Sonometric Measurements of Motor-Neuron-Evoked Movements of an Internal Feeding Structure (the Radula) in Aplysia

Irina V. Orekhova,1 Jian Jing,1 Vladimir Brezina,1,2 Ralph A. DiCaprio,3 Klaudiusz R. Weiss,1,2 and Elizabeth C. Cropper1,2

 1Department of Physiology and Biophysics and  2Fishberg Center for Research in Neurobiology, Mt. Sinai Medical Center, New York, New York 10029; and  3Neurobiology Program, Department of Biological Sciences, Ohio University, Athens, Ohio 45701


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Orekhova, Irina V., Jian Jing, Vladimir Brezina, Ralph A. DiCaprio, Klaudiusz R. Weiss, and Elizabeth C. Cropper. Sonometric Measurements of Motor-Neuron-Evoked Movements of an Internal Feeding Structure (the Radula) in Aplysia. J. Neurophysiol. 86: 1057-1061, 2001. In many systems used to study rhythmic motor programs, the structures that generate behavior are at least partially internal. In these systems, it is often difficult to directly monitor neurally evoked movements. As a consequence, although motor programs are relatively well characterized, it is generally less clear how neural activity is translated into functional movements. This is the case for the feeding system of the mollusk Aplysia. Here we used sonomicrometry to monitor neurally evoked movements of the food-grasping organ in Aplysia, the radula. Movements were evoked by intracellular stimulation of motor neurons that innervate radula muscles that have been extensively studied in reduced preparations. Nevertheless our results indicate that the movements and neural control of the radula are more complex than has been assumed. We demonstrate that motor neurons previously characterized as radula openers (B48) and closers (B8, B15, B16) additionally produce other movements. Moreover, we show that the size of the movement evoked by a motor neuron can depend on the preexisting state of the radula. Specifically, the motor neurons B15 and B16 produce large closing movements when the radula is partially open but produce relatively weak closing movements in a preparation at rest. Thus the efficacy of B15 and B16 as radula closers is context dependent.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Many extensively studied neural networks control behavior-generating structures that are at least partially internal (e.g., digestive structures). In many of these cases, it has proven to be technically difficult to monitor neurally evoked movements in situ. It is nevertheless important to do this because the muscles that control peripheral structures are often complexly intertwined, making it difficult to predict movements from data obtained in studies of individual muscles.

In this study, we used a digital sonomicrometry system that has relatively recently become commercially available to monitor movements of an internal structure that mediates feeding in the mollusk Aplysia, the radula. We evoked movements of the radula by intracellularly stimulating motor neurons. Individual radula muscles have been extensively studied in reduced preparations (e.g., Brezina and Weiss 1995; Brezina et al. 1994a-e, 1995; Cohen et al. 1978; Cropper et al. 1987a,b, 1988, 1990; Evans et al. 1996, 1999; Jordan et al. 1993; Scott et al. 1997; Vilim et al. 1994, 1996a,b; Weiss et al. 1978; Whim and Lloyd 1989, 1990). Nevertheless our sonomicrometric results indicate that overall radula movements and their neural control is more complex than has been assumed.


    METHODS
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INTRODUCTION
METHODS
RESULTS
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Preparation

Aplysia californica (200-300 g) were anesthetized with isotonic MgCl2. The buccal ganglion and the buccal mass were dissected out with only two nerves intact, the radula nerve and nerve 3 (which together contain the axons of B15, B16, B8, and B48).

Sonomicrometry

We used a commercially available (Sonometrics, London, Ontario) digital ultrasonic system (TRX-4 with 15-µm resolution enhancement). Experiments were performed with two or four omni-directional piezoelectric crystals (0.7 mm diam, attached to 42 gauge Teflon-coated copper wires; Fig. 1A). The buccal mass was anchored inside a chamber by pinning the lip tissue. In two-crystal experiments, we measured radula opening/closing by placing crystals laterally on the symmetrical radula halves. Crystals were attached to the radula in two ways. The buccal mass was placed ventral side up, and a small cut was made in the I2 muscle to get access to the inner side of the radula. A hypodermic needle was used as a guide when crystals were pushed through the inner surface of the radula to the outer surface. If necessary, VetBond (3M) was used to hold crystals in place and the opening in the I2 muscle was sealed with VetBond. Alternatively, the buccal mass was placed dorsal side up, a part of the esophagus was removed to visualize the radula halves, and crystals were glued to the outside of the radula.



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Fig. 1. A: schematic representation of crystal placement. Only the radula, accessory radula closer (ARC) muscle, and the underlying musculature are shown. Experiments were conducted with 2 or 4 crystals (). We used 2 crystals to monitor radula opening and closing. Crystals were symmetrically placed on the two radula halves (crystals 1 and 2). We used 4 crystals to simultaneously monitor radula opening/closing and radial radula movements. To monitor radial movements one crystal was glued to the radula at approximately the point where the ARC attaches (crystal 3), and the second crystal was positioned at the other end of the ARC (crystal 4). Crystal 4 is gray to indicate that it is actually implanted underneath the ARC therefore it would not be visible in the view shown. B: simultaneous sonomicrometric and video monitoring of radula movements evoked by stimulation of B8. B1: sonometric recordings of radula closings evoked by stimulating B8 for 3 s at different frequencies (indicated by the numbers above the bars). B2: a plot of video and sonometric measurements normalized as described in the text. Note that the 2 detection methods gave similar results.

In four-crystal experiments, we measured both radula opening/closing and radial movements of the radula. The third crystal was applied (through the above-mentioned cut in the I2 muscle) to the inner side of the radula near the place of the ARC muscle attachment. To place the fourth crystal, the I2 muscle was cut where it overlies the ARC. This crystal was then glued to the tissue underneath the ARC. Unless otherwise indicated, movements are expressed as means ± SE.

Video recording

In some experiments radula movements were videotaped using a camera mounted on a Wild M5 microscope and a standard videocassette recorder. To make radula movements visible, the dorsal side of the buccal mass was cut open.

Electrophysiology

Motor neurons were stimulated using standard intracellular techniques with double-barreled electrodes (beveled to 8-15 MOmega ). Unless otherwise noted, motor neurons were stimulated at 30 Hz for 3 s, which produced the largest radula movement possible. Neuronal activity was monitored via the four analog input channels of the sonomicrometry system.


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For an initial verification of the sonomicrometric recordings, we simultaneously videotaped radula movements. In these experiments, we monitored radula closing and stimulated the B8 neurons, which have been described as radula closer motor neurons (Morton and Chiel 1993). We elicited radula closings of different amplitudes by firing B8 neurons at different frequencies (Fig. 1B1). In these experiments, we normalized both the sonometric records and video records by expressing movements as a percent of the initial unstimulated length. The sonometric and video measurements of radula movements were very similar (Fig. 1B2), confirming the validity of the sonomicrometry.

The neurons that innervate the ARC muscle, B15 and B16, have also been described as radula closers (Cohen et al. 1978). Bilateral stimulation of B16 at frequencies that produce maximal muscle contractions did in fact close the radula (n = 16; Fig. 2A). In most (5 of 7) preparations, B15 also closed the radula (Fig. 2A). Radula closing movements produced by maximal stimulation of the ARC motor neurons were, however, smaller than those produced by maximal stimulation of B8 (Fig. 2A). On average B8 closing movements were 0.94 ± 0.14 mm (n = 14), B15 closing movements were 0.31 ± 0.13 mm (n = 5), while B16 closing movements were 0.23 ± 0.03 mm (n = 16). When B15 and B16 were stimulated together, closing movements were larger than when either motor neuron was stimulated alone, but were never as large as those elicited by B8 stimulation [in 2 experiments 0.39 and 0.5 mm with B15 and B16 as compared with the mean value of 0.94 mm with B8 (see preceding text)].



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Fig. 2. Movements evoked by stimulating individual radula motor neurons at 30 Hz for 3 s. Top: opening/closing movements; downward deflections are closings and upward deflections are openings. Bottom: radial movements; downward deflections are inward movements and upward deflections are outward movements. The bars under the bottom traces indicate periods of neuronal stimulation. A: recordings from a single preparation that compare movements evoked by the B8 neurons to those evoked by B15 and B16. Left: the B8 neurons produced large closings and no clear movement in the radial direction. Middle and right: in contrast, neurons B15 and B16 produced relatively small closing movements but large inward movements. B: the B48 neurons produced both large radula opening and large outward movements. Note that the radula does not return to the rest position between periods of activity in B48.

In addition to the relatively small closing movements evoked by B15 and B16, we simultaneously observed other movements that were, for the most part, not detected by the two crystals placed on the radula halves. [Previous investigators have also reported complex radula movements evoked by the ARC motor neurons that were not characterized (e.g., Cohen et al. 1978).] To characterize these movements, we utilized an extra pair of crystals that was placed so that it would detect radial radula movements (i.e., movements that would be toward or away from the center of the buccal mass in intact animals; Fig. 1A). We found that stimulation of the ARC motor neurons produced inward movements that were considerably larger than the closing movements (Fig. 2A). This was true for both B15 [inward movements were 1.35 ± 0.14 mm (n = 8); closing movements were 0.31 ± 0.13 mm (n = 5)] and B16 [inward movements were 0.56 ± 0.08 mm (n = 12); closing movements were 0.23 ± 0.03 mm (n = 16)]. In contrast, B8 stimulation produced no significant movement in the radial direction (n = 9; Fig. 2A).

This might suggest that the ARC motor neurons do not function primarily as radula closers. However, the preceding experiments were conducted in unstimulated preparations. During ingestive motor programs, activity of B8 and the ARC motor neurons is preceded by activity of the B48 neurons, which have been described as radula openers and antagonists of the ARC motor neurons (Evans et al. 1996). Thus it is possible that during feeding the radula is at least partially open when the ARC motor neurons become active.

We found that B48 stimulation produced an average opening of 1.08 ± 0.21 mm (n = 7; Fig. 2B). Moreover, in keeping with the idea that the muscles innervated by B48 act as ARC antagonists, B48 additionally produced movements that were the opposite of the radial movements produced by the ARC motor neurons, i.e., when B48 was stimulated the radula moved outward an average of 0.74 ± 0.07 mm (n = 3; Fig. 2B). Interestingly, we found that the radula did not return to the rest with respect to either movement when B48 was stimulated every 30 s [which is less often than it is likely to be active in vivo (Evans et al. 1996); Figs. 2B and 3, A2 and B2]. Thus the radula is presumably partially open when the ARC motor neurons are activated during normal feeding behavior.



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Fig. 3. Radula movements evoked by stimulating combinations of radula opener and closer motor neurons at 30 Hz for 3 s. A: upward deflections are radula openings, downward deflections are radula closings. Bars under traces indicate periods of neural stimulation. In 1 and 2, B16 and B48 were each stimulated alone. In 3, stimulation of B48 was alternated with stimulation of B16. Note that when B48 was stimulated, the radula opened and did not return to the rest position until after B16 was stimulated. B, top: upward deflections are radula openings and downward deflections are radula closings. Bottom: upward deflections are outward movements and downward deflections are inward movements. In 1 and 2, B15 and B48 were each stimulated alone. In 3, B48 and B15 were both stimulated. Note that the radula did not return to the rest position until after B15 had been stimulated.

To determine whether B48 elicited movements affect movements induced by the ARC motor neurons, we alternated B48 and B16 stimulation, starting with B48 stimulation (Fig. 3A3). Thus B48 was stimulated and the radula opened. The radula was still partially open approximately 15 s later when B16 was stimulated. Under these conditions, B16 produced a closing movement that was larger in magnitude in that it brought the partially open radula back to the rest position. To determine whether B16 stimulation could return the radula to the rest position under more physiological conditions, i.e., when the radula was open more widely, we stimulated the motor neurons so that there was no significant pause between B48 and B16 activity. We found that stimulation of B16 still returned the radula to the rest position (Fig. 3A3). We found the same to be true when we alternated stimulation of B48 with that of the second ARC motor neuron B15 (Fig. 3B3). It was true not only for opening/closing movements but also for radial movements (Fig. 3B3). Thus the starting position of the radula does in fact affect the amplitude of radula movements induced by the ARC motor neurons.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Sonomicrometry

Sonomicrometry has been used to record shape changes of structures in intact animals primarily in the cardiovascular system (e.g., Chang et al. 1996; Jones and Narayanan 1998; Kubota et al. 1998). The specific system we used is quite sensitive, and crystals are relatively small and easy to attach to peripheral structures. Because this is the first use of sonomicrometry to study feeding in Aplysia, we chose to study relatively simple movements of the food-grasping structure, the radula. Movements of the radula are central to feeding and individual radula muscles have been the subject of many studies (e.g., Brezina and Weiss 1995; Brezina et al. 1994a-e, 1995; Cohen et al. 1978; Cropper et al. 1987a,b, 1988, 1990; Evans et al. 1996, 1999; Jordan et al. 1993; Scott et al. 1997; Vilim et al. 1994, 1996a,b; Weiss et al. 1978; Whim and Lloyd 1989, 1990). Finally, activity in radula motor neurons is often used to monitor motor programs in the isolated nervous system (e.g., Church and Lloyd 1994; Hurwitz and Susswein 1996; Hurwitz et al. 1994, 1997; Kabotyanski et al. 1998, 2000; Morgan et al. 2000; Nargeot et al. 1997; Perrins and Weiss 1998; Plummer and Kirk 1990; Rosen et al. 1991; Sanchez and Kirk 2000; Susswein and Byrne 1988). Thus it is of great interest to more precisely characterize radula neuromuscular function.

Radula movements produced by B15, B16, and B8 neurons

The B8 neurons and the ARC motor neurons (B15 and B16) have been described as radula closer motor neurons (Cohen et al. 1978; Morton and Chiel 1993). Here we show that movements produced by these neurons are not identical. For example, there is an inward radial movement produced by B15 and B16 that is not observed with B8. Inward movements are likely to play different roles as the position of the radula in the buccal mass changes during a feeding cycle. If the radula is protracted, this movement may contribute to retraction. If the radula is in the neutral position, it may increase the distance between the radula and the musculature that forms the top of the buccal mass and thereby reduce the friction that the palate of the buccal mass offers. It may thus facilitate food transport by increasing the space available for this purpose.

The B8 neurons and the ARC motor neurons also differ notably in their effectiveness as closers. In unstimulated preparations, closing movements produced by the ARC motor neurons are much weaker than those produced by the B8 neurons. However, during actual feeding, the radula is unlikely to be in the rest position when the ARC motor neurons begin to fire. Instead it is likely to be partially open. When the radula is partially open, the closing movements produced by the ARC motor neurons are larger and are sufficient to return the radula to the rest position. Thus the ARC motor neurons could be important as closers especially during behaviors where food is not actually ingested and the radula does not have to close tightly. When animals swallow and grasp food, additional closure produced by the B8 neurons may become necessary.

In summary, our results indicate that the radula movements actually produced when the relatively well characterized motor neurons B8, B15, and B16 are stimulated are more complex than might appear from previous work. The ARC motor neurons produce a previously uncharacterized movement whose role in behavior may depend on the starting position of the radula. Furthermore, although the B8 neurons and the ARC motor neurons can all close the radula, their role in behavior may differ.


    ACKNOWLEDGMENTS

This work was supported by a K02 Award (MH-01267), a K05 Award (MH-01591), a T32 grant (DA-07135), an F32 Award (MH-12890), and National Institute of Mental Health Grants MH-51393 and MH-36730. The National Resource for Aplysia of the University of Miami provided some of the animals used in this study with funds from Division of Research Resources Grant RR-10294.


    FOOTNOTES

Address for reprint requests: E. C. Cropper, Dept. of Physiology and Biophysics, Box 1218, Mt. Sinai Medical School, One Gustave L. Levy Place, New York, NY 10029 (E-mail: croppe01{at}doc.mssm.edu).

Received 8 February 2001; accepted in final form 7 May 2001.


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0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society