Functional Organization of the Spinal Locomotor Network Based on Analysis of Interneuronal Activity

Abstract

Locomotion is a vital motor function for any living being. In vertebrates, a basic locomotor pattern is controlled by the spinal locomotor network (SLN). Although SLN has been extensively studied, due to technical difficulties, most data were obtained during fictive locomotion, and data about the activity of spinal neurons during locomotion with intact sensory feedback from limbs are extremely limited. Here, we overcame the technical problems and recorded the activity of putative spinal interneurons from spinal segments L4-L6 during treadmill forward locomotion evoked by stimulation of the mesencephalic locomotor region in the decerebrate cat. We found that neurons were activated and inactivated preferably within one of the four phase ranges presumably related to preparation for the limb lift-off, the limb lift-off, transition from the limb flexion to limb extension during swing, and the limb touch-down. We analyzed the activity phases of recorded interneurons by using a new method that took into account the previously ignored information about the stability of neuronal modulation in the sequential locomotor cycles. We suggested that neurons with stable modulation (i.e., small dispersion of their activity phase in sequential cycles) represent the core of SLN. Our analysis revealed groups of neurons active approximately out of phase and presumably contributing to the control of vertical (VC) and horizontal (HC) components of the step. We found that most VC- and HC-related neurons were located in the intermediate and dorsal/ventral parts of the grey matter, respectively. Our experimental data can be used as a benchmark for computational models of locomotor neuronal networks.

Keywords: decerebrate cat; locomotion; mesencephalic locomotor region; neuronal network; spinal cord; spinal neurons.

FIGURE 1

Recording of the activity of spinal neurons during MLR‐evoked locomotion. (A) Experimental design. MLR‐stim, an electrode for stimulation of the mesencephalic locomotor region. MEA, a microelectrode array for recording neuronal activity. Limb‐L, a mechanical sensor recording anterior/posterior movements of the left hindlimb (a sensor recording the right hindlimb movements is not shown). FP, a force plate. (B–D) An example of 18 spinal neurons recorded simultaneously in L6 during locomotion. Red and green, neurons with stable and unstable modulation, respectively. Blue, non‐modulated neurons. (B) Locations of the neurons. (C) Superimposed spikes of individual neurons extracted from the mass activity. (D) Activity of individual neurons (##1–18) during locomotion recorded along with hindlimb movements (Limb‐R, Limb‐L) and contact forces (Force‐R, Force‐L). Swing phases of the left hindlimb are highlighted.

FIGURE 2

Analysis of the activity of spinal neurons during MLR‐evoked locomotion. (A–E) An example of analysis of activity (neuron #4 from Figure 1B–D). For each individual step, a spike raster aligned at the swing start (A), the instantaneous firing rate versus time (B), the rate versus phase with dual‐reference phase normalization (C), and the burst phase (D) were determined. Burst phases in individual steps were used to calculate the average and SD for burst onset and offset phases (E).

FIGURE 3

Phase distribution of bursts of individual neurons recorded in L4 and L6 segments in the normalized locomotor cycle. Neurons recorded in each segment are ranked according to the value of their parameter W (integral stability of the activity phase) shown on the right panel in logarithmic scale. Bursts of individual neurons recorded in L4 and L6 are indicated by thick red and blue lines, respectively. Activity of the same neurons in the preceding and the following cycles is indicated by the corresponding pale colors. Thin black lines indicate standard deviations of the burst onset and offset phases.

FIGURE 4

Important phases of the locomotor cycle. (A–D) Distributions of phase probability density (A, B) and phase weighted probability density (C, D) for the burst onsets (A, C) and offsets (B, D) for all (L4 + L6) neurons. (E, F) Phases of peaks (parts of the curve above the average level indicated by dotted line in A–D) and major peaks (above the doubled average level indicated by dashed line in A–D) of probability (E) and weighted probability (F) distributions are shown separately for L4 and L6 neurons. Thin and thick lines and small and large diamonds indicate peaks and major peaks and the peak tops, respectively. Only significant peaks were taken into account: If a peak would disappear after the exclusion of one neuron with w = 1, this peak was considered not significant. Additionally, two neighboring peaks were fused into one if they were separated by an insignificant trough that would disappear after the addition of one neuron with w = 1. Practically all peaks fell into four phase ranges I–IV (indicated by yellow in E, F).

FIGURE 5

Phase distribution of sorted into Clusters bursts of individual neurons with stable modulation recorded in L4 and L6 segments in the normalized locomotor cycle. The mean bursts of individual Clusters with the middle point indicated (white circles) are shown by thick purple lines. Neurons of different Clusters are demarcated by dotted lines. Clusters are ordered according to the midburst phase. Numbers in circles on the left are Cluster numbers. Four phase ranges I–IV are indicated by yellow. Other abbreviations as in Figure 3.

FIGURE 6

Functional classification of Clusters. (A) Heatmap of the phase shift between midbursts of different Cluster pairs. All Clusters can be combined into four pairs of Groups active approximately out of phase (with phase shifts close to 0.5, outlined by four dashed rectangles): Gr1/Gr2, Gr3/Gr4, Gr5/Gr6, and Gr7/Gr8. (B) The paw trajectory during the locomotor cycle. Green, red, yellow, and blue parts of the trajectory are, respectively, forward (F), backward (B), upward (U), and downward (D) limb movements. (C) Groups are formed by Clusters with similar midburst phases (each color band corresponds to one Group). These Groups can be assigned to the horizontal (HC) or vertical (VC) components of the step (see explanations in the text and Figure S7). Green and red stars show the middle of the swing and stance, respectively.

FIGURE 7

Location of individual neurons of different Clusters in the spinal cord. VC network neurons and HC network neurons are shown separately on cross‐sections of the gray matter at spinal segments L4 and L6. Thick hatched lines demarcate the dorsal, intermediate, and ventral zones of the gray matter. Designations (shapes and colors) are as in Figure 6C.

FIGURE 8

Structure of the spinal locomotor network controlling forward stepping.