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First published online November 17, 2006
Journal of Experimental Biology 209, 4788-4801 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02582
Correlation of C-start behaviors with neural activity recorded from the hindbrain in free-swimming goldfish (Carassius auratus)
1 Dominick P. Purpura Department of Neuroscience, Albert Einstein College of
Medicine, Bronx, NY 10461, USA and
2 Department of Biology, Williams College, Williamstown, MA 01267,
USA
* Author for correspondence (e-mail: sweiss{at}aecom.yu.edu)
Accepted 4 October 2006
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Summary |
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 TOP Summary IntroductionMaterials and methodsResultsDiscussionReferences |
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Key words: startle, escape, Mauthner, reticulospinal, medial longitudinal fasciculus
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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). Fast startle responses have been the subject of a number of
studies related to this issue, since in species such as crayfish
(Wine and Krasne, 1972;
Beall et al., 1990;
Edwards et al., 1999),
earthworms (Drewes et al.,
1978), squid (Otis and Gilly,
1990; Neumeister et al.,
2000; Preuss and Gilly,
2000), larval zebrafish (Prugh
et al., 1982) and goldfish
(Zottoli, 1977; Eaton et al.,
1981; Canfield and Rose, 1993),
these behaviors seem to be triggered by identifiable cells.
One of the best-characterized startle behaviors in vertebrates is the
C-start exhibited by teleosts. There are two sequential stages to the C-start
(Domenici and Blake, 1997;
Eaton et al., 2001): stage 1,
where the head rotates about the center of mass and the fish's body exhibits a
curvature that resembles a C, and stage 2, during which the fish propels
forward. Sometimes the fish may also rotate in the opposite direction during
stage 2, producing a counterturn (Foreman
and Eaton, 1993). C-starts in goldfish are initiated by the
Mauthner (M) cells (Wilson,
1959; Zottoli,
1977; Eaton et al., 1981), an identifiable pair of large neurons
found in the medulla oblongata (Zottoli, 1978). These neurons have been
studied extensively and have a number of unique features that make them
amenable for the analysis of the neuronal basis of behavior
(Faber and Korn, 1978;
Zottoli and Faber, 2000;
Korn and Faber, 2005). For
example, the large extracellular field potential recorded in the vicinity of
the axon cap, a structure surrounding the M-cell initial axonal segment,
unequivocally allows identification of that cell. Consequently, metal
microelectrodes have been implanted near the axon cap, allowing this neuron's
activity to be recorded in free-swimming goldfish. It is well established that
the M-cell extracellular field potential is correlated with contralateral EMG
responses (Zottoli, 1977) and
C-starts (Eaton et al., 1981). In translucent larval zebrafish activation of
identifiable reticulospinal neurons can be monitored in vivo by
calcium imaging. These studies demonstrate that stimuli that elicit the
C-start activate a distributed population of reticulospinal neurons in
parallel with the M-cell (Gahtan et al.,
2002). However, because this imaging technique requires that the
fish be immobilized it is not yet clear how activation of these neurons
correlates with the trajectory and kinematic parameters of the escape
behavior. In addition, most of the escapes studied so far have been triggered
by abrupt stimuli, most often mechanosensory. The respective roles of the
M-cells and the other reticulospinal neurons in escapes triggered by more
gradual and longer lasting stimuli are also not yet established.
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). Various
stimuli were used to elicit fast tail flexion, which was correlated with a
single, all-or-none extracellular spike followed by a graded compound action
potential (Wilson, 1959). This
technique is attractive since it provides the possibility of recording the
collective activity of one or both M-axons and other descending activity in a
free-swimming fish. For example, recordings from the surface of the lamprey
spinal cord have revealed correlations between descending neuronal population
activity and swimming (Deliagina et al.,
2000).
The M-axons are visible in the hindbrain on the surface of the medulla
oblongata between the vagal lobes in goldfish
(Zottoli et al., 1995). At
that location the axons are approximately 150 µm below the surface. We have
taken advantage of this morphological feature to monitor M-axon activity and
that of other descending axons in the medial longitudinal fasciculus
(mlfd) in free-swimming goldfish. The results show that (1) the
activity of the two M-axons can be distinguished in field potential recordings
from the mlfd and are correlated with C-start behavior, including
its direction; (2) activity of other descending axons, which arise in the
rhombencephalic medial reticular zone, is correlated with counterturns
following the initial C-bend; and (3) diverse auditory and visual stimuli
elicit C-start behaviors triggered by the M-cell. Thus diverse stimuli elicit
a stereotypic C-start behavior initiated by the M-cell in concert with a
population of other neurons that also may function to mediate counterturns
during stage 2 of the escape.
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Materials and methods |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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). In general, the larger fish, 15-20 cm, were used
for chronic recording experiments.
Elicitation of startle responses and kinematic measurements
The circular experimental aquarium had a diameter of 76 cm and a water
depth of 28 cm. Water temperature was maintained at 18°C. C-starts were
elicited with sounds generated by software written in Igor Pro. The
computer-generated signal was amplified (Servo 120; Samson, Syosset, NY, USA)
and produced by an underwater loud speaker (UW-30; University Sound, Buchanan,
MI, USA) at the outer circumference of the tank. Sound pulses, i.e. pips, were
generated as a single cycle of a 100, 200 or 300 Hz sound wave. Because of
reverberation from the walls of the tank and continued vibration of the
speaker membrane the sound persisted for multiple cycles with successively
longer periods and more distortion. The peak pressure of the pip stimulus
occurred at the first or second cycle, depending on the location of the
hydrophone, and was 179-185 dB re: 1 µPa in the center of the tank. 200 Hz
sound waves 50 or 200 ms in duration that increased in intensity exponentially
from zero were also used to evoke escapes. These `ramped' sound waves were
used to elicit escapes because, as opposed to pips, they remain constant in
frequency and they proved ideal for studying the auditory processing
properties of the M-cell (Szabo et al.,
2006). In addition, predatory strikes by other fish that evoke
C-starts sometimes do not appear to be sudden strikes but rather consist of
progressively more powerful multiple body-bends
(Harper and Blake, 1991). The
peak of the ramped stimulus occurred at the last cycle and was 179-186 dB re:
1 µPa in intensity in the center of the tank at a distance 44 cm from the
speaker membrane. Visual looming stimuli, which also simulate object approach,
consisted of a series of computer generated black disks of increasing size
projected with a LCD projector (1024x768 pixels, 60 Hz refresh rate) on
a white screen 
16 cm above the fish
(Fig. 1A). This produces a
growing image on the retina, and its size can be quantified as the view angle
=2xtan-1(d/2xs), where
d is the diameter of the projected disk on the screen and s
is the distance from the screen to the eye. Increasing disk size simulates the
normally occurring decrease in s, during an approach. Stimuli
differed in their projected size, approach velocity and duration
(Fig. 1B), as described in
detail elsewhere (Preuss et al.,
2006). The time intervals between trials varied randomly from 2-20
min. The auditory stimulus was recorded (sampling rate, 30 s) with a
hydrophone (SQ01; Sensor Technology, Collingwood, Ontario, Canada) and the on-
and offset of the visual looming stimulus were monitored with a
photodiode.
Video recordings were made from the underside of the tank at 1000 frames
s-1 at a resolution of 512x384 pixels using a high-speed
video camera (Kodak Extapro 1000 HRC; Eastman Kodak, San Diego, CA, USA), and
were stored digitally. A 1 ms flash from a light-emitting diode outside the
tank and not seen by the fish marked the stimulus onset on the video and
served as a reference point for latency measurements. For each startle
sequence selected, 176 frames (coincident with the onset of the auditory
stimulus or 25 frames before the visual response onset) were analyzed. The
x- and y-positions of the head and the center of mass (COM)
were measured manually in ImageJ (NIH, Bethesda, MD, USA), or using the
automated movement tracking software BIOBSERVE (Bonn, Germany). Visually
evoked startles were tracked manually at a lower temporal resolution because
the LCD projector produced a flicker effect. When tracked manually, the COM
was defined as a point on the midline located between the pectoral fins.
Angular and linear displacement plots were calculated from x- and
y-positions of the head using a fixed rectilinear coordinate system.
Heading (H) is the angle, in degrees, of the midline vector
connecting the COM to the tip of the head and was calculated by the equation:
H=arctan(yhead-yCOM)/(xhead-xCOM)
at 1 ms intervals for auditory stimuli and 3-5 ms intervals for visual
stimuli. Angular velocity (dH/dt) was calculated by fitting
the fish's heading as a function of time with a 10th degree polynomial and
taking the derivative of this function. Stage 1 was defined as the interval
beginning with movement of the head and ending with forward propulsion of the
center of mass (COM) by more than 5 mm
(Nissanov et al., 1990;
Eaton and Emberly, 1991).
Other investigators (Domenici and Blake,
1997) classify the end of stage 1 by measuring the latency to the
maximal initial rotation.
Statistics and data analysis
All statistical tests were carried out using Statview 5.0 (SAS Institute,
Cary, NC, USA) All custom analysis software was written in Labview 7.0. Values
are given as the mean ± standard error of the mean (s.e.m.) unless
specified otherwise. N=number of fish; n=number of
trials.
Surgery for chronic recordings
Fish were anesthetized during the surgery with continuous perfusion of ice
water containing 60 mg l-1 MS-222 (3-aminobenzoic acid ethyl ester;
Sigma; St Louis, MO, USA). The fish were restrained in a manner similar to
that used for acute recording in vivo
(Preuss and Faber, 2003). When
the fish no longer exhibited a motor response to touch of the head and caudal
fin, the skin overlying the cranium was removed with a razor blade until the
surface of the skull was free of moisture. Two self-threading screws
(Stoelting, Wood Dale, IL, USA) were inserted in the skull. A 4 mmx3 mm
opening centered at the midline and located just rostral to the suture between
the occipital and parietal bones was drilled and lipid droplets overlying the
brain were removed by aspiration. Electrode penetration was in the
rostral-caudal direction at an angle of approximately 60-70° from the
horizontal. The electrode tip was positioned 100-300 µm lateral to the
midline and just caudal to the facial lobe, at the brain surface and lowered
to a depth <300 µm. The M-axons are visible in this region with the aid
of a dissecting microscope. Microelectrodes were prepared from Teflon coated
316 stainless steel wire 100 µm in diameter (California Fine Wire Company,
Grover Beach, CA, USA) with 0.5-1.0 mm of the tip stripped of insulation or
the tip cut with a pair of fine scissors. Both techniques yielded comparable
results. Impedance measurements ranged from 30-40 K
at 1 kHz. A
stainless steel wire stripped of insulation at its tip and wrapped around the
metal skull screws served as a reference. After positioning the recording
electrode, the cranium was filled with mineral oil. The electrode was fixed in
position with a combination of cyanoacrylate and dental cement. EMG electrodes
were prepared from 40 µm steel alloy (Stablohm 800A, California Fine Wire
Company, Grover Beach, CA, USA) with 2-5 mm of insulation removed using a
razor blade. The deinsulated tips were bent at the ends and fed into a syringe
needle. The needle, with the electrodes inside, was inserted in dorsal white
musculature two scale widths dorsal to the visible band of the posterior
lateral line and near the midpoint of the rostral-caudal axis. The electrodes
were held in place by a drop a cyanoacrylate adhesive on the epidermis with
the overlying scales removed.
Activity from the hindbrain of ten free-swimming goldfish was successfully recorded; other fish were unable to maintain equilibrium following the surgery, presumably due to inadvertent damage to the semicircular canals. Experiments were initiated 2-5 h post operatively. The leads connecting the brain and EMG electrodes were several feet in length. During the experiment wires were occasionally manually detangled. Evoked startle responses in which tangled leads restricted the fish's movement or resulted in cross-talk were excluded from analysis. Following chronic recordings, in some experiments, the fish was re-anesthetized with MS-222 and immobilized with curare. The spinal cord was exposed and extracellular responses to antidromic stimulation were recorded from the implanted electrode.
Electrophysiology
Extracellular responses were amplified 1000x with a 4-channel
differential AC amplifier (Model 1700, A-M Systems, Carlsborg, WA, USA) and
bandpass filtered from 100 Hz-20 kHz. EMG responses were amplified 100x
and bandpass filtered from 300-10 kHz. In a subset of experiments, in
curarized fish, simultaneous recordings were obtained intracellularly from the
M-cell soma and extracellularly from the hindbrain. For this purpose we used
previously described methods for the intracellular recording
(Preuss and Faber, 2003).
Briefly, an antidromic stimulus was generated by a bipolar stimulating
electrode placed on the spinal cord and M-cell responses were recorded with a
2-7 M glass micropipette containing 5 mol l-1 potassium
acetate or 3 mol l-1 KCl. Data were recorded online with a
Macintosh G4, using acquisition software developed in the laboratory for Igor
Pro (Wavemetrics, Lake Oswego, OR, USA) at a sampling interval of 10-50 µs,
and analyzed with the same software and Labview 7 (National Instruments,
Austin, TX, USA).
Morphology of the axons in the mlfd
One goldfish (5 cm body length) was anesthetized in 0.03% MS-222 and
perfused intracardially with fixative (2.5% glutaraldehyde, 1%
paraformaldehyde in 0.1 mol l-1 sodium cacodylate buffer, pH 7.4).
The brain was removed, dissected and post-fixed for 1 h, washed in 0.1 mol
l-1 cacodylate buffer, post-fixed in 2% OsO4 in 0.1
cacodylate buffer for 4 h, washed, dehydrated in a graded ethanol series,
cleared in propylene oxide and embedded in Epon. The brain was serially
sectioned (5 µm) in the transverse plane. Unstained sections were mounted
on glass slides for analysis and photographed (Zeiss Axiocam digital
camera).
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The boundaries of the mlfd extended laterally from the midline to the far edge of the M-axon and ventrally from the ventricle to the commissure that separates the dorsal from the ventral portions of the mlf, i.e. the mlfv (Fig. 2).
Cells of origin of the mlfd
To identify the somata of origin that give rise to axons in the
mlfd, Lucifer Yellow (Sigma) was injected into axons adjacent to
the M-axon in 16 fish. In order to obtain a more general view of the cells of
origin, in three experiments a population of damaged axons was filled using
dextran biotin (10 k, Invitrogen, Carlsbad, CA, USA).
In the Lucifer Yellow experiment, the M-axon was filled first, followed by
injection into one or more adjacent axons. For this purpose a glass
microelectrode (30 M) was filled with 5% Lucifer Yellow in
distilled water. In addition to visually positioning the electrode in the
M-axon, identification of the M-axon required the electrode could be raised
and lowered approximately 50 µm without change in resting potential Lucifer
Yellow was then injected (25-50 nA for 200 ms three times per second for a
minimum of 30 min). Fish were perfused with 100 ml of freshwater teleost
saline (pH 7.4), followed with 100 ml of 10% formalin in 0.1 mol
l-1 phosphate buffer (Fisher, Fairlawn, NJ, USA). The brains were
removed and placed in fresh fixative overnight, dehydrated and cleared in
methyl salicylate before observation under the fluorescence microscope. The
distance between the M-cell initial segment and the soma of the non-M-cell was
measured in 16 brain whole mounts. These measurements were used to determine
the location of the other neuron relative to a particular hindbrain segment.
The neurons were identified using previously defined descriptions
(Lee et al., 1993). After the
brains were viewed in wholemount, they were embedded in paraffin, sectioned
transversely at 15 µm, soaked in xylene and coverslips placed on top using
a nonfluorescent mounting medium (Eukitt; O. Kindler; distributed by
Calibrated Instruments).
To label multiple axons in the mlfd, including the M-axon, a blunt glass microelectrode placed over the right M-axon, was lowered 200-300 µm below the surface and withdrawn, and this protocol was repeated as the electrode was moved medially in 20 µm steps to the midline. Dextran biotin crystals on the tip of an insect pin were then applied to the lesioned region.
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80 axons on either side of the
midline. There are a number of relatively large myelinated axons medial and
ventral to the M-axon (Fig. 2).
In the one fish studied quantitatively this tract contained 32 axons with
diameters 
10 µm on the left and 35 on the right. The dextran biotin backfilling experiments revealed the segmental distribution of cells with axons in the mlfd (Fig. 3A). In order to determine the distribution of the somata of origin of these mlfd fibers, we analyzed one of the three brains that had the most intense label and in which inspection of the lesioned region confirmed it was restricted to the mlfd.
In this case, 72 somata were backfilled. They were localized to the
midbrain nucleus of the mlf and throughout the rhombencephalic medial
reticular zone (Table 1).
Twenty neurons were located in the nucleus of the mlf, two were
vestibulospinal neurons, and 50 were reticulospinal cells. Of the latter, 29
were ipsilateral to the injection site while the other 21 were located
contralaterally. Some of the neurons could be identified using published
descriptions (Lee et al.,
1993). Examples of reticulospinal neurons in different rhombomeres
are shown in the transverse section in Fig.
3Bi-iv.
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Injection of Lucifer Yellow into the axons adjacent to the M-axon often
filled both reticulospinal neurons (Fig.
4Ai) and T-reticular interneuron axons
(Kimmel et al., 1985), also
known as cranial relay neurons (Hackett
and Faber, 1983). One vestibulospinal neuron was labeled in this
limited sample. Based on the distance between the M-cell initial segment and
the soma of the non-M-cell, the 16 cells were assigned to a rhombomere
(Table 1). For example, the
reticulospinal neuron in Fig.
4Ai-iii was 794 µm caudal to the M-cell's initial segment,
placing it in r6. Two other examples of reticulospinal neurons that have
crossed axons and are located in r6 are given in
Fig. 4Bi,ii. These two, as well
as the neuron in Fig. 4Aii,iii,
might be MiD3cm the M-cell homologue located in r6
(Lee et al., 1993).
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The antidromic response in the extracellular recordings consisted of a
composite field potential with at least two components, an all or none short
latency spike-like component (662±40 µs, N=4 fish),
followed 
1 ms later by a complex series of waves
(Fig. 5B). When the intensity
of stimulation was increased a second all or none component was added to the
short latency response and there was a graded increase (not shown) in the
amplitude of the longer latency component. A field potential with these
characteristics could be measured across a 800 µm width of the hindbrain
(Fig. 5Ci) and at various
depths (Fig. 5Cii), although it
is not clear whether the blunt tipped electrode penetrated into the tissue.
The initial component had the same threshold intensity (in V) and latency as
the intracellularly recorded antidromic M-spike. The longer latency components
of the population response are most likely generated by the activation of
nearby fibers in the mlfd such as the axons of other descending
reticulospinal neurons as well as neurons postsynaptic to the M-axon, such as
the T-reticular interneuron axons.
To confirm the identification of the short latency component, the M-cells
were also stimulated orthodromically using two independent techniques. First,
the cell was depolarized past threshold using intrasomatic current injection
(Fig. 5D). The somatic M-spike,
generated in the initial segment of the axon
(Funch and Faber, 1982) and
the hindbrain field potential occurred simultaneously. Following orthodromic
stimulation of the right M-cell the micropipette was repositioned in the left
M-cell soma and when the same procedure was repeated, the orthodromic M-spike
was again associated with a hindbrain field potential. Note that in both
examples (Fig. 5D), the evoked
field potential consisted of the initial M-spike followed by a larger response
with a delay of 1.5 ms, which is likely generated by a population of
reticulospinal, motoneurons and local interneurons triggered by the M-spike.
Antidromic and orthodromic evoked extracellular M-spikes differed in amplitude
and waveform.
Second, the M-cell could also be excited orthodromically with sound stimuli in air (Fig. 5E). Loud abrupt sounds in air (peak 84 dB re: 20 µPa) resulted in 5-10 mV depolarizations in the M-cell soma but did not excite the cell to threshold. In that case, no obvious field potential was generated in the hindbrain. Chloride ions were then injected iontophoretically into the M-cell to shift this ion's equilibrium potential in the depolarizing direction. Consequently, sound evoked inhibition converted to excitation and summed with the afferent excitatory input, such that a loud sound could then excite the M-cell past threshold. The resulting M-spike and M-spike triggered activity could be detected with the hindbrain surface electrodes as distinct field potentials (Fig. 5E). The mean delay from the M-spike to the M-triggered activity was 1.58±0.11 ms (N=7).
In one experiment, the orthodromic and antidromic M-spikes were too small in amplitude to discriminate from background noise. However, the M-spike triggered activity was still present suggesting that even when the electrode was poorly positioned M-spike triggered activity could still be detected. Overall, the experiments indicate that the short latency response in the hindbrain field potential is consistently correlated with the M-cell action potential, and that the amplitude of the response allows for the discrimination of the spikes generated by the two M-axons in these acute preparations.
Field potentials associated with evoked C-starts
We analyzed a total of 185 startles in 10 fish implanted with an electrode
on the surface of the hindbrain. Startles were evoked by abrupt single cycle
sound pips, ramped sound waves, or visual looming stimuli. As described in
detail in a subsequent section, kinematic parameters calculated from a random
cross section of these startles (n=15), evoked by all three types of
stimuli, indicate that they were typical C-starts
(Table 2)
(Nissanov et al., 1990;
Eaton and Emberley, 1991;
Zottoli et al., 1999;
Preuss and Faber, 2003). Every
C-start was associated with an evoked field potential and when the stimulus
failed to evoke a startle there was also no evoked field. The amplitude and
waveform of the evoked field potential varied across fish because of
differences in electrode placement.
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The results of Fig. 5 suggest it might be possible to distinguish spikes generated by the left and right M-cells on the basis of their different amplitudes, especially if the recording electrode is not on the midline. In confirmation, the amplitude of the first component of the evoked field potential was correlated with the direction of the C-start in successive trials, in three fish (two tailed student's t-tests; P<0.0001, n=17,19,51, respectively). In two of these experiments the initial 2 and 5 trials, respectively, did not exhibit the observed relationship. This discrepancy likely resulted from changes in the position of the electrode tip. Specifically, in fish in which the electrode was implanted to the left of the midline, the first component was larger in amplitude when the fish made a C-start to the left (Fig. 6Ai,ii). The opposite was the case when the electrode was implanted to the right of the midline (Fig. 6Bi,ii). The amplitude and waveform of the first component of the field potential were independent of the type of stimulus used to evoke the C-start, e.g. pip vs ramped sound waves (Fig. 6Aiii,iv) or visual looming stimuli (Fig. 6Biii,iv). Note that the ramped stimulus is constant in frequency throughout its duration, as opposed to the pip, which is distorted (Fig. 6Aiii,iv and Fig. 7A,B). These results strongly suggest that the first component of the evoked field potential is the M-spike.
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). Furthermore,
when the different stimuli were used in the same experiment, the evoked
M-spikes were comparable, as shown in Fig.
7A,B for C-starts evoked by auditory pips and ramped sound waves,
respectively. A similar result was obtained when comparing auditory and
visually evoked C-starts. In addition, there was no stimulus-dependent
difference in the time interval from the M-spike to EMG onset for the
different stimuli (Kruskall-Wallis test, P>0.5, ANOVA
F=0.8328, d.f.=2, P=0.47; pip n=14; ramped
n=10; visual looming n=5). It remains possible that later
components of the evoked field potentials may not be entirely generated by
neural activity and may in part be generated by volume conducted muscle
activity (Fig. 7A,B). Volume
conduction of muscle activity was observed by others using comparatively
higher impedance electrodes to chronically recording from the M-cell axon cap
(Zottoli, 1977; Eaton et al.,
1981).
The delay from the M-spike to the first detectible movement averaged
8.13±0.17 ms for 185 C-starts. This value is consistent with reported
delays between the M-spike recorded from the axon cap and movement onset
(Zottoli, 1977; Eaton et al.,
1982) and thus further supports the notion that the first component of the
field potential is the M-spike. In addition, the time interval was
stimulus-independent (ANOVA; F=2.091, d.f.=4, P=0.08;
Table 3).
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Stimulus-dependent differences in the latency of the M-spike reflect
differences in how the M-cell processes these stimuli
(Preuss and Faber, 2003;
Preuss et al., 2006). In
implanted fish the latency from stimulus onset to the M-spike was
8.61±0.2 ms (n=160) in C-starts evoked by pips. To determine
whether the frequency of a pip stimulus influences the timing of the M-spike
C-starts were evoked with three different pip frequencies. While the mean
M-spike latency did decrease for pips of increasing frequency
(Table 3) the differences were
insignificant (ANOVA F=0.412, d.f.=2, P=0.66). On the other
hand the latency from the onset of the computer-triggered 200 Hz ramped sound
signal to the M-spike was 13.71±1.7 ms (n=15), which was
significantly longer than the latency of startles evoked by sound pips
(n=160, Student's t-test; P<0.0001;
Table 3). This difference was
expected, since for at least its first few cycles the ramped stimulus is much
weaker than the pip. The onset latency for the M-spike evoked by a
heterogeneous group of visual looming stimuli ranged from 32-743 ms
(n=10; Table 3),
reflecting processing time in the retina and optic tectum. In trials with a
fixed set of visual stimulus parameters (800 ms duration; angular retinal size
of the falling disk, i.e. view angle 4-90° with respect to the fish eye),
the latency was less variable, namely 731.04±12.06 ms (n=4).
The latency of the startle evoked by visual looming stimuli is optimized for
collision avoidance and depends on a dynamic scaled function of the view angle
(Preuss et al., 2006).
Kinematics of C-starts in non-implanted fish
This study used three types of stimuli, and the chronic recordings indicate
that all evoke an M-initiated C-start. If this is the case, these behaviors
should have the same kinematic properties. Therefore, 75 startle responses
evoked by diverse stimuli were analyzed in 11 non-implanted fish. Each startle
was evoked when the fish was near the middle of the tank and was filmed at
high magnification. Multiple kinematic parameters from each startle were
calculated and compared across stimulus conditions. We also examined the
distribution of individual kinematic parameters for evidence of multiple
distinct behaviors. For this purpose, a randomly selected subset of previously
analyzed visually evoked responses (Preuss
et al., 2006) were selected and reanalyzed with the new algorithm,
used to generate additional kinematic parameters.
In addition to stage 1 duration and peak angular velocity, which have been
used previously to distinguish C-starts from other startle behaviors
(Domenici and Batty, 1997;
Hale, 2002), three other
parameters were used: distance traveled by the center of mass (COM) at 70 and
100 ms, and total turn angle. The dependency of each on stimulus type examined
by MANOVA revealed a significant effect of the stimulus (Wilks' Lambda test,
F=3.404, P=0.006). However, of these five kinematic
parameters, all but the angular velocity (P<0.01) were independent
of the stimulus type. In the case of angular velocity, the mean value for
startles evoked by visual stimulation (3.03±0.10 deg. ms-1,
n=28) was significantly smaller than those of startles evoked by pips
(4.33±0.33 deg. ms-1, n=27) or by ramped 200 Hz
sound waves (4.158±0.32 deg. ms-1, n=20). This
difference was not apparent in the previous analysis of a larger group of
visually evoked startles (Preuss et al.,
2006), and it might be due to either the smaller sample size or
the fact that the visual evoked C-starts were tracked manually at a lower
temporal resolution, relative to auditory evoked C-starts that were tracked
automatically.
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Neural correlates of C-starts exhibiting counterturns
The presence of a counterturn during some C-starts raises the question of
whether activity in other reticulospinal neurons precedes that counterturn.
Comparison of the evoked fields in Fig.
6Ai,Bi indicates that the amplitudes of the M-spike and the
initial components of the field potential associated with it varied little,
with exception of the differences in the sizes of two M-spikes. However, close
inspection of the later components of the recordings suggested the responses
could be subdivided into two populations, one that did not exhibit significant
levels of late activity and one with a marked compound response at >10 ms
after the M-spike. The latter responses occurred relatively infrequently
(10% of all trials) and they could be isolated as follows: mean response
and standard deviation waveforms for left- and right-directed escapes were
calculated separately for each experiment (n=3). Individual responses
were then compared with these averages and those that deviated appreciably
from the mean ± 1 standard deviation (s.d.) for at least 5 ms were
identified and the kinematics associated with the responses were analyzed. In
the example of Fig. 8Ai, the
evoked field potential (red) is similar to the mean potential (black,
n=30 right C-starts in a single fish), and the fish executed a
typical 130° C-bend but did not exhibit a counterturn, as shown in
Fig. 8Aii. The example in
Fig. 8Bi,ii illustrates an
atypical field potential and in this case the fish performed a counterturn to
the left, presumably triggered in part by the late activity in the
mlfd. Overall, analysis of the C-start escape trajectories selected
on the basis of a long latency component of the corresponding field potential
occurred revealed that 19/22 exhibited counterturns. The others appeared to be
typical C-bends. In order to ascertain if there were other trials exhibiting
counterturns that were not associated with the atypical late activity we
reviewed video recordings and did not visually identify any trials with
counterturns besides the 19 described above.
The above results suggest that the late responses apparent in the hindbrain evoked field potential are correlated with the occurrence of the counterturn. There were no correlations between the amplitude of the late activity and the magnitude or direction of the counterturn. However, the timing of the two responses was quite consistent. First, the latency between the onset of the largest wave in the late response (Fig. 8Bii) and that of the counterturn averaged 17.5±1.87 ms. To better quantify this relation and pool data from different fish the temporal axes of individual fish were aligned according to the onset of the initial brainstem field potential. Then 18 control responses and 18 that had counterturns were separately analyzed. To account for phase cancellation of the variable late response the root mean squared (RMS) of each field potential was calculated and averaged using a bin size of 1 ms. In trials with counterturns an additional component, as compared with the controls, was present in the interval beginning 7 ms after the onset of the field potential (Fig. 9).
The 19 C-start trials exhibiting counterturns were evoked by the full complement of stimuli used. Kinematic measurements were made for 18 of these counterturn trials. The onset of the counterturn occurred at 31.2±2.44 ms, the turn angle of the counterturn was 40±7.8°, and the maximum angular velocity of the counterturn was 1.06±0.18 deg. ms-1. These values are similar to those calculated in non-implanted fish exhibiting C-starts with counterturns.
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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; Eaton et al.,
1981; Canfield and Rose, 1993).
Although this approach provides essential information, it was limited because
correlations could be drawn only for responses in one direction and because
activity of other reticulospinal neurons could not be measured. We demonstrate
here that implanting a single electrode in the hindbrain allows for the
discrimination of either one of the M-axons as well as detection of population
activity from descending axons in the mlfd in free-swimming
goldfish. Indeed, when it was possible to distinguish right and left M-axon
spikes, the direction was always predictable from the recordings. Furthermore,
the longer latency activity in the mlfd accurately predicted
whether there would be a counterturn following the initial C-bend.
Discrimination of M-axon spikes
The results presented here demonstrate that the field potential preceding a
C-start is long-lasting relative to the duration of a single M-spike.
Nevertheless, in most experiments an initial brief component could be
detected, and it could be unequivocally identified as the M-spike when the
latter was recorded simultaneously with an intracellular electrode. However,
in the absence of such a direct confirmation, it is difficult to be confident
of M-spike identification. It remains to be seen if a different electrode
design or an electrode array could improve the resolution in chronic
experiments with hindbrain recordings. Regardless, it is clear that a single
M-cell spike triggers C-starts for diverse stimuli, namely abrupt sound pips,
ramped sound waves, and visual looming stimuli. Furthermore, in each case the
hindbrain field potential that immediately follows the M-spike is generated,
at least in part, by activity in a population of axons in the mlfd
and this field was stereotyped in a given fish.
It was suggested that neural activity in addition to the M-spike might
correlate with C-starts having larger turn angles or angular velocities
(Nissanov et al., 1990).
However, the relative constancy of the hindbrain field potential, in the
absence of counterturns, suggests this activity cannot be easily correlated
with turn angle or angular velocity with our recordings. Nevertheless, the
presence of this compound field potential, as well as the late fields that
precede a counterturn, raises the question of which other neurons generate
it.
Source of mlfd activity following the M-spike
The population of neurons that direct the C-start are thought to be
localized to the rhombencephalic medial reticular zone
(Foreman and Eaton, 1993;
O'Malley et al., 1996;
Gahtan and O'Malley, 2001;
Gahtan et al., 2002;
Nakayama and Oda, 2004). This
structure has eight segments, seven of which are clearly distinguishable
(r1-r7) and an eighth consisting of a continuous column of reticulospinal
neurons (Kimmel, 1982;
Kimmel et al., 1982;
Kimmel et al., 1985;
Metcalfe et al., 1986;
Lee and Eaton, 1991;
Lee et al., 1993;
Nakayama and Oda, 2004).
Segment 4 is positioned at the level of the root of the VIIIth nerve and
contains the Mauthner cells. A pair of neurons in r5 (MiD2cm) and a pair in r6
(MiD3cm) are morphologically similar to the M-cells in larval
(Kimmel et al., 1982;
Metcalfe et al., 1986) and
adult teleost fish [zebrafish (Lee and
Eaton, 1991), goldfish (Lee et
al., 1993)]. The similarities include dorsal-ventral position,
predominant lateral and ventral dendrites, an axon that crosses the midline
and extends caudally the extent of the spinal cord
(Kimmel et al., 1982;
Nakayama and Oda, 2004).
Consequently MiD2cm and MiD3cm are designated as M-cell homologues.
Calcium imaging studies in the larval zebrafish demonstrate that a gentle
tap to the head that reliably evokes C-starts produces short-latency calcium
responses in the M-cell homologues
(O'Malley et al., 1996) and
identified neurons in all eight hindbrain segments
(Gahtan et al., 2002). It is
not clear whether the tap stimulus directly excites this widespread population
of neurons or if the population is activated, in part, by the M-cell or other
reticulospinal neurons.
Our results are consistent with the notion that M-cell activation triggers
activity in other reticulospinal neurons. That is, orthodromic activation of
the M-cell by transmembrane current injection produces a field potential
following the M-spike that is comparable to that produced by natural stimuli.
Given the location of the recording electrode, activity in the fibers of the
mlfd are likely contributors to this triggered field potential,
with a minor contribution, if any, from more distant axon tracts, such as the
ventral mlf, commissural axons, axons of the lateral longitudinal fasciculus
as well as interneurons and motoneurons. Furthermore, at the level of the
recording site, the mlfd contains the axons of neurons in all
hindbrain segments except r1, as well as midbrain neurons of the nucleus of
the mlf and local interneurons. Involvement of neurons in these brain regions
would be consistent with the observations in zebrafish
(O'Malley et al., 1996;
Liu and Fetcho, 1999;
Gahtan and O'Malley, 2001;
Gahtan et al., 2002).
Based on our field recordings the duration of neural activity, en
masse, does not exceed 8-12 ms. Furthermore, in the absence of a
counterturn, there is no additional population activity. These results are
consistent with the concept that the neural commands that determine the
complete trajectory of a C-start are issued before the onset of forward
propulsion (Eaton et al.,
1988). However, counterturns that follow the initial C-bend
require additional neural commands that occur later.
Counterturns or directional changes have been described previously
(Foreman and Eaton, 1993) and
it has been hypothesized that a population of non-Mauthner reticulospinal
neurons are responsible for their initiation
(Gahtan et al., 2002). Our
results directly confirm that counterturns are associated with late compound
field potentials in the mlfd. As with the initial C-bend, it is not
clear how angular velocity or other kinematic parameters of the counterturn
are encoded.
M-cell mediation of C-starts evoked by diverse stimuli
Our data indicate that goldfish C-starts evoked by a diverse set of stimuli
are, at least in their initial stages, fairly consistent with respect to
measured kinematic parameters and neural correlates. The similarities between
the kinematic parameter values we have calculated and those previously
published for goldfish responding to additional types of stimuli support this
conclusion (Nissanov et al.,
1990; Eaton and Emberley,
1991; Zottoli et al.,
1999; Preuss and Faber,
2003). This finding is consistent with the notion that a single
motor circuit involving the M-cell mediates all fast-starts in adult goldfish
and with evidence for multimodal inputs to the M-cell
(Zottoli et al., 1987;
Canfield, 2003;
Preuss et al., 2006;
Canfield, 2006). In contrast
other fish, such as the northern pike, exhibit not only C- but also S-starts,
in which the fish bends in to an S-shape by contracting muscle rostrally on
one side of the body and caudally on the other. The S-start is executed as
both a strike and an escape and involves different neural circuits than the
C-start (Hale, 2002).
Differences in the escape behaviors exhibited across species may be expected,
since the underlying neural circuits are thought not to be evolutionarily
conserved (Hale et al., 2002).
However, the possibility remains that certain as yet unknown stimuli may evoke
alternative escape behaviors in goldfish that may or may not be triggered by
the M-cell.
Correlating behavior with individual neurons
Bullock predicted the "list of identifiable cells in the
vertebrates will grow well beyond the Mauthner's, some Müller's, some
electromotor neurons, and some spinal premotor giant cells in teleost
fish" (Bullock,
1978). Indeed he effectively anticipated the discovery of the
clustering of identified neurons in the hindbrain of zebrafish and goldfish
(Kimmel et al., 1982;
Lee and Eaton, 1991;
Lee et al., 1993). He further
suggested that rather than look for unique, identifiable neurons, it might be
more instructive to search for groups of neurons that are indistinguishable,
for example by size and location. Perhaps such an `equivalence size class' is
represented by the axons in the mlfd. These axons might represent
functional units involved in C-starts and the contribution and timing of each
might be functions of the stimulus location
(Eaton and Emberly, 1991) and
C-start trajectory that results, including later movements such as
counterturns.
In order to test the notion that population activity recorded here might
mask a more specific population code, it might be advantageous to employ
electrodes, or electrode arrays, with better spatial resolution
(Canfield and Mizumori, 2004).
Indeed, a major advantage of this system is that a number of the
reticulospinal neurons that potentially contribute to the behavior have
already been identified in immobilized zebrafish
(O'Malley et al., 1996;
Gahtan and O'Malley, 2001;
Gahtan et al., 2002) and that
these neurons can be easily classified by the rhombomere in which they reside.
Thus single-unit recordings from these neurons may provide clues to the
specific coding strategies that determine the precise trajectory of the
escape.
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Acknowledgments |
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References |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Beall, S. P., Langley, D. J. and Edwards, D. H.
(1990). Inhibition of escape tailflip in crayfish during backward
walking and the defense posture. J. Exp. Biol.
152,577
-582.
Bullock, T. H. (1978). Identifiable and addressed neurons in the vertebrates. In Neurobiology of the Mauthner Cell (ed. D. S. Faber and H. Korn), pp.47 -131. New York: Raven.
Bullock, T. H. (1990). Goals of neuroethology. Bioscience 40,244 -249.[CrossRef]
Canfield, J. G. (2003). Temporal constraints on visually directed C-start responses: behavioral and physiological correlates. Brain Behav. Evol. 61,148 -158.[CrossRef][Medline]
Canfield, J. G. (2006). Functional evidence for visuospatial coding in the Mauthner neuron. Brain Behav. Evol. 67,188 -202.[CrossRef][Medline]
Canfield, J. G. and Mizumori, S. J. (2004). Methods for chronic neural recording in the telencephalon of freely behaving fish. J. Neurosci. Methods 133,127 -134.[CrossRef][Medline]
Canfield, J. G. and Rose, G. J. (1993). Activation of Mauthner neurons during prey capture. J. Comp. Physiol. A 172,611 -618.[CrossRef]
Canfield, J. G. and Rose, G. J. (1996). Hierarchical sensory guidance of mauthner-mediated escape responses in goldfish (Carassius auratus) and cichlids (Haplochromis burtoni). Brain Behav. Evol. 48,137 -156.[Medline]
Deliagina, T. G., Zelenin, P. V., Fagerstedt, P., Grillner, S.
and Orlovsky, G. N. (2000). Activity of reticulospinal
neurons during locomotion in the freely behaving lamprey. J.
Neurophysiol. 83,853
-863.
Domenici, P. and Batty, R. S. (1997). Escape behavior of solitary herring (Clupea harengus) and comparisons with schooling individuals. Mar. Biol. 128, 29-38.[CrossRef]
Domenici, P. and Blake, R. (1997). The kinematics and performance of fish fast-start swimming. J. Exp. Biol. 200,1165 -1178.[Abstract]
Drewes, C. D., Landa, K. B. and McFall, J. L.
(1978). Giant nerve fibre activity in intact, freely moving
earthworms. J. Exp. Biol.
72,217
-227.
Eaton, R. C. and Emberley, D. S. (1991). How
stimulus direction determines the trajectory of the Mauthner-initiated escape
response in a teleost fish. J. Exp. Biol.
161,469
-487.
Eaton, R. C., DiDomenico, R. and Nissanov, J. (1988). Flexible body dynamics of the goldfish C-start: implications for reticulospinal command mechanisms. J. Neurosci. 8,2758 -2768.[Abstract]
Eaton, R. C., Lee, R. K. and Foreman, M. B. (2001). The Mauthner cell and other identified neurons of the hindbrain escape network of fish. Prog. Neurobiol. 63,467 -485.[CrossRef][Medline]
Edwards, D. H., Heitler, W. J. and Krasne, F. B. (1999). Fifty years of a command neuron: the neurobiology of escape behavior in the crayfish. Trends Neurosci. 22,153 -161.[CrossRef][Medline]
Faber, D. S. and Korn, H. (1978). Electrophysiology of the Mauthner cell: basic properties, synaptic mechanisms, and associated networks. In Neurobiology of the Mauthner Cell (ed. D. S. Faber and H. Korn), pp.47 -131. New York: Raven.
Foreman, M. B. and Eaton, R. C. (1993). The direction change concept for reticulospinal control of goldfish escape. J. Neurosci. 13,4101 -4113.[Abstract]
Funch, P. G. and Faber, D. S. (1982).
Action-potential propagation and orthodromic impulse initiation in Mauthner
axon. J. Neurophysiol.
47,1214
-1231.
Gahtan, E. and O'Malley, D. M. (2001). Rapid lesioning of large numbers of identified vertebrate neurons: applications in zebrafish. J. Neurosci. Methods 108,97 -110.[CrossRef][Medline]
Gahtan, E., Sankrithi, N., Campos, J. B. and O'Malley, D. M.
(2002). Evidence for a widespread brain stem escape network in
larval zebrafish. J. Neurophysiol.
87,608
-614.
Hackett, J. T. and Faber, D. S. (1983). Mauthner axon networks mediating supraspinal components of the startle response in the goldfish. Neuroscience 8, 317-331.[CrossRef][Medline]
Hale, M. E. (2002). S- and C-start escape
responses of the muskellunge (Esox masquinongy) require alternative
neuromotor mechanisms. J. Exp. Biol.
205,2005
-2016.
Hale, M. E., Long, J. H., Jr, McHenry, M. J. and Westneat, M. W. (2002). Evolution of behavior and neural control of the fast-start escape response. Evolution 56,993 -1007.[CrossRef][Medline]
Harper, D. G. and Blake, R. W. (1991). Prey
capture and the fast start performance of Northern Pike Esox lucius.J. Exp. Biol. 155,175
-192.
Kimmel, C. B. (1982). Reticulospinal and vestibulospinal neurons in the young larva of a teleost fish, Brachydanio rerio. Prog. Brain Res. 57,1 -23.[Medline]
Kimmel, C. B., Powell, S. L. and Kimmel, R. J. (1982). Specific reduction of development of the Mauthner neuron lateral dendrite after otic capsule ablation in Brachydanio rerio.Dev. Biol. 91,468 -473.[CrossRef][Medline]
Kimmel, C. B., Metcalfe, W. K. and Schabtach, E. (1985). T reticular interneurons: a class of serially repeating cells in the zebrafish hindbrain. J. Comp. Neurol. 233,365 -376.[CrossRef][Medline]
Korn, H. and Faber, D. S. (2005). The Mauthner cell half a century later: a neurobiological model for decision-making? Neuron 47,13 -28.[CrossRef][Medline]
Lee, R. K. and Eaton, R. C. (1991). Identifiable reticulospinal neurons of the adult zebrafish, Brachydanio rerio. J. Comp. Neurol. 304,34 -52.[CrossRef][Medline]
Lee, R. K., Eaton, R. C. and Zottoli, S. J. (1993). Segmental arrangement of reticulospinal neurons in the goldfish hindbrain. J. Comp. Neurol. 329,539 -556.[CrossRef][Medline]
Liu, K. S. and Fetcho, J. R. (1999). Laser ablations reveal functional relationships of segmental hindbrain neurons in zebrafish. Neuron 23,325 -335.[CrossRef][Medline]
Metcalfe, W. K., Mendelson, B. and Kimmel, C. B. (1986). Segmental homologies among reticulospinal neurons in the hindbrain of the zebrafish larva. J. Comp. Neurol. 251,147 -159.[CrossRef][Medline]
Nakayama, H. and Oda, Y. (2004). Common sensory
inputs and differential excitability of segmentally homologous reticulospinal
neurons in the hindbrain. J. Neurosci.
24,3199
-3209.
Neumeister, H., Ripley, B., Preuss, T. and Gilly, W. F. (2000). Effects of temperature on escape jetting in the squid Loligo opalescens. J. Exp. Biol. 203,547 -557.[Abstract]
Nissanov, J., Eaton, R. C. and DiDomenico, R. (1990). The motor output of the Mauthner cell, a reticulospinal command neuron. Brain Res. 517, 88-98.[CrossRef][Medline]
O'Malley, D. M., Kao, Y. H. and Fetcho, J. R. (1996). Imaging the functional organization of zebrafish hindbrain segments during escape behaviors. Neuron 17,1145 -1155.[CrossRef][Medline]
Otis, T. S. and Gilly, W. F. (1990).
Jet-propelled escape in the squid Loligo opalescens: concerted
control by giant and non-giant motor axon pathways. Proc. Natl.
Acad. Sci. USA 87,2911
-2915.
Preuss, T. and Faber, D. S. (2003). Central
cellular mechanisms underlying temperature-dependent changes in the goldfish
startle-escape behavior. J. Neurosci.
23,5617
-5626.
Preuss, T. and Gilly, W. F. (2000). Role of prey-capture experience in the development of the escape response in the squid Loligo opalescens: a physiological correlate in an identified neuron. J. Exp. Biol. 203,559 -565.[Abstract]
Preuss, T., Osei-Bonsu, P. E., Weiss, S. A., Wang, C. and Faber,
D. S. (2006). Neural representation of object approach in a
decision-making motor circuit. J. Neurosci.
26,3454
-3464.
Prugh, J. I., Kimmel, C. B. and Metcalfe, W. K.
(1982). Noninvasive recording of the Mauthner neurone action
potential in larval zebrafish. J. Exp. Biol.
101, 83-92.
Szabo, T. M., Weiss, S. A., Faber, D. S. and Preuss, T.
(2006). Representation of auditory signals in the M-cell: role of
electrical synapses. J. Neurophysiol.
95,2617
-2629.
Wilson, D. M. (1959). Function of giant Mauthner's neurons in the lungfish. Science 29,841 -842.
Wine, J. J. and Krasne, F. B. (1972). The
organization of escape behavior in the crayfish. J. Exp.
Biol. 56,1
-18.
Zottoli, S. J. (1977). Correlation of the
startle reflex and Mauthner cell auditory responses in unrestrained goldfish.
J. Exp. Biol. 66,243
-254.
Zottoli, S. J. and Faber, D. S. (2000). The Mauthner cell: what has it taught us? Neuroscientist 6, 25-37.
Zottoli, S. J., Hordes, A. R. and Faber, D. S. (1987). Localization of optic tectal input to the ventral dendrite of the goldfish Mauthner cell. Brain Res. 401,113 -121.[CrossRef][Medline]
Zottoli, S. J., Bentley, A. P., Prendergast, B. J. and Rieff, H. I. (1995). Comparative studies on the Mauthner cell of teleost fish in relation to sensory input. Brain Behav. Evol. 46,151 -164.[Medline]
Zottoli, S. J., Newman, B. C., Rieff, H. I. and Winters, D. C. (1999). Decrease in occurrence of fast startle responses after selective Mauthner cell ablation in goldfish (Carassius auratus). J. Comp. Physiol. A 184,207 -218.[CrossRef][Medline]
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