Kinematics of the buccal mass during swallowing based on magnetic resonance imaging in intact, behaving Aplysia californica
1
Department of Biomedical Engineering, Case Western Reserve University,
Cleveland, OH 44106-7080, USA
2
MR Systems Department, G. E. Medical Systems Israel Ltd, Keren Hayesod
Street, PO Box 2071, Tirat Carmel 39120, Israel
3
Department of Biology, Case Western Reserve University, Cleveland, OH
44106-7080, USA
4
Department of Neurosciences, Case Western Reserve University, Cleveland,
OH 44106-7080, USA
* Author for correspondence at address 3 (e-mail: hjc{at}po.cwru.edu )
Accepted 14 January 2002
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Summary |
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Key words: feeding, mollusc, biomechanics, Aplysia californica, magnetic resonance imaging, freely moving subject, behaviour
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Introduction |
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We have focused our study on molluscan feeding behavior. Feeding behavior
has been intensively studied in a wide variety of molluscan species. A recent
review describes the analysis of the movements of the feeding musculature and
its neural control in a variety of molluscan genera, including Lymnaea,
Planobarius, Helisoma, Clione, Limax, Navanax, Pleurobranchaea and
Tritonia (Murphy,
2001). The subject that we have chosen, the marine mollusc
Aplysia californica, has been intensively studied as a model system
for understanding the neural basis of learning, memory and motivated
behaviors. The neural, biophysical and molecular mechanisms of learning and
memory identified in Aplysia californica have served as the basis for
understanding the molecular mechanisms of learning in mammals and primates
(Kandel and Pittenger, 1999
;
Silva et al., 1998
). Feeding
behavior in Aplysia californica has also been intensively studied as
a model system for understanding the modulation of behavior under the control
of motivational variables (Kupfermann,
1974
; Kupfermann et al.,
1998
). Many of the motor neurons controlling the feeding
apparatus, the buccal mass, have been identified
(Church et al., 1991
;
Church and Lloyd, 1994
;
Morton and Chiel, 1993
), as
have the interneurons that play a critical role in pattern generation and in
switching among different feeding responses, such as biting, swallowing and
rejection (Evans and Cropper,
1998
; Hurwitz et al.,
1994
, 1966,
1997
;
Hurwitz and Susswein, 1996
;
Jing and Weiss, 2001
;
Kirk, 1989
;
Rosen et al., 1991
;
Susswein and Byrne, 1988
).
Analysis of the biomechanics of feeding in Aplysia californica has
recently become a focus of interest. Earlier studies demonstrated that the
feeding musculature was subject to both extrinsic and intrinsic modulatory
influences (for a review, see Katz and
Frost, 1996) (see also Morgan
et al., 2000
). The implications of these modulatory influences for
rhythmic behavior that must occur at different speeds, and for the integration
of signals released at different frequencies of neural activation, have been
explored (Brezina et al., 2000
;
Weiss et al., 1992
). The
potentially context-dependent role of a specific muscle, the 15 or ARC muscle,
has been examined in isolated buccal masses using ultrasound and intracellular
stimulation of the motor neurons controlling the muscle
(Orekhova et al., 2001
). A
detailed Hill-type model of a muscle of the buccal mass, the I2 muscle, has
recently been described on the basis of measurements of its force/frequency,
length/tension, force/velocity and passive properties
(Yu et al., 1999
).
Furthermore, the kinematics of the buccal mass as a whole has been
characterized in juvenile transilluminated animals
(Drushel et al., 1997
) and has
served as the basis for kinematic models
(Drushel et al., 1998
). The
previous work in transilluminated juveniles indicated that the buccal mass
assumed characteristic shapes during different parts of the swallowing cycle,
but the movements of the internal musculature responsible for these shapes was
not clear.
To visualize the kinematics of the buccal musculature in intact, behaving
animals in the mid-sagittal plane, which provides maximal information about
the internal structures, we have developed a novel magnetic resonance imaging
(MRI) interface that makes it possible to obtain an intrinsic reference frame
for structures in freely moving subjects and have developed an apparatus in
which freely behaving Aplysia californica can feed and be imaged
simultaneously. Studying the kinematics of the buccal mass in intact animals
makes it possible to determine the movements of its internal musculature
within their natural context of sensory feedback and mechanical interaction
with the environment. We report that it is possible directly to monitor the
kinematics of individual muscles and the coordinated movements of many
structures of the buccal mass using this technique, and these kinematic
measurements clarify many aspects of the muscular mechanisms of swallowing in
Aplysia californica. Portions of this work have appeared in
preliminary form (Chiel et al.,
1999; Neustadter et al.,
2001
; Sutton et al.,
2000
).
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Materials and methods |
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Real-time MRI interface
A novel interface was developed to address two potential problems of
imaging freely moving subjects: (i) it is essential to maintain accurate
localization as an animal moves, and (ii) it is essential to assess the degree
of error in the mid-sagittal image, which could be para-sagittal or rotated
along the anteroposterior, dorso-ventral or medio-lateral axes. To address
both these concerns, the interface acquires three interleaved orthogonal
images. Acquiring three orthogonal images provides a rapidly updated reference
frame intrinsic to the moving subject. Furthermore, the two images orthogonal
to the mid-sagittal slice make it possible to assess whether that image is
para-sagittal or rotated. To minimize artifact in the main image due to
intersection with the orthogonal slices, despite the long relaxation time
(T1) of sea water, the tip angles for the main mid-sagittal slice,
and for the auxilliary coronal and axial slices are different (40°,
10° and 10°, respectively); for the theory underlying these choices,
see (D. M. Neustadter and H. J. Chiel, in preparation). The time between
repeated acquisitions of the main image was 310ms, and the time between
repeated acquisitions of each orthogonal image was 620ms.
The user interface runs in two modes. In the localization mode, a new image is acquired every time the user modifies a scan parameter. In the dynamic imaging mode, acquisition runs continuously. Switching between the two modes takes less than 1 s, allowing dynamic imaging to be started immediately when the required slice is located and allowing relocalization to be performed immediately when subject movement is observed. Each image, whether a main image or an orthogonal image, is reconstructed immediately upon the completion of its acquisition and immediately displayed in the appropriate window of the user interface. In addition, a schematic representation of the orientation of the main slice is displayed, and the intersection of the main slice with the two orthogonal slices can be interactively modified by the user.
Data were acquired using echo planar imaging with standard two-dimensional Fourier transform reconstruction. The Elscint 2T-Prestige whole-body MRI system was used, with a 15mT m-1 maximum gradient strength and a 30mT m-1 ms-1 maximum slew rate, allowing 64 encodings with a 1 mm pixel resolution to be acquired in 155 ms. The resolution was 1 mmx1 mm pixels using a total acquisition matrix of 64x128. Details of the imaging sequence are shown in Fig. 1A.
|
Tank and coil for MRI of Aplysia californica
The imaging tank and coil are shown in
Fig. 1B. The tank was long and
cylindrical to avoid susceptibility artifacts. The diameter of the tank also
had to be smaller than or equal to the imaging field of view to avoid folding
artifacts in the encoding direction because sea water produces a relatively
high signal. The tank is 6.4 cm in diameter, allowing 64 1 mm pixels in the
view direction with no folding, and 82 cm in length. The imaging coil is a
standard transmit/receive quadrature birdcage coil whose dimensions were
optimized to maximize signal-to-noise ratio and image uniformity. The coil is
12 cm in length and 8 cm in diameter and is permanently attached to the
imaging tank. The animal is placed inside a holding capsule
(Fig. 1C), which is inserted
into the tank, and the holding capsule is then pulled into the imaging region
using strings that exit the tank at either end. The animals used in these
imaging procedures were the largest that would fit in the holding capsule and
tank (400-580 g) to maximize buccal mass size (approximately 2.5 cmx2
cmx2 cm).
Feeding stimulus
When fed a thin strip of seaweed, 400 g animals normally ingest it at a
peak rate of approximately 0.5 cm s-1. With the placement and
imaging procedure taking up to 2-3 min, a strip of food close to 1 m in length
is required to maintain steady swallowing responses. Most seaweed stipes are
not that long or are too fragile to be cut into long strips. Instead, we used
seaweed-flavored noodles. Noodles were made out of flour, water, salt and
ground-up seaweed (dried laver) and were both stimulating to the animal and
mechanically stable under water when cut into strips with a cross section of 2
mmx2 mm. The noodles were wound around a spool mounted on the front of
the capsule holding the animal (Fig.
1C). The animal was fed the end of the strip before being moved
into the imaging region, and it continued eating throughout the imaging
procedure.
A sequence of three successive orthogonal images taken during swallowing is shown in Fig. 2. Comparisons of animals eating seaweed strips and seaweed-flavored noodles indicated no differences in interswallow intervals or willingness to feed. MRI data were also obtained from animals consuming thin strips of seaweed, making swallowing movements in response to seaweed extract, swallowing in response to polyethylene tubes with a small piece of seaweed on the end presented simultaneously with seaweed extract and swallowing in response to string wrapped with seaweed. No qualitative differences in swallowing behavior were observed in response to these different stimuli.
|
High-spatial-resolution MRI measurements
Two animals were anesthetized with an injection of 333 mmol l-1
MgCl2, and their buccal masses were imaged at high spatial
resolution. One buccal mass was imaged on the Elscint system, and each pixel
had a resolution of 0.3 mmx0.3 mm, over 96 s. The detailed parameters
for each scan are as follows: sequence type, fast spin echo; TE (time to
echo), 120 ms; TR (time to repeat), 900 ms; NEX (number of excitations), 4;
acquisition matrix, 256x256; FOV (field of view), 7.7 cmx7.7 cm;
slice thickness, 1.0 mm; tip angle, 90°. A second buccal mass was imaged
on a GE Medical Systems 1.5T Signa MRI system using a solenoid
transmit/receive coil, and each pixel had a resolution of 0.1 mmx0.1 mm.
The detailed parameters for each scan are as follows: sequence type, fast spin
echo; TE, 120 ms; TR, 3000 ms; ETL (echo train length), 16; FOV, 5; SW (slice
width), 1.5; AM (acquisition matrix), 512x512; NEX, 4. A high-resolution
image of the buccal mass is shown in Fig.
3B.
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An outline of the radular stalk was superimposed on each MR image (Fig. 4A). The mid-sagittal shape of the radular stalk outline was obtained by performing a three-dimensional reconstruction of the radular stalk shape from high-resolution MR images (Fig. 3C) and then sectioning it mid-sagittally (Fig. 3D). The resulting stalk outline is scaled from a single frame for each sequence so that it fits onto the stalk in the image, and its scale and shape are then fixed for all remaining frames in that sequence.
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To identify structures of the buccal mass in the high-temporal-resolution MR images, images were compared with histological cross sections (e.g. Fig. 3A) and with high-spatial-resolution MR images (e.g. Fig. 3B). In addition, the entire sequence of high-temporal-resolution MR images for each behavior was displayed as a QuickTime movie (version 4.1, Apple Computer, Inc., Cupertino, CA, USA). Successive images were repeatedly played to enhance and clarify changes that occurred from image to image that were often subtle but were seen clearly in moving rather than in still images.
To measure specific features, each image was imported into Paint Shop Pro (version 7.0, JASC Software, Eden Prairie, MN, USA), and the following kinematic measures were drawn on each image in different layers: (i) jaw line, (ii) radular stalk outline and radular stalk angle, (iii) lateral groove (the borders of the I1/I3/jaw musculature dorsally and ventrally), (iv) odontophore angle (the angle of the anterior edge of the I6 muscle), (v) an outline of the odontophore, excluding the base of the radular stalk if it protruded below the odontophore, and (vi) an outline of the entire buccal mass including the jaw musculature, the odontophore and the radular stalk, but excluding the pharyngeal tissue because it is only partially visible in these images (Fig. 4). Each analyzed MR image was then imported into Animation Shop (version 3.0, JASC Software), and each layer was saved as a separate TIFF image. Measurements of parameters were then performed on these TIFF images using custom-designed programs written in IDL (version 5.4, Research Systems Inc., Boulder, CO, USA; code available from the authors on request). The resulting data was analyzed and displayed using Mathematica (version 4.1, Wolfram Corporation, Inc., Champaign, IL, USA).
The feeding cycle was normalized on the basis of definitions of the
components of the swallowing cycle from our previous work (Drushel et al.,
1997,
1998
). Our initial definition
of the start of the cycle, which we designated t1, was based on the
movement from peak protraction to peak retraction, since this was an
unequivocal movement that was easy to see in the transilluminated slugs.
However, it is clear from our own work and that of others that the feeding
cycle is initiated from the onset of protraction
(Hurwitz et al., 1996
;
Hurwitz and Susswein, 1996
).
As a consequence, we begin the cycle from the onset of protraction, which was
designated t4 in our original definition of the cycle. Furthermore,
in our original study, we designated the inter-swallow interval as
t3. Examination of successive swallows imaged using magnetic
resonance demonstrated that the t3 interval was extremely variable,
whereas the other cycle durations were more consistent. We therefore excluded
the t3 interval from our analysis. The time intervals are therefore
defined as follows, using the nomenclature adopted in our original papers for
consistency: t4, start of anterior buccal mass movement to peak
protraction; t1, peak protraction to peak retraction; t2,
peak retraction to the loss of the
shape, i.e. the shape in which the
base of the elongated radula/odontophore extends ventral to the long axis of
the buccal mass (see fig. 3A in
Drushel et al., 1997
). Cycle
times are normalized to the sum of the times
t4+t1+t2.
Lengths l were normalized to 100(l-lmin)/(lmax-lmin), so that lengths ran from 0 at lmin to 100 at lmax. After normalization and averaging, data were smoothed using an interpolation function, which fitted cubic polynomials between successive data points. In addition, functions for the standard deviation of the data were constructed as follows: interpolation functions for each individual normalized data set were subtracted from the interpolation function for the averaged normalized data set, and these differences were summed, squared and divided by the number of samples minus 1 (i.e. by 4-1=3), and then the square root of the function was taken. The normalized, averaged data function is plotted, as well as the normalized, averaged data function with the standard deviation function added to or subtracted from it. This provides an indication of the dispersion around each point in the averaged function. Inferences about significant changes in kinematic variables during the swallowing cycle are drawn only if two points on the averaged curve differ by more than two standard deviations.
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Results |
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These results are consistent with observations of swallowing-like behaviors
in semi-intact and reduced preparations
(Drushel et al., 1998) and with
the changes in shape described in our studies of juvenile transilluminated
animals (Drushel et al.,
1997
). The temporal and spatial resolution of these images make it
possible to describe movements of buccal muscles whose borders are visible in
the mid-sagittal image, movements of the odontophore relative to the whole
buccal mass and movements within the odontophore itself.
Kinematics of the prow
On the anterior surface of the buccal mass is a small structure that has
not been described previously, which we refer to as the prow, since its shape
is reminiscent of the prow of a ship (Fig.
6A-C). Anatomical analysis indicated that muscle fibers run along
its anterior side (Fig. 6D) and
that it is filled with a jelly-like fluid whose consistency is similar to the
fluid filling the radular sac. Because it is water-filled, it appears as a
bright area on the anterior surface of the odontophore in the MR images (Figs
3B,
4C). The anterior surface of
the prow is rounded during protraction and during the initial phases of
retraction (Fig. 5, frames
1-10). During protraction, it is pressed up against the ventral I3 musculature
(Fig. 5, frames 3-9). As the
radular stalk protrudes through the ventral surface of the odontophore, the
prow becomes elongated and thinner (Fig.
5, frames 11-19; it is thinnest in frame 19). After a strong
retraction, the prow is the first part of the odontophore to extend anterior
to the lateral groove and into the region of the I1/I3/jaw musculature
(Fig. 5, frames 20-22).
Kinematics of buccal muscles I2 and I3
Because the lateral groove is visible in high-temporal-resolution
mid-sagittal MR images, it is possible to determine the cross-sectional length
of the I2 muscle (a thin protractor muscle that wraps around the posterior
part of the buccal mass) (Hurwitz et al.,
1996) and also to measure directly the anteroposterior lengths of
the I1/I3/jaw musculature dorsally and ventrally
(Fig. 3A,B). Thus, we can
measure the kinematics of these muscles of the buccal mass directly during a
swallowing cycle. It is possible to visualize directly the I7 muscle in the
high-spatial-resolution MR images (Fig.
3B), but it is too small to visualize directly in the
high-temporal-resolution MR images (Fig.
4).
I2 kinematics
We measured the length of the I2 muscle from the dorsal lateral groove to
the ventral lateral groove around the posterior end of the buccal mass. In the
four sequences that we measured, we observed that I2 shortened from the start
of anterior buccal mass movement to peak protraction
(Fig. 7A-E, t4 period;
Fig. 5, frames 1-9), lengthened
from peak protraction to peak retraction
(Fig. 7A-E, t1 period;
Fig. 5, frames 9-18) and then
shortened from peak retraction to the loss of the shape
(Fig. 7A-E, t2 period;
Fig. 5, frames 18-20). There is
an inflection point in the length of I2 during the middle of retraction in
three of the four swallows (Fig.
7B-E, t1 period). The inflection in the I2 length
corresponds to the images during which the lengthened radula/odontophore
rotates and moves posteriorly to the lumen of the jaws
(Fig. 5, frames 16-18). Swallow
1, which is the weakest swallow, shows little or no inflection
(Fig. 7A, t1 period).
Previous estimates of the changing I2 length based on data from
transilluminated juvenile slugs and a kinematic model of the buccal mass show
excellent correspondence to the directly measured MRI data, reaching a minimum
at the end of protraction and a maximum at the end of retraction (compare
Fig. 7E,F) [data in
Fig. 7F replotted from Drushel
et al. (1998
)].
I3 kinematics
We measured the antero-posterior length of the I3 muscle in the
mid-sagittal images. The antero-posterior lengths of the dorsal and ventral
parts of the I3 muscle behave differently during the swallowing cycle. The
primary changes in antero-posterior length of the ventral I3 are due to the
rotation of the odontophore and its effect on the `hinge', the point of
connection between the base of the odontophore (the ventral I4 muscle) and the
ventral I3 muscle. As the odontophore rotates towards the jaws during the
onset of protraction, the hinge is stretched backwards, causing the ventral
surface of the I3 muscle to lengthen (Fig.
8A-E, right panels, t4 portion of the cycle;
Fig. 5, frames 2-8). After the
peak of protraction, the odontophore rotates towards the esophagus, the
stretch is relieved, and the ventral portion of I3 shortens; it then shortens
still further with the loss of the shape after the peak of retraction
(Fig. 8B-E, right panels,
t2 portion of the cycle; Fig.
5, frames 11-19; the
shape is not large in swallow 1, so
additional shortening is not observed in the t2 portion of the cycle;
Fig. 8A, t2 portion of
cycle).
The major changes in antero-posterior length of the dorsal I3 muscle appear to reflect the extent to which the odontophore has inserted itself between the halves of the lumen of the jaw. As the odontophore pushes anteriorly into the jaw musculature, the dorsal I3 shortens (Fig. 8A-E, left panels, during the t4 portion of the cycle; Fig. 5, frames 2-9). During retraction, as the lengthened odontophore begins to rotate posteriorly and makes contact dorsally with the I3 muscle, the dorsal length of the I3 muscle is at its shortest; it then rapidly increases in length as the odontophore withdraws posteriorly from the jaw lumen (Fig. 8B-E, left panels, during the end of the t1 portion of the cycle; Fig. 5, frames 15-17; this is not seen in Fig. 8A, which shows a weaker protraction and retraction cycle).
Consistent changes are observed in the dorso-ventral length of the I3
muscle at the lateral groove during the swallowing cycle. During the
protraction phase, as the odontophore rotates towards the jaws, the I3 muscle
at the lateral groove expands (Fig.
9A-E, left panels, t4 portion of the cycle;
Fig. 5, compare frames 1 and
9). During retraction, the length of I3 at the lateral groove decreases; the
length reaches a minimum during the loss of the shape, at which time a
`pinching in' of the buccal mass at the lateral groove is sometimes visible
(e.g. Fig. 5, frames 19-21;
Fig. 9A-E, left panels,
t2 portion of the cycle).
The change in the dorso-ventral length of the I3 muscle at the jaws during the swallowing cycle is more variable than that at the lateral groove. In general, the width of the I3 at the jaws increases later than does the width of the I3 at the lateral groove [Fig. 9E, compare peak in left panel (lateral groove I3 width) during t4 with peak in right panel (jaw I3 width) during t1]. Moreover, the minimum width of the I3 at the jaws occurs after the minimum width in the I3 at the lateral groove (Fig. 9E, t2 period; compare left and right panels). In some swallows, the jaws show relatively little expansion during the entire cycle (Fig. 9C, right panel), whereas in others, significant expansion is observed (e.g. Fig. 9B, D, right panels).
Odontophore kinematics
We measured bulk movement of the entire buccal mass, movement of the
odontophore within the buccal mass, deformations of the odontophore and
motions of the radular stalk within the odontophore.
Bulk movement of the entire buccal mass
In previous studies, we observed that the line connecting the jaws and the
esophagus could serve as a fixed reference frame for overall movements of the
buccal mass (Drushel et al.,
1997). In the current study, we observed that the posterior part
of the buccal mass was free to move relative to the jaws. We therefore used
the line of the jaws as an intrinsic reference frame since it moves rigidly as
part of the anterior jaw cartilage. We observed that the motions of the jaw
line were quite variable (Fig.
10). The angle of the jaw could change during protraction or
during retraction and could increase or decrease. In one sequence, the
difference between the minimum and maximum angle was approximately 10°
(Fig. 10A); in the other three
sequences, the difference ranged from approximately 25 to 30°
(Fig. 10B-D). To correct for
these bulk rotations, all rotational and translational movements were
referenced to the jaw line in each sequence. Since we orient the jaws to the
right and the esophagus to the left, and the dorsal surface of the buccal mass
upwards and the ventral surface downwards (see
Fig. 2, sagittal section),
clockwise angles that move the odontophore towards the jaw line are reported
as positive, and translational movements of the odontophore from the esophagus
towards the jaw line are reported as positive, in units of radula stalk width
(RSW).
Movement of the odontophore within the buccal mass
At the onset of forward movement of the buccal mass, the anterior border of
the odontophore (i.e. the anterior border of the I6 muscle) rotates towards
the jaws (Fig. 11, left
panels; Fig. 5, frames 1-8).
Peak rotation towards the jaws ends before the peak of protraction in
swallowing (during the t4 period before the t1 border;
Fig. 11, left traces; note
especially the average trace, Fig.
11E, left). The odontophore rotates posteriorly relative to the
line of the jaws from before the end of protraction until after the end of
retraction (i.e. into the t2 period;
Fig. 11, left panels;
Fig. 5, frames 8-20). During
the retraction phase, there is an abrupt decrease in angle at the time that
the odontophore withdraws posteriorly from the jaws
(Fig. 11, left panels, during
the t1 period; Fig. 5,
frames 15-18).
In contrast, the translation of the most anterior tip of the odontophore
towards the jaws continues past the peak of protraction (i.e. into the
t1 period, after retraction of the posterior end of the buccal mass
has begun; Fig. 11, right
panels; Fig. 5, frame 10). The
anterior tip of the odontophore then moves posteriorly relative to the jaw
line throughout retraction and even past peak retraction (i.e. into the
t2 period when the shape is lost;
Fig. 11, right traces;
Fig. 5, frames 11-20). Note
that during retraction, at the same time that there is a rapid decrease in
angle, there is also a rapid posterior motion, which corresponds to the time
at which the odontophore withdraws from the jaws
(Fig. 11, right panels, during
the t1 period; Fig. 5,
frames 15-18). The timing of the odontophore movements does not correspond
exactly to the borders of the t4, t1 and t2 periods because
these periods are defined by the timing of movements of the posterior border
of the buccal mass, and the timing of the internal movements and deformations
of the odontophore (see below) does not exactly correspond to these
movements.
Deformations of the odontophore
We examined the changes in the dorso-ventral and antero-posterior length of
the odontophore during a swallowing cycle. To provide a reference frame for
these deformations, we rotated the outline of the odontophore so that the
anterior border of I6 was vertical, and then determined the maximum
dorso-ventral and antero-posterior lengths of the odontophore's outline in
this position. The antero-posterior length extended only to the anterior
border of I6 and excluded the changing thickness of the prow in order to
determine the changing dimensions of the solid muscle and cartilage of the
odontophore. The antero-posterior length of the odontophore increased during
protraction, as did the dorso-ventral length, but to a lesser extent
(Fig. 12, near the end of the
t4 period; compare left and right panels). The dorso-ventral length
increased and remained higher during retraction at the same time that the
antero-posterior length decreased (Fig.
12, t1 period; compare left and right panels). The
dorso-ventral lengthening of the odontophore at the beginning of the
retraction period corresponds to, and accounts for, the positive translation
of the anterior end of the odontophore after the onset of the retraction of
the posterior end of the buccal mass, which defines the onset of the
t1 period (Fig. 11E, right side, t1 period). During the loss of the shape, there
were decreases in both the antero-posterior and dorso-ventral lengths
(Fig. 12, t2 period).
The length changes are not mirror images of one another, especially during the
t2 period, suggesting that some of the changes in these specific
dimensions may be compensated for by changes in the medio-lateral width, which
is not directly measurable in the mid-sagittal MR images.
Movement of the radular stalk within the odontophore
Movements internal to the odontophore can be examined by measuring the
rotations and translations of the radular stalk within the frame of reference
of the odontophore. We measured the rotations of the radular stalk relative to
the anterior border of the I6, and the translations of the base of the radular
stalk relative to the base of the odontophore. At the onset of protraction,
the radular stalk moves into the odontophore
(Fig. 13, right panels, period
t4; Fig. 5, frames
1-6) and initially rotates posteriorly relative to the anterior margin of the
odontophore (Fig. 13, left
panels, period t4; Fig.
5, frames 1-2). During the middle of the protraction phase,
however, the radular stalk rotates slightly anteriorly and then again rotates
posteriorly relative to the anterior margin of the odontophore
(Fig. 13, left panels, middle
of period t4; Fig. 5,
frames 3-4 versus frames 5-6). During the retraction phase, the
radular stalk moves out of the odontophore
(Fig. 13, right panels,
t1 period; Fig. 5,
frames 10-18) and rotates towards the anterior margin of the odontophore
(Fig. 13, left panels,
t1 period; Fig. 5,
frames 10-15). The radular stalk begins to move back into the odontophore
before the end of retraction, whereas the translation and rotation of the
odontophore do not change direction until after the end of retraction (compare
Fig. 13, right panels,
t1 period, and Fig.
11, left and right panels, t1 period). As the buccal mass
loses its shape, the radular stalk moves back into the odontophore
(Fig. 13, right panels,
t2 period; Fig. 5,
frames 18-22) and rotates sharply posteriorly relative to the anterior margin
of the odontophore (Fig. 13,
left panels, t2 period; Fig.
5, frames 18-22).
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Discussion |
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Limitations of MRI measurements
There are several limitations of the magnetic resonance images. The spatial
resolution of the images is 1 mmx1 mm, which is small enough to resolve
the borders of large structures, but insufficient to directly resolve small
muscles such as the 17 muscle (which can be resolved in
higher-spatial-resolution MR images; Fig.
3B). Studies of a cylindrical phantom indicated that distortion
due to susceptibility artifacts was limited to 1.5 mm, which is also adequate
for the accuracy of the reported measurements. Another concern is the
possibility of blurring, which could occur if the structure moved from one
pixel to another during image acquisition. The overall frequency of the
mid-sagittal images (approximately 3Hz) was low enough that rapid movements of
the odontophore could cause blurring. The actual images are acquired in 155
ms, however, and careful examination of the higher-temporal-resolution movies
of the transilluminated slug (Drushel et
al., 1997) indicated that even the fastest overall movements of
the odontophore, such as its very rapid initial retraction, would not be rapid
enough to induce significant blurring.
Another potential limitation could be a movement of the animal that caused the main image to be para-sagittal or to be rotated relative to the mid-sagittal plane of the buccal mass. Careful analysis of the coronal and axial images just preceding and just following the sagittal images allowed us to select sequences in which this possibility was minimized (Fig. 2). Finally, an inherent limitation of the images analyzed in this paper is that they are confined to a single plane, whereas the buccal mass is a three-dimensional structure. This limitation could be overcome either by improvements in MR imaging technology allowing a high-quality full three-dimensional dataset to be acquired at the necessary temporal resolution or by developing three-dimensional kinematic models of the buccal mass that allow the third dimension to be systematically inferred from the mid-sagittal images.
Functional role of the prow of the odontophore
The fluid-filled projection at the anterior border of the odontophore
(Fig. 6), which we have called
the prow, is likely to play a significant functional role in the protraction
and anterior rotation of the odontophore. Since the prow is the first part of
the odontophore to extend into the lumen of the jaws, it may act as a wedge to
separate the apposed halves of the 13 musculature as the odontophore begins to
protract through the lumen of the jaws. Furthermore, the observation that the
prow is pressed against the ventral surface of the I3 muscle as protraction
proceeds suggests that the effective center of rotation of the odontophore
relative to the buccal mass may shift from the odontophore's ventral
connection with the I3 muscle (the `hinge') to the prow during
protraction.
Functional implications of the kinematics of I2
In this paper, we provide a first description of the in vivo
kinematics of the I2 muscle during swallowing based upon internal anatomical
borders that define its mid-sagittal extent. Our data are consistent with
estimates obtained from juvenile transilluminated slugs
(Drushel et al., 1997). It is
also possible to analyze these data with respect to values obtained from
in vitro studies of the length/tension and force/velocity properties
of I2 (Yu et al., 1999
).
Assuming that the length of I2 at the end of the t2 period is close
to the resting length of the I2 muscle, which we showed was equal to
0.86lmto (where lmto is defined as the
optimal muscle and tendon length of I2), the peak length of I2 at the end of
retraction (the end of the t1 period) is
1.09±0.08lmto (mean ± S.D., N=4),
and the minimum length of I2 at the peak of protraction (the end of the
t4 period) is 0.66±0.03lmto (mean ±
S.D., N=4). Thus, I2 is close to the peak of its length/tension curve
at the onset of protraction, providing it with the ability to protract the
odontophore strongly. In contrast, it is likely that I2 can generate no more
than approximately 40 % of its maximum force near the peak of protraction
because of its shortening (see fig.
2C in Yu et al.,
1999
).
By smoothly interpolating between the length data points and integrating
the resulting interpolated function, it is possible to estimate the velocity
of the I2 muscle during swallowing (data not shown). In general, the maximum
velocity of lengthening of I2 is observed near the end of the retraction phase
(t1 period) as the radular stalk moves out of the odontophore and the
odontophore withdraws from the lumen of the jaws
(0.52±0.24lmto s-1, mean ± S.D.,
N=4), suggesting that the maximum force in I2 could be increased by
as much as 60 % (see fig. 2D in
Yu et al., 1999), which could
serve to brake this rapid movement. The maximum velocity of shortening is seen
early in the shortening phase of protraction and during the loss of the
shape (0.45±0.06lmto s-1, mean
± S.D., N=4), suggesting that the force in I2 could be
decreased by as much as 80 % during this rapid contraction.
Functional implications of the kinematics of I3
Although the mid-sagittal view does not provide direct information about
the movements of the I1/I3/jaw musculature in the third dimension, several
inferences can be drawn from the kinematics reported in this paper: the
movement of the `hinge' point, the muscular hydrostatic properties of the
structure, the `pinch' at the lateral groove and the relative expansion of the
lumen of the jaw musculature at the lateral groove versus the line of
the jaws.
The ventral fibers of the base of the odontophore (the 14 muscles) interdigitate with the fibers of the underlying ventral I3 muscle at the lateral groove. We have used the term `hinge' to describe this interdigitation since it is clear in isolated semi-intact preparations that the odontophore rotates around this point. The kinematics of the antero-posterior length of the I3 demonstrates that the `hinge' is not a rigidly fixed point but is stretched as the odontophore rotates anteriorly or posteriorly. Over the course of a swallowing cycle, the antero-posterior length of I3 on the ventral surface of the buccal mass undergoes a 44±5 % expansion (mean ± S.D., N=4) from its minimum length at the maximum posterior rotation of the odontophore (Fig. 8, right panels, t2 period, and Fig. 11, left panels, t2 period) to its maximum length at maximum anterior rotation of the odontophore (Fig. 8, right panels, t4 period, and Fig. 11, left panels, t4 period).
Changes in the antero-posterior length of the I3 muscle on the dorsal surface of the buccal mass may reflect the muscular hydrostatic properties of the jaw musculature. The length of I3 decreased relative to its maximum length (by 28±17 %, mean ± S.D., N=4) when the tip of the odontophore was furthest anterior, i.e. most fully protracted into the lumen of the jaws, compared with when the tip of the odontophore was furthest posterior relative to the line of the jaws, i.e. most fully retracted out of the lumen of the jaws. When the odontophore is most fully protracted into the lumen of the jaws, it should maximally stretch the lumen of the jaws in the medio-lateral dimension. To maintain the volume of the structure, the musculature should compress it in its antero-posterior dimension. Conversely, when the odontophore is most fully retracted from the lumen of the jaws, the jaws should be minimally stretched in the medio-lateral dimension, leading to expansion in the antero-posterior dimension to maintain the same volume.
As the buccal mass loses its shape, the dorso-ventral length of the
I3 muscle at the lateral groove decreases
(Fig. 9, middle of t2
period, left panels). This corresponds to a pinching together that is visible
at the lateral groove (Fig. 5,
frames 19-21). This pinch may serve to aid the process of retraction by
pushing the radula/odontophore further backwards into the esophagus.
The different anatomical characteristics of the I1/I3/jaw musculature may be reflected in the relative expansions that occur in the structure dorso-ventrally at the lateral groove versus at the line of the jaws. Underlying the anterior I3 musculature are plates of jaw cartilage, joined dorsally and ventrally, which are stiff and thus presumably strongly resistant to expansion when stretched very slightly beyond their rest length. At rest, the posterior extent of the cartilaginous plates is only half the antero-posterior length of the jaw musculature (Fig. 3A; note the position of the posterior end of the cartilage in comparison with the location of the lateral groove, which marks the posterior border of the I3 musculature). The remaining section of the I1/I3/jaw musculature complex is composed solely of muscle and of pharyngeal tissue, which are presumably much more distensible.
The ratios of the dorso-ventral length of the I3 muscles at the lateral groove to the dorso-ventral length at the jaw when neither is significantly stretched (i.e. just before the onset of protraction) differ by a factor of 2.05±0.22 (mean ± S.D., N=4). From the material properties of the anterior versus posterior sections of the I1/I3/jaw musculature, one might predict that the jaw cartilage would resist expansion more than the musculature at the posterior end of muscle I3. However, since the dorso-ventral length of the jaw cartilages is much smaller to begin with, it is possible that the jaws must undergo a proportionally larger expansion. It is clear from the data that, at their maximum expansion, the dorso-lateral length of the I3 and its underlying cartilage at the jaw line are never as large as the dorso-lateral length of the I3 at the lateral groove, even at its minimum value (Fig. 9A-D; compare left and right panels). However, the relative expansion of the dorso-lateral length at the jaw line is greater than the expansion at the lateral groove: the ratio of the percentage expansion at the jaws to the percentage expansion at the lateral groove is 2.1±0.82 (mean ± S.D., N=4). It is interesting to note that there are folds in the anterior cartilage of the jaws that straighten out as the jaw is stretched, and this may allow the jaw to expand significantly before its resistance to tensile stress sharply stops its ability to expand further.
Bulk movement of the buccal mass
In previous studies of transilluminated juvenile Aplysia
californica, we observed that the line connecting the jaws and the
esophagus served as a fixed reference frame for overall movements of the
buccal mass (Drushel et al.,
1997). In the present study, we observed that the posterior part
of the buccal mass was free to move relative to the jaws, and we therefore
chose to use the line of the jaws as an intrinsic reference frame. In our
previous studies, we observed that the overall movement of the buccal mass was
relatively consistent (see fig.
4 in Drushel et al.,
1997
), whereas in this study we found considerable variability
(Fig. 10). One reason for the
difference may be that the previous studies utilized juvenile slugs that were
swallowing a cut piece of seaweed that was large and stiff relative to their
scale. As a consequence, their entire buccal mass was effectively held rigidly
around this rod so that the esophagus was constrained to rotate together with
the jaws. The large animals used in this study are swallowing a relatively
soft noodle or polyethylene tube, and the posterior part of the buccal mass is
free to move relative to the jaws. The movements of the posterior part of the
buccal mass are likely to reflect both internal deformations and the effects
of the extrinsic muscles and may, therefore, be more variable. Another
possible reason for the discrepancy between results is that we were unable to
visualize the foot and head borders of the animal clearly in these studies,
because they are essentially transparent in MRI, and movements of these
surrounding structures relative to the fixed frame of the holding capsule
would also affect the angle of the jaw line.
Kinematics of the odontophore
If the odontophore were a rigid body, its net translation and rotation
relative to the line of the jaws could be considered independently of its
deformations and the internal movements of the radular stalk. As is clear from
examination of the MR images, however, the internal expansion or contraction
of the odontophore affects both the angle that its anterior edge makes with
the jaw line and the movement of the point that is closest to the line of the
jaws. Thus, we will consider all these movements simultaneously during each
part of the swallowing cycle.
During protraction, the radular stalk translates into the odontophore (Fig. 13, right panels, t4 period) and the odontophore becomes widest in its antero-posterior dimension (Fig. 12, left panels, t4 period). The odontophore also undergoes its maximum anterior rotation during protraction, in part because of its antero-posterior expansion (Fig. 11, left panels, t4 period). As the radular stalk moves into the odonotophore and the odontophore expands at the onset of protraction, the radular stalk initially rotates away from the anterior margion of the odontophore (Fig. 13, left panels, t4 period). In each swallowing response, this is followed by a consistent rotation of the radular stalk towards the anterior margin of the odontophore, followed by a further rotation away from the anterior margin. These observations suggest that, as the odontophore moves anteriorly into the lumen of the jaws during protraction, the halves of the odontophore may be kept open by pulling the radular sac and stalk into the odontophore, and the anterior and posterior rotation of the radular stalk relative to the anterior margin of the odontophore may reflect an additional muscular contraction that prolongs the opening phase by keeping the radular sac and stalk within the odontophore despite the constricting forces of jaw musculature.
Early in retraction, the anterior translation of the odontophore reaches its peak even though the odontophore has already begun to rotate posteriorly (Fig. 11, t1 period; compare right and left panels), accompanied by a peak increase in its dorso-ventral length (Fig. 12, right panels, t1 period). After the peak anterior translation, the radular stalk translates out of the odontophore (Fig. 13, right panels, t1 period) and rotates away from the anterior edge of the odontophore (Fig. 13, left panels, t1 period). During late retraction, there is an abrupt increase in the posterior rotation and translation of the odontophore (Fig. 11, t1 period), which is associated with the very rapid posterior rotation of the elongated odontophore out of the jaw lumen. These observations suggest that the radula may begin closing at approximately the time of peak protraction, perhaps because of a contraction of the 14 muscles, which push the radular stalk out of the odontophore. The entire elongated structure rotates posteriorly, probably as a result of passive forces and/or activation of musculature at the interdigitation of the ventral 13 and 14 muscles (the `hinge'). Contraction of the 13 muscles, causing the jaw lumen to squeeze down mediolaterally upon the odontophore, and the changed shape of the odontophore induce it to `snap back' once it has rotated far enough posteriorly out of the jaw musculature (indications of the snap-back are seen in Fig. 5, frames 16-18, Figs 7E, 8E, left panels, Figs 9E, 13E, right panels, and Fig. 11E, left and right panels).
During the loss of the shape, the odontophore rotates and
translates backwards (Fig. 11,
t2 period) and contracts in both its dorso-ventral and
antero-posterior dimensions (Fig.
12, right and left panels, t2 period), and the radular
stalk both translates into the odontophore and rotates away from its anterior
edge (Fig. 13, t2
period). If the odontophore is isovolumetric, the contraction in the
dorso-ventral and anteroposterior dimensions implies that it expands in the
mediolateral dimension. This inference and the movements of the radular stalk
all suggest that, at this phase, the halves of the radula are opening. Thus,
the underlying mechanism of the
shape of the overall buccal mass is
the posterior rotation of the closed radula halves and elongated odontophore
into the esophagus, and the loss of the
shape is due to the rapid
translation of the radular stalk into the odontophore as the halves of the
radula open. We will refer to this rapid inward translation of the radular
stalk into the odontophore as the `collapse' of the elongated odontophore, but
this term is not meant to imply that the movement is passive or that the
entire structure becomes smaller in all dimensions, since it is likely to
involve active forces and an expansion of the odontophore in the medio-lateral
dimension.
One possible way in which the radular stalk could move into the odontophore
would be if it were strongly compressed by the 12 muscle. However, the radular
stalk begins to translate into the odontophore near the end of the t1
period (Fig. 13, right
panels), and the I2 muscle does not begin to shorten significantly until the
middle of the t2 phase (Fig.
7). Furthermore, studies of its activation in vivo
suggest that it does not actively contract until the onset of the protraction
phase, t4 (see fig.
11 in Hurwitz et al.,
1996) (see fig. 12
in Drushel et al., 1998
). It is
possible that the passive properties of I2, which are significant when it is
stretched beyond 1.0lmto, could contribute to the collapse
(Yu et al., 1999
). The data
suggest, however, that activation of the 17 muscle could play a more
significant role in moving the radular stalk into the odontophore
(Evans et al., 1996
).
Variability of responses and degrees of freedom in the buccal
mass
Although we have focused primarily on the common features of all four
sequences, which can be observed both in the individual responses and in the
normalized, averaged responses, the variability among the four swallowing
responses is striking. The durations of the swallows vary (swallow 1, 5.6s;
swallow 2, 6.8s; swallow 3, 7.1s; swallow 4, 6.5s) and the amplitudes of the
motions vary (for example, compare the anterior translation of the odontophore
during the protraction period in swallow 1 with that in swallow 2,
Fig. 11A,B, right traces,
t1 period). In the same animal, two swallows that immediately follow
one another can vary significantly. Swallows 1 and 2 immediately follow one
another in one animal, and swallows 3 and 4 immediately follow one another in
a second animal. The variability in the swallowing responses may be due to the
great variability observed in ingestive-like motor patterns observed in
isolated buccal ganglia, even though sensory feedback is absent, and thus may
be due to inherent properties of the central pattern generator for feeding;
see, for example, Church and Lloyd
(1994), who comment on the
great variability of patterns seen in reduced preparations and even in
isolated ganglia. It may also be due to the changing conditions of the food
and its positioning within the buccal mass, which could strongly and rapidly
modulate the feeding responses. It is known, for example, that changing the
mechanical load on the buccal mass can rapidly alter neuronal responses
(Hurwitz and Susswein, 1992
).
Finally, the variability may be due to the inherently flexible nature of the
muscle and cartilage that constitute the buccal mass, which may afford a
relatively large number of degrees of freedom that can be affected flexibly
both by changes in neural output and by interactions with the biomechanics of
food. These observations support our hypothesis that understanding the full
functionality of systems with a high level of neural and biomechanical
flexibility requires the simultaneous monitoring of both aspects of the system
in freely behaving subjects.
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Acknowledgments |
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References |
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