Elimination of Left-Right Reciprocal Coupling in the Adult Lamprey Spinal Cord Abolishes the Generation of Locomotor Activity

Abstract

The contribution of left-right reciprocal coupling between spinal locomotor networks to the generation of locomotor activity was tested in adult lampreys. Muscle recordings were made from normal animals as well as from experimental animals with rostral midline (ML) spinal lesions (~13%→35% body length, BL), before and after spinal transections (T) at 35% BL. Importantly, in the present study actual locomotor movements and muscle burst activity, as well as other motor activity, were initiated in whole animals by descending brain-spinal pathways in response to sensory stimulation of the anterior head. For experimental animals with ML spinal lesions, sensory stimulation could elicit well-coordinated locomotor muscle burst activity, but with some significant differences in the parameters of locomotor activity compared to those for normal animals. Computer models representing normal animals or experimental animals with ML spinal lesions could mimic many of the differences in locomotor activity. For experimental animals with ML and T spinal lesions, right and left rostral hemi-spinal cords, disconnected from intact caudal cord, usually produced tonic or unpatterned muscle activity. Hemi-spinal cords sometimes generated spontaneous or sensory-evoked relatively high frequency "burstlet" activity that probably is analogous to the previously described in vitro "fast rhythm", which is thought to represent lamprey locomotor activity. However, "burstlet" activity in the present study had parameters and features that were very different than those for lamprey locomotor activity: average frequencies were ~25 Hz, but individual frequencies could be >50 Hz; burst proportions (BPs) often varied with cycled time; "burstlet" activity usually was not accompanied by a rostrocaudal phase lag; and following ML spinal lesions alone, "burstlet" activity could occur in the presence or absence of swimming burst activity, suggesting the two were generated by different mechanisms. In summary, for adult lampreys, left and right hemi-spinal cords did not generate rhythmic locomotor activity in response to descending inputs from the brain, suggesting that left-right reciprocal coupling of spinal locomotor networks contributes to both phase control and rhythmogenesis. In addition, the present study indicates that extreme caution should be exercised when testing the operation of spinal locomotor networks using artificial activation of isolated or reduced nervous system preparations.

Keywords: central pattern generators; coordination; coupling; locomotion; oscillators.

Figure 1

Models of left-right pairs of spinal cord oscillators that are coupled by relatively strong reciprocal inhibition in parallel with weaker reciprocal excitation (–|●), as is the case for the lamprey spinal locomotor central pattern generators (CPGs; Hagevik and McClellan, ; MN, motoneuron pool). (A) Left and right spinal oscillators that are autonomous and not dependent on left-right reciprocal connections for rhythmogenesis and generation of rhythmic locomotor burst activity. (B) Left and right spinal oscillators that are interdependent and require left-right coupling for rhythmogenesis and the generation of locomotor burst activity.

Figure 2

(A) Diagram of a normal adult lamprey showing bilateral muscle recording electrodes at 25% body length (BL, normalized distance from anterior tip of the head; 1, 2) and 45% BL (3, 4). (B) Locomotor muscle burst activity with different cycle times (CTs). During relatively (B1) slow (CT ≈ 460 ms, Freq ≈ 2.17 Hz), (B2) medium (CT ≈ 330 ms, Freq ≈ 3.03 Hz), or (B3) fast (CT ≈ 164 ms, Freq ≈ 6.10 Hz) swimming, locomotor muscle burst activity was characterized by left-right alternation (1↔2, 3↔4) and a rostrocaudal phase lag (1→4, 2→3). Note the decrease in CT for faster swimming. Some of the muscle action potentials were clipped in (B2,B3).

Figure 3

(A) Diagram of an experimental adult lamprey showing muscle recording electrodes at 25% BL (1, 2) and 45% BL (3, 4), longitudinal midline (ML) spinal cord lesion from 13% to 35% BL (thick horizontal line), and spinal cord transection (T) at 35% BL. (B) Muscle activity. (B1) Following a ML spinal cord lesion alone, locomotor muscle burst activity during swimming (CT ≈ 211 ms, Freq ≈ 4.74 Hz) was characterized by left-right alternation of muscle activity in the rostral (1↔2) and caudal (3↔4) body as well as a rostrocaudal phase lag for ipsilateral activity (1→4, 2→3). (B2) Subsequently, following a spinal transection at 35% BL for the same animal as in “B1”, stimulation of the oral hood (bar) elicited tonic muscle activity in the rostral body (1, 2), while movements and muscle activity were absent in the caudal body (3, 4). (C) Parameters of locomotor muscle burst activity (bars = means; vertical lines = SDs) during swimming for normal animals (open bars; n = 15) and for experimental animals with rostral longitudinal ML spinal cord lesions alone (filled bars; n = 21; see “Materials and Methods” section): (C1) CTs; (C2) burst proportions (BPs) for rostral and caudal locomotor muscle burst activity; (C3) intersegmental rostrocaudal phase lags; and (C4) right-left phase values for rostral and caudal locomotor muscle burst activity. Statistics: **p < 0.01; unpaired t-tests with Welch correction, when necessary, or Kruskal-Wallis with Dunn’s multiple comparisons post-test).

Figure 4

(A) Diagram of an experimental adult lamprey showing muscle recording electrodes at 25% BL (1, 2) and 45% BL (3, 4), and longitudinal midline (ML) spinal cord lesion from 13% to 35% BL (thick horizontal line). (B) Muscle activity during swimming for three different animals. For these particular animals (n = 10; see text) there was left-right alternation of caudal locomotor muscle burst activity (3↔4), while rostral activity usually was either tonic or consisted of relatively high-frequency “burstlet” activity (1 and 2; see text and Figures 5–7 for further descriptions of “burstlet” activity).

Figure 5

(A) Diagram of an experimental adult lamprey showing muscle recording electrodes at 25% BL (1, 2), longitudinal ML spinal cord lesion from 13 to 35% BL (thick horizontal line), and spinal cord transection (T) at 35% BL. Muscle recording electrodes at 45% BL are not shown for simplicity (see Figures 3A, 4A). (B) Examples of spontaneous or sensory-evoked muscle activity for different preparations following both spinal lesions. (B1) (upper) Very slow “burst” activity (~0.25 Hz, between horizontal arrows), and the activity during the horizontal black bar is expanded (lower) showing that the longer “bursts” consist of repetitive “bustlets” occurring at relatively high frequencies (see Figures 6A1–C1). (B2–B4) Examples of episodes of “burstlet” activity for other animals. Approximate instantaneous “burstlet” frequencies (*) shown below selected parts of the recordings. Scale bar: 1.0 s (B1 upper), 100 ms (other recordings).

Figure 6

(A1–C1) Distributions of frequencies of rostral muscle “burstlet” activity (25% BL) for three experimental animals following both a longitudinal ML spinal cord lesion and spinal cord transection (A = left, rostral recording channel; B,C = right, rostral recording channel; see 1 and 2 in Figure 5A). Average frequencies: (A1) 28.4 ± 12.3 Hz (20 frequencies >50 Hz, not shown); (B1) 26.6 ± 12.8 Hz (5 frequencies >50 Hz, not shown); and (C1) 31.5 ± 13.9 Hz. N = total number of analyzed cycles for each animal. (A2–C2) Plots of BP vs. CT for the corresponding animals shown in (A1–C1) (BP1 = BP for channel 1; BP2 = BP for channel 2; see 1 and 2 in Figure 5A). Dotted lines, p values and r² values indicate results from regression analysis.

Figure 7

(A1) Raw muscle “burstlet” activity and (A2) integrated “burstlet” activity (τ = 3 ms) recorded from the right, rostral body (25% BL) following a rostral ML spinal lesion and spinal transection (T) (e.g., see channel 2 in Figure 5A). (A3) Autocorrelation of activity in “A2”. The coefficient of rhythmicity (C_r = 0.31) was calculated using the amplitude of the first trough (a2) and the amplitude of the second peak (a1) (see “Materials and Methods” section). The “burstlet” frequency was calculated from the inverse of the x-axis coordinate (time lag) for point “a1” and was equal to ~38.5 Hz. (B1) Raw muscle “burstlet” activity and (B2) integrated “burstlet” activity (τ = 3 ms) recorded from the right, rostral body (25% BL) from a different animal than in “A” following a ML spinal lesion and spinal transection. (B3) Autocorrelation of activity in “B2”. The coefficient of rhythmicity was 0.16. The frequency calculated from point “a1” was 62.5 Hz, but the mid-point of the second peak (~20 ms) corresponded to a frequency of ~50 Hz.

Figure 8

(A) Diagram of an experimental adult lamprey showing muscle recording electrodes at 25% BL (1, 2) and 30% BL (3, 4), and longitudinal ML spinal cord lesion from 13% to 35% BL (thick horizontal line). (B1) Integrated muscle burst activity (τ = 5 ms) characterized by left-right alternation (1↔2, 3↔4) and a rostrocaudal phase lag (1→4, 2→3). Note the “burstlet” activity (arrowheads) superimposed on the longer locomotor bursts. (B2) Cross-correlation of activity in (B1) indicating a phase delay of ~17 ms (1→4, upper plot, first peak) and a CT ≈ 250 ms (Freq ≈ 4.0 Hz), which corresponded to an intersegmental rostrocaudal phase lag of ~0.011.

Figure 9

(A) Diagram of experimental adult lamprey (same animal as in Figure 8) showing muscle recording electrodes at 25% BL (1, 2) and 30% BL (3, 4), longitudinal ML spinal cord lesion from 13 to 35% BL (thick horizontal line), and spinal transection (T) at 35% BL. (B1) Integrated “burstlet” activity (τ = 3 ms) and (B2) corresponding cross-correlation plot of this activity for recording channels 1→4 and 2→3. For the initial part of the recording, note the “burstlet” activity for one ipsilateral channel (1 or 2) and the relative absence of activity for the other ipsilateral channel (4 or 3, respectively). (C1) Integrated “burstlet” activity (τ = 3 ms) and (C2) corresponding cross-correlation plot for same animal as “B”.

Figure 10

(A) Diagram of computer model showing RS neurons in the brain directly activating rostral (1, 2) and caudal (3, 4) left-right pairs of spinal oscillators that were connected by net reciprocal inhibition (–●). Ipsilateral oscillators were connected by asymmetrical reciprocal excitation (–|) that was stronger in the descending direction (D_E = 1.0) than the ascending direction (A_E = 0.24), similar to that previously described (Hagevik and McClellan, ; McClellan and Hagevik, 1999). The model representing “normal” animals (see B), without a longitudinal midline (ML) spinal lesion, had intact reciprocal inhibition between the rostral pair of oscillators. For the model representing “experimental” animals (see C), with a ML spinal lesion, reciprocal inhibition between the rostral pair of oscillators was removed. (B) Rhythmic “locomotor” output waveforms generated by a model representing “normal” animals were characterized by left-right alternation (1↔2, 3↔4) and a rostrocaudal phase lag (1→4, 2→3). (C) “Locomotor” output waveforms from a model representing “experimental” animals also featured left-right alternation and rostrocaudal phase lags, but with some differences in locomotor parameters. (D) Parameters of rhythmic “locomotor” output waveforms generated by the computer models representing “normal” animals (open bars) and “experimental” animals with a rostral ML spinal cord lesion (black bars): (D1) CTs decreased by a moderate amount (~15%) for the “experimental” model (i.e., with rostral ML spinal lesion) compared to those for the “normal” model. (D2) BPs for rostral and caudal “locomotor” waveforms were modestly larger (~9% and ~2%, respectively) for the “experimental” vs. “normal” models. (D3) The rostrocaudal phase lag was substantially larger (~80%) for the “experimental” model vs. “normal” model. (D4) Right-left phase values decreased very modestly (~4.5%) following incorporation of a ML spinal lesion for the “experimental” model.