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First published online June 29, 2007
Journal of Experimental Biology 210, 2526-2539 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.003939
Modulation of locomotor activity in larval zebrafish during light adaptation
Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6058, USA
* Author for correspondence (e-mail: granatom{at}mail.med.upenn.edu)
Accepted 3 May 2007
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Summary |
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Key words: zebrafish, behavior, tracking, locomotion, light adaptation, visual startle, escape, masking
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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;
Samuel and Sengupta, 2005) but
remain elusive in vertebrates. A key factor facilitating the success of
analyses in invertebrates has been the ability to quantify directly the
initiation and modulation of intrinsic motor patterns. Objective
quantification is complicated in higher vertebrates by the enormous complexity
and diversity of the locomotor repertoire. However, the sophisticated
behavioral repertoire of adult animals is built upon a more limited set of
simple early behaviors, hard-wired into the brain during embryonic
development. These genetically encoded sensory–motor interfaces must be
sufficient to enable the developing organism to locate a secure and nourishing
environment, and to detect and avoid predators.
The relative simplicity of the juvenile brain and restricted set of
behaviors in young animals offer advantages for studying behavioral selection.
Similarly, it has long been recognized that the nervous system of fish offers
an opportunity to study fundamental neuronal pathways without the many complex
neuronal circuits elaborated in mammals
(Stahl, 1977). Thus for
studies of the neuronal basis of behavior, larval fish have the dual
advantages of a functional nervous system of limited complexity, and
phylogenetic relevance to functional neuroanatomy in mammals. Larval zebrafish
perform several locomotor behaviors, including the optomotor response
(Clark, 1981), prey tracking
(Gahtan et al., 2005;
McElligott and O'Malley,
2005), phototaxis (Brockerhoff
et al., 1995; Orger and Baier,
2005) and multiple modes of escape response
(Kimmel et al., 1974) (H.A.B.
and M.G., unpublished). These behaviors are constructed from a small
repertoire of motor patterns, including routine turns
(Budick and O'Malley, 2000),
J-turns (McElligott and O'Malley,
2005), slow scoots (Budick and
O'Malley, 2000), burst swims
(Budick and O'Malley, 2000;
Gahtan et al., 2005;
Muller and van Leeuwen, 2004),
capture swims (Borla et al.,
2002) and C-starts (Kimmel et
al., 1974). Descriptive level models of how different maneuvers
are combined into adaptive behaviors have been reported; however, direct
quantitation of motor events would facilitate elucidation of the underlying
neural circuitry.
Visually guided behaviors, including the optomotor response and predation,
have been extensively studied in zebrafish
(Neuhauss, 2003), but little
is known about how simple changes in illumination effect larval behavior.
Previous studies have described a startle response to abrupt decrements in
light (Easter and Nicola,
1997; Kimmel et al.,
1974), possibly in order to avoid looming predators. In addition
to triggering visual startle responses
(Hopf et al., 1973;
Yates, 1981), in mammals
photic stimuli trigger pupillary contraction
(Keeler, 1927) and acutely
suppress pineal melatonin synthesis (Klein
and Weller, 1972; Lewy et al.,
1980). It is now clear that locomotor activity in mammals is
controlled by both endogenous circadian rhythms and acute light exposure
(Aschoff, 1960). Light acutely
suppresses locomotor activity in nocturnal mammals, but promotes activity in
diurnal mammals (reviewed in Redlin,
2001). The direct effect of light on activity is mediated by a
non-image forming visual pathway, starting with melanopsin expressing
intrinsically photoreceptive retinal ganglion cells
(Hattar et al., 2003;
Panda et al., 2003). This
phenomenon, known as masking, can countermand circadian signals regulating
activity levels and constitutes a parallel system for matching behavioral
states with the diurnal cycle.
Most previous descriptions of the larval zebrafish motor repertoire have
relied on comparing the kinematic performance of behaviors that have been
classified by an observer (Budick and
O'Malley, 2000; McElligott and
O'Malley, 2005). Subjective classification of response types may
fail to identify behaviors distinguished by subtle differences and precludes
quantification of the large numbers of events desirable for measuring changes
in behavior elicited by experimental manipulations. We therefore sought to
automate the measurement of motor activity in zebrafish larvae and classify
responses on the basis of quantitative measures of movement kinematics. We
used automated analysis to measure behavioral responses to changes in
illumination, examining effects on motor behavior at temporal windows ranging
from milliseconds to hours. Our data show a role for direct photic modulation
of locomotor activity in zebrafish larvae, similar to masking phenomena in
higher vertebrates, and reveal a novel motor pattern elicited by sudden
decrements in light intensity.
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Materials and methods |
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Video recording
High speed video imaging was carried out with a Motionpro camera (Redlake,
Tucson, AZ, USA) at 1000 frames s–1, 512x512 pixel
resolution, using a 50 mm macro lens. Experiments were carried out at
26–28°C in a dark room with the experimental setup further isolated
by a black shroud such that light (apart from the infra-red array, below) in
the testing area was <10 nW cm–2 (designated as `darkness'
in the text). Larvae were tested in the same 6 cm dishes as they were raised,
at a density of 30/7 ml. For dark recording, larvae were illuminated using a
custom built array of 50 infra-red (880 nm peak) LEDs (remounted
R30-123-881-120AN, Ledtronics, Torrance, CA, USA), 43 mmx50 mm in size,
mounted 75 mm below the testing arena. We measured the spectrum of the
infrared LED array and found that the integral of the spectrum below 650 nm
was 55 nW cm–2. As the spectral sensitivity function in
larval zebrafish falls off after 620 nm
(Brockerhoff et al., 1997) it
is unlikely that the infra-red LED array provided significant visual
stimulation. Consistent with this, Brockerhoff et al. noted that no
optokinetic response is elicited by infrared light
(Brockerhoff et al., 1995).
Behavioral assays
Unless otherwise specified, all larvae were tested after being light
adapted for at least 3 h at 
65 µW cm–2. To ensure
even exposure to light before testing, each plate was illuminated from below
by a separate LED on a custom built light board with control of intensity
using sheets of GamColor neutral density filters (GAM, Los Angeles, CA, USA).
The light sources in all experiments were 5 mm white LEDs (Jameco Electronics,
320531, Belmont, CA, USA), with intensity controlled by adjusting voltage of
the power source and exchanging neutral density filters (Newport, Irvine, CA,
USA). For diffusion we used 3.0 mm white acrylic (ACRY2447, Modern Plastics,
Los Angeles, CA, USA) after finding minimal effect on the spectrum of
transmitted light. For `dark adaptation', larvae were maintained in constant
darkness for at least 12 h. In all assays, larvae were placed on the testing
apparatus 3 min before beginning the experiment to minimize effects of
handling and to allow larvae to adjust to any small differences in
illumination from the light board (supplementary material Fig. S1). Light
intensity was measured using either a Reed LX-1102 photometer (Calright
Instruments, San Diego, CA, USA) or an IL-1400A-SEL033FW radiometer
(International Light, Peabody, MA, USA). Where appropriate, the magnitude of
changes in illumination is indicated by the log of the ratio of the final
intensity and the initial intensity (logI). A Stamp BS2sx
microcontroller (Parallax, Rocklin, CA, USA) was used to coordinate activation
of the video and light stimuli. In all behavioral assays, the duration of
video recordings was either 400, 500 or 1000 ms (as indicated in the figures),
either immediately after the stimulus or at designated time points. The
apparatus for eliciting and measuring acoustic startle responses will be
described elsewhere (H.A.B. and M.G., unpublished), but briefly, responses
were elicited using a minishaker (4810, Bruel and Kjaer, Naerum, Denmark),
controlled by a digital-analogue card (PCI-6221, National Instruments, Austin,
TX, USA). Circuit diagrams and pBASIC programs will be provided upon request.
Details of the behavioral assays used are described in the figure legends.
Kinematic analysis
All kinematic analysis software was written in the IDL Development
Environment (ITT Visual Information Solutions, Boulder, CO, USA). Analysis of
video sequences is accomplished in four steps.
). A bandpass filter is applied to each image in the stack so
that the head of each fish is converted to a roughly circular shape indicating
a local maximum (supplementary material Fig. S2A). This position is
137±42 µm (N=120 larvae, mean ± s.d.) caudal to the
midpoint of the eyes (and approximately 380 µm caudal to the tip of the
snout) and is not significantly influenced by pigmentation pattern
(supplementary material Fig. S2B) or vigorous movement (supplementary material
Fig. S2C). The coordinates of all local maxima are identified in the first
frame of the recording, then each maxima is individually tracked by finding
the nearest maxima in subsequent frames.
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) is the integral angle traversed between
the initial head orientation (Oi) and the orientation at the peak
of the first curvature sinusoid (Op in
Fig. 1Bii, equivalent to the
C-bend angle at the end of stage 1 for C-starts). Distance (d) is the
total path length traveled by a larva during a movement episode measured by
summing the movement of the head position from frame to frame
(Fig. 1Biii). Displacement
(
) is the straight-line change in the head position of the larva from
the first to the last frame. Trajectory (
) is the vector angle of
movement, relative to the initial orientation of the fish. This is calculated
by taking the angle between the vector (V) from the initial head
position to the final head position and the initial orientation
(Oi) of the larva (V–Oi in
Fig. 1Biv). Other measurements
include `duration', the interval in ms from the start of movement to the peak
of the first sinusoid, the `maximal angular velocity', the greatest change in
head orientation until the peak of the first sinusoid, the `swim yaw', the
mean amplitude of head swings during swimming after the first bend and
counterbend (calculated by taking the average change in head orientation
during each half-cycle) and the `swim rhythm', the average peak-to-trough
duration in ms of sinusoids following the initial peak/trough pair. This
latter measurement is inversely proportional to tail beat frequency: tail beat
frequency=1000/(2x swim rhythm). Bend angle and amplitude are used to
classify movement type, as described in the text.
Larvae engaged in swimming at the beginning of a video recording (in the first 10 ms) were excluded from analysis, as were larvae initiating movement in the last 20 ms, as insufficient frames remain to determine motor pattern type. This typically resulted in elimination of 5–10% of traces from further analysis. Motor pattern kinematics cannot be measured if larvae begin a movement bout lying on their side. As recordings are made from above, the visibility of both eyes is a good surrogate for vertical posture. To find the eyes, a second bandpass operation is performed in the region centered on each head and local maxima recorded. Maxima anterior to the midpoint of the head are counted. Only larvae with two anterior local maxima are further analyzed. Generally 2–3% of larvae are excluded by this procedure. Analysis software is available upon request.
Laser ablations
To visualize reticulospinal neurons, a 50% solution of
fluorescein-conjugated dextran 10 K Mr (Invitrogen,
Carlsbad, CA, USA) in 10% Hanks' saline was pressure injected into the ventral
spinal cord of 4 d.p.f. larvae. The next day, larvae were treated briefly with
0.03% tricaine (3-aminobenzoic acid ethyl ester, Sigma, St Louis, MO, USA) and
mounted in methylcellulose or 2% low-melt agarose. Ablations were performed
using a Micropoint pulsed nitrogen laser (Photonic Instruments, St Charles,
IL, USA) mounted on a compound microscope (Carl Zeiss, Thornwood, NY, USA)
with a 63x water lens. Cells were pulsed for 30 s at 10 Hz. Larvae were
remounted after 6 h and inspected. As previously reported, after ablation the
Mauthner axon stump was clearly visible in many instances
(Liu and Fetcho, 1999),
whereas following unsuccessful ablations fluorescence returned to the Mauthner
cell. Larvae were individually tested for dark-flash and acoustic startle
responses on 6 d.p.f. At the end of the experiment, larvae were fixed in 4%
paraformaldehyde and stained with 3A10 antibody (1:50, kind gift of Dr T.
Jessell) (Hatta, 1992) and
Alexa-594 conjugated goat anti-mouse antibody (Invitrogen) to confirm complete
elimination of both Mauthner cells in lesioned larvae. This procedure resulted
in successful lesion of 80% of Mauthner cells targeted.
Statistical analyses
All Student t-tests are two-tailed, independent sample, assuming
equal variance unless noted and were performed using Excel (Microsoft,
Redmond, WA, USA). Analysis of variance (ANOVA) and non-linear regression
analysis was carried out using SPSS 14.0 (SPSS, Chicago, IL, USA). P
values were subjected to Bonferroni correction to maintain alpha=0.05.
Gaussian fitting was carried out using D. Lindler's xgaussfit tool
for IDL. Spectral analysis of ultraradian time series was performed using the
fast Fourier transform implementation in IDL. Time constants were estimated by
fitting data with a single exponential function using CurveExpert v1.3
(Hixson, TN, USA). Values are means ± s.e.m., except where otherwise
noted.
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Results |
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), by adding the angles made by the head and tail with the
mid-body segment (Fig. 1Bii).
Plotting the curvature of a fish over time reveals smooth oscillations
reflecting the sinusoidal motion of swimming
(Fig. 1C).
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) and
15.7±3.7 ms in our measurements at 28°C), the accuracy is
sufficient to ensure measurement of the bend amplitude at the sinusoid peak
and thus distinguish turns from smaller movements. This shows that movement
episodes can be reliably identified by the computer, permitting automated
analysis of kinematics, using the primary measurements of position,
orientation and curvature.
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). Forward directed swim bouts have been previously
referred to as `slow' or `burst' swims, depending on the vigor of the
side-to-side movements (Budick and
O'Malley, 2000; Ouagazzal et
al., 2001; Thorsen et al.,
2004). However, as behavioral measurements suggest that the
intensity of rhythmic activity during swimming constitutes a continuous
variable (H.A.B. and M.G., unpublished), here we will simply refer to forward
movements as `scoots' (Fig.
2A). `Routine turns', here referred to as `turns', are initiated
with large changes in body curvature and head angle
(Fig. 2B). Indeed, the
histogram of initial bend amplitudes for 4199 movement episodes initiated by
larvae in the absence of extraneous stimulation reveals a bimodal
distribution, consistent with the presence of scoots and turns
(Fig. 2C). Scatter analysis of
a separate 1681 spontaneous movement episodes shows that larvae initiating
locomotor activity with small bend amplitudes (less than 35°) also tend to
show only slight changes in head orientation (less than 20°), allowing the
two types of movement to be distinguished on the basis of these two criteria
(Fig. 2D).
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Analysis of kinematic parameters of movement episodes automatically
classified as scoots or turns affirms that these represent distinct locomotor
behaviors. Scoots are almost completely forward movements
(Fig. 2E, trajectory
17±23°, mean ± s.d., N=672), whereas turns cause
larvae to swim at an angle to their initial orientation (62±30°,
N=1009) and result in a greater displacement
(Fig. 2F, 1.57±1.17 mm
for turns versus 0.91±0.57 mm for scoots, independent sample
t-test P<10–10). The second peak of the
sinusoid, representing the counterbend for turns, is also different for scoots
and turns (Fig. 2G,H).
Following these first two components of the sinusoid, turns are followed by an
average of 3.6±2.0 tail beats, and scoots by 2.8±1.8 movements.
The mean yaw of scoots is slightly but significantly smaller than for turns
(Fig. 2I). In addition, the
average duration of tail flips (denoted `rhythm') is slightly less in turns
(Fig. 2J). Mean swim rhythm for
turns and scoots is 12.6±2.7 ms and 13.7±1.7 ms, respectively,
yielding tail-beat frequencies of 39.6 Hz and 36.4 Hz, respectively, in
agreement with previously reported values
(Borla et al., 2002;
Budick and O'Malley, 2000;
Muller and van Leeuwen, 2004).
These results demonstrate that the two major locomotor maneuvers reported by
human observers can also be identified on the basis of their distinctive
kinematic properties, permitting automated analyses of behavior.
Responses of zebrafish larvae to transient changes in irradiance
We first considered the effect of transient increases in lighting for
larvae pre-adapted to 20 µW cm–2 of white light. A 500 ms
`light flash' of 200 µW cm–2 evoked an increase in turn
responses from 19.6±4.4% initiations per 400 ms window in baseline
controls to 57.4±6.8% in the light-flashed groups (N=8 each
condition, two-tailed t-test, P=0.0064), but no changes in
the frequency of turn initiations from 1 s to 5 min after the stimulus
(Fig. 3A). Scoot initiations
were reduced immediately after the stimulus, likely because the larvae were
instead initiating turns, but thereafter returned to baseline levels and
remained stable (data not shown). More intense light elicited a greater
increase in turn initiations (Fig.
3B; N=5 each intensity, one-way ANOVA
F(4,20)=16.4, P<0.001). A similar pattern was
seen for brief decrements in illumination (`dark flashes' of 1 log unit
intensity). A significant spike in turn initiations was observed immediately
after the stimulus, from 24.9±3.6% initiations per 500 ms window in
baseline controls to 86.6±5.2% in the dark-flashed groups
(N=10 each condition, two-tailed t-test,
P<10–10), with the frequency of turns returning
to baseline at 1 s through 5 min after the stimulus
(Fig. 3C). Scoot initiations
were not affected. The acute increase in turn initiations was proportional to
the magnitude of the change in illumination
(Fig. 3D; N=6 each
intensity, one-way ANOVA F(5,30)=31.6,
P<0.001). Thus, both sudden increases and reductions in irradiance
provoke immediate turn responses.
Because abrupt sensory stimuli in a variety of modalities elicit startle
responses in larval zebrafish, we next asked whether visually evoked turn
responses show kinematic similarities to startle responses. Scatter analysis
showed that turns evoked by dark flashes (N=506) have markedly larger
bend angles relative to their angular velocity than acoustic startle responses
(N=269) (Fig. 4A).
Only six out of 166 routine turns achieved bend angles over 100°
(Fig. 4A), and five of these
instances had an angular velocity similar to that of acoustic startle
responses, suggesting that the larvae may have responded to environmental
cues. Acoustic and touch-evoked startle responses in zebrafish larvae are
initiated within 15 ms of the stimulus
(Liu and Fetcho, 1999). In
contrast, turns elicited by light flashes are initiated at a latency of
183±93 ms (Fig. 4B) and
turns initiated in response to dark flashes are even more delayed, with a mean
latency of 408±105 ms (Fig.
4C). Response latencies are somewhat variable; in other
experiments, we found that dark-flash turn latencies varied with the degree
and duration of light adaptation and could be as short as 150 ms (data not
shown). However, in every experiment we observed responses with latencies
exceeding 500 ms. The protracted latency of visually evoked turns is
unexpected and suggests that the function of these responses may not be to
escape from predators.
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The escape response in adult fish is directed away from threatening visual
stimuli (Dill, 1974;
Domenici, 2002). We analyzed
turn direction according to initial orientation. Surprisingly, larvae
responded to dark-flash stimuli by turning towards the position of the dimmed
light (Fig. 4H). Binning
orientations by quadrant, for larvae initially facing the light with their
right eye, 83.9±2.5% of turns were to the right, compared to
30.2±7.6% rightward turns for larvae facing the light with their left
eye (mean ± s.e.m., t-test,
P=1.8x10–4; five groups tested with 20 dark
flashes each). Hence dark-flash responses differ from visual escape responses
in the direction of their trajectory.
High-performance acoustic startle responses in zebrafish larvae are mediated by the bilateral Mauthner cells (H.A.B. and M.G., unpublished). The slow kinematics of dark-flash responses suggest that Mauthner cells may not be involved in this motor pattern. To test this idea, we laser ablated both Mauthner cells (Fig. 5A) and asked whether dark-flash responses were impaired. As expected, lesion of both Mauthner cells completely eliminated short latency startle responses to acoustic stimuli (Fig. 5B). In contrast, both responsiveness (Fig. 5C; two-tailed t-test, P=0.89) and kinematics (Fig. 5D; two-tailed t-test, P>0.1 for all measures) of C-bend responses to dark-flash stimuli were indistinguishable in ablated larvae and controls, arguing against involvement of Mauthner cells in the performance of the dark-flash response. As dark-flash responses differ from acoustic startle responses in lacking Mauthner cell dependence, and show distinct kinematic properties from routine turns, they constitute a novel motor pattern. We refer to these maneuvers as O-bends, reflecting the near circular shape achieved by the larvae, and their appearance during light-off stimuli (Fig. 4I).
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Zebrafish larvae express rhythmic locomotor activity controlled by a
circadian clock (Cahill et al.,
1998; Prober et al.,
2006). It is possible that the changes we observed in locomotor
activity following light and dark shifts were secondary to phase shifting of
the intrinsic oscillator. To address this possibility, we measured locomotor
activity over 24 h using ultraradian cycles consisting of 1 h light and 1 h
dark. Such short light:dark cycles preclude circadian entrainment in diverse
species (Aschoff, 1999)
including fish (Sanchez-Vazquez et al.,
1996; Sanchez-Vazquez and
Tabata, 1998). Consistent with activity levels being directly
modulated by light, spectral analysis of the time series obtained revealed a
harmonic peak for both scoots and turns at 121 min. Non-linear regression
using a sinusoidal model confirmed that a significant component of variance in
activity levels was accounted for by a periodic factor of close to 120 min
(scoots: 119.9±0.91 min, r2=0.510; turns:
120.6±1.08 min, r2=0.427; estimate ±95%
confidence interval). Thus, under such conditions, larvae exhibited cyclic
motor activity, with initiations of both scoots and turns being maximal during
light periods (Fig. 6Di). The
mean frequency of turn initiations during light periods was 14.0±0.58%
(per 400 ms window), significantly higher than the initiation frequency of
7.7±0.8% during dark periods (two-tailed t-test,
P<10–10). Scoot initiations were 17.3±0.5%
in light versus 9.4±0.5% during dark episodes (two-tailed
t-test, P<10–10). Combining data from
all cycles confirmed that behavioral adaptation to onset of illumination is
rapid, occurring within 5 min, whereas motor activity gradually declines
starting 5–10 min after the onset of darkness
(Fig. 6Dii). These results
argue that the effect of light on activity levels in zebrafish larvae is
direct, similar to masking stimuli in mammals.
We next sought to measure the kinetics of dark and light adaptation. Measurement of locomotor activity during the first 7 min of dark adaptation (Fig. 6E) showed that the initiation frequency of scoots was transiently elevated, peaking 1 min after dark adaptation (12.6% initiations per 400 ms window for controls in constant illumination versus 26.7% for larvae exposed to dark, two-tailed t-test, P<10–4). Apart from the spike in turns in response to the change in illumination, turns remained constant over the first 7 min of dark adaptation. In contrast, measurement of locomotor activity in the first 7 min of light adaptation (Fig. 6F) demonstrated that after an acute spike in both scoots and turns provoked by the change in lighting, there was a lag phase of just 1 min before locomotor initiations rapidly increased to levels characteristic of light adapted larvae. After 2 min of light adaptation, initiations of both scoots and turns were significantly elevated above controls (scoots: 1.6% initiations per 400 ms window for controls in constant darkness versus 14.3% for larvae exposed to light; two-tailed t-test, P=0.012; turns: 1.1% for controls versus 11.9% for larvae exposed to light; two-tailed t-test, P=0.0018). Changes in locomotor activity during light and dark adaptation therefore have distinct time courses. Locomotor activity rapidly increases during light adaptation after a short lag phase. In contrast, dark adaptation triggers a biphasic behavioral program. For the first few minutes, net locomotor activity increases. After 5–10 min of constant darkness, locomotor activity begins to decline, reaching baseline levels within 30 min of the onset of darkness.
In the course of experiments, we noticed that dark-adapted larvae showed
little responsiveness to dark flashes. This enabled us to measure the time
course of acquisition or loss of responsiveness to dark flashes as an
alternate measure of the kinetics of light and dark adaptation. Light-adapted
larvae shifted to darkness did not lose responsiveness to dark flashes for the
first 3 min, but thereafter rapidly lost responsiveness with a time constant
of 10 min (Fig. 7A). Thus,
the temporal course of dark adaptation as assessed by locomotor activity and
dark flash responsiveness is broadly similar.
During light adaptation from darkness, maximal dark-flash responsiveness
was achieved after 20 min of light adaptation, with a time constant of 7
min (Fig. 7B). In contrast,
larvae already adapted to light quickly adjusted to brighter illumination.
After a shift to more intense illumination, light adapted larvae responded to
`dim flashes' (down to the original level of illumination) after just 1 s of
adaptation and reached maximal adaptation after 60 s
(Fig. 7C). Thus the slow
kinetics for recovery of O-bend responsiveness during adaptation from darkness
cannot be accounted for by postulating that dark flash responsiveness is
generally slow to adapt to changes in light levels. The differential time
course of changes in locomotor activity and O-bend responsiveness during light
adaptation from darkness argues that behavioral light adaptation is not a
unitary process, but rather involves changes in several regulatory
circuits.
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Analyzing larvae recorded in groups makes it possible to examine large
numbers of motor events and facilitates quantitative analysis of behavior. A
limitation of this technique is that the resolution of individual larvae is
reduced and finer aspects of motor control cannot be examined. For example, we
are not able to measure the contribution of pectoral fins to forward
propulsion and braking (Budick and
O'Malley, 2000; Thorsen et
al., 2004), nor of fine tail movements for reorientation during
predatory strikes (McElligott and
O'Malley, 2005). A further limitation is that a single camera
mounted from above can only record movements in the horizontal plane. On the
other hand, automated measurement of behavior allows motor patterns to be
classified on the basis of kinematic features. An observer-independent
approach can reveal unanticipated motor patterns. The large turns recorded
during dark flashes are kinematically distinct from other types of motor
patterns involving turn movements, being large angle, but slow performance,
suggesting that they form a distinct maneuver within the larval motor
repertoire.
We found that zebrafish larvae show elevated locomotor activity during
periods of bright illumination. This is likely to reflect a trade-off between
requiring light to feed (Clark,
1981; Gahtan et al.,
2005; McElligott and O'Malley,
2005) and the risk of predation. Thus, elevated locomotor activity
in the light enlarges the area searched for food, while during darkness, when
larvae do not efficiently feed, reduced locomotor activity minimizes the
chances of encountering and attracting the attention of predators
(Munk and Kiorboe, 1985). It
is therefore advantageous for zebrafish larvae to synchronize activity levels
to the diurnal cycle. The daily rhythm of activity in mammals is controlled by
both the endogenous circadian system and external light cues
(Aschoff, 1960). While
circadian control of activity has been well documented in fish, fewer studies
have explicitly addressed whether light stimuli can directly modulate fish
behavior. Acute effects of light on adult fish behavior have been described
for fry retrieval and fanning behavior
(Reebs, 1994), feeding and
locomotor activity (Sanchez-Vazquez et
al., 1996; Sanchez-Vazquez and
Tabata, 1998). Moreover, we interpret data in a recent study on
hypocretin/orexin control of sleep/wake behavior in larval zebrafish as
showing that light exposure during circadian night induces elevated locomotor
activity [fig. 5D in Prober et
al. (Prober et al., 2006)].
Our demonstration that light exposure directly modulates locomotor activity in
larval zebrafish throughout the circadian cycle will facilitate molecular
genetic analyses of masking responses.
Direct photic control of activity may serve to fine-tune the inaccurate
free-running circadian periods measured in a variety of fish species including
larval zebrafish (Cahill et al.,
1998; Hurd and Cahill,
2002). In larval zebrafish, locomotor activity is subject to
circadian control from the onset of spontaneous movement at day 4, with
maximal activity during early subjective day
(Hurd and Cahill, 2002). Our
results demonstrate that, as in mammals, light/dark cues can override activity
levels set by the endogenous clock. In mammals such masking stimuli are most
effective when they coincide with the activity period set by the circadian
clock (Aschoff, 1999;
Redlin and Mrosovsky, 1999).
Further experiments will be required to determine whether masking stimuli and
circadian rhythms interact in a similar way in larval zebrafish.
Following the onset of darkness, larvae showed a transient elevation in
scoots prior to a gradual drop-off in motor activity. As many diurnal fish
species engage in cover-seeking activity at dusk
(Helfman, 1986), it is
possible that the hyperactivity we observed is aimed at finding shelter prior
to night. However, the onset of night is not the only condition in which a
larva may find itself in darkness. Accidental navigation under debris may also
occlude light. Dark induced hyperactivity may therefore serve to facilitate
navigation back to areas of illumination if the darkness is not due to
nightfall.
We propose that the O-bend responses to dark flashes may also serve to
maintain larvae in well-lit environments. An alternative hypothesis is that
abrupt reductions in illumination represent the shadow of a potential
predator, and that the large angle turns elicited are a precursor to the adult
zebrafish visual startle response (Easter
and Nicola, 1997). Two lines of evidence argue that O-bend
responses are navigational rather than defensive. First, we found that
responses to light occlusion are made towards the direction of the occluded
light. This would displace the larva towards a potential predator. In
contrast, escape trajectories in adult fish displace fish away from predators
(Dill, 1974;
Domenici, 2002). The visual
startle response in adult fish is generally elicited using a looming stimulus
in which rapid expansion of an object in the visual field simulates predator
approach. Zebrafish larvae also respond to looming stimuli by turning away
from the potential threat (H.A.B. and M.G., unpublished). It therefore seems
unlikely that larvae interpret sudden light occlusion as a potential threat.
Instead, a rapid drop in light intensity may constitute a distinct cue
occurring when a larva strays into a shaded environment. A 180° turn would
reorient the larva towards the well-lit region from which it came.
Second, stimulation of the optic nerve in adult fish can bring the Mauthner
cell to threshold (Zottoli et al.,
1987) and visual stimuli can elicit C-starts with similar
kinematics to acoustic/vibrational startle responses
(Dill, 1974;
Eaton et al., 1977). Our data
show that larval responses to abrupt light decrements are not mediated by the
Mauthner cell and show distinct kinematics from acoustic startle responses.
Dark-flash responses have a broad latency distribution, with responses often
being initiated several hundred ms after the stimulus. By comparison, acoustic
startle latencies in larvae range from 4–12 ms. The long latency and
slow performance of responses to sudden light occlusion are inconsistent with
a primarily defensive role. Escape responses to head-touch stimuli in larval
zebrafish are mediated by the Mauthner cell together with its segmental
homologs (Liu and Fetcho,
1999). Although we cannot exclude the possibility that the
Mauthner homologs mediate dark-flash responses, escape responses initiated by
these neurons have much greater angular velocity than dark-flash responses. It
is likely that O-bend responses are triggered by a distinct cohort of
reticulospinal neurons. Thus, in contrast to previous proposals that the
larval `visual startle' response is the precursor to the adult zebrafish
escape response (Easter and Nicola,
1997), we suggest that O-bend responses are not mediated by the
Mauthner circuit, are primarily navigational, and constitute one of several
mechanisms by which zebrafish larvae maintain themselves in an illuminated
region conducive to successful feeding.
Prolonged dark adaptation had two behavioral effects, reducing locomotor
activity and eliminating dark-flash responsiveness. The kinetics of dark
adaptation were similar for these two processes. Both behaviors were
maintained for the first 3 min of darkness then declined over the course of 30
min. In contrast the time course for recovery of responsiveness to dark
flashes during light adaptation (time constant=7 min) is slower than the
recovery of locomotor activity (turns; time constant=4 min) or recovery of the
optokinetic response (time constant=3 min)
(Page-McCaw et al., 2004). A
variety of mechanisms operating on different timescales are known to
participate in light adaptation (Dunn and
Rieke, 2006; Pugh et al.,
1999). Thus, the rapid normalization of sensitivity to dark
flashes after a shift to higher light levels may involve biochemical processes
in photoreceptors such as phosphorylation of cone opsins
(Kennedy et al., 2004),
whereas retinal network and possibly central adaptations are likely involved
in the slower adjustments to and from darkness.
Work on behavioral choice in invertebrates is beginning to shed light on
how the nervous system produces behavior. One reason for the productivity of
these studies has been the focus on describing circuits that activate
intrinsic and stereotyped motor patterns
(Briggman et al., 2005;
Gray et al., 2005).
Goal-directed behaviors are then understood as the outcome of sequential
activation of elements of the motor repertoire. Here we take a similar
approach in a vertebrate model, the larval zebrafish, showing that motor
patterns can be reliably measured and distinguished. Visual stimuli
differentially activate and modulate elements of the motor repertoire.
Following light extinction, larvae execute large angle turn responses toward
the vanished light source, then show transient locomotor activation before
slowly settling into a hypoactive state. After the onset of illumination,
larvae rapidly increase baseline activity levels. We propose that these
patterns of motor activation all serve to maximize time spent in well-lit
environments suitable for feeding. These results provide a foundation for
future studies examining neural mechanisms controlling the expression of motor
patterns in larval zebrafish.
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Acknowledgments |
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Footnotes |
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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=7.9; peak 2,
µ=59.6, 
*time/b3+b4), such that
b3 is the periodicity of the function (see text). (Dii) Mean initiation
frequency for scoots (open circles) and turns (closed circles) for each time
point during a 2 h period averaged over all 12 cycles. (E) Turn initiations
show a transient increase for 5 s following the switch to sustained darkness
(Ei, open circles, N=30 groups) compared to larvae maintained in
constant illumination (200 µW cm–2, closed circles,
N=30 groups). No change in turn initiations occurs over the next 7
min. Immediately after light extinction, scoot initiations (Eii) are slightly
reduced; however, after 60 s, scoots show a transient but highly significant
increase above baseline levels. For each group of 400 ms recordings were
collected at the indicated time points (*P<0.05,
t-test versus constant light). (F) Behavioral light
adaptation begins 60 s after dark-adapted embryos are exposed to bright light
(140 µW cm–2, open circles, N=20 groups). After
an initial spike in turns (Fi) elicited by the abrupt change in illumination,
there is a lag of approximately 1 min in which turn initiations remain at
similar levels to larvae maintained in constant darkness (closed circles,
N=20 groups). Thereafter turn initiations rapidly climb to
light-adapted levels. Scoot initiations (Fii) show a similar pattern, with an
acute spike following light onset, a lag phase of 60 s, then a rapid increase
to normal light-adapted levels (*P<0.05,
t-test versus constant dark).