Lateral Turns in the Lamprey. I. Patterns of Motoneuron Activity

Patriq Fagerstedt and Fredrik Ullén

Nobel Institute for Neurophysiology, Department of Neuroscience, Karolinska Institutet, SE-171 77 Stockholm, Sweden


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Fagerstedt, Patriq and Fredrik Ullén. Lateral Turns in the Lamprey. I. Patterns of Motoneuron Activity. J. Neurophysiol. 86: 2246-2256, 2001. The activity of motoneurons during lateral turns was studied in a lower vertebrate, the lamprey, to investigate how a supraspinal command for the change of direction during locomotion is transmitted from the brain stem and integrated with the activity of the spinal locomotor pattern generator. Three types of experiments were performed. 1) The muscular activity during lateral turns in freely swimming adult lampreys was recorded by electromyography (EMG). It was characterized by increased cycle duration and increased duration, intensity, and cycle proportion of the bursts on the side toward which the animal turns. 2) Electrical stimulation of the skin on one side of the head in a head-spinal cord preparation of the lamprey during fictive locomotion elicited asymmetric ventral root burst activity with similar characteristics as observed in the EMG of intact lampreys during lateral turns. The cycle duration and ventral root burst intensity, duration, and cycle proportion on the side of the spinal cord contralateral to the stimulus were increased; hence a fictive lateral turn away from the stimulus could be produced. The fictive turn propagated caudally with decreasing amplitude. The increase in burst duration during the turn correlated well with the increase in cycle duration, while changes in contralateral burst intensity and burst duration did not co-vary. Turning responses varied depending on the timing (phase) of the skin stimulation: stimuli in the first two-thirds of a cycle evoked a turn in the same cycle, whereas stimuli in the last third gave a turn in the following cycle. The largest turns were evoked by stimuli in the first third of a cycle. 3) Fictive turns were abolished after transection of the trigeminal nerve or a rhombencephalic midline split, but not in a rhombencephalic preparation with transected cerebellar commissure. High spinal hemisection was sufficient to block turning toward the lesioned side, while turns toward the intact side remained. Taken together these findings suggest that the reticulospinal turn command is essentially unilateral and generated in the rhombencephalon.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Locomotion allows an animal to move around in its environment to search for food, to find a mate, or to escape predators. Spinal circuits generate the basic locomotor movements while supraspinal centers adapt these to the needs and goals of the animal by sending commands for alterations in speed, postural corrections, and steering (Grillner 1981; Orlovsky et al. 1999).

Lampreys, like elasmobranchs and teleosts, swim forward by propagating waves of lateral flexion rostrocaudally along their bodies (Gray 1933a; Grillner 1974; Grillner and Kashin 1976; Wallén and Williams 1984). During turns in the horizontal plane (lateral turns) the muscular activity on the turning side increases, producing a larger force against the water on that side, and thus a turn toward the activated side (Gray 1933b; McClellan and Hagevik 1997). Neural correlates of locomotion (fictive swimming) can be elicited in the lamprey in the brain stem-spinal cord preparation by bath application of excitatory amino acids to the spinal cord or by electrical or chemical stimulation of the brain stem (Cohen and Wallén 1980; Grillner and Wallén 1980; McClellan and Grillner 1984; McClellan and Hagevik 1997; Poon 1980; Wallén and Williams 1984). In intact lampreys, mechanical or electrical stimulation to the skin of the head evokes lateral turns away from the stimulus (McClellan 1984). In brain stem-spinal cord preparations of adult lamprey, the same stimuli evoke an asymmetric ventral root burst pattern followed by brief episodes of swimming activity (McClellan 1984). In the head-spinal cord preparation of the larval lamprey, fictive locomotion was elicited by stimulation of the brain stem. On this background electrical stimulation of the skin on one side of the head elicited modifications of ventral root burst activity with changes in timing and intensity of the burst resembling in vivo turns (McClellan and Hagevik 1997).

Skin stimulation activates trigeminal sensory afferents (McClellan 1984), which in turn evoke excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs, respectively) in ipsi- and contralateral reticulospinal (RS) neurons (Rovainen 1967; Viana Di Prisco et al. 1995; Wickelgren 1977). RS neurons constitute the main descending system in the lamprey (Bussières 1994; Ronan 1989; Rovainen 1967, 1974; Swain et al. 1993). They activate ipsilateral motoneurons directly and also influence bilateral motor output indirectly through the segmental spinal networks (Buchanan 1982; Ohta and Grillner 1989; Rovainen 1974). There are also crossing excitatory connections from the brain stem to spinal cord segments (e.g., from Mauthner cells) (Rovainen 1974).

Here we describe the activity of motoneurons during lateral turns and in the accompanying paper (Fagerstedt et al. 2001) the corresponding supraspinal commands. Three sets of experiments were performed in this study. First, we investigated the EMG pattern of the turn in freely swimming lamprey. Second, we have developed a head-spinal cord preparation of the adult lamprey, in which turn-like asymmetric ventral root activity (fictive lateral turns) could be elicited by skin stimulation. By using this preparation, we examined the relationships between the turn characteristics and the phase of skin stimulation in the locomotor cycle. Third, the course of descending pathways responsible for elicitation of turns was investigated in lesion experiments. Preliminary accounts of these results have appeared in abstract form (Fagerstedt et al. 1998; Ullén et al. 1998).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals

Adult river lampreys (Lampetra fluviatilis, 20-30 cm total length, n = 7) were used in in vivo experiments, and American silver lampreys (Ichthyomyzon unicuspis, 15-25 cm total length, n = 30) were used in in vitro experiments. They were obtained from commercial suppliers in Sweden and Iowa, respectively. Animals were kept in aerated fresh water aquaria at 5°C in 12 h light/12 h darkness until used.

Surgical procedures

Animals were anesthetized by immersion in cold water containing tricaine methane sulfonate (MS-222, Sigma; 200 mg/l water) adjusted to pH 7.4. Insertion of electromyographic (EMG) electrodes for the in vivo experiments was performed in a chamber with a mixture of anesthetic and fresh water. Bipolar EMG electrodes were made from a wire with two insulated, stainless steel leads, 100 µm in diameter. The wire was threaded through the barrel of a 22-gauge hypodermic needle. The tips (0.5 mm) of the wires were stripped of insulation, and spread 0.5-1.0 mm apart and bent into a hook, which was then pulled back and nestled into the tip of the barrel of the needle. Using these needles, EMG electrodes were implanted in the muscle bilaterally, caudal to the last gill, and immediately rostral to the anterior dorsal fin. The cables from all four electrodes were wired together to form one bundle originating at the start of the dorsal fin. The animal was then transferred to the experimental aquarium containing fresh water, until it recovered from the anesthesia. All surgical procedures were performed according to principles of laboratory animal care, as approved by the Swedish National Board for Laboratory Animals-CFN (LSFS 1988:45), Swedish law (SFS 1988:534/539/541), and the European Communities Council (directive 86/609/EEC). The experiments were approved by Stockholms Norra Djurförsöksetiska Nämnd.

For in vitro experiments the head and the rostral half of the spinal cord with the underlying notochord, usually 20-50 segments long, were dissected in cold oxygenated saline of the following composition (in mM): 91 NaCl, 2.1 KCl, 2.6 CaCl2, 1.8 MgCl2, 20 NaHCO3, 0.5 L-glutamine, and 4 glucose, buffered to pH 7.4 by bubbling with 95% O2-5% CO2 (Wickelgren 1977). The preparation was pinned down in a silicone elastomer (Sylgard, Dow Corning, Midland, MI)-lined cooling chamber (8-12°C) perfused with the cold oxygenated saline. A plastic barrier at segment 2-6, sealed with agar, was used to divide the chamber into a brain and a spinal cord pool (Fig. 1B).



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Fig. 1. Experimental arrangements. A: arrangement for simultaneous recording of electromyogram (EMG) and movements in freely swimming lampreys. EMG and video recordings were synchronized by pulses (1 Hz) recorded simultaneously by both systems (Synch). An asterisk indicates the starting position of the lamprey. B: the in vitro preparation positioned in a chamber divided into two pools by an agar barrier. The rostral pool contained the brain and was perfused with normal Ringer solution. The caudal pool contained the spinal cord and was perfused with a Ringer solution containing D-glutamate to elicit fictive locomotion. Two bipolar insulated silver wire stimulation electrodes were used to stimulate the skin of the head, one on each side. Fictive locomotion and fictive turning movements evoked by the skin stimulation were recorded by two pairs of left and right extracellular glass pipette electrodes positioned on one rostral and one more caudal pair of ventral roots. One pair of caudal extracellular glass pipette electrodes was positioned on the left and right spinal cord surface to allow recording of activity descending from the brain stem and rostral spinal cord. C: classification of the fictive turning cycle and measured parameters. The duration of each duty cycle (CD) was measured between the onsets of the ventral root bursts ipsilateral (L rostral VR) to the stimulated side (L stim). The cycle in which the stimulus train began was defined as the turn cycle (Cycle 0). The cycle was further subdivided into four functional phases; the ipsilateral burst (iBD), the interburst interval between the ipsilateral and the contralateral bursts (IB1), the contralateral burst duration (cBD), and the interburst duration between the contralateral burst and the ipsilateral burst of the succeeding cycle (IB2). Turning responses were grouped in 3 bins for analysis of phase dependence of different turning parameters on the timing of stimulus onset during the swim cycle (phase 1 = iBD, phase 2 = IB1 and the 1st half of cBD, and phase 3 = 2nd half of cBD and the IB2).

Swimming trials

Turns in intact animals were recorded in a shallow (8 cm deep) L-shaped aquarium with a white fluorescent lamp positioned along one of the walls as illustrated in Fig. 1A. The water temperature in the aquarium was 7-10°C. The behavior of the animals was recorded with a video camera (25 frames/s), and the locomotor activity was recorded with EMG electrodes (see above).

After being transferred to the aquarium, the animals spontaneously assumed a quiescent position, attached to the bottom of the aquarium with their sucker mouth. As a rule, the passive animals could be moved gently with the hand to the indicated starting position (marked by an asterisk in Fig. 1A). Locomotion was evoked by illuminating the tail of the animals, which contains dermal photoreceptors (Ullén et al. 1993; Young 1935), with an optical guide (8 mm diam, 90 W white lamp) held 1-2 cm away. As the animals swam along the illuminated side of the aquarium, they typically performed a lateral turning movement away from the light (negative phototaxis).

In vitro experiments

Fictive locomotion was induced by addition of D-glutamate (0.5-2 mM; Tocris Cookson, Bristol, UK) to the oxygenated saline in the spinal cord pool (Fig. 1B). Locomotor activity was recorded from a pair of rostral (segments 10-20) and caudal (segments 20-35) ventral roots by gently pressing the tips of Ringer solution-filled glass electrodes against the roots. Two additional electrodes were used to record activity from the surface of the caudal end of the spinal cord (usually segments 35-50). Asymmetric ventral root activity was evoked by DC electrical stimulation (train of 2-ms pulses, 10-50 Hz, 250-1,500 ms duration) of the skin to one side of the head by bipolar silver wires (0.5 mm diam), insulated except for the tips. Due to variability in excitability between preparations and adaptation of the stimulated skin, the current strength (10-1,000 µA) was varied between trials. Recordings were digitized on-line (sampling period 480-1,000 µs) using Axoscope 7 software and a Digidata 1200 A/D board (Axon Instruments, Foster City, CA).

Data processing and analysis

Each video record of a swimming lamprey was first analyzed qualitatively, and trials with turning movements were selected. The video records were analyzed frame by frame, and the interval during which a turn occurred was indicated on the EMG record. The time of onset and termination of EMG and ventral root bursts were detected by a semi-automatic selection algorithm (Datapac 2000; Run Technologies, Laguna Hills, CA). Onset and termination points were used to calculate burst duration and cycle duration (BD and CD in Fig. 1C). Burst proportion was calculated as the ratio between burst duration and cycle duration. Burst intensity was calculated as burst amplitude, defined as the area of the rectified burst, divided by burst duration. The delay/overlap between ventral root bursts was calculated for in vitro turns only and labeled interburst duration 1 and 2 (IB1 and IB2 in Fig. 1C).

The cycles containing the maximum body curvature in the in vivo records, and the onset of the stimulus train in the in vitro records, respectively, were labeled cycle 0. The five cycles before (-5 to -1) and after (1 to 5) cycle 0 were analyzed for each stimulus train. All parameters were normalized against the mean value of that of the cycles preceding cycle 0. All statistical analyses used ANOVA (* P < 0.05 and ** P < 0.01).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In vivo turns

When a swimming lamprey approached the illuminated area of the aquarium, it turned away from the light (Fig. 1A). Figure 2A shows the EMG activity during a turn to the left (the turning side). The burst of left rostral EMG activity just preceding the point of maximum body curvature (defining cycle 0) is increased in duration leading to an increased cycle duration. This was followed by an increased burst duration and intensity on the nonturning side, here termed rebound, further increasing the cycle duration.



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Fig. 2. Lateral turns in freely swimming lamprey. A: EMG activity in right and left myotomes corresponding to segments 15 (R rostr and L rostr, respectively) and 30 (R caud and L caud, respectively) during a turn to the left. The turn cycle (Cycle 0) is defined as starting with the burst occurring during the point of maximum body curvature (mc) at the rostral electrode pair. B-E: changes in turn parameters in the rostral segment during 14 turns in 7 animals. B: the cycle duration increased significantly during cycle 0. C: the rostral EMG burst duration increased significantly on the turning side during cycle 0, while that of the contralateral side was unchanged. D: the rostral EMG burst proportion increased on the turning side and decreased on the contralateral side. E: the rostral EMG burst intensity increased significantly on the turning side.

Averaging 14 turns from 7 animals around the point of maximum body curvature showed an increased cycle duration during turns (Fig. 2B), which usually began with a burst on the turning side. The duration and intensity of this burst was increased (Fig. 2, C and E). In some experiments (cf. Fig. 2A) it was followed by an increased burst duration and intensity on the opposite side, but no significant increase of the mean could be observed. The burst proportion on the turning side was increased, while the burst proportion on the opposite side was decreased, except in cases with a rebound, where it could be unchanged or increased instead (Fig. 2D). All turn-related changes in burst intensity and duration were larger in the rostral segments compared with the caudal ones (data not shown).

In vitro turns

A brain-spinal cord preparation was placed in a two-pool experimental chamber (Fig. 1B), where rhythmic symmetric left-right alternating ventral root activity (fictive locomotion) was induced by bath application of D-glutamate (see METHODS). Short trains of electrical impulses (see METHODS) to the skin on the left side of the head (L skin stim) evoked a transient asymmetry in the ventral root bursting pattern (Fig. 3A). The first ventral root bursts on the contralateral side to the stimulus (R rostr VR and R caud VR) after the beginning of the stimulus train were prolonged and increased in intensity. The contralateral response was followed by larger and longer-than-normal ipsilateral bursts (L rostr VR and L caud VR in Fig. 3A). Stimulation of the skin on the right side of the head modulated the ventral root output in the opposite way (Fig. 3B). Ventral root responses were associated with a bilateral increase of activity recorded from the surface of the caudal part of the spinal cord (data not shown). There was no significant difference in the amplitude of the wave on the two sides, and the latency of the wave corresponded to a minimum conduction velocity of 3 m/s of a putative descending pathway.



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Fig. 3. Asymmetric ventral root responses (fictive turns) to electrical skin stimulation during fictive locomotion. A: stimulus of the left side of the head (L skin stim). A short stimulus train evoked increased burst duration and burst intensity in the contralateral ventral roots (R rostr VR and R caud VR). The cycle period and the burst proportion on the contralateral side were increased, while the ipsilateral burst proportion was decreased (L rostr VR and L caud VR). The following cycle was also prolonged due to longer-than-normal ipsilateral VR bursts, with ipsilateral bursts that had larger burst proportion than normal and increased intensity. Effects in the rostral roots were larger than in more caudal ones. B: stimulus to right side of the head (R skin stim) evoked similar changes in the opposite direction.

A quantitative analysis performed on ventral root responses to skin stimulation (n = 195) in seven animals is shown in Fig. 4. Duty cycles were defined as starting with the onset of a ventral root burst ipsilateral to the stimulated side, and cycle 0 was defined as the cycle in which the stimulus train began (see Fig. 1C). With this procedure the response never occurred earlier than cycle 0. All responses to stimulation were further divided into three groups in Fig. 5 depending on whether the onset of the stimulation occurred during the ipsilateral burst (phase 1), or during the first or second half of the contralateral burst (phases 2 and 3, respectively; see Fig. 1C). The mean duration of cycle 0 was prolonged (Fig. 4A). The increase was mainly due to an increase in burst duration on the side contralateral to skin stimulation (Fig. 4B). The preceding ipsilateral burst was slightly prolonged or unchanged depending on whether the stimulation started early or late in cycle 0 (Fig. 5B). The interval between the contralateral burst of cycle 0 and ipsilateral burst of cycle 1 was shortened (IB2 in Fig. 4C). Cycle 1 was also prolonged (Fig. 4A), and began with a longer-than-normal ipsilateral burst (Fig. 4B) followed by a contralateral burst, the length of which was also dependent on stimulus phase (Fig. 5B). Consequently, the burst proportion was increased on the contralateral side and decreased on the ipsilateral side in cycle 0 (Fig. 4D). The burst intensity (Fig. 4E) was increased on both sides, but the increase was significantly larger on the side contralateral to the stimulus. The ipsilateral burst proportion and intensity was also increased during cycle 1, while the contralateral one remained unchanged (Fig. 4, D and E). The phase analysis showed that stimulus trains beginning in phase 1 and 2 increased the cycle duration in the cycles 0 and 1 (Fig. 5A), the contralateral burst duration, proportion, and intensity in cycle 0 (Fig. 5B), and the ipsilateral burst duration in cycle 1 (Fig. 5C). Stimulation trains given early in the swim cycle (in phase 1), also produced a marked increase in the ipsilateral burst intensity in cycle 0 (Fig. 5E). Stimulation later in the cycle, that is after the midpoint of the contralateral burst (phase 3) had its main effect on cycle duration and burst duration and intensity delayed by one cycle, occurring in cycle 1 rather than in cycle 0. 



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Fig. 4. Quantitative analysis of fictive turns (n = 195 from 7 experiments). All values normalized to the mean of cycle -5 to -1. A: the mean cycle duration increased during cycle 0 and 1. B: the mean contralateral rostral and caudal burst VR duration were significantly increased during cycle 0. In the succeeding cycle the mean ipsilateral rostral and caudal burst VR duration were increased (rebound). C: the mean rostral interburst duration was significantly decreased during fictive turns. D: average contralateral burst durations increased more than the cycle duration during cycle 0, leading to an increased average contralateral, and a decreased average ipsilateral burst proportion. In cycle 1, the mean ipsilateral burst proportions were increased. E: the mean burst intensities were increased on both sides during fictive turns, with the largest increases on the contralateral side during cycle 0, and on the ipsilateral side during cycle 1.



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Fig. 5. Correlation analysis of fictive turn parameters. A: there was a significant correlation (R2 = 0.2352, P < 0.0001) between the increase in cycle period and the increase in contralateral burst duration for cycle 0 (n = 195, 7 animals). B: burst intensity increase and burst duration increase on the contralateral side during cycle 0 did not show any correlation in 6/7 experiments (data from single experiment shown), suggesting an independent control of these parameters.

All changes in the rostral segments were mirrored in more caudal segments, although with a smaller amplitude (Figs. 3, A and B, and 4, A-E). Turn-like changes in both rhythm and burst intensity could be observed down to the 50th segment but was less clear or absent further caudally (one preparation, tested to segment 70). These transient changes in fictive locomotor activity resemble the asymmetric muscle activity observed in the intact adult (see Fig. 2) (McClellan 1984) and in larval lampreys during in vivo turns (McClellan and Hagevik 1997), and the response is thus by us labeled "fictive turn."

The amplitude of the ventral root response to skin stimulation differed between trials and between preparations (data not shown). The increase in cycle duration in cycle 0 correlated positively with an increase in burst duration on the contralateral side (R2 = 0.2352, n = 195 in 7 preparations; Fig. 6A), the normalized burst duration increasing around twice as much as the normalized cycle duration. In some preparations, different stimuli could produce either a strong increase in burst duration on the contralateral side, or a strong increase in the burst intensity without a proportional increase in the burst duration, suggesting that these two characteristics may vary independently (R2 = 0.0002, n = 82; Fig. 6B).



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Fig. 6. Rostral ventral root response to skin stimulus in different phases of the swim cycle recorded in the rostral ventral roots. A-D: the mean cycle duration (A), mean contralateral (B), and ipsilateral burst duration (C), as well as the contralateral burst intensity (D) were increased during cycle 0 when the skin stimulus was given in the first two-thirds of cycle (phase 1 and 2), and in cycle 1 when the stimulus was given in the last third (phase 3). E: the mean ipsilateral burst intensity was increased during the turn cycle (cycle 0) when the stimulus was given in the first third of the cycle (phase 1) and during cycle 1 (as a rebound) when the stimulus was given in the late two-thirds.

Pathway for the lateral turn command

The pathway for fictive turns evoked by skin stimulation was investigated by transecting different cranial nerves on the stimulated side and by making lesions in the brain stem and spinal cord (Fig. 7, A and E). The skin stimulus was transmitted to the brain stem in trigeminal afferents since transection of the trigeminal (lesion 4 in Fig. 7E; not illustrated), but no other cranial nerve on the stimulated side abolished fictive turns (n = 3) (see also McClellan 1984). Rhombencephalic midline lesions (lesion 5 in Fig. 7E) also blocked the fictive turns (n = 4, data not shown) demonstrating that the signals need to cross the midline at the rhombencephalic level. In these preparations, skin stimulation occasionally caused increased ipsilateral activation of the ventral roots. Fictive turns could be evoked in rhombencephalic preparations [transection of the brain stem at the rostral border of the rhombencephalon (lesion 1 in Fig. 7A), n = 6; Fig. 7B], showing that pathways through mesencephalon are not necessary to generate turning responses. Similarly, a section of the cerebellar and posttectal commissures (lesion 2 in Fig. 7A) did not affect fictive turning (n = 4; Fig. 7C).



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Fig. 7. Effect of brain and spinal cord lesions on fictive turns. A: permissive lesions. B: fictive turn evoked in a rhombencephalic preparation. C: fictive turn in preparation with transected cerebellar commissure. D: a spinal hemisection ipsilateral to the stimulated side did not block contralateral increases in burst duration and intensity. E: brain stem and spinal cord lesions affecting turn responses. F: after a spinal hemisection contralateral to the stimulated side only a slight activation of the ipsilateral side could be observed.

To elucidate the role of ipsilateral and contralateral components of the descending commands at the spinal level, hemisections of the rostral spinal cord (segments 1-6) were performed either on the side ipsilateral (lesion 3 in Fig. 7A) or contralateral to the stimulated side (lesion 6 in Fig. 7E). Hemisections ipsilateral to the stimulated side did not abolish fictive turns (n = 3; Fig. 7D). The cycle duration (Fig. 8A), the contralateral burst duration (Fig. 8B), and intensity (Fig. 8C) were increased during cycle 0. On the side ipsilateral to the stimulated skin, there was no increase in the mean burst duration (rebound) during cycle 1 after the ipsilateral lesion (Fig. 8B), although rebounds were observed in a few experiments. The descending wave of activity recorded from the surface of the caudal spinal cord was practically abolished on the lesioned side (data not shown) indicating that most of this activity originated above the lesion (i.e., in the brain stem). A hemisection of the spinal cord on the side contralateral to the stimulation blocked the generation of turns (n = 3; Fig. 7F). No increase in contralateral burst duration, or in the cycle duration could be observed during cycle 0 (Fig. 8, D and E). The increase in burst duration on the ipsilateral side during the following cycle (rebound) was also absent (Fig. 8E). There was still, however, an increase in the burst intensity on the contralateral side, albeit smaller than in the control, while on the ipsilateral side the increase in burst intensity was greater than normal, especially during cycle 1 (Fig. 8F).



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Fig. 8. Quantitative analysis of fictive turns after rostral spinal hemisections in 3 animals. A-C: lesion in the 1st segments of the spinal cord ipsilateral to the stimulus. The amplitude of the changes in cycle duration (A), burst duration (B), and burst intensity (C), in lesioned preparations (solid lines; n = 22 trials) and in control preparations (dashed lines; n = 49) were not significantly different during cycle 0. There was no significant increase of the burst duration (B) during cycle 1 (rebound) on the ipsilateral side in the lesioned preparations. D-F: with contralateral lesions (solid lines; n = 28), however, fictive turns were significantly attenuated. There was no turn-like increase in cycle period (D), or burst duration (E) on the lesioned side. Contralateral lesions produced a stronger increase in burst intensity during cycle 1 (F), than in ipsilateral lesions (C) and controls (dashed lines).

In five preparations, spinal hemisections were performed at a level between the rostral and caudal pair of the recorded ventral roots. The propagation of the turn was undisturbed by lesion on the side ipsilateral to the stimulus, suggesting that local interactions between the two sides of the spinal cord are not a necessary part of the descending turn command (data not shown). Lesions on the contralateral side blocked the fictive turn in more caudal segments, demonstrating that a perturbation of the alternating ventral root bursting pattern in the form of fictive turn does not traverse a spinal hemisection (data not shown). These results suggest that axons running in the spinal cord contralateral to the stimulus are necessary for transmitting the turn command caudally.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study we utilize the possibility of eliciting asymmetric spinal motor activity by electrical skin stimulation to one side of the head, first demonstrated by McClellan and co-workers (McClellan 1984; McClellan and Hagevik 1997), to investigate the descending pathways and phase dependence of descending commands for lateral turns in the adult lamprey. We have shown that the EMGs underlying lateral turns in freely swimming adult lampreys are characterized by an increased cycle duration during the turn, as well as an increased burst duration, burst proportion, and burst intensity on the side of the turn (Fig. 2). These changes in muscular activity are similar to those observed during lateral turns in intact larval lampreys (McClellan and Hagevik 1997), thus indicating that the motor pattern for lateral turning is conserved during metamorphosis from the larval to the adult stage, and is similar to that of teleosts (Gray 1933b).

Fictive turns evoked by electrical skin stimulus

Brief electrical skin stimulation to one side of the head of adult lamprey brain-spinal cord preparations during fictive locomotion evoked a transient asymmetry in the alternating ventral root burst activity. The changes in ventral root activity included an increased cycle period with increased burst duration, burst proportion and intensity on the side contralateral to the stimulus, followed by an increased burst duration and proportion on the ipsilateral side (rebound; Figs. 3 and 4). The largest responses were observed in the rostral segments of the spinal cord, then decreasing in size caudally, and present at least down to the 50th segment.

This asymmetric motor output, here labeled fictive turn, is very similar to the changes in EMG activity recorded during lateral turns and would thus direct the body of the lamprey away from the skin stimulus (present study and McClellan 1984). The rebound was less evident during in vivo turns, but was observed in some experiments (cf. Fig. 2A, and in intact larval lampreys) (McClellan and Hagevik 1997). Similar responses to electrical skin stimulation were also observed in the larval lamprey in vitro (McClellan and Hagevik 1997) where fictive locomotion was induced by chemical microstimulation of the brain stem. Both these in vitro studies show that it is possible to modulate the spinal locomotor central pattern generator (CPG) to transiently switch from symmetric activity ("rectilinear swimming") to an asymmetric activity ("turning" away from a stimulus), i.e., that fictive turns can be generated without sensory feedback from the movement performed.

Lateral turns are characterized by changes both in timing (cycle duration, burst duration) and in intensity of the locomotor bursts. We found that electrical skin stimulation could evoke both responses with large increases in burst intensity combined with a small prolongation of the burst duration, as well as responses with small burst intensity increases coupled to a large increase in burst duration (Fig. 6B). This suggests that certain turn characteristics can vary independently of each other. Whether different turn strategies are used in different behavioral contexts remains to be investigated.

Pathway of the descending turn command

Neither transection of the brain stem rostral to the rhombencephalon, nor a transection of the cerebellar commissure affected fictive turns elicited from a trigeminal stimulus (Fig. 7, A-C). A hemisection of the spinal cord on the same side as the stimulus also failed to block the descending turn command (Fig. 7D). Fictive turns were significantly attenuated, however, by a contralateral hemisection of the spinal cord (Fig. 7, E and F), showing that the pathway for fictive turns crosses over to the contralateral side in the rhombencephalon. This was verified by a performing a midline split of the rhombencephalon, which abolished fictive turns. Lesions of the ipsilateral trigeminal nerve, but not sectioning of any other cranial nerve, also blocked the response, showing that the turning response is dependent on trigeminal afferent pathways. Previously it was shown that trigeminal afferents activated by skin stimulation of the head, both can initiate locomotion and elicit avoidance responses, through activation of descending pathways originating in the brain stem (McClellan 1984; McClellan and Grillner 1983). Trigeminal afferents originate from dorsal cells in the medulla and rostral spinal cord and the trigeminal ganglia where the central projections terminate in the sensory part of the ipsilateral trigeminal nucleus (Koyama et al. 1987; Matthews and Wickelgren 1978). They synapse on excitatory and inhibitory relay neurons, some of which have ipsilateral descending projections to the spinal cord (Ronan 1989). Trigeminal nerve stimulation evokes polysynaptic mixed IPSP-EPSPs in both ipsilateral and contralateral reticular nuclei (Rovainen 1967; Viana Di Prisco et al. 1995; Wickelgren 1977). The fact that turns could be generated in rhombencephalic preparations shows that these bilateral trigemino-reticular pathways are likely to carry the signals eliciting a turn (Fig. 7).

The RS system is the main descending system in the lamprey and is composed of hundreds of mainly ipsilaterally projecting neurons in four bilateral reticular nuclei, and also the Mauthner and accessory Mauthner neurons, which project to the contralateral spinal cord (Bussières 1994; Nieuwenhuys and Nicholson 1998; Swain et al. 1993). Our results further demonstrate that unilateral pathways at the level of the spinal cord, running on the side contralateral to the stimulus, are both necessary and sufficient for transmitting the descending turn command. Spinal hemisections ipsilateral to the stimulated side between the two pairs of ventral root electrodes did not block the propagation of the turn caudally, revealing that the descending turn command is not dependent on local interactions between the left and right subdivisions of the spinal CPG. The rebound could partly be caused by descending commands on the side ipsilateral to the stimulus since it was attenuated after high ipsilateral spinal hemisections (Figs. 7 and 8). Spiking activity of reticulospinal neurons on the ipsilateral side during the rebound was also seen occasionally (see Fagerstedt et al. 2001). It is not clear, however, whether it is part of the descending turn command or a spinal rebound mechanism since a similar phenomenon was observed after stimulation of long axons in the contralateral half of the isolated lamprey spinal cord (Rovainen 1985). The function of the rebound activity during turning behavior is unclear, but it has been speculated that it could serve as restoring force after the turn (McClellan and Hagevik 1997).

Phase dependence of fictive turns

Voluntary movements, ongoing activity, and evoked reflexes can be in conflict with each other and must either be integrated, or the conflict must be resolved at some level in the control hierarchy (i.e., motoneurons, the CPG, or the level of descending command neurons). In vivo, feedback from ongoing locomotor activity is presumably also important to integrate the turn with locomotion, by switching behavior in the optimal phase of the movement, and with the correct amplitude and direction.

We have shown that the changes in the characteristics of a fictive turn depended on in which phase of the swim cycle the stimulus was given (Fig. 5). This dependency on the phase of stimulus could arise at several levels of the lamprey CNS. The skin stimulus is first relayed to the trigeminal nucleus. Trigeminal neurons may receive phasic information from the spinal cord (Dubuc et al. 1993; Vinay et al. 1998a,b), which could modulate the transmission of the turn command. The reticulospinal neurons in the different reticular nuclei receive phasic ascending signals from the locomotor networks in the most rostral spinal cord during fictive locomotion (Dubuc and Grillner 1989; Kasicki and Grillner 1986; Kasicki et al. 1989). In this preparation, however, where the agar barrier is located over segments 2-6, intracellular recordings from these cells indicate that the amplitude of the phasic modulation is low (Fagerstedt et al. 2001).

Phasic dependence on stimulation could also arise in the spinal cord. The different locomotor interneurons and motoneurons are rhythmically modulated during locomotion (Buchanan and Cohen 1982; Buchanan and Kasicki 1995; Buchanan et al. 1989). A descending command would have a greater effect if it arrives when the neurons are in their depolarized phase (on the active side of locomotor network), than if they occur on the inactive, hyperpolarized side.

Integration of the descending turn command

To change behavior from rectilinear swimming to horizontal turning, either separate mechanisms in the spinal cord should be activated, or the activity of the neuronal networks controlling rectilinear swimming should be modified. Changes in the activity of motoneurons could be caused either by a direct effect on motoneurons exerted by descending pathways, or indirectly, by affecting the locomotor interneurons, or by a combination of both. The changes in cycle duration and burst duration, however, require modulation of the activity of locomotor interneurons, i.e., the CPG. RS neurons have been shown to monosynaptically excite both spinal motoneurons and interneurons constituting the locomotor CPG (Ohta and Grillner 1989). The lamprey locomotor CPG can be modeled as two hemi-segments coupled by reciprocal inhibition (Buchanan 1986; Buchanan and Grillner 1987). Results from computer modeling studies indicate that excitation by descending pathways of the crossing inhibitory interneurons plays an important role for the increase of cycle and burst duration during turning (Kozlov et al. 2001).


    ACKNOWLEDGMENTS

We thank S. Grillner, G. N. Orlovsky, and D. Parker for valuable comments on the manuscript.

Support by grants from the Swedish Medical Research Council (MFR 3026), the Swedish Research Council for Engineering Sciences (TFR 282-96-905), Karolinska Institutet, Åke Wibergs Stiftelse, Magnus Bergwalls Stiftelse, and the Swedish Brain Foundation is gratefully acknowledged.


    FOOTNOTES

Present address and address for reprint requests: F. Ullén, Neuropediatrics, Dept. of Woman and Child Health, Astrid Lindgren Children's Hospital, SE-171 76 Stockholm, Sweden (E-mail: Fredrik.Ullen{at}neuro.ki.se).

Received 21 June 2000; accepted in final form 11 June 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
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RESULTS
DISCUSSION
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0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society