Preferred locomotor phase of activity of lumbar interneurons during air-stepping in subchronic spinal cats

Abstract

Spinal locomotor circuits are intrinsically capable of driving a variety of behaviors such as stepping, scratching, and swimming. Based on an observed rostrocaudal wave of activity in the motoneuronal firing during locomotor tasks, the traveling-wave hypothesis proposes that spinal interneuronal firing follows a similar rostrocaudal pattern of activation, suggesting the presence of spatially organized interneuronal modules within the spinal motor system. In this study, we examined if the spatial organization of the lumbar interneuronal activity patterns during locomotor activity in the adult mammalian spinal cord was consistent with a traveling-wave organizational scheme. The activity of spinal interneurons within the lumbar intermediate zone was examined during air-stepping in subchronic spinal cats. The preferred phase of interneuronal activity during a step cycle was determined using circular statistics. We found that the preferred phases of lumbar interneurons from both sides of the cord were evenly distributed over the entire step cycle with no indication of functional groupings. However, when units were subcategorized according to spinal hemicords, the preferred phases of units on each side largely fell around the period of extensor muscle activity on each side. In addition, there was no correlation between the preferred phases of units and their rostrocaudal locations along the spinal cord with preferred phases corresponding to both flexion and extension phases of the step cycle found at every rostrocaudal level of the cord. These results are consistent with the hypothesis that interneurons operate as part of a longitudinally distributed network rather than a rostrocaudally organized traveling-wave network.

Fig. 1.

Single unit activity and EMG activity during a bout of air-stepping. Top: example of a well-isolated single unit examined in this study. The raw voltage trace from a single channel is shown with spikes from a well-isolated unit marked by red circles. Superimposed spike waveforms from this unit are shown (inset, top left). Middle: the spike rate of the above unit. The spike rate was estimated by convolving a 100-ms Gaussian kernel with the spike train. Bottom: corresponding EMG activity envelopes (see materials and methods) from 7 muscles, 3 flexors (1 bifunctional), and 4 extensors, from each hindlimb. The 3 flexor muscles are plotted against blue background. Vertical black lines indicate the onset of right soleus activity. A right step cycle is defined as the period between the onsets of soleus activity (i.e., period between 2 vertical lines). For this unit, the spike rate is nearly constant before the onset of locomotion. In contrast, phasic modulation of the spike rate is apparent for the duration of air-stepping.

Fig. 2.

Fluorescent photomicrograph of a 25-μm-thick transverse spinal cord section taken at the caudal end of the L3 spinal segment in 1 animal. The electrode arrays were coated with DiO (Molecular Probes, Eugene OR) and inserted at 2,000-μm depth with the center of the two arrays aligned on the cord midline (the distance between the 2 electrodes array was ∼1,500 μm). Electrodes tracks can be seen extending to the dorsal surface of the cord with the fluorescence being strongest at the dorsal aspect. The depth of the most ventral signs of fluorescence and the medio-lateral positions of the tracts match the stereotaxic coordinates (the most ventral fluorescence for the array on the right was on an adjacent section). The yellow box indicates the assumed region covered by the electrode array on the left based on the distances between shanks and sites. Rexed laminae for the L3 segment are overlaid in gray and indicate that recording sites were indeed located within the deep intermediate horns (laminae V–VII).

Fig. 3.

Determining preferred phase of firing of an isolated unit with circular statistics using the unit shown in Fig. 1 as an example. A step phase value is calculated for each spike in a given spike train (illustrated in red). The step phase value is the ratio between the relative time of occurrence from the onset of its containing step and the duration of the entire step cycle. A histogram of the step phase values for all spikes in a spike train is generated with 50 equal size bins from 0 to 1 (i.e., the range of the step phase value). Since stepping is a repetitive act and each bin of the histogram represents the spike counts from particular phases of a step cycle, the histogram values can be interpreted as the magnitude of 50 vectors pointing in equally spaced direction around a circle. This interpretation is best represented by a polar plot as shown on the right. The vector average of these vectors produces a mean vector whose direction is the preferred phase for a unit. To summarize, the preferred phase is the point within the step cycle where a unit, on average, fires maximally.

Fig. 4.

A: preferred phase values (relative to the right step cycle) of all units from 5 animals (different markers) with units' colors based on the side of the spinal cord they were recorded from (left in red; and right in yellow). Twenty-bin histograms of pooled phase values of units from the left (red) and right (gray) side of the spinal cord are shown (inset). B: the preferred phases were widely distributed throughout the right step cycle as seen in B showing the rank-ordered preferred phases and angular deviations of each unit. The horizontal axis indicates the phase value relative to the right step cycle. Note that preferred phases of units recorded from the left side of the cord are localized in the middle of the right step cycle (see preferred phase histogram in A and the rank order plot in B), whereas units from the right side are localized near the beginning and end of the right step cycle. C: polar representation of pooled preferred phases with respect to a normalized right step cycle (from 0 to 1) from all animals, regardless of their hemispheric location. D: polar representation of preferred phases from units recorded on the left (red circles) and right (yellow circles) side of the spinal cord. The average preferred phase of units from left side is 0.55 ± 0.16, as indicated by the red arrowhead. The average preferred phase of units from right side is 0.9 ± 0.21, as indicated by the yellow arrowhead. Average angular deviation for all units was 0.53 ± 0.11 with over 40% of the units having an angular deviation less than 0.5 and fewer than 12% having an angular deviation greater than 0.65.

Fig. 5.

Twenty-bin histograms of preferred phases from units recorded from the left (red) and right (gray) side of the spinal cord are shown for each of the 5 animals. Units' preferred phases segregated based on the side of the cord from which they were recorded for each of the 5 animals.

Fig. 6.

Preferred phases of muscle activity. A: example of the wrapped Gaussian fitting procedure used to determine the mean phase of muscle activity. Least square fitting was performed on the average EMG activity histogram profiles for 4 exemplar muscles, 1 flexor and 1 extensor muscle from each hindlimb. The mean phase of activity was determined relative to the step cycle of the hindlimb ipsilateral to the unit's hemispheric location. B: polar representation of the average (black tick mark) and standard deviation (indicated by the angular width of the wedge) of mean phase of activity of a single flexor and extensor muscle from the ipsilateral and contralateral side with respect to the hemispheric location of the unit. The average mean phases of ipsilateral flexors and extensors were 0.79 (0.081 SD) and 0.19 (0.077 SD), respectively. The average mean phases of contralateral flexors and extensors were 0.29 (0.079 SD) and 0.70 (0.079 SD), respectively. C: preferred phases of muscle activity for all extensors and flexors across all cats. The dots indicate the individual preferred phases of the average binned EMG activity profile for each of the muscles recorded. The wedges indicate the range of preferred phases for each of the muscle groups (ipsi- and contralateral flexors and extensors). Flexors and extensors showed similar preferred phases of activity for both the ipsi- and contralateral hindlimbs.

Fig. 7.

Scatter plots of the mean phases of 1 flexor and 1 extensor activity for the ipsilateral and contralateral hindlimbs vs. the preferred phases of ipsilateral units are plotted separately on the left. These individual scattered plots are combined to generate the summary plot on the right. Unity lines are also shown for reference. Muscle activity showed no correlation with neuronal preferred phases for any of the muscles studied as shown for the individual plots or the combined plot. Muscle color legend as in Fig. 6.

Fig. 8.

Histograms of the coefficients of determination (R², i.e., squared Spearman correlations values) between each spike train and all of the corresponding ipsilateral or contralateral flexors or extensors activity (P < 0.05 for all correlation values shown). The coefficient of determination values between the vast majority of the units and any of the muscles studied were weak (≤0.5) with few units exhibiting large values (>0.5) for any muscle. The activity of 14 units was found to correspond well (coefficient of determination >0.05) with the activity of the ipsilateral extensor. These results indicate that very few interneurons were tightly tuned to the muscular activity of the flexors and extensors sampled in this study.

Fig. 9.

A representative example of the single units' preferred phases from 1 animal plotted against their rostrocaudal recording locations. The plot on the left shows the relationship when preferred phase is measured relative to the left step cycle, and the plot on the right is for when the phase is measured relative to the right step cycle. Preferred phases did not correlate with rostrocaudal localization for the units studied.

Fig. 10.

Binned (5-mm bin size) rostrocaudal distribution of the number of ipsilateral units with preferred phases in the 1st (<0.3), 2nd (>0.3 and <0.6), and 3rd (>0.6) portion of the ipsilateral step cycle. Interneurons located ipsilaterally largely fall within the 1st and last third of the ipsilateral step cycle for all 5 animals, whereas interneurons located contralaterally fall within the middle third of the step cycle.