1The Nobel Institute for Neurophysiology, Department of Neuroscience, Karolinska Institutet, S-171 77 Stockholm, Sweden; 2A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119 899, Russia; 3Institute of Neurobiology, Sun Juan, Puerto Rico 00901; and 4Institute of Information Transmission Problems, Russian Academy of Sciences, Moscow 101447, Russia
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ABSTRACT |
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Deliagina, T. G.,
G. N. Orlovsky,
A. I. Selverston, and
Y. I. Arshavsky.
Neuronal Mechanisms for the Control of Body Orientation in
Clione I. Spatial Zones of Activity of Different Neuron
Groups.
J. Neurophysiol. 82: 687-699, 1999.
The marine mollusk Clione limacina,
when swimming, can stabilize different body orientations in the
gravitational field. Here we describe one of the modes of operation of
the postural network in Clionemaintenance of the
vertical, head-up orientation. Experiments were performed on the
CNS-statocyst preparation. Spike discharges in the axons of different
types of neurons were recorded extracellularly when the preparation was
rotated in space through 360° in different planes. We characterized
the spatial zones of activity of the tail and wing motor neurons as
well as of the CPB3 interneurons mediating the effects of statocyst
receptor cells on the tail motor neurons. It was found that the
activity of the tail motor neurons increased with deviation of the
preparation from the normal, rostral-side-up orientation. Their zones
of activity were very wide (~180°). According to the zone position,
three distinct groups of tail motor neuron (T1-T3) could be
distinguished. The T1 group had a center of the zone near the
ventral-side-up orientation, whereas the zones of T2 and T3 had their
centers near the left-side-up and the right-side-up positions,
respectively. By comparing the zone of activity with the direction of
tail bending elicited by each of the groups, one can conclude that
gravitational reflexes mediated by the T1, T2, and T3 groups will evoke
turning of the animal toward the head-up orientation. Two identified
wing motor neurons, 1A and 2A, causing the wing beating, were involved
in gravitational reactions. They were activated with the downward inclination of the ipsilateral side. Opposite reactions were observed in the motor neurons responsible for the wing retraction. A presumed motor effect of these reactions is an increase of oscillations in the
wing that is directed downward and turning of Clione
toward the head-up orientation. Among the CPB3 interneurons, at least four groups could be distinguished. In three of them (IN1, IN2, and
IN3), the zones of activity were similar to those of the three groups
(T1, T2, and T3) of the tail motor neurons. The group IN4 had the
center of its zone in the dorsal-side-up position; a corresponding group was not found among the tail motor neurons. In lesion
experiments, it was found that gravitational input mediated by a single
CPB3 interneuron produced activation of its target tail motor neurons in their normal zones, but the strength of response was reduced considerably. This finding suggests that several interneurons with
similar spatial zones converge on individual tail motor neurons. In
conclusion, because of a novel method, activity of the neuronal network
responsible for the postural control in Clione was
characterized in the terms of gravitational responses in different
neuron groups comprising the network.
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INTRODUCTION |
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Control of body orientation and equilibrium is a
vital motor function of the brain. The functional organization of
nervous mechanisms responsible for the maintenance of a preferred body posture has been analyzed in considerable detail both in lower and
higher vertebrates including humans (for recent reviews see Horak and Macpherson 1996; Macpherson et al.
1997
; Orlovsky 1991
; Platt 1993
).
Much less is known, however, about the neuronal organization of the
postural control mechanisms. The principal difficulty with these
studies is that the postural control system, especially in higher
vertebrates, is extremely complex. It includes numerous sensory and
motor centers located in different parts of the brain, and the
integrity of these centers is necessary for normal function. This
problem hinders progress in the analysis of postural mechanisms at the
network and cellular level.
The invertebrates present many more opportunities for the analysis of
neuronal networks controlling different motor behaviors including the
maintenance of body orientation in space. The most extensive studies in
this area were carried out on the crayfish (Takahata and Hisada
1982a,b
; Takahata et al. 1985
). It was shown that the dorsal-side-up orientation in swimming animals is controlled by input from two statocysts. Gravitational reflexes were characterized for different spatial orientations of the crayfish. However, a sufficiently complete description of the postural network in
crustaceans is still lacking, primarily because of the difficulties in
the identification of individual neurons involved in postural control.
In the present study, we investigated the neuronal mechanisms for
postural control in the pteropod mollusk Clione limacina. The advantages of this animal model are the following: when swimming, Clione exhibits a very distinct spatial orientation behavior
(Arshavsky et al. 1991b; Panchin et al.
1995a
); all principal classes of neurons comprising the
postural network in Clione and their connections have been
identified (Panchin 1997
; Panchin et al.
1995a
,b
); and we have developed a novel method for the in vitro
study of neuronal correlates of postural activity (Deliagina et
al. 1998a
).
Clione is a planktonic animal. In the sea and in the
aquarium at lower water temperature, Clione usually can be
found oriented vertically, with its head up. Clione swims
upward or maintains itself at a particular depth by continuous beating
of two wings (Arshavsky et al. 1985a,b
; Satterlie
and Spencer 1985
; Satterlie et al. 1985
). Any
deviation from the vertical orientation evokes a set of corrective
motor responses (specific movements of the tail and wings) aimed at
restoration of the initial orientation. These reflexes are driven by
input from two statocysts. After removal of both statocysts,
Clione is not able to maintain any definite orientation and
continuously loops in different planes (Panchin et al.
1995a
).
The activity of the gravitational postural control system of
Clione can be subjected to modifications related to
different forms of behavior (Arshavsky et al. 1991a,
1993a
,b
). The main mode of activity is stabilization of the
head-up orientation (see preceding paragraph). The system also can
switch to stabilization of the head-down orientation (Panchin et
al. 1995a
). This mode is observed when Clione leaves
water layers with temperature >12-15°C and swims downward.
Head-down swimming also is observed at a certain stage of hunting
behavior. Horizontal swimming can also be observed under certain
conditions (Deliagina, Arshavsky, and Orlovsky, unpublished
observations). A different modification is a complete inhibition of
postural activity during the defense reaction. In the present paper, we
describe the mode of operation of the postural system when head-up
orientation is stabilized. The other modes will be considered in the
following papers of this series.
The main neuron groups participating in the gravitational reflexes in
Clione and their interconnections were identified earlier by
means of paired recordings (Panchin et al. 1995a,b
).
They are shown schematically in Fig.
1A. The statocyst internal
wall is lined with 9-11 statocyst receptor cells (Tsirulis
1974
). These cells are mechanoreceptors responding to the
pressure exerted by the statolith (Stl). The statocysts are located on
the dorsal surface of the pedal ganglia. The statocyst receptor cells
send their axons to the cerebral ganglia where they affect at least two
classes of the cerebropedal interneurons, CPB2 and CPB3. Excitatory, inhibitory, or mixed postsynaptic potentials can be recorded in these
interneurons when individual statocyst receptor cells are stimulated.
The CPB3 interneurons (their estimated number is 10-20) send their
axons to the pedal ganglia; the morphology of different types of CPB3
cells is shown schematically in Fig. 1, B-D. In the pedal
ganglia, the CPB3 neurons affect the tail motor neurons (Fig.
1A) in which they can evoke excitatory postsynaptic
potentials (EPSPs), inhibitory PSPs (IPSPs), or mixed PSPs. Different
groups of tail motor neurons evoke bending of the tail in different
directions as was shown in the experiments with electrical stimulation
of tail nerves (Panchin et al. 1995a
). Thus the
statocyst receptor cells, the CPB3 interneurons and the tail motor
neurons constitute the chain of the gravitational tail reflexes.
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The statocyst receptor cells also initiate activity in the chain of gravitational wing reflexes. They affect a group of cerebro-pedal interneurons (CPB2), which project to the wing motor neurons (Fig. 1E). By affecting the wing motor neurons, the CPB2 interneurons produce asymmetry in the locomotor beating of the wings.
It was demonstrated directly in the in vitro experiments that two of
the neuron classes shown in Fig. 1A, namely the statocyst receptor cells and the tail motor neurons, respond well to natural gravitational stimulation, i.e., a change in the spatial orientation of
the statocysts (Arshavsky et al. 1991a,b
; Panchin
et al. 1995a
). For the tail motor neurons, this was
demonstrated by recording the activity of their axons in the tail
nerves when the CNS-statocysts preparation was turned by forceps from
the dorsal-side-up position to the ventral-side-up position. With this
method, however, gravitational reflexes only could be characterized
very roughly for two spatial orientations, dorsal and ventral side up.
Nevertheless, these experiments led to formulation of the following
hypothesis about the organization of gravitational reflexes
(Panchin et al. 1995a
). When the head-up orientation is
stabilized, any deviation from this orientation results in excitation
of the statocyst receptor cells in the lowest part of the statocyst
cavity. These cells, via the CPB3 interneurons, will excite tail motor
neurons projecting to the opposite side of the body wall. These motor
neurons will elicit tail bending in the direction opposite to the
initial body sway. The deviated tail, like the rudder of boat, will
produce rotation of the animal toward the vertical until the normal,
head-up orientation is restored.
In the present study, we tested the validity of this hypothesis about the organization of the postural control system in Clione. For this purpose, we characterized the spatial zones of activity of different groups of tail motor neurons and compared them with the motor effect produced by activation of each particular group. We also characterized the spatial zones of activity of some CPB3 interneurons and compared them with the zones of tail motor neurons. In addition, we investigated the role of wings in postural stabilization. We characterized the spatial zones of activity of different groups of wing motor neurons and compared them with the motor effect produced by activation or inhibition of each particular group. This study led us to a conclusion that postural reflexes in Clione, mediated by identified moto- and interneurons, are able to compensate for any possible deviation of the animal from the normal orientation.
A brief account of these studies has been published in an abstract form
(Deliagina et al. 1998b).
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METHODS |
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Experiments were carried out at the White Sea Marine Biological
Station Kartesh. Mollusks were collected locally and kept in
aquaria at 5-12°C. All the experiments described in the present paper were performed at temperatures in the experimental chamber of
5-12°C, when the postural network was presumably tuned at
stabilization of the head-up orientation (Panchin et al.
1995a).
Preparation
All experiments were performed on an in vitro preparation of the
CNS (without buccal ganglia) isolated together with the statocysts (Fig. 2A). In
Clione, tail muscles are innervated by motor axons through
bilateral nerves N2(1), N2(2), N3, and N4 originating primarily from
the pedal ganglia; a small number of axons in N2 come from the pleural
ganglia. Retrograde staining through these nerves has shown that there
are ~30 cells on each side of the CNS (presumed tail motor neurons).
They are located preferentially in the pedal ganglia and, to a lesser
extent, in the pleural and cerebral ganglia (Panchin et al.
1995a; Deliagina, Arshavsky, and Orlovsky, unpublished data).
In preliminary experiments, we recorded gravitational responses from
the axons of efferent neurons (presumed tail motor neurons) in all tail
nerves. It was found that the most pronounced and persistent responses
occurred in the left and right nerves N2(1) and N3. These two pairs of
nerves were subjected to the most detailed analysis. Experiments with stimulation or transection of these nerves (Panchin et al.
1995a
) have shown that both N3 nerves elicit ventral tail
flexion, whereas each of the N2(1) nerves elicits tail flexion in the
ipsilateral-dorsal direction.
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Each of the wings in Clione is innervated by ~20 motor
neurons, with their axons passing through the wing nerve
(Arshavsky et al. 1985a,b
; Satterlie
1993
; Satterlie and Spencer 1985
;
Satterlie et al. 1985
). Among these motor neurons, two
cells, 1A and 2A, are very large and elicit a strong motor response.
The 1A motor neuron innervates the dorsal aspect of the wing and fires
in the "dorsal phase" of the locomotor cycle, whereas the 2A motor
neuron innervates the ventral aspect of the wing and fires in the
"ventral phase" of the cycle. When recording extracellularly from
the wing nerve, the large-amplitude discharges of these two locomotor
motor neurons can be recognized easily. In the present study, these discharges were used to monitor the gravitational influences on the
locomotor system. In addition, we studied the gravitational effects on
the motor neurons responsible for the wing retraction. These neurons
can be recognized according to their tonic discharge and the absence of
rhythmic input from the locomotor rhythm generator (Huang and
Satterlie 1990
).
The influences of the statocysts on the tail and wing motor neurons are
mediated by different groups of cerebropedal interneurons. Of these
groups, the CPB3b and CPB3c neurons affecting the tail motor neurons
have their axons passing through the subpedal commissure (Fig. 1,
C and D) (Panchin et al. 1995b).
To record the gravitational responses of these neurons, the subpedal
commissure (Fig. 2A) was cut, and discharges in the axons of
interneurons were recorded from one or both stumps of the commissure.
Recording chamber
The method for recording activity in the nerves, used in the
present study, represents an elaboration of the method described earlier (Deliagina et al. 1998a). A 35-mm polystyrene
petri dish (Falkon 3801) was used as a recording chamber (Fig. 2,
B and C). Five silver wires (0.5 mm diam) were
inserted into the chamber through its bottom. These wires were used to
make contact with the electrodes
small pieces of the filter paper
positioned on the bottom of the chamber. The electrodes were soaked in
and covered with a thin layer of sea water. The isolated CNS was
positioned with its dorsal side up on a larger filter paper electrode,
and its rostrocaudal axis was aligned with the diameter of the chamber as shown in Fig. 2B. Then the electrodes and the CNS were
covered with a thin layer of paraffin oil. The nerves chosen for
recording were pulled carefully through the oil and placed on the
corresponding electrodes. The chamber then was filled completely with
paraffin oil and the lid was put on and tightly closed. No air was left in the chamber. The electrodes were connected to the inputs of the
amplifiers. Up to four nerves could be recorded simultaneously. Spike
discharges were recorded extracellularly in the axons; for this
purpose, AC amplifiers with the bandwidth of 50-5,000 Hz were used.
Usually the spike amplitude was ~1 mV.
Natural gravitational stimulation
The chamber with the preparation was mounted on a platform that
allowed rotation of the chamber over a range of 0-360°. Depending on
the initial orientation of the chamber in relation to the platform, three different modes of rotation could be employed. In Fig.
3, the left panels (A,
1-3) show these three modes of rotation of the chamber, whereas
B, 1-3, shows a movement of Clione if these modes of rotation were applied to the whole animal. For simplicity, we
shall use the terms describing orientation of the whole animal (head
up, dorsal side up, etc.) when characterizing spatial orientation of
the preparation. Figure 3, A1 and B1, shows
rotation in the sagittal planethe sagittal sway (pitch) characterized
by the angle
. Figure 3, A2 and B2, shows
rotation in the frontal plane
the lateral sway characterized by the
angle
. Finally, Fig. 3, A3 and B3, shows the
third mode of rotation used in this study; that is, rotation around the
longitudinal axis of the animal, the axis being situated
horizontally
the horizontal roll characterized by the angle
.
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Rotation of the recording chamber was performed in steps (Fig. 3C); each step was 45°, the transition from one position to another took ~1 s, and each position was held for ~4 s. As a rule, each test was repeated several times with alternating rotations in the opposite directions. Figure 3C schematically shows the test with two full turns in opposite directions. In addition to the 360° rotation, trapezoid angular movements between two positions, with an amplitude of ±90°, were employed (Fig. 3D). In this case, one of the two positions was usually chosen in the center of the angular zone of activity of the tested neuron group (see RESULTS). Rotation of the chamber was performed manually and recorded by a potentiometric transducer.
In the figures, the following designations for the spatial orientation
of the preparation are used. For sagittal sway, = 0°
corresponds to the normal (vertical, head-up) orientation; positive
values of
correspond to ventral (forward) sway; negative values of
correspond to dorsal (backward) sway (see also Fig. 11F). For lateral sway,
= 0° corresponds to the
head-up orientation; negative values of
correspond to right sway;
positive values of
correspond to left sway (see also Fig.
11E). For horizontal roll,
= 0° corresponds to
the dorsal-side-up orientation of the preparation; negative values of
correspond to right roll; positive values of
correspond to left roll.
To make the presentation of results more convenient and to avoid possible confusion caused by similar angular measures for the orientations in different planes of rotation, special designations were given to the six "basic" orientations. They are: head up (H), tail up (T), dorsal side up (D), ventral side up (V), left side up (L), and right side up (R). The intermediate positions were designated by indicating the closer basic orientations, like HR, HL, DR, etc.
In the present study, it was found that the most typical pattern of
gravitational response in the tail and wing motor neurons, as well as
in the cerebropedal interneurons, was their activation within a limited
angular zone and silence outside the zone. Only in a few cases the
neurons with gravitational input were not silent outside the zone. To
characterize the zones, we used the following valuesthe width of the
zone (z) and the center (c) (Fig. 3C). These values were measured for all tested neurons except for the cases
when the spikes of individual neurons were difficult to distinguish
because of their high-frequency. In these cases, z and
c were measured for a whole group of simultaneously active neurons. The values of z and c then were averaged
separately for each neuron group over all experiments. Altogether, the
zones were measured in >40 experiments. Each experiment lasted for a few hours, but in some cases pronounced gravitational responses could
be recorded for a much longer time,
2 days.
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RESULTS |
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Gravitational reactions of tail motor neurons
Up to 10 units of different spike amplitude and shape could be distinguished when recording from the tail nerves, N2(1) or N3. About half of them responded to gravitational stimuli. Figure 4A shows a response in LN2(1) to roll tilt (transition from the right-side-up orientation to the left-side-up orientation). With a higher time resolution (Fig. 4B), four units (1-4) could be distinguished. In the L position, the firing frequency of units 1-4 was ~5, 15, 40, and 10 Hz, respectively. The value of firing frequency of 5-15 Hz was most characteristic for the responses of tail motor neurons observed in different experiments. In the R position, these units were silent.
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When stimulated by stepwise rotation, the tail motor neurons were activated in a specific zone of angles (Fig. 4C). In addition to a static response, that is, a difference in the rate of continuous firing observed in two different positions held for a long period of time, some neurons also exhibited a dynamic response, that is, a reaction to a change of orientation, which usually lasted for 1-2 s. As shown in Fig. 4A, the static response gradually decayed with a time constant of 1-2 min.
Figure 5, A and B, shows responses in two symmetric tail nerves, LN2(1) and RN2(1), to lateral and sagittal sway. Neurons in LN2(1) were activated with left sway, in the TL, L, and LH positions. On the contrary, RN2(1) was activated mainly with right sway, in the LH, H, HR, R, and RT positions. Activation of LN2(1) and RN2(1) with the ipsilateral-side-up orientation was also characteristic of their testing by the horizontal roll (see Figs. 4C and 5E). Sagittal sway usually evoked activation of neurons in both nerves in approximately the same zones, around the D position. Figure 5B illustrates a case when neurons responded in the H, HD, and D positions. In some experiments, however, sagittal sway elicited rather weak activation of LN2(1) and RN2(1). From Figs. 4C and 5, B and E, one also can see that, in addition to the units with a highly pronounced gravitational input, a few units are firing without any obvious relation to their angular position.
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In contrast to the motor neurons from the left and right nerves LN2(1) and RN2(1), which received different gravitational inputs, neurons from another pair of nerves, LN3 and RN3, received largely identical inputs. Figure 5C shows responses in LN3 and RN3 to sagittal sway. All units in these nerves had their zones centered close to the ventral-side-up orientation (position V). The zones of smaller units occupied the DT, T, TV, and V positions; these zones were wider than the zones of larger units which occupied the TV and V positions (see also Fig. 9A). Horizontal roll (Fig. 5D) also revealed a similarity of gravitational inputs to the motor neurons from LN3 and RN3: the zones of neurons in both nerves were centered close to the V position. Lateral sway did not evoke activation of LN3 and RN3 in any zone (not illustrated).
Rotation in opposite directions did not reveal any significant directional sensitivity of the tail motor neurons. This is illustrated in Fig. 5E for the LN2(1) and RN2(1) nerves tested by horizontal roll. For example, the zones of smaller units in LN2 occupied the DL, L, and LV positions in both turns, whereas the zones of larger units occupied the L and LV positions. In some experiments, however, a difference in the zone size and position could be observed in successive tests. This most likely was caused by spontaneous changes in the excitability of neurons constituting a chain of gravitational reflexes. Sometimes spontaneous bursts of activity in the tail nerves, not caused by gravitational stimulation, were observed which considerably hampered the analysis of gravitational responses.
Three principal patterns of gravitational response have been found in tail motor neurons that allowed us to classify the neurons in three groups. A few neurons with weakly pronounced response were excluded from the analysis. The diagrams in Fig. 6 (top) show the angular zones of activity for different groups of motor neurons averaged over all experiments (n = 12-42 for different groups). Group T1 includes motor neurons from the left and right nerves N3. Group T2 includes motor neurons from the left nerve N2(1). Finally, group T3 includes motor neurons from the right nerve N2(1). The horizontal bars indicate an average width of the zones, whereas the black circle in the center of each bar indicates the center of the zone, with the SD value shown by the horizontal lines.
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The angular zones of all the three groups of tail motor neurons were ~180° in width when tested by sagittal sway, by lateral sway, or by horizontal roll. The center of the T1 zone, when tested by sagittal sway and horizontal roll, was very close to the V position. The centers of T2 and T3 zones, when tested by lateral sway and horizontal roll, were very close to the L and R positions, respectively. The centers of the T2 and T3 zones, tested by the sagittal sway, were close to the D position. On the basis of the data presented in A-C, a schematic spatial reconstruction of the zones was done (see DISCUSSION and Fig. 11, A-C).
As shown in Fig. 6, dispersion of the center of the zones, characterized by the SD value, ranged from 16 to 28° for different zones. This dispersion was most likely caused by two factors-the individual variability between the animals and the variability in positioning the CNS in the recording chamber. A dispersion of the width of zones also was measured (not illustrated in Fig. 6). Its value appeared considerably larger than the dispersion of centers (the range was 18-45°). The most likely explanation for this finding was that the width of the zone depended on the excitability in the chain of gravitational reflexes to a larger extent than the position of the center. In this relation, one should mention that in the course of individual experiments, spontaneous variations of the width of the zones often were observed.
When recording from other tail nervesN1, N2(2), and N4 (Fig.
2A)
a few units with position-dependent activity could be
observed in some experiments. The angular zones of these units did not differ from the zones of groups T1, T2, or T3 described above. Responses in the N1, N2(2), and N4 nerves were not systematically studied, however.
Gravitational reactions of wing motor neurons
Lateral tilt of the CNS, caused by lateral sway or by horizontal
roll, evoked reactions of wing motor neurons. Figure
7 shows gravitational responses in the
left and right wing nerves (LNW and RNW) evoked by the horizontal roll.
Two larger units in each of the nerves represent discharges of the
large locomotor wing motor neurons 1A and 2A. These neurons receive
alternating periodical inputs from the locomotor rhythm generator.
Because of these inputs, the discharges of 1A and 2A alternate, as
shown clearly in Fig. 7B at higher recording speed. The
present study has shown that both rhythmic locomotor input and
gravitational input converge on the 1A and 2A motor neurons. Because of
the gravitational input, the 1A and 2A motor neurons exhibited their
locomotor rhythmic spike activity only within a specific angular zone
(Fig. 7A). For the left wing, the zone occupied the D, DR,
and R positions. For the right wing, the zone occupied the VL, L, and
LD positions. Besides the locomotor motor neurons 1A and 2A, one also
could see, in each of the wing nerves, the spikes of at least one
nonlocomotor, tonic motor neuron. The zone of activity of this motor
neuron in the LNW occupied the VL and L positions. In the RNW, the zone occupied the DR and R positions. These tonic motor neurons are most
likely responsible for the wing retraction (Huang and Satterlie 1990; Deliagina, Arshavsky, and Orlovsky, unpublished data). A strict reciprocity between responses of the locomotor and tonic motor
neurons of the two wings is shown in Fig. 7B where the
horizontal roll was applied in a trapezoid manner and the preparation
was moved between the centers of the left and right zones. Neither locomotor nor tonic wing motor neurons responded to rotation in the
sagittal plane (not illustrated).
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The diagrams in Fig. 6 (middle) show the angular zones of activity of the wing motor neurons averaged over all experiments (n = 10-22 for different groups). The neurons were classified into four groups according to their gravitational input. The group W1 included the 1A and 2A locomotor motor neurons of the left wing; the group W2 included the 1A and 2A motor neurons of the right wing. The left and right tonic wing motor neurons constituted the groups W3 and W4, respectively. The zones of all four groups of wing motor neurons were ~100° in width when tested by the lateral sway or by the horizontal roll; that is narrower than the zones of tail motor neurons. The centers of W2 and W3 zones were very close to the L position, whereas the centers of W1 and W4 zones were very close to the R position.
As shown in Fig. 6, dispersion of the center of the zone, characterized by the SD value, ranged from 11 to 34° for different zones and was thus similar to the SD value for the centers of tail motor neuron zones. The dispersion of the width of the zones was larger, it ranging from 19 to 58°. A schematic spatial reconstruction of the zones of wing motor neurons will be given in DISCUSSION (Fig. 11D).
Gravitational reactions of CPB3 interneurons
Gravitational inputs to the tail and wing motor neurons are
mediated by the cerebropedal interneurons, the morphology of which is
shown schematically in Fig. 1, B-E. Two types of these
interneurons (CPB3b and CPB3c) have their axons in the subpedal
commissure (SPC) and could be recorded from the stumps of the
transected commissure (see METHODS and Fig. 2A).
Units with different discharge patterns were recorded with this
methodposition-sensitive and tonic units. The former were considered
as the discharges of the CPB3b or CPB3c interneurons according to the
following criteria (Panchin et al. 1995a
): they were
recorded from the subpedal commissure, they transmitted gravitational
signals, and they evoked gravitational responses in the tail motor
neurons (see next section). The spike amplitude in these neurons was
~200 µV, which is considerably smaller than the amplitude of spikes
of motor neurons in the tail and wing nerves. Unfortunately the
presence of larger tonic units in the SPC in some experiments hampered
detection of smaller spikes of the CPB3 interneurons.
Up to four CPB3 interneurons, with different angular zones of activity,
could be identified in each (left or right) stump of the SPC. Figure
8A shows discharges of the
four CPB3 interneurons recorded from the left part of the transected
SPC in different positions of the preparation. The interneurons 1-4
fired in the V, L, R, and D positions, respectively. In addition,
interneuron 4 fired in short bursts during transition from the R to the
L position and from the L to the V position, when the preparation rapidly passed the D position. Units recorded in the right stump of the
SPC also had their maximal activity in the V, L, R, and D positions,
respectively (not illustrated). The firing frequency of interneurons
usually was 10-15 Hz (which is similar to the frequency of motor
neurons), but in some cases it was higher (50 Hz).
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The angular zones of activity of individual interneurons were difficult to measure in the cases when different neurons fired with the spikes of similar amplitude. However, in some experiments, the differences in amplitude were considerable, like in the experiment illustrated in Fig. 8, B and C. In this particular case, only two units were seen in the right SPC, the large one responding in the V position and the small one responding in the D position. When tested by the horizontal roll (0-360°), the zone of the larger unit occupied the V, VL, R, and VR positions, whereas the zone of the smaller unit occupied the L, LD, D, and DR positions (Fig. 8C). When tested by sagittal sway, the larger unit also appeared active in and around the V position and the smaller unit in and around the D position (not illustrated).
The diagrams (Fig. 6, bottom) show the angular zones of activity of the four groups of cerebropedal interneurons recorded in the SPC. Each group contained two cells, one recorded from the left stump of the SPC and one recorded from the right stump. The zones were averaged over all experiments (n = 8-24 for different groups). The IN1, IN2, IN3, and IN4 groups had their centers of zones close to the V, L, R, and D positions, respectively. All the zones were 135-170° in width. By comparing the diagrams of Fig. 6, top and bottom, one can conclude that the zones of activity of the CPB3 interneurons (groups IN1, IN2, and IN3) resemble the zones of activity of the tail motor neurons (groups T1, T2, and T3, respectively) except that they are narrower and IN2 and IN3 are not activated during sagittal movements. However, the zone of the IN4 group has no corresponding zone among the zones of tail motor neurons.
Action of CPB3 interneurons on tail motor neurons
Two types of experiments were carried out to estimate the effects produced by the CPB3b and CPB3c interneurons, through their axons in the SPC, on the tail motor neurons. First, we compared the gravitational responses in the tail nerves before and after transection of the SPC and found no marked difference (not illustrated). This finding indicates that gravitational input to tail motor neurons, mediated by the CPB3a interneurons (Fig. 1B) as well as by the axons of the CPB3b and CPB3c interneurons before they enter the SPC (Fig. 1, C and D), is considerably stronger than input mediated by the axons that crossed the SPC.
A contribution of the axons crossing the SPC, however, could be revealed in a different set of experiments illustrated in Fig. 9. After transection of all connections of the right pedal ganglion with the rest of the CNS except for the subpedal commissure (Fig. 9C), a position-dependent activity could still be observed in the right N3 nerve. As shown in Fig. 9B, the T1 group still could be activated by sagittal sway, and the activity occurred approximately in the normal zone, that is, in and around the V position, though the strength of the response was considerably reduced as compared with control (Fig. 9A). Such a response was observed in four of seven experiments. Because only one of the CPB3 interneurons, projecting through the SPC, has the zone of its activity in and around the V position (unit 1, Fig. 8) and the response disappeared after complete isolation of the pedal ganglion, this finding indicates that this particular interneuron, even when it is active alone, makes a noticeable contribution to the activation of the tail motor neurons in their normal zones.
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Equivalence of the left and right statocysts
In behavioral experiments, it was found that Clione was
able to stabilize its orientation close to the normal (head-up)
orientation after removal of any one of the two statocysts
(Panchin et al. 1995a). In the present study, we
recorded gravitational responses of tail motor neurons, wing motor
neurons, and CPB3 interneurons before and after removal of the left
statocyst (n = 4) or right statocyst (n = 4). It was found that the position and width of the angular zones of
activity in the inter- and motor neurons persisted after removal of any
of the statocysts. The intensity of firing within the zone, however,
was reduced slightly in some experiments. Figure
10 illustrates the case when removal of
the left statocyst had practically no effect on the response to
horizontal roll in the LN3 and LN2(1) nerves. Similar results were
obtained for the wing motor neurons. In experiments with recording from the SPC (n = 4) it was found that all four groups
(IN1-IN4) of CPB3 interneurons retained their zones of activity after
removal of one statocysts, though the frequency of discharge within the zones was somewhat reduced.
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These results, taken together, suggest that the equivalence of two statocysts is based on the fact that individual cerebropedal interneurons receive similar gravitational inputs from the left and the right statocysts.
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DISCUSSION |
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Spatial zones of activity of different groups of motor neurons
In the present study, the spatial zones of activity have been characterized for different groups of motor neurons and interneurons involved in the control of body orientation in Clione. This was made possible by a novel method, which allows us to record gravitational reactions in an isolated CNS-statocyst preparation.
Three modes of rotation of the preparation in space were employed in the present study (Fig. 3, A and B). Sagittal sway (deviation from the vertical orientation forward or backward, Fig. 3, A1 and B1) and lateral sway (deviation to the left and to the right, Fig. 3, A2 and B2) revealed the zones of activity in two orthogonal vertical planes. Horizontal roll (rotation around the longitudinal axis positioned horizontally, Fig. 3, A3 and B3) allowed us to characterize the activity of neurons caused by a 90° deviation from the normal orientation in different directions. These tests revealed three principal groups of tail motor neurons with different spatial zones of activity (Fig. 6). Group T1 (motor neurons in the left and right N3 nerves) responded preferentially to the backward sway with the center of the zone located close to the ventral-side-up (V) position. This group exhibited no activity with lateral sway in any direction. On the contrary, groups T2 and T3 [motor neurons in the nerves LN2(1) and RN2(1)] responded preferentially to lateral sway. Group 2 had the center of the zone located close to the left-side-up (L) position, whereas group 3, near the right-side-up (R) position. The zones of groups T2 and T3 extended also to the dorsal-side-up (D) position, but these groups exhibited no activity in the V position, in which group T1 had its zone center.
To represent the relationships between the zones of activity of
different groups of tail motor neurons, we made their schematic three-dimensional reconstruction (Fig.
11, A-C) on the basis of the one-dimensional zones (Fig. 6). On these graphs (Fig. 11,
A-C), the orientation of Clione in relation to
the gravity force was represented by a radius-vector originating from
the center of the sphere (shown in A). As in Fig. 6, the
angles and
show deviations of Clione from the
vertical orientation for the sagittal and lateral sway, respectively.
At
= 0°,
= 0° Clione
is oriented vertically, with its dorsal side positioned toward the
reader. Positive values of
correspond to deviations toward the
dorsal side up (forward sway); positive values of
correspond to
deviations toward the right side up (left sway). The arcs drawn on the
sphere by a thick line show the angular width and position of the
one-dimensional (1-d) zones revealed by sagittal sway (the arc along
the
-meridian), by lateral sway (the arc along the
-meridian),
and by horizontal roll (the equatorial arc). (For the T2 and T3 groups,
the arcs along the
-meridian occurred on the opposite side of the
sphere and were not shown). The thin lines connect the extreme points of the 1-d zones to show the presumed borders of the three-dimensional (3-d) zones. From Fig. 11, A-C, one can see that the T1-T3
zones are very wide, each of them occupies approximately one-half of the sphere, and the zones of different groups considerably overlap with
each other.
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The three-dimensional reconstruction of the zones for wing motor neurons was performed in the same way and is presented in Fig. 11D. Groups W2 and W3 of wing motor neurons had a center of their zone close to that of the group T2 of tail motor neurons. Similarly, groups W1 and W4 had a center of their zone close to that of the group T3 of tail motor neurons. A size of the zones of wing motor neurons, however, was smaller than that of tail motor neurons.
Correlation between motor responses evoked by different groups of motor neurons and their zones of activity
Effects on the postural orientation produced by different groups
of the position-sensitive tail and wing motor neurons have not been
measured directly. However, these effects can be estimated on the basis
of morphological data as well as on the basis of experiments with
electrical stimulation of the nerves or their transection
(Panchin et al. 1995a). The left and right nerves N3
innervate the ventral aspect of the tail. Stimulation of each of these
nerves evokes a ventral tail flexion. After transection of these
nerves, no spontaneous ventral flexion of the tail was observed in
otherwise intact Clione. One thus can suggest that the tail
motor neurons of group T1, with their axons in the LN3 and RN3 nerves,
elicit the ventral tail flexion. The left and right nerves N2(1)
innervate the left-dorsal and the right-dorsal aspects of the tail,
respectively. Stimulation of LN2(1) evokes left-dorsal tail flexion,
whereas stimulation of RN2(1) evokes right-dorsal tail flexion. After
transection of these nerves, no spontaneous dorsal and lateral flexion
of the tail was observed. One thus can suggest that the tail motor
neurons of the group T2, with their axons in the LN2(1) nerve, elicit
the left-dorsal tail flexion. Similarly, the tail motor neurons of
group T3, with their axons in the RN2(1) nerve, elicit the right-dorsal
tail flexion.
The present study also has shown that gravitational input induces
left-right asymmetry in the activity of wing motor neurons. The effect
of this asymmetry on postural orientation was not measured directly but
can be estimated on the basis of simple considerations. A unilateral
activation of the locomotor motor neurons 1A and 2A will result in an
increase of the amplitude of wing beating (Arshavsky et al.
1985a,b
; Satterlie 1993
; Satterlie and
Spencer 1985
; Satterlie et al. 1985
). One can
suggest that this asymmetry in the beating of two wings will produce a
force turning Clione in the contralateral (in relation to
the "stronger" wing) direction. Simultaneous activation of the
retractor motor neuron of the contralateral wing will reduce
oscillations of that wing, which will also promote the turning of
Clione toward the "weaker" wing.
Figure 11, E, 1-3, and F, 1-3, shows schematically the postural corrective responses caused by deviation of Clione from the vertical in the frontal and sagittal planes as well as the groups of motor neurons eliciting these responses. With the left sway (E2), Clione occurs in the zone of activation of the T3 group of tail motor neurons, as shown in C. This group evokes tail flexion to the right (E2), which will elicit turning of Clione toward the vertical orientation. With a larger left sway, Clione is then also in the zone of activation of the W1 and W4 wing motor neurons, as shown in D. These motor neurons evoke asymmetry in the wing beating, with the prevalence of the left wing, which also will result in the turning of Clione toward the vertical orientation. Thus the tail and the wing motor responses supplement each other when restoring the normal body orientation.
With the right sway (E3), Clione occurs in the
zone of activation of the T2 tail motor neurons, as well as of the W2
and W3 wing motor neurons. These motor neurons will evoke corrective motor responses turning Clione to the left and thus
restoring the normal body orientation. Finally, the sagittal body sway
with the dorsal (F2) or ventral (F3) side up
moves Clione into the zones of activation of the T2 and T3
groups, or T1 group, respectively (A-C). These groups evoke
the dorsal (F2) or the ventral (F3) tail flexion,
respectively, which will result in the turning of Clione
toward the vertical orientation. Because the zones of tail motor
neurons are very wide and together cover the whole sphere (A-C), any deviation of Clione from the normal
orientation will result in specific group(s) of the motor neurons being
activated, and an adequate motor response (tail flexion), aimed at
restoration of the vertical orientation, will be generated. This notion
was supported in the experiments on the isolated CNS with an
artificially closed feedback loop (Deliagina et al.
1998a). The robotics system, driven by signals from the T2 and
T3 groups of tail motor neurons, was able to compensate for large
postural disturbances (
180°).
Sensorimotor transformations performed by a population of cerebropedal interneurons
According to the anatomic data, each of the statocysts of
Clione contains 9-11 receptor cells (Tsirulis
1974). By recording responses in the statocyst nerve to
rotation of the statocyst in different planes, we found that the
maximal angular dimension of the zones of activity (receptive fields)
in individual receptor cells is about 135° (Deliagina, Arshavsky, and
Orlovsky, unpublished data). In the present study, it was found that
the zones of activity of CPB3 interneurons have a similar size (Fig.
6). The simplest explanation for this finding is that the individual
interneurons are driven by inputs from single receptor cells of a statocyst.
The persistence of gravitational responses in the CPB3 interneurons, in the tail motor neurons (Fig. 10) and in the wing motor neurons after removal of one statocyst, found in the present study, suggests that two homologous receptor cells, from the left and right statocysts, converge on the same cerebropedal interneurons involved in the gravitational reflexes.
All three groups of tail motor neurons (T1-T3) have their counterparts among the interneurons (groups IN1-IN3) with approximately the same position of the zone of activity (Fig. 6). In lesion experiments, it also was found that the zone position of tail motor neurons persisted after reduction of the number of CPB3 interneurons projecting on them, though the value of response within the zone reduced (Fig. 9). These findings suggest that a few interneurons with similar spatial zones converge on individual tail motor neurons.
Comparison with postural systems in other species
In terrestrial species including humans, control of body posture
and equilibrium is usually based on integration of sensory inputs of
three modalitiesvestibular, visual, and somatosensory (for a review,
see Horak and Macpherson 1995
). In aquatic animals, which do not contact the substratum, somatosensory information plays a
minor role. In the goldfish and some other bony fishes, vestibular
input to the postural system is of primary importance. Nevertheless,
deprived of this input, some fishes are able to stabilize their
orientation in space relaying exclusively on visual input (Graf
and Mayer 1983
; von Holst 1935
). In the lamprey
(a lower vertebrate), vestibular input is the dominating one; when deprived of this input, the animal is not able to stabilize its orientation in space (Ullén et al. 1995a
).
Nevertheless, visual input can, to some extent, affect the orientation
stabilized by the vestibular-driven postural system (Ullén
et al. 1995b
).
In Clione, gravitational input from the statocysts to the
postural control network completely determines postural orientation and
equilibrium (Panchin et al. 1995a). This allowed us to
focus in the present study exclusively on the gravitational mechanisms for postural control and use the in vitro preparation of the CNS where
the postural network is driven by only one gravitational input.
In all species that are able to stabilize their orientation in space, a
deviation from the desired body orientation evokes corrective motor
responses aimed at restoration of the initial orientation. These
responses, as well as the underlying activity of motor neurons (usually
recorded as the electromyographic responses), have been studied in many
species including humans (see e.g., Amblard et al.
1988). The general conclusion from these studies is that the
principal characteristics of the motor response (i.e., the content of
the activated muscle groups and the temporal pattern of their
activation) strongly depend on the direction and value of the deviation
from the preferred orientation (see e.g., Macpherson et al.
1997
). This conclusion was confirmed in the present study. It
was found that the spatial pattern of the corrective motor response (a
selected group of motor neurons) is determined by the direction of the
deviation from the vertical orientation, whereas the value of the
response may depend on the value of deviation.
In different species including humans, which stabilize their
orientation in space during locomotion, postural and locomotor mechanisms strongly interact with each other (see e.g.,
Hirshfeld and Forssberg 1991; Nashner
1980
; Nashner and Forssberg 1986
). In the
present study, it was found that the locomotor mechanisms in
Clione are actively used by the postural system: lateral
deviations from the vertical orientation induced a large asymmetry in
the periodical locomotor commands sent to the muscles of the left and
right wing (Fig. 7). This asymmetry was caused by gravitational input
addressed primarily to the wing motor neurons but not to the
rhythm-generating (CPG) interneurons because the locomotory rhythm
persisted almost unchanged in different positions (Fig. 7B).
A tendency to preserve the basic locomotor pattern during postural
corrections is characteristic also for other species (see e.g.,
Nashner and Forssberg 1986
; Orlovsky
1972
).
An important feature of the postural control systems in different
species is their remarkable flexibility, that is, an ability to
stabilize different orientations (see e.g., Orlovsky
1991). The flexibility of the postural control system of
Clione and the underlying neuronal mechanisms will be
described in the following papers of this series.
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ACKNOWLEDGMENTS |
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This work was supported by grants from National Institute of Neurological Disorders and Stroke (NS-38022), Howard Hughes Medical Institute (International Research Scholars Grant 75195-544801), Swedish Medical Research Council (11554), and Royal Swedish Academy of Science (Research Grant for Swedish-Russian scientific cooperation).
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FOOTNOTES |
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Address for reprint requests: T. G. Deliagina, The Nobel Institute for Neurophysiology, Department of Neuroscience, Karolinska Institute, S-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 21 January 1999; accepted in final form 12 April 1999.
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REFERENCES |
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