Recovery of C-starts, equilibrium and targeted feeding after whole spinal cord crush in the adult goldfish Carassius auratus
Department of Biology, Williams College, Williamstown, MA 01267, USA
* Author for correspondence (e-mail: steven.j.zottoli{at}williams.edu)
Accepted 21 May 2003
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Summary |
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Key words: C-start, startle response, functional recovery, behavioral recovery, Mauthner cell, regeneration, spinal cord crush, goldfish, Carassius auratus
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Introduction |
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Goldfish display a rapid response to vibratory stimulation in which the
animal's body typically forms a C shape (Eaton et al.,
1977,
1981
). This C-start is
ideally suited for quantitative studies of behavioral recovery after spinal
cord injury because (1) C-starts have been extensively characterized in
normal animals (e.g. Eaton et al.,
1988
; Foreman and Eaton,
1993
; see review by Eaton et
al., 2001
), (2) much of the neuronal network that controls
C-starts has been described (Faber
and Korn, 1978
; Fetcho and
Faber, 1988
; Faber et al.,
1989
,
1991
;
Fetcho, 1991
) and (3) the
neurons that initiate C-starts are identifiable and accessible
(Bartelmez, 1915
;
Furshpan and Furukawa, 1962
;
Zottoli, 1978
).
Preliminary reports indicate that recovered C-starts differ from
those of sham-operated control fish for up to 12 months after spinal cord
injury (Zottoli and Freemer,
1991; Zottoli et al.,
1994
; Zottoli and Faber,
2000
). The present study provides a quantitative approach to
analyze these C-start differences and also provides qualitative
descriptions of the recovery of equilibrium and targeted feeding.
There is a vast literature on behavioral regeneration after spinal cord
injury of many vertebrates (see Larner et
al., 1995), including the larval and adult lamprey (Cohen et al.,
1988
,
1989
;
McClellan, 1994
), amphibians
(e.g. Davis et al., 1990
),
fish (Bernhardt, 1999
;
Doyle et al., 2001
) and
mammals (e.g. Schwab and Bartholdi,
1996
). However, there are few preparations that provide the
ability to determine the underlying neuronal basis for that recovery. The
identification of neurons that are responsible for the return of
C-starts will provide the unique opportunity to analyze the mechanisms
underlying behavioral recovery at the cellular level.
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Materials and methods |
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The fish were between 6 months and 1.5 years in age (Hunting Creek Fisheries, Inc., personal communication).
Choice of wound type and site: whole spinal cord crush at the
spinomedullary level
Spinal cord crush was chosen because it is most similar to the type of
injury that might occur naturally. One distinct advantage of crush wounds is
minimal bleeding compared with cut wounds.
The spinomedullary level (SML) was chosen as a site to crush the whole spinal cord in order to maximize behavioral deficits. After spinal cord crush and recovery from the anesthetic, experimental fish lay on their sides with no movement caudal to the wound; i.e. fin, trunk and tail movements caudal to the wound were abolished by this wound while those movements that control vital functions rostral to the wound (i.e. respiration and feeding from the bottom of the tank) were spared. The progressive return of behavior could be unambiguously documented by visual observation after SML crushes.
The SML level was also chosen to allow visibility of the spinal cord during and after the crush operation. As described below, the spinal cord was exposed in the brain case. This approach did not require damage to either muscle or vertebrae and allowed the cord to be fully exposed and, as a result, visible with the aid of a dissecting microscope.
Spinomedullary crush technique
Forty-five goldfish had a spinal cord crush at the SML (i.e. the junction
area between the spinal cord and medulla). Eight sham-operated goldfish served
as controls for the effects of the surgical procedures alone. All spinal cord
crushes were performed by one experimenter (i.e. S.J.Z.). A holding
temperature of 22.4°C was chosen because preliminary results had indicated
that goldfish may not regain C-starts at 16°C
(Zottoli and Faber, 1977).
Fish were initially anesthetized in 0.03% ethyl-m-aminobenzoate (Sigma-Aldrich
Co., St Louis, MI, USA) until breathing ceased and were transferred to an
operating chamber where chilled water containing 0.012% of anesthetic was
re-circulated through the mouth and over the gills (the chilled water reduced
the gill temperature of the fish from approximately 22°C to 10°C). A
hole was drilled in the skull to expose the area from the caudal portion of
the corpus cerebellum to the spinal cord. Overlying muscle, cartilage and fat
were removed to expose the SML, and care was taken not to damage the posterior
semicircular canals. The spinal cord was crushed at the SML. The tips of the
forceps (No. 5 Dumont forceps) were lowered on either side of the spinal cord
until they touched the floor of the brain case. The forceps were moved
rostrally until they were at the junction of the vagal lobes and medulla
oblongata (i.e. the SML). The tips were then closed tightly and held together
for approximately 2 s. This crush procedure was then repeated. When the first
crush was made, the anesthetized fish moved slightly, giving a preliminary
indication that the brain tissue had been damaged. Although the crush did not
result in disconnection of the spinal cord from the medulla oblongata, a
distinct line was evident where the crush had been made. Very little bleeding
resulted from this operation. The brain was protected from osmotic shock by
covering it with a Vaseline-paraffin oil mixture to a level just below the
skull. A piece of thin plastic the size of the hole was placed on the mixture.
Thirty-gauge stainless steel wire was looped through two small accessory holes
drilled on either side and rostral to the operation hole. The wire was twisted
together caudally where a loop was made on one of the ends. The caudal loop
was anchored to musculature just behind the skull with silk suture thread. The
twisted wire and string acted as a secure framework for the vinyl polysiloxane
impression material (Imprint, 3M) used to `cap' the skull. After the
operation, the re-circulating anesthetic solution was replaced with
conditioned tap water and the fish recovered, initiating breathing in
approximately 10-15 min. Fish were returned to their home tanks and monitored
closely for 30-60 min.
Behavioral observations
General observations
Experimental and sham-operated control fish were observed to determine
their general health, whether there were any noticeable movements caudal to
the wound site at rest, and their position in the tank relative to the
vertical plane. The fish were observed daily for the first 10 postoperative
days to carefully monitor the effect of the operation. Following the 10th
postoperative day, observations occurred three times per week until 128
postoperative days. Observations were then made weekly until 168 postoperative
days and a final observation was made 190 days postoperatively. Testing for
C-starts occurred at random times during the day, and, in general, a
set of six trials took approximately 1 h to complete.
Targeted feeding
During the preoperative, three-week acclimatization period, fish were fed
10-12 mini-pellets of food three times a week. All fish easily located and
targeted pellets floating on the surface at each feeding session during the
3-week period. The pellets were consistently consumed within a 2-h period, and
the tanks were then cleaned.
During this preoperative period, fish were tested once for their ability to target the floating pellets on the water surface during a 4-min test period. The test was divided into two parts: (1) five food pellets were placed on the water surface within a plastic floating ring, 5.1 cm in diameter, and fish were given 2 min to target food within the ring, and (2) if a fish failed to target the pellets in the ring within 2 min, the ring was removed, five additional pellets were added and the fish's ability to target free-floating pellets for another 2 min was observed. A fish met the targeting criterion if it touched and/or ate one of the floating pellets within the 4-min test period.
Postoperatively, this test was done weekly for both experimental and sham-operated animals at one of the normal feeding sessions until the 146-158th postoperative day.
If six or more pellets had not been consumed within approximately 2 h, the remaining pellets were removed and the fish was provided with TetraMin Tropical Flakes (Tetra GMBH, Melle, Germany), which sank to the bottom of the tank. Fish were observed to ensure they were eating some of this food from the tank bottom. Two hours after the addition of flake food the tanks were cleaned to reduce the accumulation of organic matter in the tank.
Equilibrium
The position of experimental and sham-operated fish relative to the
vertical plane was noted during the 190-day postoperative interval. Each fish
was categorized as being on its side (i.e. the sagittal plane of the fish was
perpendicular to the vertical plane of the tank), tilted (partial equilibrium)
or upright (full equilibrium; the sagittal plane of the fish was parallel to
the vertical plane of the tank). A transition from one of these categories to
another required the change to be observed in two consecutive observation
periods. When this condition was met, the first observation date was used as
the transition or recovery date.
Fourteen fish that regained full equilibrium and the eight sham-operated control fish were tested in water circulated at two different speeds to determine their ability to maintain equilibrium while swimming between 167 days and 169 days postoperatively. A circular, Plexiglas tank, 10 cm high and 23 cm in diameter, was filled with conditioned tap water at 22°C to a depth of 8 cm. The tank was placed on a stir plate, and a magnetic stir bar (9.5 mm in diameter; 58 mm in length) was placed in the center. A fish was placed in the tank and, after a 2 min acclimation period, the stir bar was rotated at a slow speed (estimated water speed, 6.1 cm s-1) for 2 min and then a faster speed (estimated water speed, 36 cm s-1) for 2 min. Fish tended to swim near the edge of the tank to avoid the stir bar.
C-starts
Fish were tested preoperatively for their ability to respond to a vibratory
stimulus with a C-start. One set of six trials with an inter-trial
interval of at least 2 min was given preoperatively 4-11 days prior to the
spinal cord crush. Fish were screened during preoperative testing to meet the
following three criteria: (1) each fish had to respond to the stimulus with
C-starts in at least three of six trials, (2) at least one
C-start had to be to the left and one to the right and (3) the fish
silhouette had to be compatible with the software thinning algorithm (e.g.
some fish had silhouettes that made it difficult for the software
analysis).
Postoperatively, a block of six trials with an inter-trial interval of at least 2 min was given every 14 days for up to 138-158 days postoperatively. Fish were tested with a block of six trials one final time between 182 and 195 postoperative days. Fish were fed and their tanks were cleaned after testing.
Our behavioral testing system for delivering the vibratory stimulus is
similar to that described by Eaton and colleagues
(Wieland and Eaton, 1983;
Eaton et al., 1988
) except for
the following modifications: (1) a circular arena was used instead of a square
one to prevent fish from settling into corners, (2) the water depth was
reduced by 5.1 cm to a final depth of 7.7 cm to restrict vertical movement of
the fish and (3) the stimulus was more intense (i.e. 600 µm vertical tank
movement compared with 3-6 µm used by
Eaton et al., 1988
) to ensure
the delivery of a supra-threshold stimulus. We describe some of the general
features of the test tank, the stimulus and imaging system below.
Fish were placed in a circular arena, 20.3 cm in diameter and 10 cm deep to restrict movement. The arena was centered in a tank with opaque sides and a clear bottom, 43.5 cm square and 23.5 cm in depth. The tank and arena were filled to a depth of 7.7 cm with conditioned tap water. The water in the test tank was equilibrated to the temperature at which the animals were held. The central arena was aerated, and fish were allowed to acclimate for at least 10 min before testing.
Fish were allowed to orient randomly in the test tank prior to stimulation with an abrupt vibratory stimulus. The vibratory stimulus was created by lifting the test tank with a solenoid and was delivered when the fish was stationary with its body oriented radially in the circular arena. The solenoid was separated by 0.69 mm (a feeler gauge was used) and was triggered by computer with 1.5 waves of the 60 Hz linevoltage. The fish tank was lifted approximately 600 µm.
Two cameras were located below the test tank to record fish movement within
the arena (Fig. 1). Fish were
videotaped with a conventional video camera/VCR system. In addition,
silhouettes were captured by a customized matrix camera, consisting of a 10
000-pixel array of photodiodes (EG&G Reticon Camera/Controller
MC521/RS521; EG&G Reticon, Gaithersburg, MD, USA). The matrix camera and
solenoid-activated vibratory stimulus were triggered at the same time, and
silhouettes were stored on computer memory every 2 ms for a total of 104 ms
(i.e. 52 images; Wieland and Eaton,
1983; Eaton et al.,
1988
).
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Determination of the probability of eliciting a C-start
Observation of videotapes provided a preliminary screening for the
occurrence and direction of C-starts. Since fish were stimulated when
their bodies were straight and after they had come to rest, the identification
of a response coupled to the stimulus was usually clear. The responses were
categorized as one of the following. (1) Full body response, involving the
whole body; the fish typically formed a C shape and, during these
responses, were displaced from their original location. (2) Partial body
response; movement of head structures (i.e. operculi, eyes, mouth), fins and
upper trunk. The fish formed a shallow C shape that resulted in minimal
displacement of the fish from its original location. (3) Head and fin
response; movement of the head and fins occurred with no apparent movement of
the trunk and tail. (4) No response.
Two different experimenters independently categorized these responses based on single frame analysis of the videotapes. In cases of disagreement, one of the investigators (i.e. S.J.Z) re-observed the trial and made a final judgement regarding the category.
Computer software (i.e. KNOWAL;
Nissanov, 1991) was used to
analyze all category 1 and 2 responses to determine whether they met the
criteria for a C-start. The 52 matrix camera silhouettes
(Fig. 2A) were each reduced to
a midline 1 pixel thick (Fig.
2B) using a thinning algorithm. The rostral 40% of each midline
was then converted to a regression line
(Fig. 2C;
Nissanov, 1991
). These
regression lines were used to determine whether significant axial movement had
occurred based on the following criteria: (1) the angle of the linear
regressions between the start silhouette (fish silhouette before the onset of
significant movement; each silhouette represents 2 ms) and start + 1
silhouette is greater than 3°, (2) no directional reversal past the start
position occurs within the four silhouettes subsequent to the start silhouette
and (3) if the angle between the start and start + 2 silhouettes is less than
10° then start + 3 cannot be situated between start and start + 2
silhouettes. Computer analysis of some trials was not possible, and occurrence
of a startle response was determined from the videotapes.
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Determination of C-start kinematic parameters
The C-start has been divided into two stages
(Blaxter et al., 1981;
Eaton et al., 1991
). During
stage 1, there is a major contraction of the body musculature on one side
resulting in a characteristic C-like shape. Stage 2 is characterized by
forward propulsion that may be associated with a turn. These two stages are
the focus of this paper even though they may be followed by other movements,
including swimming. Stage 1 and stage 2 kinematic parameters were
automatically calculated by the computer from regression lines of the
midlines, representing the head and rostral trunk.
Fig. 2C provides a graphic
representation of most of the following kinematic parameters.
The stage 1 parameter measured is stage 1 latency or start latency. This is the latency from the onset of the vibratory stimulus to the beginning of the response. Regression lines were used to determine whether significant movement had occurred based on the criteria discussed above. The latency from activation of the solenoid to sound pressure onset was 4.4 ms, as determined with a hydrophone. In addition, the matrix camera started filming 0-2 ms after camera activation. The maximum latency, as determined by the computer, was therefore adjusted by subtracting 2.4 ms (i.e. computer-determined latency - 4.4 ms + 2 ms).
Stage 2 parameters are as follows:
The goldfish C-start not only has axial body movements but also
includes closure of the jaw, adduction of the operculi and retraction of the
eyes (Diamond, 1971;
Hackett and Faber, 1983
).
These cranial components of the C-start were not analyzed in this
study.
Statistical analyses
All data are reported as means ± S.D. For statistical
analyses, data were organized into six, 25-day intervals (T1-T6) encompassing
a total of 150 postoperative days. C-starts from eight sham-operated
control animals and 11 experimental fish that recovered equilibrium and
C-starts were analyzed. One fish that had not recovered equilibrium but
had recovered C-starts and one other fish that had recovered a
C-start on the 190th postoperative day were not included.
Comparisons of the probability of eliciting a
C-start
The probabilities of eliciting a C-start were calculated for each
fish at each of the last three time intervals (T4-T6). The proportions were
arcsine transformed and analyzed using a repeated-measures analysis of
variance (RM-ANOVA). In addition, the individual control and experimental
probabilities in the longest postoperative interval (i.e. 126-150 days) were
arcsine transformed and compared using an unpaired t-test.
Comparisons of C-start kinematic parameters
C-start kinematic parameter values analyzed in this study include
stage 1 and stage 2 latency, angle at the beginning of stage 2, escape
trajectory angle, center of mass movement and linear velocity of the center of
mass movement. C-starts that did not have a second stage were not used
in this analysis. To determine whether kinematic parameters differed over the
last three time intervals (T4, 76-100 days; T5, 101-125 days; T6, 126-150
days) or by treatment (i.e. control vs experimental) and to determine
whether there was a possible interaction between the two, a multivariate
analysis of variance (MANOVA) was run on mean parameter values determined for
each fish at each 25-day time interval. The treatment was the only significant
factor. Since time had no significant effect on parameter values, we chose to
use a reduced data set to minimize the effect of an unequal number of
responses between fish. Specifically, for each parameter analyzed, we chose
the first response at a given 25-day time interval by a given fish and
discarded the rest of the values for that fish at that interval. Thus, a
maximum of three responses was chosen for each fish (i.e. one from each of the
last three time intervals; in some cases, less than three responses were
available since the fish may not have responded in one or more of the time
intervals). Means were calculated for control and experimental fish at each
interval and were compared using a MANOVA. Since the MANOVA indicated an
effect of treatment, one-factor ANOVAs were run to determine what control
parameters (all T4-T6 values) differed from the corresponding experimental
parameters (all T4-T6 values). The P values were adjusted with a
standard Bonferroni adjustment to correct for type I error. A significance
level was set at P=0.05.
Re-crush of spinal cords in fish that had recovered
C-starts
In order to determine whether regeneration across the crush wound was
responsible for behavioral recovery, the spinal cord was re-crushed 198-200
days after the original operation in five fish that had recovered
C-starts, equilibrium and targeted feeding. The crush was at the same
location (i.e. SML) and the procedure was identical to the original operation.
General behavioral observations were made up to 10-12 days following the
second operation. Fish were tested for their ability to respond to a vibratory
stimulus with a C-start. One set of six trials was given 2-5 days after
the re-crush with the standard stimulus strength. A second set of six trials
was given 10-12 days after the re-crush with a greater stimulus strength.
Histological procedures: completeness of the wound
After spinal cord crush there was no evidence of movement caudal to the
wound in any of the fish. To determine whether the crush wound actually
damaged all nerve fibers, SML crushes were performed on three goldfish as
described above. After recovery from the anesthetic, the fish were placed in
their home tank. One of these fish displayed movement caudal to the wound site
and was not used for histological observation.
The brains of the remaining two fish were re-exposed under anesthesia eight
days postoperatively, and a whole spinal cord cut was made 1-2 mm caudal to
the original crush. Biocytin (Sigma Chemical Co.), re-crystallized on the tip
of 45-gauge stainless steel wire, was introduced into the cut wound. After the
biocytin dissolved, the wire was removed, the skull was re-sealed and, after
recovery from the anesthetic, the animals were placed in their home tank for 2
days. The fish were then anesthetized and perfused with fixative consisting of
4% paraformaldehyde, 1% glutaraldehyde in 0.1 mol l-1 phosphate
buffer (pH 7.4). Brains were removed and placed in fresh fixative for 1 h,
after which they were rinsed three times in phosphate buffer. Brains were
stored in phosphate buffer for one week and then placed in 30% sucrose in 0.1
mol l-1 phosphate buffer 24 h before frozen sectioning. Brains were
cut frozen in the transverse plane at 60 µm. The sections were stored in
0.1 mol l-1 phosphate buffer and then processed with a procedure
modified from Bass et al.
(1994). Specifically, sections
were: (1) incubated for 10 min in a hydrogen peroxide-methanol-phosphate
buffer mixture to reduce endogenous peroxidase, (2) rinsed three times in
buffer, (3) incubated for 30 min in 0.4% Triton X in buffer, (4) incubated for
3 h in an avidin-biotinylated horseradish peroxidase complex (Elite Kit,
Vector Laboratories, Burlingame, CA, USA), (5) rinsed twice in phosphate
buffer, (6) incubated for 3-5 min in 0.05% diaminobenzidine, 0.01% hydrogen
peroxide in phosphate buffer, (7) rinsed twice in phosphate buffer and (8)
stored in phosphate buffer until mounting on chrom-alum-coated slides. After
the sections dried on the slide, they were dehydrated, placed in xylene and
then coverslipped with Eukitt (O. Kindler, Freiburg, Germany).
The serial sections were studied to determine whether all nerve processes were separated at the wound site. Specifically, if a wound was complete, one would expect no biocytin-labeled fibers to be present rostral to the wound.
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Results |
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General behavioral status of fish up to 190 postoperative days
All eight sham-operated control fish survived the 190-day duration of this
study. These fish remained healthy, maintained equilibrium at rest and, while
swimming, were able to target food pellets from the water surface and
displayed C-starts in response to the vibratory stimulus.
All 45 experimental fish were on their sides on the bottom of the tank immediately following spinal cord crush and recovery from anesthesia. Fish displayed movements of the eyes, operculi and jaws rostral to the crush wound. However, there was no evidence of movement caudal to the wound in any of the fish. However, movement could be evoked caudal to the wound by gently lifting the animals out of their tanks; this movement is thought to be due to spinal reflexes.
Twenty-five of the 45 experimental fish survived the course of this study. Of these surviving fish, 12 regained equilibrium and C-starts, two regained equilibrium but not C-starts, 11 did not regain equilibrium. Of the 11 that did not regain equilibrium six had no apparent body abnormalities, while five had body abnormalities that are believed to hinder behavioral recovery (Fig. 4A). Specifically, four had the caudal half of their trunk and tail bent upwards and one had a bloated air bladder and was floating. One of these fish did recover a C-start while on its side. Twenty fish died during this study. The distribution of the number of fish that died over the 190-day postoperative interval (Fig. 4B) reveals two clusters, one between 3 and 52 days and another between 91 and 162 postoperative days.
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The postoperative interval at which C-starts, equilibrium and targeted feeding returned varied between fish. Nonetheless, fish regained behaviors in a sequential manner with pectoral and/or pelvic fin movements appearing first, followed by targeted feeding, partial equilibrium, full equilibrium and finally C-starts (Fig. 5).
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Recovery of targeted feeding
Preoperatively, all fish demonstrated the ability to target food pellets
within a 4-min test period (i.e. either in the floating ring within 2 min or
free floating in the subsequent 2 min of the test). Postoperatively, seven of
eight sham-operated control fish met the targeting criterion on the first test
day (3 days postoperative) while the 8th fish targeted food on the 2nd test
day (10 days postoperative).
Twenty-two of the 25 experimental fish that survived the 190-day postoperative interval met the targeting criterion. The earliest recovery of the targeting criterion occurred on postoperative day 15 while the longest occurred at day 109 (Fig. 5). The three fish that did not meet the criterion were fish that never regained equilibrium; however, eight other fish that did not regain equilibrium were capable of targeting food pellets.
All eight sham-operated control fish and 19 of 22 experimental fish targeted pellets in the ring during the first 2 min of the test at least once during the 190-day postoperative interval.
Recovery of equilibrium
Sham-operated control animals were upright and swimming immediately
following the operation and recovery from anesthetic (i.e. about 10-20 min
after being returned to their home tanks). Thus, the operation itself did not
result in damage to the spinal cord or semicircular canals. In addition, the
`cap' used to cover the skull wound did not hinder the ability of the fish to
maintain full equilibrium.
Experimental fish were upright preoperatively. Immediately after recovery from crush wounds, all experimental fish were lying on their sides on the bottom of their tanks. Fourteen of 25 experimental fish gradually recovered full equilibrium (Fig. 5) during the 190-day postoperative interval. Three other fish regained partial equilibrium (i.e. they remained tilted) during this interval.
The 14 fish that regained full equilibrium and the eight sham-operated control fish were tested in water circulated by a stir bar at two different speeds to determine their ability to maintain equilibrium while swimming. Seven sham-operated control fish swam into the water current for the majority of the test while one swam with the current at both stir bar speeds. While swimming into the water current at both test speeds, the seven control fish maintained their position in the water column (i.e. did not drift backwards) and occasionally moved forward against the current.
At the slower stir bar speed, 10 of the 14 experimental fish that had regained full equilibrium and swam into the water current were able to hold their position while the other four fish were unable to maintain their position and drifted backwards. Two of the 14 experimental fish lost the ability to maintain full equilibrium at the slower stir bar speed while 13 of the 14 fish were unable to maintain equilibrium at the faster speed.
Recovery of C-starts
All C-starts that were classified as a full body response (category
1) and could be analyzed met software criteria for a C-start (see
Materials and methods). Three of 23 trials that were classified as partial
body responses (category 2) met software criteria for a C-start (i.e.
in 20 trials fish did not move enough for the computer to calculate a start
frame).
Probability of eliciting a C-start response to a vibratory
stimulus
Sham-operated control fish responded with a C-start to a vibratory
stimulus in at least 75% of the trials on average throughout the six 25-day
time intervals used for analysis (T1-T6;
Fig. 6). By contrast, a
C-start could not be elicited to a vibratory stimulus in experimental
fish for the first 50 postoperative days. Two of the 12 fish that eventually
regained C-starts displayed this response by 75 postoperative days, and
11 of the 12 fish regained C-starts by 150 postoperative days
(Fig. 6). The 12th fish
regained a C-start on the 190th day.
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The probability of eliciting a C-start was compared over T4-T6 (T4, 76-100 days; T5, 101-125 days; T6, 126-150 days). A RM-ANOVA revealed a significant effect of treatment (i.e. control vs experimental) but not time. Comparison between experimental and control fish proportions at the 126-150-day interval indicated that the probability of eliciting a response was significantly greater in control animals (P<0.0001, N=8 for control; N=11 for experimental; t-test).
Comparison of C-start kinematic parameters
C-start kinematic parameters were compared over T4-T6 for 11 of the
12 fish that recovered C-starts. A MANOVA indicated a significant
effect of treatment (i.e. control vs experimental) but not time.
Although there was a diversity in the trajectories of recovered
C-starts, as shown for three fish in
Fig. 7, statistical analyses
indicate that recovered C-starts were slower and less robust than those
elicited preoperatively or those of sham-operated control fish. A comparison
between preoperative C-starts and those elicited 95-109
dayspostoperatively in Fig. 8
highlights some of the most dramatic differences in C-starts that we
encountered. Stage 1 and 2 latencies for experimental fish were significantly
longer than the corresponding latencies of control fish (P<0.0001,
N=8 for control; N=11 for experimental; P<0.008
with Bonferroni adjustment). In addition, all other stage 1 and stage 2
response parameters, including the angle at the beginning of stage 2, escape
trajectory angle (ETA), center of mass movement and linear velocity of the
center of mass movement, were significantly smaller in experimental animals
when compared with the corresponding control parameters (P<0.0001,
N=8 for control; N=11 for experimental; P<0.008
with Bonferroni adjustment). For example, ETAs of 100° occurred in all
eight sham-operated control fish and in 65.7% of the analyzed C-starts
(69/105) but only occurred in two of the 11 experimental fish and in 2.5% of
recovered C-starts (2/79). The largest control ETA was 199°,
compared with 105° for an experimental fish. The pooled control and
experimental kinematic data (data from all responses between 75 and 150
postoperative days) for stage 1 latency, center of mass movement and the
linear velocity of the center of mass movement are shown in
Fig. 9. In addition,
Table 1 contains mean control
and experimental values for all kinematic parameters. These means were
calculated by first averaging values from all trials of an individual fish for
a particular parameter and then calculating a mean value for control and
experimental groups. Therefore, the means in
Fig. 9 (all trials) and those
in Table 1 (mean of means)
differ. Finally, the means in Table
1 were not those used to test for significant difference as
described in the Materials and methods.
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Both sham-operated control fish and experimental fish had a limited number of C-starts with no second stage (i.e. the center of mass did not become displaced 0.75 cm from its position at the start) between 75 and 150 postoperative days. Two of eight sham-operated control fish each had one C-start with no second stage. Six of 11 fish that regained C-starts and equilibrium had trials with no second stage. Examples of control and experimental C-starts with no second stage are shown in Fig. 10.
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Loss of recovered behaviors after spinal cord re-crush
Five fish were chosen from the 11 that had regained C-starts,
equilibrium and targeted feeding by the 150th postoperative day. A re-crush of
the original wound site resulted in the loss of these behaviors. Photographs
in Fig. 11 provide a
comparison of equilibrium before and after re-crush for one fish. Three of the
five fish had limited pectoral fin or caudal fin movement 10-12 days after the
re-crush.
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Discussion |
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Effectiveness of the stimulus in eliciting a C-start
after spinal cord crush
The type and/or amplitude of the stimulus was critical for the successful
elicitation of recovered C-starts. After an SML crush, C-starts
could not be elicited for six months postoperatively with a sound pulse
consisting of two cycles of a 200 Hz sinusoidal signal delivered by an
underwater loudspeaker (Zottoli et al.,
1989; Universal model UW-30; see
Zottoli, 1977
for details). By
contrast, the vibratory stimulus used in this study was effective in eliciting
C-starts as early as 64 postoperative days. The ability to elicit
C-starts at short postoperative intervals most likely resulted from the
stimulus amplitude (600 µm displacement of the test tank), which is well
above threshold levels determined for control fish
(Eaton et al., 1988
; 3-6 µm
displacement).
Completeness of a crush wound
One disadvantage of a crush wound as compared with a cut wound is that
there is no way to determine whether the wound is complete at the time of
injury. After a cut wound, it is possible to use a probe to confirm that the
proximal and distal pieces of spinal cord are completely separated
(Pearcy and Koppányi,
1924); such an approach is not possible after a crush wound.
Only fish that showed no spontaneous movement below the level of the SML
crush site within the first 10 postoperative days were used in this study. It
is generally accepted that paralysis caudal to a wound site is a good
indicator of the completeness of the wound
(Keil, 1940;
Tuge and Hanzawa, 1937
). For
example, when the spinal cord was not completely cut at the high cervical
level (level D; equivalent to the SML), Japanese rice minnows (Oryzias
latipes) remained upright (Tuge and
Hanzawa, 1935
). However, behavioral evidence alone is not
sufficient to determine the extent of a wound.
Our histological results indicate that spinal cord crush damages and
ultimately results in separation of all descending axons at the wound site.
However, an occasional afferent process may be spared. The effectiveness of an
SML crush on damaging descending axons is supported by studies in which the
Mauthner axon was filled with Lucifer yellow either rostral or caudal to the
wound site 30-62 dayspostoperatively. In all cases, the axon had separated and
retracted from the crush site and no longer extended across the wound (Zottoli
et al., 1987,
1988
).
Although it is difficult to compare behavioral studies that differ in wound
level and type of wound, fish species and temperature, the similarity in the
time course of recovery between cut and crush wounds lends support to the
complete nature of our wound. The recovery of swimming in goldfish after whole
cord transection at the high thoracic level, in which separation of the cord
was confirmed, is maximized between 2 and 2.5 months
(Koppányi and Weiss,
1922; Pearcy and
Koppányi, 1924
;
Bernstein, 1964
), an interval
at which the majority of fish in this study were able to target food from the
water surface. In Japanese rice minnows, movement caudal to a spinal cord
transection at the high cervical level (equivalent to the SML) appeared
between 15 and 30 postoperative days (Tuge
and Hanzawa, 1935
), which corresponds to our observations of
movement caudal to the wound site occurring as early as 15 postoperative days.
In addition, we found that full equilibrium returned as early as 60
postoperative days, which would explain why Tuge and Hanzawa
(1935
) did not observe upright
posture of fish for up to 40 days postoperatively.
Recovery of behavior was due to morphological regeneration across the
wound site
Recovered C-start, equilibrium and targeted feeding behaviors were
lost when the spinal cord was re-crushed. This result indicates that
morphological regeneration of nervous tissue across the original wound site
was responsible for the return of these behaviors. It is interesting that a
few fish had limited pectoral fin or caudal fin movement after the re-crush.
These fin movements could have resulted from an incomplete wound or from
extraspinal pathways. A spinal cord cut rather than a crush should be
performed on fish that have recovered behaviors in the future to distinguish
between these two alternative explanations.
All fish did not recover C-starts, full equilibrium or
targeted feeding after spinal cord injury at the SML level
Mortality of experimental fish resulted from whole spinal cord crush since
no sham-operated control fish died during the 190-day postoperative interval.
Seven fish died 3-52 dayspostoperatively (mean, 22 days). Since bleeding was
minimal after the spinal cord crush and no control animals died as a result of
the operation, this short-term death was most likely due to infection related
to the crush.
An additional 13 fish died 91-162 days postoperatively (mean, 133 days). None of these fish recovered equilibrium and, as a result, were provided with food that sank to the bottom of the tank. Although all of these fish were observed to ingest the food, seven of nine fish that were still alive after 121 postoperative days were noticeably emaciated. We suggest that these fish died due to lack of sufficient nutrition even though five of the nine fish were capable of targeting food pellets on the water surface. Gavage feeding may be an effective way to decrease mortality in this group of fish.
The mortality occurring over a 5-6-month postoperative interval in two
separate studies, using identical protocols to those used in this study, was
similar to that reported here (i.e. 44.4%). Specifically, the mortality was
32.2% in one study (N=31; 5 months postoperatively;
Zottoli et al., 1994) and
36.7% in the other (N=49; 6 months postoperatively; S. J. Zottoli and
J. E. Nierman, unpublished observations). Tuge and Hanzawa
(1935
) reported a somewhat
higher mortality (65.6%) of those fish that had spinal cord transections at
the high cervical level and survived for approximately 2 months
(Tuge and Hanzawa, 1937
).
All fish did not regain targeted feeding or full equilibrium. Approximately half (six of 11) of those fish that did not recover equilibrium had either a bent trunk (N=5) or an over-inflated swim bladder (N=1). Even though regeneration of nervous tissue could potentially support the return of equilibrium in these cases, it would be impossible for the fish to maintain equilibrium due to body abnormalities. The other five fish that did not regain equilibrium had no noticeable morphological restrictions that could explain the lack of behavioral recovery.
Two to three months after spinal cord transections at the high thoracic to
cervical levels, many adult fish appeared `normal', while others had partial
or no behavioral recovery (Tuge and
Hanzawa, 1937; Pearcy and
Koppányi, 1924
). There are many factors that might account
for the lack of recovery of behavior after spinal cord injury. The age of the
fish, subtle differences in the crush wound or the wound level may limit the
return of behavior. In addition, regenerating central nervous system (CNS)
neurons are known to make inappropriate pathway choices into the peripheral
nervous system just caudal to an SML crush
(Bentley and Zottoli, 1993
;
Zottoli et al., 1994
). This
inappropriate pathway choice may limit, delay or prevent the return of
behavior caudal to the wound.
Recovered equilibrium differed from that in sham-operated control
fish
Those goldfish that recovered equilibrium appeared `normal' when left
undisturbed in their home tanks. When they were challenged to swim in a water
current, all but one experimental fish was unable to maintain full
equilibrium. By contrast, sham-operated control fish were able to maintain
full equilibrium under the same conditions. Differences in the amount of
muscle (see below), in muscle fatigue or in the regenerated neuronal circuitry
of experimental compared with control fish may contribute to the inability of
experimental fish to maintain equilibrium.
Recovered C-starts differed from those in sham-operated
control fish
Significant differences exist between C-starts of experimental fish
and sham-operated controls. The differences include lower probability of
response, longer stage 1 and 2 latencies, smaller turning angles and shorter
distances traveled and velocities attained by the center of mass. We
hypothesize that these differences can be explained by changes in neuronal
circuitry that occur as a result of regeneration.
The Mauthner cell (M-cell) initiates C-starts in goldfish when
exposed to a vibratory stimulus similar to that used in this study
(Eaton et al., 1981).
Morphological and physiological studies indicate that it is unlikely that
M-cells are involved in the return of C-starts during the 190
postoperative days of this study.
After spinal cord cut or crush at the SML, the goldfish M-cell undergoes an
axon reaction both morphologically
(Zottoli et al., 1984) and
physiologically (Faber, 1984
;
Faber and Zottoli, 1981
;
Titmus and Faber, 1990
;
Titmus et al., 1986
). The
proximal M-axon retracts about 1.5 mm from a crush site
(Agostini and Zottoli, 1986
). A
few M-cells die after long postoperative intervals following SML crush
(15.6°C holding temperature; Zottoli
et al., 1984
), and others display abortive regeneration
(Zottoli et al., 1988
).
However, the majority of M-axons sprout extensively both rostrally and
caudally for distances up to 5 mm within the CNS at 22°C
(Zottoli et al., 1988
). Few of
the caudally projecting axons cross the wound site and those that do tend to
be within or directed towards the first ventral root
(Bentley and Zottoli, 1993
;
Zottoli et al., 1994
).
Intracellular stimulation of M-axons in goldfish that have recovered
C-starts did not elicit electromyogram (EMG) responses caudal to the
wound in many cases, which supports the morphological findings. In general,
the trunk EMG responses that were evoked in some cases were significantly
smaller than those of controls and are thought to have a minimal contribution
to the recovered response (Zottoli et al.,
1989). Therefore, the M-cell may contribute to the recovery of
C-starts in some fish but non-M-cells must underlie the recovery in
most cases.
The regeneration of non-M-cells such as the M-cell homologues
(Lee et al., 1993) may
underlie the recovery of C-starts. These neurons are active during
C-starts in zebrafish (Danio rerio L.) larvae
(O'Malley et al., 1996
;
Liu and Fetcho, 1999
) and are
thought to elicit non-M-cell C-starts in adult goldfish when M-cells
are ablated (Eaton et al.,
1982
; DiDomenico et al.,
1988
; Zottoli et al.,
1999
). In addition, non-M-cell C-starts
(Zottoli et al., 1999
) and
those C-starts that return after spinal cord crush are both
characterized by a significantly lower probability of response and a longer
latency from stimulus to response when compared with M-cell initiated
C-starts of control fish. Retrograde labeling of axons that have
regenerated across an SML crush would help determine whether the M-cell
homologues or other non-M-cells are potential candidates for the recovery of
C-starts.
The long-term fate of regenerating M-cells in the recovery of
C-starts is not clear at this time. A decrease in C-start
response latency in one goldfish between 2.5 and 12 months postoperatively
indicates the possible plasticity in regenerated neuronal connections that may
involve the contribution of additional cells such as M-cells
(Zottoli et al., 1994).
Although there is no overlap between stage 1 latencies of experimental and sham-operated control fish, there is substantial overlap between other kinematic parameters (see Fig. 9B,C). Thus, many recovered C-starts have kinematic values that are comparable with those of controls. However, on average there were smaller turning angles and shorter distances traveled and velocities attained by the center of mass of recovered C-starts compared with control ones. Such a difference may result from changes in muscle mass after injury. Prior to recovery of movement, the trunk and tail musculature are not used except for occasional reflex responses evoked by the experimenter during routine handling and during tank cleaning. In a separate study, using identical protocols to those used in this study, fish weight normalized to the original weight prior to the crush decreased from 1.0 to 0.85±0.06 (mean ± S.D., N=31; J. E. Nierman, unpublished observations) one month postoperatively. Those fish that recovered equilibrium weighed 0.91±0.06 (N=7) of their original weight and returned to their original weights on average by six months postoperatively (1.0±0.07; N=7). Those fish that did not recover equilibrium did not gain back their lost weight during the same six month interval (0.84±0.12; N=16). Since recovered C-starts in this study could be elicited as early as 2 months postoperatively, the reduced muscle mass may influence some of the kinematic parameters measured. However, there was no effect of time on these parameters and, therefore, it is unlikely that muscle mass had a major effect on C-start kinematics.
Can the recovery of C-starts be explained by
compensatory mechanisms?
Axonal regeneration across a crush wound could result in innervation of
targets that would provide an alternative compensatory movement to that
normally occurring in control animals. Axial motoneurons are responsible for
the major components of C-starts (Fetcho,
1991,
1992
). If regenerating axons
predominantly innervated fin motoneurons rather than axial motoneurons, their
activation would result in a propulsive movement of the fish. This propulsive
movement might be interpreted as a C-start that is slower and less
robust than control C-starts. However, EMG responses of trunk
musculature occur during recovered C-starts in free-swimming fish (S.
J. Zottoli, unpublished observations). Therefore, if compensatory mechanisms
exist, they do not appear to play a major role in the return of
C-starts.
Conclusions
Numerous studies have shown that adult teleost fish can undergo behavioral
recovery after spinal cord injury (see
Koppányi, 1955;
Zottoli et al., 1994
).
However, few studies on teleost fish (however, see
Doyle et al., 2001
) have
provided the quantitative rigor in the analysis of behavioral recovery that
has been the hallmark of swimming studies on the larval (e.g.
Davis et al., 1993
;
McClellan, 1994
) and adult
(e.g. Cohen et al., 1989
)
lamprey. Our results provide the first quantitative description of the
recovery of C-starts in adult teleost fish after spinal cord injury.
M-cells in adult goldfish are known to initiate C-starts and after
spinal cord injury can readily regenerate. However, morphological and
physiological evidence indicates that M-cells would not contribute
significantly to most recovered responses during the 190-day postoperative
interval of this study. Therefore, recovery of C-starts does not
involve restitution of the original patterns of neuronal connections. The
identification of neurons that underlie the return of C-starts will
provide the unique opportunity to analyze the mechanisms underlying behavioral
recovery at the cellular level.
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Acknowledgments |
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