Asymmetrical Effect of GABA on the Postural Orientation in Clione

T. G. Deliagina,1,2 G. N. Orlovsky,1 A. I. Selverston,3 and Y. I. Arshavsky3,4

 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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 gamma -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.


    METHODS
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INTRODUCTION
METHODS
RESULTS
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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, 10-3 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 employed---a 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.


    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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Fig. 1. A and B: swim trajectories of animal 1. A: trajectories before GABA injection. B: trajectories after GABA injection. Final points of all trajectories are combined in A and B. A dorsal view of Clione is superimposed on 1 of the trajectories in B. C: mean values of the tilt angle for animals 1-5 (triangle ) before (control) and after injection (GABA). Bars show the tilt angle for animal 1 (mean ± SD).

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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Fig. 2. Gravitational responses of the tail motoneurons recorded from the left and right nerves, [LN2(1) and RN2(1)] and from the right stump of the subpedal commissure (RSPC) before and after GABA application. The preparation was rotated in the frontal plane. A and B: full turn rotation. C-E: trapezoid rotation. Designations for different positions of the preparation are given in terms of the position of the whole animal: T, tail up; R, right side up; H, head up; L, left side up.

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 × 10-4M) 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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Fig. 3. A: effect of GABA on gravitational responses in the left and right N2(1). Spike frequency was calculated for motoneurons in LN2(1) at the L position, and in RN2(1) at the R position, before and after GABA application. The mean frequencies (average over all experiments) before GABA application were taken as a unit (control) and used to normalize the values obtained after GABA application. B and C: principle of operation of the postural system in Clione. Abscissa---the lateral tilt of the animal, ordinate---activity of the right and left groups of tail motoneurons (arbitrary units). The black and white arrows indicate the corrective motor responses caused by the right and left groups of motoneurons, respectively. B: no GABA action. The network stabilizes the head-up orientation. C: under the effect of GABA, the equilibrium point is shifted to the left.


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ABSTRACT
INTRODUCTION
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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).


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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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ABSTRACT
INTRODUCTION
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