Alpha-conotoxin ImI disrupts central control of swimming in the medicinal leech

Abstract

Medicinal leeches (Hirudo spp.) swim using a metachronal, front-to-back undulation. The behavior is generated by central pattern generators (CPGs) distributed along the animal's midbody ganglia and is coordinated by both central and peripheral mechanisms. Here we report that a component of the venom of Conus imperialis, α-conotoxin ImI, known to block nicotinic acetyl-choline receptors in other species, disrupts swimming. Leeches injected with the toxin swam in circles with exaggerated dorsoventral bends and reduced forward velocity. Fictive swimming in isolated nerve cords was even more strongly disrupted, indicating that the toxin targets the CPGs and central coordination, while peripheral coordination partially rescues the behavior in intact animals.

Figure 1

Swimming in intact leeches. a. Free swimming in control (saline-injected) leeches. a1. Video frames of a typical swimming leech (at 0.1-s intervals). Dots indicate the dorsal side of the animal’s head. a2. Path followed by the control leech shown in a1, as extracted by the “Wormfinder” algorithm. The animal’s location and shape in sequential frames (16.7 ms intervals) are plotted as colored lines with small dots marking the head. Color indicates time; the color bar applies to b2 and c2 as well. The head of the leech at the end of the clip is indicated by a black star. a3. Abstraction of the path shown in a2. Lines show the actual trajectory of the head (magenta) and the tail (cyan), as well as the same data after filtering out the undulations of the head (red), tail (blue), and center (black). Dots along the black curve indicate 0.1-s intervals; arrowheads indicate the direction of locomotion. b. After injection with ImI. b1. Video frames. b2–b3. Examples of paths followed by three ImI-injected leeches, using the same conventions as in (a).

Figure 2

Propagation of dorsoventral waves along the bodies of swimming leeches. a. Peaks (gray) and troughs (black) in a normally swimming leech progressed from head to tail. When the leech turned toward its ventral side (middle of the graph), the wave temporarily lacked a trough. b. Propagation of peaks and troughs in an ImI-injected animal. Note the stationary trough (black dots) near the middle of the graph. c. Cycle periods (i.e., the time separating frames when peaks or troughs passed an animal’s midpoint) for ImI-injected animals (black, N = 3) compared with controls (gray, N = 4). d. Propagation delays (expressed as time per percent body length) of swim waves passing through the body of ImI-injected animals (black) and controls (gray). *,p < .05. e. Forward velocities of ImI-injected animals (black), straight-swimming control animals (gray) and corner-turning control animals (white, N = 3). *, p < .05; **, p < .01; ***, p < .001; n.s., not significant; unpaired t-tests.. All error bars represent sample standard deviation (SSD).

Figure 3

ImI disrupted fictive swimming in isolated nerve cords. a. Spike trains recorded simultaneously from motor neurons DE-3 in several ganglia extending from anterior segment M3 to posterior segment M16 along an isolated nerve cord bathed in saline (top) and in 10 μM ImI (bottom). Traces above rasters show corresponding raw voltage data from one electrode. Arrows mark the time and location of the stimulus. Solid lines below records indicate portion of response that is fictive swimming; dotted lines indicate portions that are not. b. Average duration of responses to electrical stimulation in saline and in ImI. c. Average number of recognizable fictive swim cycles in response to a single stimulus, in saline (gray) and in ImI (black). Paired t-tests for N = 30 anterior (M3–8), 27 middle (M9–12), and 15 posterior (M13–16) ganglia from 19 leeches (significance as in Figure 2). d. Cycle periods during fictive swimming in saline (gray) and ImI (black). Data shown are mean of the median period for each preparation with SSD across preparations. e. Burst propagation delay between neighboring ganglia in saline (gray), and ImI (black). f. Spike trains recorded from motor neuron DE-3 in ganglion M10 during swimming in an intact leech, first in control conditions and then after injection with ImI. g. Dorsal and ventral motor neuron bursts from ganglion M11 of an isolated nerve cord in saline and in ImI. h. Quality of dorsal–ventral coordination (Q_DV, see text). The total number of recorded ganglia is indicated above each bar. (Due to unfavorable anatomy, no recordings were obtained from ventral excitor motorneurons from posterior ganglia in ImI.) i. Histograms of ISIs of DE-3 in M6 in the first 30 s after stimulation (6 stimuli in saline, 12 in ImI; one typical preparation). Note logarithmic axes.