Article Figures & Data
Figures
- Figure 1.
Overt crawling behavior after complete transection of the M2/M3 interganglionic connectives. A,B, Movement raster plots depicting elongation (E, black boxes) and contraction (C, gray boxes) movements over time for individual leeches that received a sham surgery (A) and an M2/M3 transection surgery (B). Movements are shown for time points before the surgery (Pre), 1, 7, and 21 days after surgery. C, Graph of crawling activity at several time points after surgery for a random subset of the leeches used in Figs. 2–6 (n = 10 for each group). Error bars denote the standard deviation. *, statistically significant differences (p < 0.01). D, Schematic depicting a truncated view of the CNS of a leech. All nerve recordings showing fictive locomotor activity were obtained from the dorsal posterior nerve root (DP nerve), which contains the axon of the motoneuron DE-3. Although all 21 segmental ganglia have DPs, only the DP associated with mid-body ganglion M8 (DP(8)) is illustrated here. Diagonal hash marks denote ganglia that were removed to simplify the cartoon.
- Figure 2.
Fictive crawling-like activity of an in vitro whole recovered nerve cord treated with dopamine. A, Left, schematic of a nontransected control isolated nerve cord preparation. Traces, three dorsal posterior (DP) nerve root recordings from a control nerve cord treated with 100 µM dopamine. Inset, time-expanded view of several bursts from the traces showing intersegmental coordination among the ganglia. Boxes denote the source of the time-expanded traces. B, Left, schematic depicting a recovered whole nerve cord (M3-TG) from an M2/M3 transected leech that recovered crawling behavior. Traces, three DP nerve recordings of a recovered whole cord treated with 50 µM dopamine. Insets, time-expanded views of several time-spans of the recording presented. Boxes denote the sources of the time-expanded traces. Note: The largest unit in all DP recordings is the dorsal longitudinal excitor-3 motoneuron (DE-3).
- Figure 3.
Quantification of whole recovered cord data. A, Boxplots of the mean cycle period of DE-3 bursting of isolated whole nerve cords treated with dopamine. White boxes are from controls (n = 10) and black boxes from M2/M3 transected and recovered leeches (n = 15). Note: The periods of the three DP nerves in the controls were isochronous, so only one box is shown. For preparations from transected and recovered leeches, each channel analyzed is shown. Lines within the boxes denote the median, and the box range denotes the 25th–75th percentiles. Error bars denote the entire range of the dataset. No statistical differences were found between the groups (n.s., Kruskal–Wallis H test, p > 0.5) B, Boxplots showing the frequency of observing crawl-specific coordination of individual DE-3 bursts. White boxes are from untransected control preparations. The black boxes depict the frequency of coordination between two adjacent ganglia (Adj.), ganglia within 2–3 segments from one another (Nearby), and among all ganglia recorded in a preparation (ALL). Box plot parameters are the same as in A. *, statistically significant difference between the control and transected/recovered groups (p < 0.01). No significant differences were observed among the transected recordings (n.s.; Kruskal–Wallis H test, p > 0.5).
- Figure 4.
Dopamine-induced rhythmic activity in M3–M7 ganglion chain preparations of transected and recovered leeches. A, B, Left, schematic representations of the preparations showing dopamine-treated ganglia (gray shading). Traces, extracellular recordings from DP nerves in M3, M4, and M5 ganglia. Gray shading denotes dopamine-treated ganglia, which correspond with the schematics. Right, boxplots showing the frequency of single DE-3 bursts exhibiting crawl-specific coordination for pairs of DP nerve recordings of non-transected controls (white boxes) and transected/recovered (black boxes) preparations. Lines within the boxes denote the median, and the box range denotes the 25th–75th percentiles. Error bars denote the entire range of the dataset. A, DP activity from preparations where only M3 was treated with dopamine of a nontransected control (top traces) and a transected/recovered leech (bottom traces). B, DP activity of preparations where both M3 and M4 were treated with dopamine of controls (top traces) and transected/recovered (bottom traces) leeches. In DP(6) of the non-transected control, no DE-3 bursts were observed, but occasional DE-3 spikes were generated. All dopamine concentrations were 50 µM.
- Figure 5.
Dopamine-induced rhythmic activity in M3–M7 ganglion chain preparations of transected and recovered leeches. Left, schematic representation of the preparation showing all ganglia were dopamine-treated (50 µm; gray shading). Traces, extracellular recordings of DP nerve of M3, M4, and M6. Top traces show DP activity from a untransected control, and bottom traces are from an M2/M3 transected and recovered leech. Gray shading denotes dopamine-treated ganglia. Right, boxplots showing the frequency of single DE-3 bursts exhibiting crawl-specific coordination for pairs of DP nerve recordings. White boxes are from nontransected controls and black boxes are from transected/recovered preparations. Lines within the boxes denote the median, and the box range denotes the 25th–75th percentiles. Error bars denote the entire range of the dataset.
- Figure 6.
Dopamine-induced rhythmic activity in M8-M12 chain preparations from the middle body region of transected and recovered leeches. Left, schematic representation of the preparations. The middle ganglion (M10) was dopamine-treated (50 µm; gray shading). Traces, extracellular recordings of DP nerves of M9, M10, and M11. Top traces show DP activity from a nontransected control, and bottom traces are from a transected/recovered leech. Gray shading denotes dopamine-treated ganglia. Right, boxplots of the frequency of single DE-3 bursts exhibiting crawl-specific coordination for pairs of DP nerve recordings of nontransected controls (white boxes) and transected/recovered (black boxes) preparations. Lines within the boxes denote the median and the box range denotes the 25th–75th percentiles. Error bars denote the entire range of the dataset.
- Figure 7.
Overt crawling behavior after a surgery combining denervation of the lead ganglion (M3) with a complete transection of the M2/M3 interganglionic connectives. A, B, Movement rasters depicting elongation (E, black boxes) and contraction (C, gray boxes) movements over time for an individual receiving an M3-denervation only surgical procedure (A) and a leech undergoing M3-denervation combined with M2/M3 connective transection (B). Movements are shown for time points before the surgery (Pre), and 4, 8, and 25 days after surgery. C, Graph showing the percentage of leeches exhibiting crawling behavior for M3-denervation only (n = 8), M2/M3 transection only (n = 10), and M3 denervation combined with M2/M3 transection (n = 11). Additional quantitative analyses are presented in the body of the Results section.
- Figure 8.
Laser-scanning confocal micrographs of Cy3-labeled Neurobiotin nerve-root back-fills of the lead ganglion (M3) from transected and denervated leeches that recovered (A) and did not recover (B). A, Micrograph of the lead ganglion (M3) of a leech that received a surgery combining M3-denervation and M2/M3 connective transection and exhibited recovered crawling behavior. Back-fill procedure was performed 29 days posttransection (8 days postrecovery). Right, inset is a magnified view of the gray box. B, Micrograph of the lead ganglion (M3) of a leech that received the same M3 nerve-root denervation and M2/M3 connective transection surgery, but did not recover crawling after 40 days. Right, inset is a magnified view of the gray box. Dotted lines in A and B denote edges of the M3 ganglion, connectives, and nerve roots.
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