1The Nobel Institute for Neurophysiology, Department of Neuroscience, Karolinska Institute, SE-171 77 Stockholm, Sweden; and 2A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119 899, Russia
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ABSTRACT |
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Deliagina, T. G.,
P. V. Zelenin,
P. Fagerstedt,
S. Grillner, and
G. N. Orlovsky.
Activity of Reticulospinal Neurons During Locomotion in the
Freely Behaving Lamprey.
J. Neurophysiol. 83: 853-863, 2000.
The reticulospinal (RS) system is the main
descending system transmitting commands from the brain to the spinal
cord in the lamprey. It is responsible for initiation of locomotion,
steering, and equilibrium control. In the present study, we
characterize the commands that are sent by the brain to the spinal cord
in intact animals via the reticulospinal pathways during locomotion. We
have developed a method for recording the activity of larger RS axons
in the spinal cord in freely behaving lampreys by means of chronically
implanted macroelectrodes. In this paper, the mass activity in the
right and left RS pathways is described and the correlations of this
activity with different aspects of locomotion are discussed. In
quiescent animals, the RS neurons had a low level of activity. A mild
activation of RS neurons occurred in response to different sensory
stimuli. Unilateral eye illumination evoked activation of the
ipsilateral RS neurons. Unilateral illumination of the tail dermal
photoreceptors evoked bilateral activation of RS neurons. Water
vibration also evoked bilateral activation of RS neurons. Roll tilt
evoked activation of the contralateral RS neurons. With longer or more
intense sensory stimulation of any modality and laterality, a sharp,
massive bilateral activation of the RS system occurred, and the animal
started to swim. This high activity of RS neurons and swimming could
last for many seconds after termination of the stimulus. There was a
positive correlation between the level of activity of RS system and the
intensity of locomotion. An asymmetry in the mass activity on the left
and right sides occurred during lateral turns with a 30% prevalence (on average) for the ipsilateral side. Rhythmic modulation of the
activity in RS pathways, related to the locomotor cycle, often was
observed, with its peak coinciding with the electromyographic (EMG)
burst in the ipsilateral rostral myotomes. The pattern of vestibular
response of RS neurons observed in the quiescent state, that is,
activation with contralateral roll tilt, was preserved during
locomotion. In addition, an inhibition of their activity with
ipsilateral tilt was clearly seen. In the cases when the activity of
individual neurons could be traced during swimming, it was found that
rhythmic modulation of their firing rate was superimposed on their
tonic firing or on their vestibular responses. In conclusion, different
aspects of locomotor activityinitiation and termination, vigor of
locomotion, steering and equilibrium control
are well reflected in the
mass activity of the larger RS neurons.
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INTRODUCTION |
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The reticulospinal (RS) system plays a predominant
role in the control of posture and locomotion in all vertebrates. This paper describes the activity of the RS system during locomotion in the
lamprey, a phylogenetically ancient vertebrate. The lamprey is used
extensively as an animal model for studying basic neural mechanisms
controlling motor behavior (see e.g., Grillner et al. 1995; Wallén et al. 1992
). In the lamprey,
the RS system is the main descending system projecting from the brain
stem to all parts of the spinal cord. The vestibulospinal system is
much less developed, projecting only to the anterior part of the spinal
cord (Ronan 1989
; Rovainen 1979
). In
principle, signals from the brain to the spinal cord also can be
transmitted in part by propriospinal systems (see e.g., Shik
1993
) but the knowledge on these systems in the lamprey is
rather limited (see, however, Rouse and McClellan 1997
).
The RS system in the lamprey is composed of several hundred neurons
located in the following four bilateral reticular nuclei of the brain
stem: the mesencephalic reticular nucleus (MRN) and the anterior,
middle, and posterior rhombencephalic reticular nuclei (ARRN, MRRN, and
PRRN, respectively) (Bussiéres 1994; Nieuwenhuys 1972
; Ronan 1989
;
Swain et al. 1993
). The majority of the RS neurons are
glutamatergic (Brodin et al. 1988
; Ohta and
Grillner 1989
). They exert an excitatory action on their
targets throughout the whole extent of the spinal cord and thus
activate the segmental locomotor networks to initiate swimming. These
studies also suggest that the intensity of locomotion is determined by the degree of activation of RS neurons (Grillner et al.
1995
; McClellan and Grillner 1984
; Ohta
and Grillner 1989
).
When swimming, the lamprey performs numerous maneuvers related to
steering and equilibrium control. These modifications of the locomotor
pattern are most likely caused by the corresponding modifications of
the activity of RS system. All sensory inputs (vestibular, visual,
somatosensory), affecting postural orientation and steering in the
swimming lamprey, strongly modify the activity of RS neurons
(Deliagina et al. 1992, 1993
, 1995
; Ullén
et al. 1996
, 1998
). In addition, many RS neurons exhibit
rhythmical modulation in response to "efference copy" signals
arriving from the spinal cord during swimming (Kasicki et al.
1989
; Vinay and Grillner 1992
) and therefore are
involved in an internal feedback loop. The RS system thus can be
considered as a multifunctional system, affecting different aspects of
the locomotor activity of the lamprey.
Information on the activity of RS system in the lamprey has been obtained mainly in in vitro experiments, using preparation of the isolated brain stem, with or without the spinal cord, in which many inputs to RS neurons were eliminated. A goal of the present study was to investigate the activity of RS system in the behaving intact lamprey.
When studying the activity of central neurons in intact mammals, the
bony skull usually is used as a rigid platform for securing the
recording microelectrode (see e.g., Drew et al. 1986).
In the lamprey, however, this method is difficult to apply because the
cartilage tissue surrounding the brain is comparatively soft. To
overcome these difficulties, we have developed a novel method for
recording activity of RS neurons from their axons by means of
macroelectrodes implanted in close proximity to the spinal cord.
The following problems are considered in the present paper:
1) correlations between the mass activity of RS system and
the locomotor activity elicited by different sensory inputs. It is known that swimming in the lamprey can be evoked by a number of physiological stimuliby tactile stimulation of different parts of the
body (McClellan 1984
, 1988
; McClellan and
Grillner 1983
), by eye illumination (Ullén et al.
1993b
), by illumination of the tail region where dermal
photoreceptors are located (Ullén et al. 1993b
),
by water vibration (Currie 1991
), and by vestibular stimulation, that is, inclination of the animal (Orlovsky et al. 1992
). These different types of sensory stimuli were employed in the present study. 2) Modifications of the mass RS
activity related to changes in the intensity of locomotion and to the
changes in the animal's orientation. 3) Reflection of
different aspects of locomotor control in the activity of individual RS
neurons. Activity of individual RS neurons in the intact nonswimming
lamprey is considered in the accompanying paper (Deliagina and
Fagerstedt 2000
).
Brief accounts of this study have been published in abstract form
(Deliagina et al. 1997; Orlovsky et al.
1997
).
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METHODS |
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Experiments were carried out on 10 adult (25-35 cm in length) intact lampreys (Lampetra fluviatilis), which were kept in an aerated freshwater aquarium at 7°C, with a 12 h:12 h light:dark cycle.
Electrodes
The activity of RS neurons was recorded from their axons in the
spinal cord by means of chronically implanted electrodes. The idea
underlying this novel method was to use not micro- but macroelectrodes
(thin wires) oriented in parallel to the long spinal axons (Fig.
1A). If the length of the
electrode is close to the longitudinal extent of the axonal membrane
excited by the propagating action potential, the whole electrode occurs
positioned in approximately equipotential points in the moment when the
excited membrane area opposes the electrode (Fig. 1B). No
current will flow along the electrode, and it will record the
extraaxonal potential with the same amplitude as a microelectrode
positioned at the same distance from the axon. An advantage of the wire
electrode is its mechanical stability, a very low resistance
(<103), and a low noise level (a few
microvolts). In a thinner axon (Fig. 1C), the excited part
of the fiber will be shorter, the electrode will be positioned along
points with different potentials, and a considerable shunting effect
will be caused by currents flowing along the electrode. In addition to
this effect, thinner axons provide smaller membrane currents as
compared with the thicker axons. Thus the wire electrode can serve as a
filter for recording spikes almost exclusively from the larger fibers
situated parallel with the electrode.
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The large fibers in the lamprey spinal cord are the RS axons of the
Mauthner cells and Müller cells from MRN, ARRN, and MRRN; the
middle-size fibers are the RS axons originating from MRRN and PRRN
(Fig. 1D) (Ohta and Grillner 1989;
Rovainen 1982
). Most of these fibers have conduction
velocity ranging from 2 to 5 m/s (Ohta and Grillner
1989
; Rovainen 1978
). With a spike duration in
these axons equal to ~1 ms, the length of the excited axonal segment
will be 2-4 mm. To record from these axons, we used electrodes with a
length of 3 mm. They were made of 75-µm silver wire coated with a
Teflon insulation, except for at their 3-mm-long active part. This part
was glued to a plastic plate, 6 mm long and 0.25 mm thick (Fig.
1E). The width of the plate was equal to or slightly larger
than that of the spinal cord (1.7-2 mm). Similar wires were glued to
the opposite side of the plate. This allowed us, by using a bipolar
recording technique and differential amplifiers, to substantially
reduce the artifacts caused by the electrical activity of the
surrounding muscles. The thin silver wires were then soldered to
thicker (0.25 mm), flexible, Teflon-coated copper wires that conveyed
signals to the amplifier input cable.
The distance between the left and right electrodes was chosen so that
each of them occurred positioned above the center of the corresponding
half of the spinal cord (Fig. 1D). Experiments described in
the accompanying paper (Deliagina and Fagerstedt 2000)
have shown that the electrodes can record activity in the larger axons
at a distance of
400 µm. With a width of the spinal cord of
~1,500 µm (Fig. 1D), one can suggest that each of the electrodes will record activity of the larger axons over almost the
whole cross-section of the ipsilateral half of the spinal cord.
To record separately from the left and the right RS pathways, a longitudinal plastic wall was glued to the plate with the electrodes (Fig. 1E). This wall then was positioned in the split made between the two halves of the spinal cord, and thus the left and right RS pathways were isolated electrically from each other in the area of the electrode. In most experiments, this isolation was practically complete, and each electrode recorded exclusively the ipsilateral RS activity. In some experiments, however, the isolation was incomplete and secured only a 5- to 10-fold reduction of the amplitude of the contralateral spikes (see e.g., Fig. 9A).
Implantation of electrodes
Implantation of the electrodes was performed under MS-222 (Sandoz) anesthesia (100 mg/l). The animal was positioned in the bath with the anesthetic solution that covered the gills whereas the surgical field was above the water level. The plate with electrodes was implanted at the level of the last gill through a longitudinal cut performed along the midline of the dorsal aspect of the body so that the spinal cord was exposed. The layer of fat covering the spinal cord was removed, and the midline split of the spinal cord extending for ~5 mm was performed. The plate was positioned on the dorsal surface of the spinal cord, with the longitudinal wall inserted into the split (Fig. 1F). In addition to the differential recording technique (see preceding text), two more methods were used to reduce the artifacts caused by the electrical activity of the muscles surrounding the electrodes. First, in all experiments, before implanting the electrodes, the dorsal and ventral roots were cut bilaterally throughout 10-15 spinal segments, symmetrically in relation to the site of electrode implantation, and the corresponding myotomes thus were denervated. We did not observe any significant changes of locomotor performance after such surgery. Second, in five experiments, the implanted plate with the electrodes, together with the adjoining segments of the spinal cord, were wrapped in a strip of thin (20 µm) plastic film (Fig. 1F) to isolate the electrodes electrically from the surrounding muscles. An indication that there was no electromyographic (EMG) contamination was the absence of any voltage when recording, during vigorous swimming, between the left and right electrodes facing dorsally (Fig. 1F). The wound was then closed and sutured so that the flexible copper wires were fixed tightly between the two sides of the wound (Fig. 1F).
In a few experiments, an additional plate with electrodes was implanted
at the level of the third gill, so that the distance between the two
plates was 20-30 mm. This allowed us, by comparing the moment in time
of spike occurrence in the two electrodes, to determine the direction
of spike propagation in the axons and to calculate their conduction
velocity (for details, see Deliagina and Fagerstedt
2000).
Experimental setup
In most experiments, the lamprey was freely moving in a shallow
aquarium (80 × 80 or 50 × 30 cm) (Fig.
2A). Movements of the animal
were videorecorded (25 frames/s). The implanted electrodes for
recording the activity in the RS pathways were connected, via a long
flexible cable, to the inputs of AC amplifiers. Via the same cable, two
bipolar EMG electrodes (flexible wires, 150 µm diam, implanted
bilaterally in the myotomes of the gill region) also were connected to
the amplifier inputs. The electrophysiological and video recordings
were synchronized by pulses (1 Hz) recorded simultaneously by both
systems (Fig. 2A, Synchro). Different sensory stimuli were used to evoke locomotionpinching the head or the tail,
illumination of the eye or tail by means of a fiber optic system (90 W), or water vibration (50 Hz) produced in a close proximity to the
head of lamprey.
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In some experiments, a special setup was used that allowed us to apply
vestibular stimuli during the locomotor-like activity of the lamprey
(Fig. 2B). The animal was mounted on a platform so that a
part of its body (~20% of the total length), including the
denervated segments, was fixed gently between the two plates of the
holder. This part of the body thus was immobilized, whereas the
anterior and posterior parts could move. The locomotor-like activity
appeared either spontaneously or it could be evoked by tactile
stimulation. During this activity, the anterior and posterior parts of
the body performed periodical lateral undulations characteristic of
swimming, as shown by the bilateral arrows in Fig. 2B.
Because of the fixation of the midbody area, the lamprey was not able to produce a roll tilt by itself, but the tilt could be imposed by
rotating the platform manually. Usually alternating 90° tilts to the
right and to the left were performed. In animals exhibiting locomotor-like activity, the body was rigid enough so that an imposed
tilt caused an inclination in all parts of the body. In quiescent
animals, additional mechanical restraints, connected to the rotating
platform, allowed a transmission of rotation to all parts of the body
(see also Deliagina and Fagerstedt 2000).
Processing of data
Signals from the implanted electrodes were amplified by conventional AC amplifies, digitized with a sampling frequency of 10 kHz and recorded to the disk of an IBM compatible computer by means of data acquisition software (Digidata 1200/Axoscope, Axon Instruments, Foster City, CA). The mass RS activity (multiunit spike trains) then was presented in the form of temporal histograms (100, 200, or 500 ms binwidth) using software analysis (Datapac III, Run Technologies, Laguna Hills, CA). Small waveforms were excluded from the analysis by adjusting the threshold to a level slightly above the noise level.
Videorecordings were analyzed frame by frame, and the behavioral state of the animal was classified into one of the three categories: attached to a substrate, lateral turn (left or right), and rectilinear swimming. By using the synchronizing markers, electrophysiological recordings were aligned with data obtained from videorecordings.
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RESULTS |
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Activity of spinal axons in nonswimming and swimming animals
Most of the time the lampreys can be found in a quiescent state,
attached to the bottom or walls of the aquarium by their sucker mouth.
The activity in spinal axons, recorded in the attached animals, was
rather low (Fig. 3A, beginning
of the recording). Sensory stimuli could evoke an increase of the
axonal activity. Illumination of one of the eyes evoked activation of
spinal axons on the ipsilateral side (middle part of Fig.
3A, see also Fig. 4). The
spikes recorded by the rostral electrode were followed by the spikes
recorded by the caudal electrode with a delay of a few milliseconds
(Fig. 3B), demonstrating that the spikes were generated by
descending axons with a conduction velocity of a few meters per second.
The overwhelming majority of axons recorded in the present study and in
a related study (Deliagina and Fagerstedt 2000) had
conduction velocities from 2.5 to 5 m/s, which is characteristic of
larger RS neurons
Müller cells and Mauthner cells from MRN, ARRN
and MRRN, as well as of midsize RS neurons from MRRN and PRRN
(Ohta and Grillner 1989
; Rovainen 1978
).
These descending axons have a conduction velocity exceeding that of
propriospinal axons and thus must originate in the brain stem reticular
nuclei. The frequency of discharge in individual descending axons,
caused by visual stimuli, usually did not exceed 3-5 Hz (see also
Deliagina and Fagerstedt 2000
).
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Illumination of the eye (Fig. 3A) elicited in the attached
lamprey a roll tilt toward the light source. This movement is termed an
attached-state dorsal light response; it is caused by asymmetrical contraction of muscles around the mouth (Ullén et al.
1993a). With prolonged stimulation, an "explosive" increase
of axonal activity usually occurred, followed by a detachment of the
lamprey from the substrate and swimming (swim in Fig.
3A). During swimming, the axonal activity was so high that
individual spikes were difficult to identify. In a few cases, grouping
of spikes into bursts was observed in some parts of recording, as
illustrated in Fig. 3C; the bursts in the caudal electrode
were delayed by a few milliseconds in relation to the bursts in the
rostral electrode. These delays were similar to those observed in the
attached state (Fig. 3B), suggesting that the high level of
activity recorded during swimming was caused, at least partly, by the
same group of larger RS neurons. In a few cases, we managed to trace
the activity of the same individual neurons both in the quiescent state
and during swimming. Their firing frequency increased considerably
during swimming, sometimes
10-12 Hz (see Fig. 9).
Further evidence that the high level of mass activity, recorded by the implanted electrodes, was caused by discharges in descending reticulospinal axons rather than in the ascending axons, was obtained in the experiment of Fig. 4A. It shows an activation of the spinal axons during swimming evoked by eye illumination. The lamprey subsequently was spinalized at five segments rostral to the recording electrodes, and locomotor activity then could be evoked caudal to the lesion by pinching the tail (Fig. 4B). Despite intense locomotor movements, no axonal activity comparable in size to that of Fig. 4A was recorded in the spinalized preparation.
A sharp activation of RS neurons at the onset of locomotion was always bilateral. This bilateral pattern of activity was observed with all types of stimuli eliciting locomotion, that is with unilateral eye illumination (Fig. 4, A, C, and D), tactile stimulation of the head (Fig. 5), vibration of the water (Fig. 7), illumination of the tail dermal photoreceptors (Fig. 8A), and lateral tilt of the animal (not illustrated), or when locomotion started spontaneously (not illustrated).
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Correlation between RS activity and locomotor activity
We tried to find correlations between the level of the mass RS activity and different characteristics of locomotion. One of these is the moment when the animal detached from the substrate and started swimming. For the trial illustrated in Fig. 5, A and B, the level of RS activity on the two sides at the moment of detachment is indicated by arrows. In repeated tests in the same animal or in different animals, the detachment always occurred at the rising phase of RS activity, when this activity reached 53 ± 26% (mean ± SD; n = 30) of the plateau level. At the end of the swim episode, the lamprey usually attached itself to the substrate when the level of RS activity was very low, as illustrated in Fig. 5.
The correlation between the mass RS activity and the vigor of swimming
was studied in experiments by loading the lamprey (Fig. 6C). The experimenter could
hamper the forward progression of the animal by pulling a lead attached
to the lamprey's body. In a part of these experiments, the applied
force was measured by the transducer (F in Fig. 6B). The
loading resulted in an increase of the mass RS activity, whereas
unloading resulted in a decrease of this activity (Fig. 6, A
and B). Two major values characterizing the vigor of
locomotionthe amplitude and the frequency of locomotor EMG
bursts
increased markedly when the load was applied (Fig. 6A). A strong positive correlation between the vigor of
locomotion and the mass RS activity was observed in all tested animals
(n = 5). Thus the lamprey tries to continue the forward
swimming and to overcome the load.
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In some cases, locomotion started with a lateral turn followed by a rectilinear swimming. A characteristic feature of the turn was a prolonged unilateral muscle activity (Fig. 7A, bottom 2 traces). The turn usually was associated with some prevalence of the mass RS activity on the ipsilateral (to the turn) side over the activity on the contralateral side (Fig. 7A, histograms). We calculated the degree of asymmetry in the left and right RS activity during a turn. For this purpose, the activity on each side during the turn was normalized to the mean level of activity during the rectilinear swimming that followed the turn. The ratio (asymmetry in Fig. 7B) of the activity on the ipsilateral and the contralateral side then was averaged over the duration of turn. Seven turns in three animals were used; the turning angles were between 90 and 135°. In six cases, the ipsilateral RS activity during the turn was larger than the contralateral one (asymmetry >1, Fig. 7B). The asymmetry averaged over all the turns was 1.3 ± 0.3 (Fig. 7C).
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During swimming, rhythmic modulation of the mass RS activity was observed in all experiments. The degree of modulation strongly differed between the experiments, however; it also could change spontaneously during a trial or even completely disappear to reappear some cycles later. The modulation is well seen in Figs. 4A, 8, and 9 but not in the histograms probably because the binwidth (100 or 200 ms) was comparable with the locomotor cycle duration (200-300 ms, Fig. 6A). The activity on a given side usually had a peak coinciding with the burst of EMG activity in the ipsilateral muscles in the anterior part of the body, as indicated by bilateral arrows in Fig. 8A. The rhythm of modulation fluctuated along with the rhythm of locomotor body undulations (Fig. 8B).
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In most cases, individual neurons could not be distinguished in the mass RS activity occurring during locomotion. In three experiments, however, activity of individual neurons could be traced, as illustrated in Fig. 9, where two larger units [unit 1 (right) and unit 2 (left)] are clearly seen both before (A) and during swimming (B). In both trials, vestibular reactions were elicited in the RS neurons by tilting the animal (for methods, see Fig. 2B). At rest, there was no activity in either neuron (beginning of the recording in Fig. 9A). Both neurons exhibited vestibular reactions and were activated with contralateral tilt. During locomotion, the activity of both neurons increased markedly, and their firing rate became rhythmically modulated (beginning of the recording in Fig. 9B). The two RS neurons on the right and left side tended to be active in the same phase of the locomotor cycle. In most cases, however, RS neurons in opposite sides exhibited anti-phase modulation. The pattern of vestibular responses, that is activation with contralateral tilt, was preserved during locomotion (Fig. 9B). In addition, a depression of activity with ipsilateral tilt can be clearly seen. Rhythmic modulation of the activity of these RS neurons was superimposed on their vestibular responses. Qualitatively similar results have been obtained in all three experiments with vestibular stimulation during locomotion.
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DISCUSSION |
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The macroelectrodes used in the present study allowed us to
record activity almost exclusively from the large and midsize spinal
axons that have a conduction velocity >2 m/s (see also Deliagina and Fagerstedt 2000). We suggest that
"filtration" of the larger axons was caused by two factors: a
shunting effect of the wire electrode for slowly propagating spikes and
smaller extraaxonal currents in smaller axons than in larger axons.
In the spinal cord of the lamprey, there are ~20 larger RS axons
(10-50 µm diam) on each side; they have a conduction velocity of
2-5 m/s. These axons belong to the large cellsMüller cells and
Mauthner cells from MRN, ARRN, and MRRN
and to the middle-size cells
from MRRN and PRRN (Fig. 1D) (Ohta and Grillner
1989
; Rovainen 1978
, 1982
). Most RS neurons are
much smaller and have a very low conduction velocity in their axons
(Bussiéres 1994
; Kasicki et al.
1989
; Ronan 1989
). Most likely they did not
contribute significantly to the sample of neurons recorded in this
study. Thus all the results and conclusions of this paper relate to a small subpopulation of larger RS neurons with comparatively high conduction velocity.
Correlation between the level of RS activity and the vigor of locomotion
The main result of the present study is that initiation of
locomotion is always associated with a strong bilateral activation of
the RS system. This bilateral activation occurred irrespective of the
modality and laterality of the applied sensory stimulus. The RS
activity considerably outlasted the duration of the sensory stimulus,
and swimming continued as long as a high level of RS activity was
present. A similar bilateral activation of RS neurons in response to a
unilateral sensory input (illumination of tail dermal photoreceptors)
was observed earlier in a semi-intact preparation of the larval lamprey
(Deliagina et al. 1995).
A question of fundamental interest is where and how the unilateral
sensory signals of different modalities are transformed into a
bilateral, long-lasting activity. One possibility would be that this
occurs within the RS system itself. There is no evidence to suggest
interconnections between the left and right subdivisions of the RS
system, however. Furthermore in this and previous studies, it has been
shown that some sensory inputs, such as vestibular and visual signals,
can elicit a selective unilateral excitation of RS neurons without
exciting their contralateral partners (Deliagina et al. 1992,
1993
; Orlovsky et al. 1992
). This applies to
both nonswimming and swimming lampreys (see Fig. 9). The transformation from a unilateral to a bilateral signal therefore probably will take
place at a prereticular level. One of the sites of this transformation is the mesencephalic locomotor region located in the brain stem between
pons and mesencephalon in higher vertebrates (Jordan 1986
, 1991
; Shik et al. 1966
); this region has
bilateral projections to RS neurons and evokes symmetrical locomotion
in response to a unilateral stimulation (Orlovsky
1970a
,b
). In the lamprey, an analogous region has been
described (Sirota et al. 1995
). A second area found in
the lamprey is located in diencephalon (El Manira et al.
1997
). In the transformation from a short stimulus to the long-lasting response of RS neurons, plateau potentials generated by
some RS neurons in response to sensory stimuli (Viana Di Prisco et al. 1997
) and possibly also other brain stem neurons may
play a role.
In the present study it also was found that the level of mass RS
activity strongly correlated to the vigor of locomotion as characterized by the frequency of body undulations, the amplitude of
EMG bursts and the propulsive force developed by the animal (Fig. 6).
This finding suggests that the RS system in the lamprey and other
vertebrates is responsible not only for the central initiation of
locomotion but also for the regulation of the frequency and amplitude
of oscillations generated by the spinal locomotor network (Drew
et al. 1986; Jordan 1991
; McClellan
1988
; McClellan and Grillner 1984
; Ohta
and Grillner 1989
; Orlovsky 1970b
).
Correlation between the asymmetry in RS activity and the lateral turns
Lateral turns in the lamprey are caused by a strong and prolonged
contraction of body musculature on the side of the turn. The wave of
contraction subsequently propagates along the body toward the tail
(McClellan and Hagevik 1997; Ullén et al.
1998
; Zelenin et al. 1997
). In the present
study, we have found that lateral turns were associated with an
asymmetry in the mass RS activity on the left and right sides (Fig. 7).
This asymmetry was comparatively small (1.3 ± 0.3), however, in
contrast to the asymmetry in motor output
during the turn, the EMG
activity was only present on the ipsilateral side of the body. A
possible explanation for this discrepancy could be that the turns are
generated by the spinal locomotor network that generates rectilinear
swimming and that this network is sensitive to any asymmetry in
descending commands. Two lines of evidence support this suggestion.
First, the symmetry of motor output during fictive swimming generated by the spinal cord can be strongly affected by stimulation of a single
RS neuron (Buchanan and Cohen 1982
). Second, a small asymmetry in the mass activity of the left and right RS pathways, caused by electrical stimulation, can result in a generation of a pure
unilateral motor output (Zelenin et al. 1997
). In both cases, the "amplification" of the asymmetry occurred in the spinal cord, and it is most likely based on the operation of the system of
reciprocal inhibition between the two hemi-segments (Cohen and
Harris-Warrick 1984
; Grillner and Wallén
1980
; Wallén et al. 1993
).
Correlation between vestibular responses in RS neurons and postural corrections
Behavioral experiments (Ullén et al. 1995a,b
)
have shown that the swimming lamprey maintains its dorsal-side-up
orientation due to the activity of the postural control system driven
by vestibular input. A deviation from the normal orientation elicits a
set of corrective motor responses including a lateral body flexion and a twisting of the body and dorsal fins. These motor responses are
caused by the RS system. The present study demonstrated that lateral
tilt of the lamprey during locomotor-like activity (Fig. 2B)
evokes activation of contralateral RS neurons (Fig. 9B).
This pattern of response was similar to that observed in the
nonswimming lamprey (Fig. 9A) (see also Deliagina and
Fagerstedt 2000
) and in an in vitro preparation consisting of
the brain stem and labyrinths (Deliagina et al. 1992
).
The activation of the contralateral RS neurons caused by lateral tilt
presumably presents the commands addressed to the spinal cord where
they evoke postural corrections. It remains unclear, however, how these
commands affect the spinal network. That these commands are sufficient
for postural stabilization was indicated by model experiments
(Zelenin et al. 1998
), using a technique of artificial
feedback as originally developed for Clione by
Deliagina et al. (1998)
. In these experiments, the
activity of RS neurons, recorded by implanted electrodes in the intact lamprey during its locomotor-like activity, was used to control an
electromechanical robot rotating the animal in the roll plane. This
"hybrid" system was able to stabilize the dorsal-side-up orientation of the lamprey.
Rhythmic modulation of RS neurons
In most experiments, we observed a periodical modulation of the
mass activity in RS pathways linked to the locomotor cycle with the
peak activity coinciding with the burst of EMG in the ipsilateral
rostral myotomes (Fig. 8). The lack of modulation observed in a part of
recordings could be related to the fact that individual RS neurons may
discharge in different phases of the swim cycle. This modulation was
most likely caused by efference copy signals coming to the brain stem
from the spinal locomotor CPG because a similar pattern of modulation
was observed also during fictive swimming (Dubuc and Grillner
1989; Kasicki and Grillner 1986
; Kasicki
et al. 1989
). Rhythmical modulation of the activity in RS
pathways also was observed in walking cats (Drew et al.
1986
; Orlovsky 1970b
). The functional role of
this modulation is not clear, however, primarily because the spinal targets of RS neurons are not well characterized. It was suggested that
the modulation can perform a gating function, linking the supraspinal
commands to a specific phase of the locomotor cycle (Arshavsky
et al. 1986
). Evidence in favor of this suggestion was obtained
in the present study. As shown in Fig. 9B, signals about
lateral tilt are transmitted to the spinal network by the RS neurons 1 and 2 only in a certain phase of the locomotor cycle and are not
transmitted in the opposite phase.
Multifunctional role of RS neurons
A question of fundamental interest concerns the distribution of
functions between different subdivisions of RS system. Are different
functionsinitiation of locomotion, steering, and postural stabilization
controlled by different groups of RS neurons or does
each neuron participate in the control of different functions? There is
overwhelming evidence that most RS neurons are multifunctional. First,
a multitude of inputs from the cranial nerves, from the spinal cord
afferent pathways and from the locomotor CPG, converge on RS neurons
(see e.g., Deliagina et al. 1992
, 1993
; Dubuc et al. 1992
; Kasicki et al. 1989
; Vinay et
al. 1998
). The present study has shown (see Fig. 9B)
that one and the same RS neuron responds faithfully to lateral tilt,
efference copy signals from the spinal CPG, and is activated during the
initiation of locomotion. Second, stimulation of individual RS neurons
during fictive swimming affected both the locomotor frequency and the
symmetry of segmental motor output (Buchanan and Cohen
1982
), suggesting that they could be involved both in the
initiation of swimming and in the generation of postural corrections.
Experiments with microstimulation of the brain stem reticular formation
during fictive swimming (Wannier et al. 1998
) also have
shown that most of the stimulated sites evoke asymmetry in the
segmental motor output, and a half of them also affect the locomotory
rhythm. No sites affecting only the frequency of the locomotory rhythm
have been found.
It seems thus most likely that the brain stem-spinal cord interaction
does not depend on "private labeled lines" for each type of motor
behavior (locomotion, steering, postural control); this interaction
rather depends on shared communication lines. Decoding of the commands
transmitted by the RS system takes place in the spinal cord. The
operation of the decoding mechanisms is not clear. One can suggest that
decoding is based on the comparison of signals delivered via different
subdivisions of the RS system. In particular, for extracting
information about lateral tilts, the activities in the left and right
RS pathways have to be compared (Deliagina 1997;
Deliagina et al. 1993
); the summated activity in these
pathways may carry information on the intensity of locomotion.
The polyfunctional organization of RS system discussed in the preceding text presumably accounts for the great difficulty that investigators have had in understanding this system while investigating anesthetized preparations or only one type of behavior.
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ACKNOWLEDGMENTS |
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The authors express their sincere gratitude to Dr. F. Ullén for valuable comments on the manuscript.
This work was supported by an International Research Scholars grant from the Howard Hughes Medical Institute, a Royal Swedish Academy of Sciences research grant for Swedish-Russian scientific cooperation, Swedish Medical Research Council Grants 11554 and 3026, and the Curt Nilsson Foundation.
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FOOTNOTES |
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Address for reprint requests: T. G. Deliagina, The Nobel Institute for Neurophysiology, Dept. of Neuroscience, Karolinska Institute, SE-171 77 Stockholm, Sweden.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 5 May 1999; accepted in final form 5 October 1999.
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REFERENCES |
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