Abstract
As the first step in investigating the Pacific bonito (Sarda chiliensis) as a model
system for studying the acquisition of swimming behavior and underlying neural activity in
teleost fish, we studied the changes in the swimming behavior, anatomy of motoneurons,
and muscle activation throughout the larval developmental period. The larval period lasts
just 12 days, during which there is rapid development of locomotion. At 12 days post
fertilization (dpf), bonito undergo a rapid metamorphosis to the juvenile stage. The short,
12-day developmental period suggests rapid development and organization of the
motoneuron and motor system. Swimming behavior was videotaped for five fish in each
day, and analyzed by determining a time budget for active time and determining maximum
body flexion exhibited by each fish. Motoneurons were fluorescently labeled with Dil and
muscle activity was recorded using electromyography. Locomotion is initially produced
using the trunk and develops to include use of the full tail and tip of the tail. Early in
development, larval bonito spend the majority of their time inactive but sustained
swimming periods become more common as the fish develops. Maximum body flexion
did not appear to change with development. The number of motoneurons increased from
less than 10 to over 30 cell bodies per segment. We also observed two types of
motoneurons which differed in morphology. Cell bodies also appeared to develop in tiers
until they were of the same size. Motoneuron development first took place in more rostral
segments of the fish with the number and complexity of the motoneurons decreasing
toward caudal segments. Signals obtained from EMG recordings appeared to correspond
to the basic swimming modes of rapid bursts and longer duration swimming using tail
beats. We cannot draw a direct correlation between swimming behavior, motoneuron
anatomy, and muscle activity based on the data obtained thus far, however, we have shown
that development of bonito swimming behavior is associated with rapid development of the
motoneuron system and muscle activity which correlates to swimming mode.
Introduction
The neural circuitry underlying swimming movements has been well studied in
cyclostomes and embryonic and larval amphibians, but little is known about the neural
basis of the swimming motor pattern generator in teleost fish (Yoshida et al., 1996). The
zebrafish (Danio rerio) has been used as a model system for swimming behavior and
nervous system development due to its rapid development and transparent larvae, making it
easy to visualize motoneurons. Preliminary observations of the Pacific bonito (Sarda
chiliensis) suggested that bonito would serve as an ideal model system in which to study
the anatomical, mechanical, and behavioral processes underlying the acquisition of
swimming behavior in teleosts. Bonito larvae, like zebrafish, are transparent. They hatch
within 2 days after fertilization at 22-23°C and undergo rapid growth throughout a larval
life lasting only 11 or 12 days post fertilization (dpf) before undergoing a rapid
metamorphosis to the juvenile stage. The short larval period suggests rapid development
and organization of the spinal cord motoneurons and axial motor systems. The purpose of
this study was to begin the investigation of the bonito as a model system for studying the
acquisition of swimming behavior and underlying neural activity in teleost fish.
In order to produce swimming behavior an optical stimulus, such as a rotating
striped drum, is often used (Harden Jones, 1963). This type of optical stimulus has also
been used to study the development of vision in zebrafish, where the rotation of a striped
drum evokes an optokinetic response (OKR) (Easter and Nicola, 1996). Behavior of a fish
can be quantified using a time budget, which accounts for all activity of the fish in a given
time period. The most basic time budget divides behavior into two categories; active and
inactive. The maximum flexion a fish can perform may also be important because in order
to produce undulatory movements, the fish must be flexible in lateral directions (Videler,
1993). In order to investigate the neural circuitry responsible for producing swimming
behavior, we studied the motoneuron development during the larval period and
electromyography (EMG) recordings.
We began our investigation of the changes associated with the development of
swimming activity in larval bonito by examining swimming behavior, labeling
motoneurons using the fluorescent dye Dil, and using EMG analysis to study muscle
activity. We show that development of bonito swimming is associated with rapid
development of the motoneuron system and muscle activity which correlates to the
swimming mode exhibited.
Materials and Methods
Animals
Larval bonito were obtained courtesy of the Monterey Bay Aquarium. Day 2
through 6 fish used in the behavior and motoneuron dye injections were fertilized on 25
April and raised at 26.5°C +-0.5°. The fish used for days 7 through 13 in the behavior
and in the motoneuron dye injections were obtained from a separate hatching. These fish
were fertilized on 30 April and raised at 22.5°C +-0.5°. The fish used in the EMG
recordings were fertilized on 19 May and raised at 22.5°C +0.5°. On day 7 of
development for this population, the population died out and we were unable to obtain fish
to finish the time course of development for EMG recordings. The motoneuron dye
injections for the day 4 fish was also obtained from this last population, since earlier
attempts for motoneuron labeling had been unsuccessful.
Error may have occurred in experiments performed on days 2 through 6 fish, which
were raised at a higher temperature the other stocks of bonito larvae observed (26°C versus
22°C). The effects of temperature on bonito larval development have not been fully
investigated, though preliminary studies indicate that under optimal feeding conditions
increased temperature may lead to more rapid behavioral development.
Swimming Behavior
A rotating drum was constructed using a plastic bucket with a 29 cm diameter. The
interior was painted white, and the exterior wrapped in black gaffing tape. Black stripes
were placed on the interior of the drum using black electrical tape. Each black stripe was
1.9 cm wide, separated by a white stripe 2.6 cm in width, resulting in a total of 20 stripes
18° apart (Fig. 1). The drum was attached to a rotating base, controlled by a DC motor.
The speed and direction of rotation could be varied. The bottom of the drum was painted
white, with a hole in the center in which an 9 cm diameter black-bottomed petri-dish
containing the fish were placed. The fish were filmed using a Sony CCD camera mounted
above the dish. Recordings were made on a Panasonic Time Lapse Video Cassette
Recorder (6740).
Five fish were placed in the petri-dish for each swimming experiment. For days 2
through 6, each experiment lasted 2 hours. This was broken down into 12 ten-minute time
periods. The observation began with a period in which the drum was stationary, followed
by counterclockwise and clockwise rotations of the drum. The counterclockwise and
clockwise rotation periods were separated by a period in which the drum did not rotate.
The drum was rotated at three speeds in both the counterclockwise and clockwise
directions: 2 to 3°/8, 4 to 5%/, and 6 to 7°/s. Beginning with the day 7 fish, we reduced the
duration of the experiment because we noticed that the fish were becoming tired by the end
of the 2 hour period. The revised protocol lasted a total of 25 minutes and was repeated for
a total length of 50 minutes. The 25 minute period was broken down into 5 five-minute
time periods, beginning with a the stationary drum and alternated with counterclockwise
and clockwise rotations at 6 to 7%/s periods, and ending with the a period in which the drum
did not rotate.
Behavior was observed qualitatively by viewing the tapes. We also looked for
optokinetic response by counting the number of counterclockwise and clockwise turns
during each time period in the day 7 and day 8 fish. A time budget for the fraction of time
spent active for a 2 minute period was determined, as well as the maximum body flexion
that each fish exhibited during a 2 minute time period.
A time budget of activity was determined for a 2 minute time period while the drum
was stationary. A fish was considered to be active if any part of its trunk or tail was
moving. A fish undergoing no translation but snapping its jaw was considered inactive
while a fish undergoing no translation but flicking its tail was considered active. Each fish
was followed for the 2 minute period and the amount of time spent active was determined
using a stop watch.
Maximum body flexion was measured by observing each fish individually during a
2 minute time period when the drum was stationary. When a turn which included a large
body flexion was observed, the behavior was retraced frame by frame on the VCR and
traced onto acetate paper. Body flexion angle was measured by determining the angle
between lines drawn between the head of the fish and the tip of the tail. Fig. 2 shows
examples of tracings of fish and how the angle of flexion was determined. The smaller the
angle, the greater the flexion of the fish.
Motoneuron Dye Injections
The long-chain dialklycarbocyanine lipophilic tracer, Dil, was used at a
concentration of 2.5 mg/mL in dimethylforamamide (DMF) to visualize motoneurons. Fish
were anesthetized in ice-water before being fixed in 4% formaldehyde for 2 hours. Fixed
fish were injected with Dil using microelectrodes approximately 1 um in diameter made
from 1.5 mm capillary tubes. Fish were injected 2 to 5 times in the ventral musculature and
stored overnight in a 4°C fridge. The days 2 through 5 fish were stored in distilled water
until we noticed that the osmolarity of the distilled water differed from that of the fish and
was reducing the number of successful injections. The remaining fish were generally
stored in phosphate buffer solution (PBS), which was used to dilute the fixative and would
not disrupt osmotic balance, and occasionally kept in the 4% formaldehyde. The injections
were visualized using an Olympus fluorescence microscope at 4x, 1Ox, 20x, and 40x
magnification. Successful stains were filmed using a camera attached to the microscope
and a Sony VCR (SLV-373UC). Images of labeled motoneurons were digitized using the
Mega Vision 1024XM System v.4.3 and Adobe Photoshop 2.5.
EMG Recordings
EMG recordings were obtained using a tungsten microelectrode approximately
1 um in diameter. The signal was fed through an amplifier with a gain of 10000 and a
1 kHz filter and then digitized using a Sony DAT Recorder (670). Analysis of the EMGs
was performed using Dempster software. The fish were placed in a drop of agarose in a
small petri-dish of distilled water and then penetrated with the electrode. They were also
viewed through a light microscope for movements which could be correlated with EMG
recordings.
Results
Staging
Bonito development was broken down into seven stages. In Stage 1, the Yolk-Sac
stage, the fish were characterized by lack of eye pigments, a transparent head, and an
internalized oil droplet in a large yolk-sac (Fig. 3A). Stage 1 encompassed day O (day of
fertilization) to 2 dpf. In Stage 2, the Eye Pigment stage, the fish developed retinal
epithelium pigments by 3 dpf, proto-pectoral fins, and by 4 dpf, a functioning jaw (Fig.
3B). Stage 2 ranged from 3 dpf to 5 dpf. During Stage 3, the Predator stage, the bonito
began feeding on artemia, giving them an orange gut by late 5 dpf. Yellow and black spots
appeared on the skin, and teeth appeared at 6 dpf. Stage 3 included 5 dpf to 9 dpf (Fig.
30). Morphological changes during Stage 4, the Constant Swimmer stage, included
increased definition of trunk structure, spots on the skin covering the head and gut,
development of cartilage or bone in the head, greater definition in the nasal area, and clear
myotome definition by 11 dpf (Fig. 3D). Stage 4 ranged from 9 dpf to 11 dpf. Stage 5,
the Metamorphosis stage, was characterized by a 2-fold increase in total length, increased
coloration on the gut and snout, reflective eyes and gut, the appearance of a bony dorsal
fin, development of bone in the trunk, and definition of the caudal fin, muscle structure,
and nasal area (Fig. 4). Stage 5 consisted of 12 dpf. Stage 6, the Pre-Juvenile stage, was
marked by increased trunk coloration and steady growth. Stage 6 consisted of 13 dpf to 14
dpf. At Stage 7, the fish were completely opaque and approximately 2 cm in length. Stage
7 generally began at 15 dpf.
Swimming Behavior
Stage 1 fish spent the majority of their time inactive, interrupted by short bursts of
activity. These short periods of activity typically consisted of short, rapid forward
swimming propulsions or quick tight circling, both produced primarily by movements of
the trunk. By Stage 2, the fish were swimming more consistently and began to perform
graded turns, where the head moved first followed by the rest of the body. Locomotive
behavior appeared to involve use of the full tail as well as the trunk. Sustained swimming
periods were more common by Stage 3. These periods of steady swimming were often
interrupted by short, rapid forward swimming propulsions. Turns were consistently
performed by movement of the head first followed by the tail. In Stage 3, a new behavior
termed "hovering" was exhibited, where a fish displayed repeated tail flicks but did not
translate in any direction. Locomotion appeared to be controlled both by use of the entire
trunk and the tip of the tail alone. Stage 4 fish swam nearly constantly, with short periods
of inactivity. Hovering was performed using the tip of the tail, and accelerated swimming
was achieved using the full tail and trunk.
Optokinetic Response
Attempts to quantify an optokinetic response in the day 7 and 8 fish were
unsuccessful. It did appear that older fish which were stationary oriented towards the
rotating drum by pivoting in place, although they did not respond to the drum in a
stereotyped manner if they were actively swimming.
Activity
A trend was seen for increase in activity with development (Fig. 5). Peaks of
activity were seen on days 3, 8, and 12, but the overall time spent active increased over the
12-day period.
Maximum Body Flexion
No significant trend was seen in the change of the maximum body flexion for the
bonito throughout the 12 day developmental period studied (Fig. 6). The data was
quantified using regression analysis (n-5, r-0.26, F-18.4), which indicated that the
difference of the degree of flexion throughout the larval developmental period was not
statistically significant.
Motoneuron Dye Injections
Over the larval developmental period, the number of cell bodies per segment
increased from less than 10 to over 30. We noted that motoneurons could be classified into
two different types, dependent on morphology. Type A, which was seen more
consistently, was characterized by uniform branching of cell bodies off the main ventral
root, while Type B motoneurons were characterized by more erratic branching off the main
ventral root and greater secondary branching (Fig. 7).
We also noted that motoneurons appeared to occur in tiers, with only one tier
occurring in the Stage 1 fish (Fig. 8A). Beginning with Stage 3, two tiers of the Type A
motoneuron cells were seen, with the second tier of cells being smaller in size (Fig. 8B).
In a mid Stage 3 fish, three tiers of cells were observed. The third tier of cells occurred just
caudal to the gut and was composed of small Type B cells (Fig 8C, Fig. 9). We were not
able to identify a third tier of cells in later stage fish. By the end of Stage 4, the tiers of cell
bodies were no longer distinctly layered, nor was there a distinct difference in the size of
the cell bodies (Fig. 8D).
Motoneuron development first took place in the more rostral segments of the fish,
with the number and complexity of the motoneurons decreasing towards the caudal
segments. At later stages, the number and complexity of the caudal motoneurons increased
(Fig. 10).
EMG Recordings
Late Stage 1 fish displayed a quick burst of electrical activity. Each spike lasted
about 7 ms and occurred at a frequency of 142 Hz. The total duration of this activity
reached about 360 ms, preceded by a movement artifact of approximately 170 ms. The
amplitude of the spikes within the burst was fairly constant (Fig. 11).
Electrical activity generated by early Stage 2 fish consisted of two signals followed
by a movement artifact. The first signal was more complicated in nature and consisted of at
least three main spikes, all of which may contain overlapping signals. The overall signal
lasted approximately 30 ms with a frequency of 33 Hz. Individual spikes within the signal
lasted approximately 10 ms with a frequency of 100 Hz. Approximately 50 ms later, this
signal was followed by a second spike, which was simpler, consisting of a single spike
which lasted about 5 ms with a frequency of 200 Hz and had an amplitude about half that
of the first signal. The movement artifact which followed the second signal lasted about 30
ms (Fig. 12).
A more complicated swim burst pattern was seen with early Stage 3 fish. Each
spike was more complicated in structure, appearing to consist of a primary and secondary
spike which overlapped. The individual signals lasted approximately 10 ms, with a
frequency of 100 Hz. The entire duration of the pattern lasted just over 100 ms, with the
amplitudes of the spikes in a ration of 3:4:5:6. These swim burst patterns were correlated
with head movements (Fig. 13, Fig. 14).
Swim bursts recorded from middle Stage 3 fish were variable in duration, lasting
300 to 650 ms. The first swim burst pattern was also more complicated, consisting of two
consecutive spikes which were repeated approximately 50 ms after the previous spikes
ended. The first of the two spikes was larger and relatively unitary in appearance. The
second spike was only about half the amplitude of the first spike, but more complicated,
appearing to consist of two overlapping signals. Each event lasted approximately 10 ms,
with a frequency of 100 Hz. The entire duration of the burst was approximately 650 ms
(Fig. 15). The second swim burst recorded was simpler, consisting mostly of unitary
spikes, although some spikes were complicated by underlying signals. The spikes lasted
about 5 ms, had a high frequency of 200 Hz, and relative amplitudes in a 2:3:5 ratio. The
entire duration of the pattern was approximately 300 ms (Fig. 16). Series of action
potentials were also recorded, with each individual action potential lasting less than 10 ms.
à frequency of 100 Hz. The first two spikes were separated by only about 15 ms, with a
longer separation time of about 50 ms between the remaining spikes. The signals had a
ration of amplitudes of 1:3, with the middle signal having the largest voltage (Fig. 17).
Discussion
Swimming Behavior
Qualitative observations of swimming behavior indicated that active locomotive
behavior increased with time. The gradual change to more complete use of the trunk and
tail for swimming seems to indicate a developmental period in which larval bonito gains
greater control over their muscle groups to produce more adult-type swimming.
The time budget of activity clearly showed a trend of increased activity with age
(Fig. 3). This falls into place with our general observation of increased activity with age,
and implies that the larval fish are developing swimming behavior over time to reach adult
swimming behavior. The peak of activity seen on day 8 may have been due to the hoverins
behavior. Hovering was first exhibited on day 8, and the fish seemed to spend more time
hovering on the first day of demonstrating the behavior than on consecutive days. The
drop in activity seen on day 13 may have been a result of metamorphosis, which generally
occurs on day 12. Fish that did not undergo metamorphosis on day 12 may have begun to
die.
Although there did appear to be a slight trend for increased flexion over
de velopment, further data collection would be necessary to quantify the significance of this
trend since our data should that the difference in flexion was not significant. Past studies
have hypothesized that fish may use bending stiffness of their bodies to modulate their
swimming behavior (Long and Nipper, 1996), which implies that as a fish gains greater
control over its locomotion its flexibility may change. Our data did not show a significant
change in maximum body flexion. However, we did not account for muscle development.
which is occurring concurrently. Greater muscle development may allow fish to modulate
their flexion.
Motoneuron Dye Injections
While we were able to identify two different types of motoneurons occurring in the
bonito (Fig. 7), we were not able to determine the exact areas innervated by individual
motoneurons. It is possible that the Type A and Type B motoneurons correspond to the
primary and secondary motoneurons characterized in the zebrafish (Myers et al., 1986). It
was shown in zebrafish that the three primary motoneurons precisely innervate cell-specific
subsets of contiguous muscle fibers in mutually exclusive regions (Westerfield et al.
1986), which could also occur with the two types of bonito motoneurons. However, to
determine if either of these possibilities are plausible, more detailed experiments would be
needed to determine if this is the case. If Type A and Type B motoneurons corresponded
to the primary and secondary motoneurons identified in zebrafish, they could be activated
differently dependent on the swimming mode as was demonstrated in the zebrafish for fast
and slow swimming (Liu and Westerfield, 1987) or act synchronously as in the zebrafish
escape response (Fetcho and O'Malley, 1995).
The number of motoneurons we visualized throughout the larval period was greater
than the 3 primary motoneurons identified in the zebrafish (Myers et al., 1986; Westerfield
et al., 1986). All of the motoneurons that were seen may not have been primary
motoneurons, nor were we able to identify specific motoneurons and track their
development, so a direct comparison cannot be drawn to the zebrafish. It does seem,
however, that motoneuron development is occurring more rapidly in the bonito than in the
zebrafish.
The development of cell body tiers (Fig. 8) throughout Stages 1 through 4 seemed
to begin with a single tier in Stage 1 which expanded to two tiers seen in early Stage 3.
The third tier of cells in mid Stage 3, which consisted of Type B cells, may correspond to
innervation of motoneurons into different muscle groups. This could result in the finer
locomotive behavior seen in Stage 3, where the tail is used in addition to the trunk and
turns are initiated by the head followed by the tail. By late Stage 4, the obvious tiering of
cell bodies disappeared, with the cell bodies becoming more evenly layered and equal in
size. If the various tiers of cells had innervated different muscle groups, more uniform
distribution and size could indicate equal control over all muscle groups producing
locomotive behavior. This could correspond to our observations that early in development,
locomotive behavior is dependent on movements of the trunk and evolves to consist of use
of the trunk and tip of the tail. Again, we could not specifically determine innervation of
motoneurons into specific muscle groups. However, development of the more complex
tiering of cells through Stages 1 to 4 does correlate with the notion that the nervous system
of the larval bonito is undergoing rapid morphological and organizational changes before
the onset of metamorphosis, which occurs in Stage 5.
Our observation that motoneuron development first takes place in more rostral
segments and then in the more caudal segments in later stages (Fig. 10) seems to indicate
that larval bonito have less control over their caudal segments earlier in development,
consistent with observations that Stage 1 fish use their trunk, or more rostral segments, for
locomotion. Later in development, the number and complexity of the caudal motoneurons
increased, indicating that the fish would have greater control over their tails for use in
locomotive behavior. This is also consistent with the observation that later stage bonito use
their tail in locomotive behavior.
EMG Recordings
The high frequency burst of electrical activity seen in Stage 1 fish (Fig. 11) may
correspond to the quick burst of swimming activity observed during behavior. The
signals produced by the early Stage 2 fish (Fig. 12) differ from the activity seen in the
Stage 1 fish, consisting of less spikes overall which were about equal to or lower in
frequency. This signal probably constitutes a different swimming pattern than that in the
Stage 1 fish, possibly corresponding to the slower tail beat used in locomotion rather than a
quick burst of rapid swimming.
The bursts of electrical activity seen in early Stage 3 fish (Fig. 13, Fig. 14) were
observed immediately after head jerks. These head jerks would probably have resulted in
full body movements if the fish had not been embedded in agarose, indicating that this
swimming behavior is similar to the burst of swimming seen in the Stage 1 fish rather than
the simpler tail beat. The signals obtained from the early Stage 3 fish were more
complicated and lasted longer than the Stage 1 fish, but lower in frequency, indicating a
more sustained period swimming with more motoneurons firing. The decrease in
frequency could be due to the fact that while the Stage 1 spikes were more unitary and
easily distinguished from each other, the signal in question was more complicated and
probably consisted of multiple spikes whose individual frequencies could not be
determined.
Both the bursting pattern (Fig. 15, 16) and the slower tail beat (Fig. 17) were seen
in the mid Stage 3 fish. Again, the burst pattern consisted of more complicated signals than
the quick burst patterns seen in younger fish, but were comparable in frequency to the
signal seen in the early Stage 3 fish. The duration of the pattern varied from approximately
300 ms in one instance to 650 ms in another, possibly indicating that the fish has the ability
to regulate the length of the sustained swimming period. Variation in the duration of the
pattern is similar to studies performed on the larval angelfish (Pterophyllum scalare), where
it was shown that the duration of bursts in older fish were more variable suggesting that the
force of the tail beat could be regulated flexibly as the fish developed (Yoshida et al.,
1996). The series of action potentials in Fig. 17 most likely correspond to the slower tail
beat patter, similar to the one seen in the early Stage 2 fish. The greater number of action
potentials indicate more tail beats over a longer duration than was seen in the early Stage 2
fish. Increase in the complexity, length, and number of signal pulses with age has been
seen in both larval angelfish (Yoshida et al., 1996) and in Xenopus (Sillar et al, 1993).
The electrical signals recorded from the Stage 1 through 3 fish could coincide with
the behavior observations of the bonito. Early in development, the fish display mostly
short periods of quick forward propulsive activity, which could correspond to the high
frequency, short burst of electrical activity. Later in development, the fish are able to
maintain longer periods of swimming, along with longer bursts of rapid activity. The
slower more sustained tail beats seen in steady swimming could possibly correspond to the
singular action potentials and the bursts of rapid forward propulsion could correspond to
the more complicated burst patterns.
Our EMG recordings cannot be correlated directly with specific behavior patterns or
firing of specific motoneurons because typically, a directly correlatable behavior was not
observed and insertion techniques were not optimal. Use of the single-insertion electrode
makes it impossible to determine the source of the electrical signal. Furthermore, because
the fish was much smaller in size relative to the electrode, it was difficult to determine
where exactly in the fish the electrode was inserted and stereotyped activity produced by
each motor unit which could have been recorded is unknown.
Conclusion
We cannot draw a direct correlation between swimming behavior, motoneuron
anatomy, and muscle activity based on the data obtained thus far. However, it does appear
that rapid development of locomotion is accompanied by rapid development of
motoneurons, in terms of number, complexity, and organization. EMG recordings seem to
correspond to the basic swimming modes of rapid bursts and longer duration swimming
using tail beats. Our study only begins to scratch the surface of the full-scale investigation
of bonito development. Further studies are needed to continue the development of the
bonito model system.
Acknowledgments
Iwould like to thank my partner in carrying out this project, Linda Nevin, along
with Stuart Thompson, Matt McFarlane and Christian Reilly, for their assistance in the
project. Greatly appreciated technical assistance was provided by John Lee. Larval bonito
were husbanded by David Cripes and Timothy Cooke, who generously provided them to
us for use in experimentation.
Literature Cited
Easter, S.S, Jr., and Nicola, G.N. 1996. The Development of Vision in the Zebrafish
(Danio rerio). Dev. Biol. 180: 646-663.
Fetcho, J.R., and O'Malley, D.M. 1995. Visualization of Active Neural Circuitry in the
Spinal Cord of Intact Zebrafish. J. Nuerophsyiol. 73: 399-406.
Harden Jones, F.R. 1963. The Reaction of Fish to Moving Backgrounds. J. Exp. Biol.
40: 437-446.
Liu, D.W., and Westerfield. 1988. Function of Identified Motoneurones and
Co-ordination of Primary and Secondary Motor Systems During Zebra Fish
Swimming. J. Physiol. 403: 73-89.
Long, J.H., Jr., and Nipper, K.S. 1996. The Importance of Body Stiffness in Undulatory
Propulsion. Amer. Zool. 36: 678-694.
Myers, P.Z., Eisen, J.S., and Westerfield, M. 1986. Development and Axonal Outgrowth
of Identified Motoneurons in the Zebrafish. J. Neurosci.. 6(8): 2278-2289.
Sillar, K.T., and Wedderburn, J.F.S. 1993. Control of Locomotor Movements During
Vertebrate Development. Int. Union Physiol. Sci./Am. Physiol. Soc. 8: 107-111.
Videler, J.J. 1993. The structure of the swimming apparatus: body axis and fins.
pp. 41-70. in Fish Swimming, Chapman and Hall, New York.
Westerfield, M., McMurray J.V., and Eisen, J.S. 1986. Identified Motoneurons and Their
Innervation of Axial Muscles in the Zebrafish. J. Neurosci. 6(8): 2267-2277.
Figure Legend
Fig. 1 Rotating striped drum used in the swimming behavior experiments.
Fig. 2 (A) Method used for determining maximum body flexion. Each fish represents
is a single field at 60 Hz. Translation is not represented, only change in body
shape of the fish. One fish from each stage is represented. (B) Representation
of the angle of flexion, measured between the head and the tip of the tail.
Fig
Photos of fish from each stage. Scale bars are 1 mm. (A) Stage 1. (B) Stage 2.
(C) Stage 3. (D) Stage 4.
Fig. 4 Photos of 12 day old bonito. Scale bars are 1 mm. (A) Before metamorphosis.
(B) After metamorphosis.
Fig. 5 Scatter plot of fraction of time spent active for each fish in a two-minute period.
Boxes are means for each day with standard deviations.
Fig. 6 Scatter plot of maximum body flexion observed in a two-minute period for each
fish. Greater flexion translates into a smaller angle.
Fig.
7 Comparison of Type A and Type B motoneurons for Stages 1 and 4.
(A) Stage 1, Type A motoneuron. (B) Stage 4, Type A motoneuron.
(C Stage 1, Type B motoneuron. (D) Stage 1, Type B motoneuron.
Fig. 8 Comparison of tier development for Stages 1 through 4.
(A) Stage 1. Only one distinguishable tier. (B) early Stage 3. Two tiers of
motoneuron cells are visible, both Type A. (C mid-Stage 3. Three tiers of
motoneuron cells are visible. The third tier consists of small Type B cells.
(D) Stage 4. Tiers are no longer distinguishable from each other.
9 40x view of the third tier seen in mid-Stage 3. (A) Focus on the upper two tiers
Fig.
of cell bodies. (B) Focus on the third tier of cell bodies.
Fig. 10 Spinal cord comparison of Stage 2 and 3 fish. Rostral is to the right and ventral
is down. (A) Stage 2. The majority of motoneuron development is in the
rostral segments, with fewer motoneurons caudally. (B) early Stage 3. Caudal
motoneurons have increased in number and complexity. (C) mid Stage 3. The
number and complexity of all motoneurons has increased.
Fig. 11 Burst pattern from Stage 1. (A) Overview of the entire signal, with baseline
shown at the beginning and end. (B) Close-up of the spikes seen in the bursting
pattern.
Fig. 12 Action potentials from Stage 2.
Fig. 13 Burst pattern in an early Stage 3 fish. (A) Overview of the entire signal, with
baseline shown at the beginning and end. (B) Close-up of the spikes seen in
the bursting pattern.
Fig. 14 Burst pattern from early Stage 3 fish. Similar to the one shown in Fig. 13.
Fig. 15 Burst pattern from mid Stage 3 fish. (A) Overview of the entire signal, with
baseline shown at the beginning and end. (B) Close-up of the spikes seen in
the bursting pattern.
Fig. 16 Simpler burst pattern from mid Stage 3 fish. (A) Overview of the entire signal,
with baseline shown at the beginning and end. (B) Close-up of the spikes seen
in the bursting pattern.
Fig. 17 Action potential series in a mid Stage 3 fish.
Fig. 1
18
1925
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