Spatial Orientation in the Lamprey : I. Control of Pitch and Roll

The spatial orientation of fish and lampreys can be characterized by three angles. The roll angle in the transverse plane and the pitch angle in the sagittal plane determine the animal’s orientation in the gravity field, while the yaw angle characterizes the direction of swimming in the horizontal plane. Fish have numerous effector organs to stabilize and change their orientation in space: tail, dorsal and ventral fins, paired pectoral and pelvic fins, and the body musculature. Lateral movements of the body and tail, and asymmetrical braking with the pectoral fins, are the main means of performing a turn in the horizontal plane (Gray, 1933, 1968; Harris, 1936). In elasmobranchs, paired fins can be used for stabilizing and changing the body orientation in the sagittal and transverse planes (Gray, 1968). Vestibular reflexes, aimed at stabilizing the dorsal-side-up orientation, and using the pectoral fins as effector organs, have been characterized in the dogfish (Timerick et al. 1990).

Lampreys have no paired fins, and therefore have to use their body, tail and dorsal fins to control their spatial orientation. Early studies (de Burlet and Versteegh, 1930) demonstrated the crucial role of vestibular reflexes, since unilaterally labyrinthectomized lampreys rolled continuously around their longitudinal axis during swimming, whereas bilateral labyrinthectomy completely abolished their ability to control orientation in any plane. The aim of the present paper was to investigate (1) whether both pitch and roll orientation are actively and independently stabilized by the vestibular system, and (2) to determine which motor patterns are used to change orientation in the transverse and sagittal planes. The respective roles of the fins and the tail in postural control were investigated with specific lesions. Video recordings of intact and lesioned animals were used to characterize the trajectories of swimming under different conditions and to characterize the strategies for turns. The behavioural deficits of labyrinthectomized lampreys (de Burlet and Versteegh, 1930) were re-examined more carefully, using frame-by-frame analysis of the recordings. In the second paper of the series (Ullén et al. 1995), the role of visual information in the control of body orientation is considered. These behavioural studies complement our electrophysiological investigations (Deliagina et al. 1992a,b, 1993a; Orlovsky et al. 1992), with the overall goal of elucidating how the central nervous system of the lamprey uses vestibular and visual information to control spatial orientation during swimming.

Adult river lampreys (Lampetra fluviatilis L., body length 25–40 cm) were collected in the autumn from the river Ljusnan (Sweden), during their upstream migratory phase, and kept in aerated freshwater aquaria at 4–10 ˚C with a 12 h:12 h light:dark cycle (illumination between 08:00 and 20:00 h). Experiments were performed in the daytime or evening (10:00–20:00 h), between October and March, during which months the animals entered the terminal spawning phase. In total, 54 intact and lesioned lampreys were studied. Permission to perform the lesions was granted by Stockholms Norra Djurförsöksetiska Nämnd. The following lesions were employed (all performed under MS222 anaesthesia, Sandoz).

(1) Spinalization, either immediately caudal to the brainstem (segments 1–3; N=5) or at a level immediately rostral to the anterior dorsal fin (segments 44–46; N=3). (2) Removal of all fins (N=3). (3) Amputation of the tail at the level of the anal opening (N=3). (4) Forcing the tail into a dorsally flexed position (N=6). This was accomplished by surgically inserting a longitudinal thread under the dorsal skin of the tail and tightening the thread until the tail assumed a dorsally flexed orientation (see Fig. 6B,C). The thread extended from either the rostral end of the anterior dorsal fin (N=3) or the middle of the posterior fin (N=3) to the end of the tail. (5) Unilateral (N=6) and bilateral (N=6) labyrinthectomy. This was performed by cutting the lateral wall of the vestibular capsule and removing the vestibular apparatus with a pair of fine forceps. (6) A combination of bilateral labyrinthectomy and blinding, by bilateral enucleation (N=3). (7) A combination of unilateral labyrinthectomy with spinalization at a level immediately rostral to the anterior dorsal fin (segments 44–46; N=3). All experiments were performed 1 day after surgery. In some cases, white ligatures were placed in the dorsal midline of the gill region to be used as markers to facilitate the analysis of the transverse orientation of the animal.

Three different aquaria were used (Fig. 1). The water temperature in the aquaria was 10–11 ˚C. The behaviour of the animals was recorded with a video camera (25 frames s^-1) and analyzed frame by frame. Swimming trajectories in the sagittal plane, and the animal’s pitch angle under different experimental conditions, were studied in a large but narrow aquarium (Fig. 1A,B; 140 cmX16.5 cmX60 cm deep). A mirror was mounted at an angle of 45 ˚ above the water surface to enable simultaneous recording of both lateral and dorsal views of the movements. The aquarium was divided into two by a vertical wall, which had a small (3 cm diameter) hole 18 cm under the water surface. The smaller right-hand part contained a cage made of wide-meshed metal netting (41 cm×16 cm×2.5 cm deep), at the level of the hole. For each test, the lamprey was initially placed in the closed cage. After the animal had come to rest in the cage, it was gently moved by hand to a straight position with its head facing the hole in the vertical wall. The hole was opened and the swimming response recorded. Locomotion was evoked by photostimulation of the trunk and tail with a 60 W lamp, located 10 cm from the cage. In this way, the dermal photoreceptors of the tail, known to activate locomotion (Ullén et al. 1991, 1992, 1993; Young, 1935), were stimulated. The cage was usually oriented horizontally, but in some experiments it was inclined at either +15 ˚ or -15 ˚ relative to horizontal (Fig. 1A).

The left-hand part of the large aquarium (Fig. 1A) was also employed to record free swimming without external stimuli (periods of 5 or 10 min of spontaneous locomotion). When recordings at a higher magnification were required, a smaller aquarium (Fig. 1C,D; 60 cmX35 cmX30 cm deep) was used. The recordings permitted assessment of movement patterns associated with changes in pitch (spontaneous turns upwards and downwards) in intact animals and alterations of swimming behaviour in lesioned animals. Swimming of unilaterally labyrinthectomized animals was studied in a larger, flat aquarium (80 cmX80 cmX8 cm deep, see Fig. 7B). To exclude visual influences on the orientation of the lampreys (e.g. negative phototaxis; Ullén et al. 1995), diffuse background illumination was used in all experiments.

When leaving the cage, the lampreys usually (99 %, N=174, 10 animals) swam along descending trajectories towards the bottom of the aquarium (see Fig. 3). In Fig. 2, the vertical (Fig. 2A) and lateral (Fig. 2B) projections of the most rostral point of the head are indicated with black dots for each consecutive frame. In Fig. 2B, the trajectory of the most caudal point of the tail is also indicated with asterisks and, for a few selected frames, the outline of the lamprey’s body is presented. The five following main characteristics were observed.

First, in 78 % of all tests (N=174, 10 animals), the pitch angle of swimming,, varied by less than 7 ˚, i.e. the swimming trajectory was practically linear in the sagittal plane (Fig. 2B; see also Figs 3A, 4A). In all cases, the speed of swimming at the end of the trajectory was considerably higher than the initial speed (between 1.4-and 4.2-fold increase), measured immediately after the animal had left the cage. For the trajectory shown in Fig. 2, the speed approximately tripled (0.31 m s^-1 at frame 10, compared with 0.94 m s^-1 at frame 30). The mean initial speed (N=34, four animals) was 0.38±0.17 m s^-1, and the mean final speed was 0.79±0.29 m s^-1 (mean ± ^S.^D.). In 22 % of the tests (N=174, 10 animals), the lampreys spontaneously turned abruptly upwards or downwards during swimming, as in the trajectories marked with a cross in Fig. 3A.

Second, the trajectory of the tail was always below that of the head (Fig. 2B). At higher speeds, there was a ventral flexion of the tail, in the region of the posterior dorsal fin (see, for example, frame 32 in Fig. 2B), whereas the head trajectory coincided with the body axis. At lower speeds, the whole body axis formed an angle with the trajectory of swimming (frame 19 in Fig. 2B).

Third, the relationship between the speed of swimming and was investigated for four animals (N=34). Both initial and final speed were positively correlated with (r=0.52, P<0.002 and r=0.75, P<0.0001, respectively); that is, higher speeds tended to be associated with more horizontal swimming.

Fourth, in open water, lampreys always swam with the dorsal side up. This orientation was maintained at any pitch angle. When lampreys swam along a wall, however, they sometimes oriented their belly towards the wall. Orientation was estimated from the position of markers inserted into the dorsal skin, or from the position of the dorsal fins, as seen in the vertical projection.

Fifth, lampreys showed no preferential direction of swimming in the horizontal plane. The animals continued to swim along the aquarium midline or, after a while, turned smoothly to the left or the right. Changing direction in the horizontal plane did not alter their orientation in the sagittal or transverse planes, indicating that the yaw control system can operate independently of the pitch and roll control systems (see Fig. 2).

Repeated testing of the same animal, with the cage oriented horizontally, yielded trajectory angles clustered relatively close to the average value. Fig. 3A shows the set of trajectories obtained with animals 1 and 4. The average pitch for each animal is indicated with a black arrowhead (not-straight trajectories, marked with a cross, were excluded). The average pitches for all tested animals (N=10) are shown in Fig. 3B. In four animals, the difference between the highest and the lowest value was less than 15 ˚.

To test further whether pitch is actively stabilized during swimming, trajectories with different initial pitch inclinations were investigated. Fig. 4A shows a set of trajectories obtained for the same animal when the cage was alternated between a 15 ˚ upwards (dashed lines) and 15 ˚ downwards (solid lines) orientation. Despite a difference in initial angle of 30 ˚, the trajectories largely overlapped. In Fig. 4B, the average angles of the trajectories with an upward (dashed arrows) and a downward (solid arrows) orientation of the cage are compared for all six animals tested. The difference in average angle was always less than 4 ˚ (mean 2.3 ˚), i.e. considerably less than the difference in initial inclination (30 ˚). Thus, the pitch angle was almost independent of the initial orientation of the animal.

The lampreys usually only performed turns upwards or downwards when hitting a wall, the bottom of the aquarium or the water surface. Forty turns in the vertical plane (in five animals) were spontaneously performed in open water, that is without any mechanical interaction with a surface. The majority of these (35) were downward turns. Two strategies were observed. At higher swimming speeds (0.5–1 m s^-1), the turn was usually accomplished by a ventral flexion of the whole body into an arc (Fig. 5A), so that the trajectory of swimming coincided with the longitudinal body axis of the animal. A second strategy, in which sharp turns were performed by ventrally flexing the body locally in the region of the gills (Fig. 5B), was observed at lower speeds, as when the lamprey came out of the cage in the large aquarium (see the two rightmost trajectories, marked by crosses, in Fig. 3A). Such turns were also observed in the middle of the aquarium (see the trajectory marked by an arrow in Fig. 3A).

Upward turns in open water were observed in only five cases. In the example shown in Fig. 5C, a dorsal flexion of the whole body into an arc was used; this was the most common strategy. Near a wall, both upward and downward turns occurred, as illustrated for an upward turn in Fig. 5D. Hitting the wall (frame 1) evoked a complicated behaviour (frames 3–12). The anterior part of the body was initially flexed strongly laterally (frame 3). During the turn, this lateral flexion was continuously propagated towards the tail (compare frames 3, 7 and 12). A simultaneous roll turn was also propagated caudally. This can be seen from the gradual appearance of more caudal areas of the white belly. Evidently, this behaviour was, to a large extent, triggered by mechanical interaction with the wall, along which the lamprey moved.

The ability to swim with the dorsal side up persisted even after removal of both dorsal and tail fins (N=3; Fig. 6A). The fins are therefore not essential for stabilization of the dorsal-side-up orientation during swimming under these conditions. Corrective movements made by the tail probably play a crucial role in maintaining the dorsal-side-up orientation. The tail is always deflected ventrally, both in intact lampreys (see Fig. 2B) and in animals without fins (Fig. 6A). The possible significance of this was investigated by forcing the tail into a dorsally flexed position using a longitudinal thread inserted into the dorsal superficial myotomes (Fig. 6B,C). In three animals, the thread was positioned so that the part of the body between the middle of the posterior dorsal fin and the tail was flexed. This led to a considerable disturbance of postural control. In Fig. 6D, the lateral projection of such a swimming trajectory is shown, with the outline of the body indicated for frame 17. Dots indicate frames in which the animal had a normal, dorsal-side-up orientation, whereas crosses indicate frames in which the animal was tilted by more than 45 ˚. Periods with normal orientation thus alternated with periods with considerable tilt. Interestingly, these animals had a marked ventral flexion just rostral to the anterior dorsal fin (Fig. 6D). If the surgically implanted thread extended further inside the body, from the rostral end of the anterior dorsal fin to the tail fin, the animals (N=3) lost control over their posture during swimming and continuously oscillated around the longitudinal axis.

Three animals, spinalized immediately rostral to the anterior dorsal fin, similarly lost the normal ventral flexion of the tail. Instead, they exhibited a slight ventral flexion of the body immediately rostral to the lesion. Similar results were obtained when the tail had been amputated just caudal to the anal opening (N=3). These two groups of animals, in which the normal ventral flexion of the caudal body was severely disturbed but not completely abolished, showed a movement pattern during locomotion similar to that of animals with a forced dorsal flexion of the tail region. The normal dorsal-side-up orientation during locomotion was frequently interrupted by episodes of tilted orientation.

Ventral flexion of the tail, which is an effector organ for the vestibuloreticulospinal system (see Discussion), thus appears to play a crucial role in the active stabilization of the dorsal-side-up orientation. Two other motor patterns affecting the degree of roll have been revealed in labyrinthectomized lampreys (see below). In addition, passive mechanical properties of the lamprey body may, to some extent, also contribute to the maintenance of normal orientation. The high-spinal lamprey, deprived of locomotor activity and righting reflexes, assumed a dorsal-side-up orientation when left to sink without any active movements (Fig. 6E). This is probably due to a roll evoked by the high hydrodynamic resistance of the dorsal fins.

To examine the contribution of the vestibular system to spatial orientation, unilaterally and bilaterally labyrinthectomized animals were studied (Fig. 7). After unilateral labyrinthectomy (N=6), a continuous rotation around the longitudinal axis occurred during swimming (see also de Burlet and Versteegh, 1930). The lesioned side always rolled downwards as the animal passed the normal dorsal-side-up orientation (if viewed from the front, left-side labyrinthectomy resulted in a clockwise rotation, right-side labyrinthectomy in a counter-clockwise rotation). Fig. 7A shows a sequence of swimming (lateral view), in which the outline of the body and the border between the dark back and the white belly are indicated, to illustrate the sequential rotation of the body. Between frames 1 and 25 (1 s) the animal rolled through 360 ˚. During free swimming, the body of unilaterally labyrinthectomized animals had a weakly helical shape, so that the sagittal plane of the tail region was rotated approximately 90 ˚ in relation to the sagittal plane of the head region (frames 10 and 13 in Fig. 7A). The undulatory locomotor waves were superimposed on the helical body shape. The swimming trajectory was not a straight line, but a helix, with a period equal to one body turn, as clearly seen in a vertical projection (Fig. 7B). When unilaterally labyrinthectomized lampreys ‘swam’ against the wall of the aquarium, they still rolled continuously, but without forward progression (Fig. 7C). In this case, no twisting of the body was observed and the rolling was probably due to asymmetrical tail movements (see Discussion).

Bilaterally labyrinthectomized animals (N=6) lost all control of their orientation during free swimming. They were never able to follow linear trajectories and turned continuously in all planes. Fig. 7D,E shows the top and side views, respectively, of a typical trajectory during 4 s of free swimming, during which three complete ‘loops’ were performed in both planes. Similar trajectories were observed in animals in which a bilateral labyrinthectomy was combined with bilateral enucleation (N=3). Therefore, the destabilization of the postural control system is not a consequence of visual input. Occasionally, prolonged periods (up to 3 min) of backward swimming were observed in three labyrinthectomized animals. This will be reported separately in a subsequent paper.

In the majority of locomotor responses, the swimming trajectory formed an almost straight line in the sagittal plane, i.e. the animal maintained a certain pitch angle (β) while swimming. Two main explanations for this finding can be considered: either the initial direction of swimming is maintained passively as a result of the hydrodynamic properties of the lamprey body, or a particular pitch orientation is continuously stabilized by the central nervous system. Several observations support the second hypothesis. (1) The same value of β was generally maintained, despite large changes in swimming speed along the trajectory. Without active stabilization of pitch, β would be expected to depend largely on the swimming velocity. At low speeds of active swimming, such as in the initial part of the trajectories, the vertical downward component due to passive sinking in the gravity field should give higher values of β. However, in cases where the trajectory was weakly convex, β was always slightly higher at the end of swimming, when the speed was highest (see Figs 2B, 3A). (2) The different values of β obtained with repeated testing of the same animal clustered very close to the mean value, even when there was a 30 ˚ difference in initial orientation between trials (Figs 3, 4), i.e. the lampreys tended to maintain a certain preferred pitch over several trials. The evidence, taken together, shows that the postural control system in the lamprey, driven by vestibular input, can be set to stabilize different pitch angles. This stabilized pitch angle or ‘zero point’ in the postural control system can apparently be maintained over a period of at least several minutes, a characteristic property of many gravity orientation systems in both vertebrates and invertebrates (Orlovsky, 1991). Recordings from reticulospinal neurones (which constitute the main descending pathway in lamprey; Brodin et al. 1988; Rovainen, 1967, 1979, 1983) in the brainstem–labyrinth preparation in vitro have shown that responses to roll are very consistent, whereas responses to pitch can change with time, suggesting a recalibration of the pitch control system (Deliagina et al. 1992a).

Turns upwards and downwards were performed by flexing the body in the sagittal plane. This implies that brainstem centres can exert differential control over the dorsal and ventral parts of the myotomes. For low-speed downward turns, a local activation of the ventral musculature in the gill region is required. Wannier et al. (1992) have shown that a subgroup of reticulospinal neurones project only to rostral segments. These neurones may be responsible for local flexions in this region. Among cells projecting to more caudal regions are neurones affecting only the dorsal or ventral parts of the myotomes. They may be responsible for ventral and dorsal flexions of the whole body.

Maintenance of the correct orientation in the transverse plane depends entirely on the continuous operation of vestibular reflexes. The observation that the dorsal-side-up orientation is stabilized at different pitch angles implies that the roll and pitch control systems can operate independently (for a discussion of the independence of roll and pitch signals, see Mittelstaedt, 1975). The change in orientation (lateral-side-up) sometimes seen when a lamprey swims along a vertical wall may depend on tactile stimulation from the wall.

Removing all the fins did not significantly disturb the dorsal-side-up orientation during swimming, and the lamprey was still able to follow a linear trajectory (Fig. 6A). Fin movements are therefore not crucial for basic postural control in the lamprey. Nevertheless, it appears likely that the dorsal fins are important for the finer adjustment of movements. Lamprey dorsal fin motoneurones are activated in antiphase with the somatic motoneurones of the same segment during locomotion, thus counteracting tilting of the fin (Buchanan and Cohen, 1982; Shupliakov et al. 1992). Illumination of one eye in lampreys evokes twisting of the anterior dorsal fin, so that the caudal part is flexed contralaterally (Ullén et al. 1995). It appears very likely that this fin response contributes to the roll of the animal seen in the dorsal light response (Ullén et al. 1995) and, therefore, that fin movements, at least in some situations, play a role in stabilization of the dorsal-side-up orientation.

The lesion experiments strongly suggest that the ventrally flexed tail is crucial for maintenance of the dorsal-side-up position in lampreys. Three possible explanations in which ventral deflection of the tail is essential for the control of roll can be formulated. (1) The tail acts as a rudder. If the tail is flexed laterally during forward swimming, the opposing water stream will exert pressure upon it and thus evoke a roll turn. Unilaterally labyrinthectomized animals, however, continue to rotate even when they are not moving forwards. Since this ‘rudder mechanism’ requires forward progression, it cannot be important for roll turns. (2) The roll turn starts in the anterior part of the body and then propagates so that progressively more caudal segments perform the turn, until the wave of rotation reaches the tail. In this case, the ventrally flexed tail, working against the high water resistance, could prevent rotation of the posterior trunk while the anterior part rotates. In unilaterally labyrinthectomized animals swimming against a wall, however, there was no asynchrony in the rotation of the rostral and caudal body parts (see Fig. 7C), indicating that this mechanism, if used, is of minor importance. (3) Active movements of the tail generate a reactive force against the water resistance, thus rotating the body around its longitudinal axis (the side of the body towards which the tail is flexed would roll downwards). This hypothesis is consistent with all observations.

The last hypothesis also corresponds to results obtained while studying vestibulospinal reflexes in lampreys. A roll tilt will elicit excitation of the contralateral reticulospinal neurones (Deliagina et al. 1992a) which, in turn, evoke excitation of motoneurones on the same side (Deliagina et al. 1993b). Any deflection (roll) from the normal dorsal-side-up orientation will thus evoke a deflection of the tail towards the opposite side and, since the tail is flexed ventrally, a compensatory torque around the longitudinal body axis, restoring the normal orientation. Twisting of the body into a helical shape, which was observed in unilaterally labyrinthectomized animals, is probably an additional strategy used to perform roll turns during swimming. At least three different mechanisms for performing roll turns, with different effector organs, appear to be used by lampreys: (1) lateral movements of the ventrally flexed tail; (2) twisting of the body; and (3) twisting of the anterior dorsal fin (Ullén et al. 1995).

In summary, the present work shows that the pitch and roll angles of a swimming lamprey are actively and independently stabilized by its vestibular system. Whereas the roll control system normally stabilizes an orientation with the dorsal side up, switches in the preferred angle of the pitch control system are common. In effect, therefore, the two vestibular control systems continuously compare their current ‘set-point’ with the spatial orientation of the lamprey and generate a correcting motor command whenever the roll or pitch angle of the animal deviates from the ‘desired’ value. These correcting manoeuvres (i.e. turns in the transverse and sagittal planes) have a common requirement for specific control of different groups of myotomal motoneurones, both dorsoventrally and rostrocaudally, and of the fin motoneurones.

The role of vision in postural control, as well as other responses to homogeneous visual stimuli, will be considered in a companion paper (Ullén et al. 1995).

This work was supported by the Swedish Medical Research Council (project no. 3026), Karolinska Institutets fonder, Karolinska Institutet visiting scientist fellowship and the Royal Swedish Academy of Sciences (research grant for Swedish–Russian scientific cooperation).