1The Nobel Institute for Neurophysiology, Department of Neuroscience, Karolinska Institute, SE-171 77 Stockholm, Sweden; 2A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia; 3Institute for Nonlinear Science, University of California at San Diego, La Jolla, California 92093-0402; 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. Asymmetrical Effect of GABA on the Postural Orientation in Clione. J. Neurophysiol. 84: 1673-1676, 2000. The marine mollusk Clione limacina, when swimming, normally stabilizes the vertical body orientation by means of the gravitational tail reflexes. Horizontal swimming or swimming along inclined ascending trajectories is observed rarely. Here we report that GABA injection into intact Clione resulted in a change of the stabilized orientation and swimming with a tilt of ~45° to the left. The analysis of modifications in the postural network underlying this effect was done with in vitro experiments. The CNS was isolated together with the statocysts. Spike discharges in the axons of two groups of motoneurons responsible for the left and right tail flexion, as well as in the axons of CPB3 interneurons mediating signals from the statocyst receptors to the motoneurons, were recorded extracellularly when the preparation was rotated in space. Normally the tail motoneurons of the left and right groups were activated with the contralateral tilt of the preparation. Under the effect of GABA, the gravitational responses in the right group of motoneurons and in the corresponding interneurons were dramatically reduced while the responses in the left group remained unchanged. The most likely site of the inhibitory GABA action is the interneurons mediating signals from the statocysts to the right group of tail motoneurons. The GABA-induced asymmetry of the left and right gravitational tail reflexes, observed in the in vitro experiments, is consistent with a change of the stabilized orientation caused by GABA in the intact Clione.
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INTRODUCTION |
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The marine mollusk Clione
limacina presents a unique opportunity for studying the neuronal
networks controlling such complex behaviors as spatial orientation.
Normally Clione maintains a vertical, head-up body
orientation (posture). Stabilization of the vertical orientation in any
plane is primarily based on two antagonistic gravitational tail
reflexes. Each reflex chain includes three cellular groups activated in
succession: the statocyst receptor cells, the cerebro-pedal
interneurons (CPB3), and the tail motoneurons (Deliagina et al.
1999; Panchin et al. 1995
). Any deviation from the vertical leads to a unilateral activation of motoneurons, bending
the tail, and restoration of the normal orientation (Deliagina et al. 1998
). When the water temperature is raising,
Clione turns with its head down. This new posture is
stabilized as a result of a reconfiguration of the postural network
that leads to a reversal of the gravitational tail reflexes
(Deliagina et al. 1998
, 2000b
).
A third mode of postural activity in Clione is swimming
along an inclined ascending trajectory. If the angle of inclination is
small, such swimming would allow Clione to perform
long-distance migrations. However, this mode of postural activity in an
aquarium was observed only occasionally, and until recently we could
not find a way to induce such a postural orientation. Here we report that we have found a regular way to deviate the longitudinal axis of
Clione from the vertical by injecting the -aminobutyric
acid (GABA) into the intact Clione
the injection causes a
leftward inclination of the animal. We also describe the GABA-induced
changes in activity of the postural network responsible for this phenomenon.
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METHODS |
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Experiments were carried out at the Marine Biological Station
Kartesh. In behavioral experiments, Clione was
placed close to the bottom of the aquarium (50 cm in depth), and its
subsequent swimming was videorecorded (25 frames/s). When the mollusk
reached the water surface, it was again moved to the bottom. The
segments of swim trajectories where Clione was orientated
with its dorsal side pointing toward the camera were used for measuring
the lateral tilt. The recordings were performed before and after
injection of GABA (0.15 ml,
103 M) into the hemocoel.
The body volume of Clione is ~1.5 ml, and thus the
estimated concentration of GABA in the hemolymph was 10
4 M. GABA at such a
concentration decreased pH of seawater by <0.2.
The methods used in electrophysiological experiments were described
earlier (Deliagina et al. 1999). In brief, the CNS was isolated together with the statocysts and positioned in the recording chamber on the electrode (a piece of the filter paper soaked in the sea
water). The nerves used for recording were positioned on other
electrodes. Activity of the tail motoneurons was recorded from the left
and right nerves N2(1) responsible for the lateral tail flexion.
Activity of CPB3 interneurons was recorded from a stump of the
transected subpedal commissure. The chamber was filled with paraffin
oil and tightly closed.
For gravitational stimulation of the statocysts, the chamber was
positioned so that the longitudinal axis of the preparation (which
corresponds to the longitudinal axis of the animal) was oriented
vertically and then rotated in the frontal plane of the preparation
(lateral tilt). Two modes of rotation were employeda full turn
rotation (Fig. 2, A and B), and periodical
trapezoid tilts (Fig. 2, C-E).
For GABA application, the chamber was opened and the layer of seawater covering the CNS was replaced by the GABA solution by means of a syringe needle. Despite the fact that this procedure was repeated several times, we could not completely avoid a dilution artifact, and the real concentration of GABA was probably lower than that in the replacing saline. All experiments were performed at temperature of 5-10°C when the postural network is tuned to stabilize the head-up orientation.
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RESULTS |
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Behavioral experiments
Injection of GABA was performed in nine animals. Before injection,
most trajectories of active swimming were close to the vertical. The
initial effect of GABA was a protraction of the tentacles and swimming
in circles (Arshavsky et al. 1993; Norekian and
Satterlie 1993
). This behavior lasted for 5-10 min. Then
swimming along ascending trajectories appeared. When swimming,
Clione was tilted to the left, which resulted in the
leftward inclination of the trajectories. This behavior continued up to
2 h. It was analyzed in detail in five animals. Swim trajectories
in animal 1 before GABA injection (Fig.
1A) were close to the
vertical, with a small tilt (12 ± 9°; mean ± SD) to the
right (control in Fig. 1C). Trajectories of the same animal
after GABA injection (Fig. 1B) were tilted to the left; the
mean value of tilt angle was 40 ± 13° (GABA in Fig.
1C). This behavior continued ~2 h, after which a normal
pattern gradually returned. In animals 2-5, the mean tilt
angle before injection was close to 0°. After GABA injection, the
ascending trajectories in these animals also occurred tilted to the
left, with the mean values of tilt angle grouped around 45°. The mean
values of tilt angle for all animals before and after GABA injection
are indicated by triangles (1-5) in Fig. 1C.
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Electrophysiological experiments
As shown in our previous studies (Deliagina et al. 1998,
1999
), at low temperature (5-10°) tail motoneurons from the
left (L) and right (R) nerves N2(1) are activated with the
contralateral tilt and have their maximal response when the ipsilateral
side points upward. This result was confirmed in the present study. Figure 2A shows the activity
of motoneurons from LN2(1) and RN2(1) in different positions of the
preparation throughout the full turn before GABA application. The
LN2(1) motoneurons were activated in and around the left-side-up (L)
position, whereas the RN2(1) motoneurons
in and around the
right-side-up (R) position. One of the CPB3 interneurons (a
high-frequency unit in the RSPC trace, indicated by the arrow) had a
pattern of gravitational responses similar to that of RN2(1)
motoneurons.
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The GABA application strongly reduced responses of the motoneurons from
RN2(1) and of the interneuron from RSPC but did not affect the
responses of motoneurons from LN2(1) (Fig. 2B). This asymmetrical effect of GABA is shown more clearly in Fig. 2,
C-E, where trapezoid tilts were used for gravitational
stimulation. Before GABA application (Fig. 2C), the LN2(1)
motoneurons were active in the L position, whereas the RN2(1)
motoneurons and the corresponding interneuron were active in the R
position. Application of GABA (5 × 104M) caused a
considerable reduction of the firing rate in the gravitational responses of the RN2(1) motoneurons and of the interneuron but did not
affect the responses of the LN2(1) motoneurons (Fig. 2D). With an increase of GABA concentration, gravitational responses of the
RN2(1) motoneurons and of the interneuron almost completely disappeared, whereas responses of the LN2(1) motoneurons reduced only
slightly (Fig. 2E). Washing the preparation resulted in a restoration of the initial pattern of the gravitational responses (not
illustrated). Similar results were obtained in all experiments (n = 10). To evaluate the effect of GABA, we calculated
a spike frequency in the LN2(1) while the preparation was in the L
position, and in RN2(1) while the preparation was in the R position.
These measurements were performed before and after GABA
(10
3 M) application. The
mean frequencies before GABA application were taken as a unit (control
in Fig. 3A) and then used to
normalize the values obtained after GABA application. As shown in Fig.
3A, application of GABA caused a fourfold reduction of the
response to contralateral tilt in the right group of motoneurons but
had practically no effect on the response in the left group.
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DISCUSSION |
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Previously it was shown that the postural network in
Clione has two distinct modes of activity, that is,
stabilization of the head-up and head-down orientation (Panchin
et al. 1995). Transitions from one mode to the other are caused
by a dramatic reconfiguration of the network, which resulted in the
reversal of the gravitational tail reflexes (Deliagina et al.
1998
, 2000b
). In the present study, we have shown that
the head-up mode of activity can also be subjected to graded changes
under the effect of GABA, and we suggest a mechanism for these changes
to occur.
The tail is the main effector organ for performing postural corrections
in Clione. Flexion of the tail in any plane causes turning
of the animal in that plane (Panchin et al. 1995). Tail flexions in the frontal plane are caused by the left and right groups
of N2(1) motoneurons that mediate the antagonistic gravitational reflexes (Deliagina et al. 1999
). Therefore
Clione will maintain that particular orientation in space at
which the LN2(1) and RN2(1) motoneurons are equally active. Only at
this orientation, the tail will not be flexed and Clione
will swim along a rectilinear trajectory. This principle of operation
of the postural control system is illustrated schematically in Fig.
3B where the activities of the left and right groups of
motoneurons are plotted against the tilt angle. The left tilt causes
excitation of the right group of motoneurons, whereas the right tilt
activates the left group of motoneurons; the maximal activity in each
group occurs at 90° contralateral tilt. The postural corrective
responses caused by the right and left groups of motoneurons are shown
in Fig. 3B by the black and white arrows, respectively. The
system will be in equilibrium at the point of intersection of the two
activity curves. This occurs at 0° (the head-up orientation) because
of the symmetry of the two curves under normal conditions.
Under the effect of GABA, the gravitational response for the right
group of motoneurons decreases, and the point of intersection of the
two curves now occurs not at 0° but at some angle of the left tilt
(Fig. 3C). This will be a new equilibrium point of the system. Only in this position, no postural corrections will be generated, which qualitatively explains the main behavioral effect of
GABA, that is a leftward inclination of swim trajectories. Thus graded
changes of the orientation of Clione are caused by a shift
of the equilibrium point in the control system, a principal discussed
earlier in relation to the postural system of the lamprey (Deliagina and Fagerstedt 2000a) and man (Feldman
1986
).
Under the effect of GABA, gravitational responses were inhibited both
in the right group of tail motoneurons and in the corresponding CPB3
interneurons. This finding suggests that the interneurons are the main
target of GABA action. In previous studies, it was found that
temperature-dependent reconfigurations of the postural network also
occur at the level of CPB3 interneurons (Deliagina et al. 1998,
2000b
). One cannot exclude, however, that the effect is partly
caused by the action of GABA on the statocyst receptor cells or on the
neurons projecting onto them (Arshavsky et al. 1993
).
The CNS of Clione contains ~30 GABAergic neurons
(Arshavsky et al. 1993). It is challenging to reveal
which of them are responsible for inhibition of the right chain of
gravitational tail reflexes and if there is also a system inhibiting
the left chain. In mammals, two groups of neuropeptides have been found
that produce opposite asymmetrical effects on the muscular tone of the
hindlimbs (Bakalkin 1989
; Bakalkin and
Kobylyansky 1989
).
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ACKNOWLEDGMENTS |
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This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-38022, Howard Hughes Medical Institute Grant 75195-544801, Swedish Medical Research Council Grant 11554, and the Royal Swedish Academy of Science.
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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 (E-mail: Tatiana.Deliagina{at}neuro.ki.se).
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 27 January 2000; accepted in final form 18 May 2000.
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REFERENCES |
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