The crawling motor pattern.

A. Schematic description of a leech crawling step that results from coordinated waves of elongation (i-ii) and contraction (iii-iv) phases, anchored on front and rear suckers. B. Intracellular recording of CV and extracellular recording of DE-3 (in DP nerve) motoneurons during a dopamine-induced crawling episode in an isolated midbody ganglion. Recording diagram on the left. C. Putative neuronal circuitry underlying crawling and the recurrent inhibitory circuit in a midbody ganglion. Units C and E correspond to contraction and elongation units of the segmental oscillator, respectively. DE-3 and CV are examples of motoneurons involved in each phase. NS neuron is connected to DE-3 and CV (Rela and Szczupak, 2003; Rodriguez et al., 2009) through chemical and electrical synapses; the + and - symbols indicate the polarity at which the rectifying synapse conducts.

NS activity correlates with the crawling motor pattern.

A. NS intracellular recording, DP extracellular recording and DE-3 binned firing frequency (bFF) during a crawling episode. NS recording was resampled to match the data rate of DE-3 bFF. Recording diagram on the left. The blue vertical line marks the beginning and the blue dotted line the end of each DE-3 burst. Different crawling features and NS hyperpolarization amplitude (NS hyp amplitude) are indicated. B. Cross-correlograms of the DE-3 bFF curves and NS resampled traces. Each gray trace corresponds to an individual experiment. The black curve corresponds to the mean cross-correlogram. (n = 23 crawling episodes containing 5 cycles, obtained in 23 ganglia from 18 leeches). C. Successive cycles of the NS recording shown in panel A, segmented at the times indicated by the dotted lines and aligned by the times indicated by solid blue lines indicated in A. D. NS hyperpolarization amplitude as a function of DE-3 Burst FF. E. Like in D, as a function of DE-3 burst duration. F. Like in D, as a function of interburst lag preceding DE-3 bursts. G. Like in D, as a function of the interburst lag after DE-3 bursts. For D-G, n = 19 crawling episodes, with 5 cycles each. In D to G, the correlation was tested using linear mixed-effects models, contemplating different experiments as a random variable. In D and G, p>0.05, and in E and F, p<0.05. The lines correspond to regressions obtained from the linear mixed-effects model. The coefficients were 0.374 ± 0.089 mv/s for E and 0.122 ± 0.034 mv/s for F.

Effect of NS on crawling.

A. Intracellular NS and extracellular DP recordings during a dopamine-induced crawling episode. A +7 nA pulse was injected in NS at the time indicated by the thick line. Segments above the recordings mark three epochs: previous (pre), during (test) and after (post) the pulse. B. Crawling period (end-to-end), DE-3 mFF, burst duration and duty cycle for cycles in the three epochs marked in A, for control experiments where no current was injected in NS (gray violins, n= 120 cycles from 26 episodes in 26 ganglia from 16 leeches) and for experiments where NS was depolarized (light blue violins, n= 92 cycles from 19 episodes in 19 ganglia from 15 leeches); each point corresponds to the mean value during the epoch for each individual experiment. Bars provide the mean ± SEM. *** p<0.001 (interaction between epoch and treatment factors). C. Mean DE-3 mFF for the three epochs from each individual experiment for control (CTRL) and depolarizing (DEPO) tests. Lines link dots that correspond to each individual experiment. *** p<0.001 (pairwise simple contrasts, Δ pre-test = -15.591 ± 1.037 Hz and Δ test-post = 16.648 ± 0.919 Hz).

Profile of motoneuron activity during crawling.

A.i. Representative example of a spike sorting analysis implemented in paired extracellular recordings of DP and PP nerves during a crawling episode. Each color dot singles out a distinct active unit along the recordings. A.ii. Spikes waveforms corresponding to spikes detected in the DP and PP nerves in Ai. A.iii. bFF calculated for each unit identified in A. The horizontal dotted lines indicate bFF = 0 Hz. Scales on the right are in Hz. B. Raster plot showing mean bFF along a cycle, set from the first spike of a DE-3 burst to the first spike in the next burst, obtained from 147 rhythmic units identified in DP, PP and AA nerves (n = 46 ganglia in 24 animals). The values are the mean of 4 crawling cycles for each unit. The units were classified into four groups, indicated on the left. The nerves from which the motoneuron activity was recorded are indicated on the right. The gray scale on the right indicates the bFF value in Hz. C. Mean bFF for all the units included in each group. The shaded area represents the standard error of the mean.

Analysis of leech crawling.

A. Schematic representation of a leech with nine points as painted on their dorsal midline. The sections between points 4-5 and 5-6 comprise ganglia 8-15 that were used for the electrophysiological analysis. B. Relative position of midbody ganglia 1–21. Mean and standard error of the mean are shown (n = 3) (Kearney et al. 2022). C. Representative example of the changes in total length and section 4-5 of a leech during four steps as the animal was crawling. The limits of each step are indicated by black vertical lines (see Materials and Methods); red and blue vertical lines mark the start of elongation and the end of contraction, respectively; black dashed vertical lines indicate the maximal section length. Light red and blue shades indicate elongation and contraction phases, respectively. D. Superimposed length curves of a section across random multiple steps (n= 15 of 76 total steps for this animal) of one leech. E. Duty Cycle of the elongation and contraction phases measured in crawling behavior or in fictive crawling. Behavioral variables were calculated as the duration of each phase relative to the sum of elongation and contraction phases (dynamic step in C), excluding isometric phases. Points represent the mean for each leech, while bars and lines indicate the mean ± SEM across all leeches (n = 249 steps from 8 leeches). For the Duty Cycle of DE-3 and Anti-Phase units, points correspond to the mean for each ganglion, with bars and lines showing the mean ± SEM across all ganglia (n = 4 cycles for each DE-3 or Anti-Phase motoneuron, 6 ganglia, 6 leeches).

Effect of NS on the different phases of crawling.

A.i. Mean bFF along cycles in pre, test, and post epochs for each unit within the four groups described in Figure 4. For each unit type the upper line of graphs presents control experiments, where no current was injected in NS, and the bottom line, depolarization experiments, where 5-7 nA current steps were injected in NS during the test epoch. Thin lines show the mean bFF (including 4 - 6 cycles) for each experiment, and thick lines show the overall mean. A.ii. Superimposition of the overall bFF mean of the three epochs. The shaded area presents the standard error of the mean. B & C. Maximum bFF (Max bFF) and Relative Half Width (Relative HW) measured in each individual unit. A-C are plotted for each of the four groups of units indicated. Lines connect the mean values across each epoch within the same experiment. Mixed-effects models with two fixed factors were performed; on the left of the B and C panel, # indicates a significant interaction between treatment and epoch factors; * indicates significant differences for pairwise simple contrasts between epochs, within each treatment. *, ** and *** (or #) indicate p<0 .05, p<0.01 and p<0.001, respectively. Supplementary Table 2 describes the statistical models performed. For DE-3, n= 26 control and 20 depolarization ganglia, from 46 ganglia; for In-Phase, n= 38 units for control and 21 units for depolarization, from 30 ganglia; for Anti-Phase, n= 18 units for control and 7 units for depolarization experiments, from 22 ganglia; and for In-Phase-Early-Onset, n= 15 units for control and 8 units for depolarization experiments, from 23 ganglia.

NS in the crawling circuitry.

Putative neuronal circuitry as described in Figure 1C, including the connection between the oscillatory circuit and NS.

Statistical Models Performed for the Effect Analysis of NS depolarization on crawling features and DE-3 activity during the fictive behavior.

Statistical Models Performed for the Effect Analysis of NS depolarization on different groups of units activity during crawling.