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
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ABSTRACT |
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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.
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INTRODUCTION |
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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.
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METHODS |
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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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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 M). 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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RESULTS |
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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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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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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.
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DISCUSSION |
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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.
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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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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REFERENCES |
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