (bursting cell in top panel).

Figure 2. Comparison of LMC and MMC locomotor oscillations

A) Single optical section of Hb9:GCaMP6f expressing motorneurons in L2. Example LMC and MMC neurons are highlighted in cyan and green, respectively. Lateral is up rostral is right. Scale bar 100 μm. B) Raw imaging signals from the motorneurons in A. Following neurochemical induction of fictive locomotor activity (10 μM NMA and 20 μM 5HT) fluorescence oscillations in LMC (cyan traces) and MMC (green traces) alternate with the electrically recorded activity in the contralateral L2 ventral root (Bottom, black trace). Traces from LMC and MMC neurons highlighted in A are bold. Scale bar 100% ΔF/F. C) Left) Expanded single locomotor cycle with overlaid imaging traces and contralateral ventral root electrical activity. L2 Imaging oscillations alternate with the contralateral L2 ventral root bursting. Right) Polar plot of imaging signal phase calculated relative to bursting in the contralateral L2 ventral root. Points are individual L2 motorneurons. D) Schematic of motorneuron positions and amplitude correlations in a single L2 optical section. Points represent motorneuron soma positions colored according to relative strength of their correlations to LMC (cyan) or MMC (green). A majority of motorneurons are more strongly correlated within a motor column than across motor columns. E) Example traces from pairs of LMC and MMC neurons numbered in D. Amplitude modulation patterns for motorneurons within the same column were more similar than those across motorneuron columns.

Figure 3. LMC and MMC display distinct phase patterns along the rostral caudal axis

A) Hb9:GCaMP6f images from lumbar segments L2 –L5 highlighting LMC (L2-L3 cyan; L4-L5 orange) and MMC (green) motorneurons. Scale bar 100 μm. B) Fluorescence intensity was measured across the population of neurons comprising each motor column. The phase of LMC motorneurons changes at the L3-L4 border (cyan to orange), while MMC neurons retain a similar phase along the lumbar enlargement. Below, LMC and MMC bursts superimposed over the contralateral L2 ventral root recording. C) Phase analysis of individual motorneuron imaging signals in the upper lumbar spinal cord. A majority of L2-L3 LMC motorneurons are flexor active with phase values centered around 0 radians. (top). Similarly, a majority of MMC motorneuron are flexor active (bottom). A common color coding scheme is used for all remaining figures. Rhythmic neurons with phase values in the flexor range (0 ± 1 radians) are colored cyan for LMC and light green for MMC. Rhythmic neurons with phase values in the extensor range (π ±1 radians) are colored orange for LMC and dark green for MMC. Rhythmic neurons with phase values outside the flexor and extensor ranges are colored grey. D) Phase analysis of individual motorneuron imaging signals in the lower lumbar spinal cord. A majority of L4-L5 LMC motorneurons are extensor active with phase values centered around π radians (77.5 +/− 21.7%; orange, top). Fewer MMC neurons are present in L4-L5 lumbar levels than L2-L3, perhaps representing the transition from cells that control axial muscles to those involved in tail movements. A small but increasing fraction of extensor-active MMC cells are detected in L4-L5 (dark green) (14.7 +/− 12%) relative to L2-L3.

Figure 4. Intracolumnar position predicts LMC motorneuron activity

A) Single optical section of Hb9:GCaMP6f expressing L4 motorneurons. Neurons in the LMCl and LMCm are highlighted in cyan and orange, respectively. MMC neurons are highlighted in green. Scale bar 100 μm. B) Locomotor activity traces from motorneurons highlighted in A. Two distinct phase groups are detected in the LMC. Flexor active the LMCl alternates with extensor active LMCm. MMC neurons (green) burst in phase with the LMCl (cyan). C) Scatterplot of motorneuron phase versus medio-lateral position separates three distinct motorneuron populations: LMCl, LMCm and the MMC. Horizontal lines are mean +/− sd of medio- lateral position for the phase categories. D) Polar plot of phase analysis from the motorneurons in A. Two phase groups characterize L4 motorneuron activity, the LMCl and MMC (cyan, green) are in phase (flexor active), whereas the LMCm is shifted ~0.5 cycles (extensor active). Inset: overlaid signals from time series in panel B highlighting relative phases in a single locomotor cycle.

Figure 5. Lumbar motor column structure

A–C) Lumbar motor column structure revealed by whole mount Hb9 antibody staining in wild type, Foxp1 ^MN, and Lhx3^ON spinal cords. D–F) Plots of motorneuron density on the medio-lateral axis in the mid lumbar spinal cord. Peaks in the density plots reflect the columnar organization of motorneuron somata detected by Hb9 immunostaining. G–I) Lumbar motor column structure revealed by ChAT immunostaining of transverse sections in wild type, Foxp1 ^MN, and Lhx3^ON spinal cords. Scale bar 100 μm. J–L) Wild type spinal cords are characterized by two distinct motor columns spanning the lumbar enlargement, with a larger number of motorneurons in the LMC relative to the MMC. In the Foxp1 ^MN spinal cord MMC cells are unaffected but LMC motorneurons settle more medially an in ectopic position (HMC*) and are absent from lateral positions. Motorneuron in Lhx3^ON spinal cords form distinct lateral (LMC*) and medial (MMC*) columns although their relative sizes are altered.

Figure 6. Locomotor network activity is preserved for motorneurons in ectopic positions

A) Neurochemically evoked fictive locomotor bursting recorded from the ventral roots in a wild type e18.5 spinal cord. Rhythmic bursts of activity alternate between ipsi and contralateral ventral roots (iL2, cL2). B) Neurochemically evoked alternating, rhythmic bursting is retained in Foxp1 ^MN spinal cords. C) Wild type and Foxp1 ^MN spinal cord fictive locomotor activity was not significantly different in cycle period (wild type 4.04 ± 0.9 s; Foxp1 ^MN 4.0 ± 0.46 s, p=0.96) or in cycle variability (wild type 0.16 ± 0.016: Foxp1 ^MN 0.08 ± 0.006; p=0.12; coefficient of variation). D) In Foxp1 ^MN spinal cords ipsi and contralateral L2 bursting alternates (blue) with mean phases clustered around 0.5 cycles, similar to wild type bursting (black). Points represent average values from ~20 cycles in single spinal cords. E) Image of GCaMP6f expressing Foxp1 ^MN motorneurons in L4. GCaMP6f signals from the highlighted Foxp1 ^MN motorneurons revealed coordinated, network driven oscillations in Foxp1 ^MN independent of their medial-lateral positions. F) Similar fractions of wild type and Foxp1 ^MN motorneurons are rhythmically active during neurochemically induced locomotor activity (88.4 ± 10.5 % wild type; 94.2 ± 3.3% Foxp1 ^MN; p=0.09). G) Phase distributions of L2-L5 HMC* and MMC in Foxp1 ^MN spinal cords. Similar proportions of HMC* and MMC are co active with L2 with phase values between 0±1 radians. (p=0.21).

Figure 7. Imaging Lhx3^ON locomotor activity

A) Single optical section of Hb9:GCaMP6f expressing Lhx3^ON L2 motorneurons. Example LMC* (cyan) and MMC* (green) neurons are highlighted. Scale bar 100 μm. B) Locomotor activity traces from the image in A. Fluorescence oscillations of LMC* (cyan) and MMC* (green) alternate with contralateral L2 ventral root activity (black). Cells highlighted in A are in bold. Inset, single locomotor cycle from imaging traces and contralateral L2 ventral root recording showing the stereotypical anti-phase relationship between ipsi and contralateral L2 activity patterns. C) Phase distributions of wild type and Lhx3^ON L3-L4 MMC neurons. Similar to the wild type MMC, a majority of Lhx3^ON MMC* motorneurons are flexor active (in phase with L2 imaging signals), with phase values clustered near zero. D) Comparison of wild type and Lhx3^ON LMC phase distributions. The activity of L3-L4 LMC motorneurons in the wild type spinal cord coalesces into flexor and extensor active populations. Flexor and extensor active LMC* are found in similar proportions to wild type. In the Lhx3^ON spinal cord increased numbers of intermediate phase LMC* (neither flexor or extensor) were observed relative to wild type LMC (black bars). Wild type, median 1.97 %, range 0–9.4% of LMC; Lhx3^ON, median 17.63%, range 1.7–24.4%, p=0.047. E–F) Reconstructions of motorneuron activity phase in L3 (top panels), and L4 (bottom. panels). Points are individual motorneuron positions colored by activity phase. Reconstructions are aligned in the medial-lateral axis relative to the lateral edge of the spinal cord. Flexor phase: LMC = Cyan, MMC=green, extensor phase: LMC=Orange, intermediate phase=Black. E) In the wild type cord, flexor active motorneuron (cyan) are generally lateral to extensor motorneurons (orange). F) In the Lhx3^ON spinal cord, the mediolateral segregation of flexor and extensor neurons is lost. Intermediate phase neurons (black) are also intermingled with flexor (cyan) and extensor (orange) active LMC*. Reconstructions are aligned in the medial-lateral axis by the lateral edge of the spinal cord. G–H) Summary histograms of L3-L4 motorneuron positions and activity classifications. In the wild type spinal cord, the positions of flexor active LMCl (cyan) are shifted laterally relative to extensor active LMCm (orange, * p=0.0063). In the Lhx3^ON spinal cord, flexor, extensor and intermediate active motorneurons are intermingled with similar positions on the medio-lateral axis (ns, p=0.21). I) Specific modules of the locomotor CPG have distinct dependencies on motorneuron position and identity. Core features of the CPG network, rhythmic drive and left-right coordination, are wired independently of motorneuron identity and position, however, normal musculotopic motorneuron activity patterns are not preserved in the absence of proper LMC identity in Lhx3^ON mutants. Loss of lateral motorneuron identity generates new activity patterns (black) potentially from abnormal mixing inputs onto motorneurons.