The descending control of locomotor behaviors is an area of
neurobiology with many unanswered questions. In the case of
mammals, the millions of descending bers that project into
the spinal cord, together with their numerous origins, pose a
considerable problem. Understanding just one subset of these
descending neurons, for example the reticulospinal neurons, is
difficult because of the variety of reticulospinal cell types and
their intermingling with other cell types in the brainstem
(Brodal, 1981; Siegel and Tomaszewski, 1983). This problem
is reduced in scope in shes because of the reduced numbers
of nerve cells and nuclei that project to the spinal cord, but
even in these simpler systems we do not have a cellular-level
understanding of the neural control systems. A recent technical
advance in this area has been the use of uorescent Ca
2+
indicators to label larval zebra sh neurons retrogradely. This
permits optical recording of neural/Ca
2+
activity in the spinal
cord (Fetcho and O Malley, 1995) and brainstem (O Malley et
al., 1996) of intact larval zebra sh. This same technique
facilitates the laser-ablation of speci c neurons, after which
behavioral de cits can be quanti ed using high-speed
behavioral recordings (Fetcho and Liu, 1999). In addition to its
transparency, the larval zebra sh ( Brachydanio rerio) is
relatively simple (for a vertebrate animal), and many of the
neurons that project from the brain into the spinal cord can be
individually identi ed, both in histological preparations
(Metcalfe et al., 1986; Bernhardt et al., 1990; Eisen, 1999) and
in vivo, using confocal microscopy (Fetcho and O Malley,
1997; Fetcho et al., 1998). These optical approaches have
recently been applied to studies of the escape behavior.
The Mauthner cell is a command neuron that, in teleost sh,
triggers an escape response each time it res an action potential
(Zottoli, 1977; Kimmel et al., 1980; Eaton et al., 1981; Faber
et al., 1989). The involvement of two other reticulospinal
neurons in the escape behavior (cells MiD2cm and MiD3cm)
was rst suggested on the basis of their anatomical similarity
to the Mauthner cell (Metcalfe et al., 1986). This anatomical
similarity, together with quantitative electromyographic and
kinematic analyses, led to the proposal that these cells provide
directional control of the escape response (Foreman and Eaton,
1993). Optical recordings of the neural activity of these
cells during escape responses (O Malley et al., 1996) and
subsequent laser-ablation experiments (Liu and Fetcho, 1999)
con rmed the hypothesis of Foreman and Eaton (1993) and
demonstrated that these neurons play a controlling role in this
2565
The Journal of Experimental Biology 203, 2565—2579 (2000)
Printed in Great Britain ' The Company of Biologists Limited 2000
JEB2652
Larval zebra sh ( Brachydanio rerio) are a popular
model system because of their genetic attributes,
transparency and relative simplicity. They have
approximately 200 neurons that project from the
brainstem into the spinal cord. Many of these neurons can
be individually identi ed and laser-ablated in intact larvae.
This should facilitate cellular-level characterization of the
descending control of larval behavior patterns. Towards
this end, we attempt to describe the range of locomotor
behavior patterns exhibited by zebra sh larvae. Using
high-speed digital imaging, a variety of swimming and
turning behaviors were analyzed in 6- to 9-day-old larval
sh. Swimming episodes appeared to fall into two
categories, with the point of maximal bending of the larva s
body occurring either near the mid-body (burst swims) or
closer to the tail (slow swims). Burst swims also involved
larger-amplitude bending, faster speeds and greater yaw
than slow swims. Turning behaviors clearly fell into
two distinct categories: fast, large-angle escape turns
characteristic of escape responses, and much slower
routine turns lacking the large counterbend that often
accompanies escape turns. Prey-capture behaviors were
also recorded. They were made up of simpler locomotor
components that appeared to be similar to routine turns
and slow swims. The different behaviors observed were
analyzed with regard to possible underlying neural control
systems. Our analysis suggests the existence of discrete sets
of controlling neurons and helps to explain the need for the
roughly 200 spinal-projecting nerve cells in the brainstem
of the larval zebra sh.
Key words: locomotion, swimming, zebra sh, Brachydanio rerio,
kinematics, prey capture, neuron.
Summary
Introduction
LOCOMOTOR REPERTOIRE OF THE LARVAL ZEBRAFISH: SWIMMING, TURNING
AND PREY CAPTURE
SETH A. BUDICK AND DONALD M. O MALLEY*
Department of Biology, 414 Mugar Hall, Northeastern University, Boston, MA 02115, USA
*e-mail: domalle@lynx.neu.edu
Accepted 7 June; published on WWW 9 August 2000

2566
behavior. Of particular signi cance is that these studies
provided direct evidence that serially homologous neurons in
successive hindbrain segments (i.e. the Mauthner cell,
MiD2cm and MiD3cm) contribute to a common behavior, the
escape response. Six other sets of potential segmental
homologues were also described by Metcalfe et al. (1986). This
may, therefore, be a general means by which brainstem
neurons are functionally organized, especially since the
hindbrain is relatively well-conserved across vertebrate species
(Fraser et al., 1990; Guthrie, 1995; Bass and Baker, 1997).
Young larval zebra sh (less then 7 days old) appear to have
approximately 200 neurons that project from the brain into
the spinal cord. These include roughly 100 reticulospinal
neurons, approximately 30 IC (ipsilateral caudal) neurons, and
approximately 20 each of T-reticular neurons, vestibulospinal
neurons and nucleus medial longitudinal fasciculus (MLF)
neurons (Metcalfe et al., 1986; Kimmel et al., 1985; E. Gahtan,
personal communication). Apart from the three speci c pairs
of neurons discussed above, the precise functional role of the
remainder of these 200 or so neurons is not known. But
descending locomotor control signals must be sent to the spinal
cord either through these neurons or perhaps through other as
yet unidenti ed descending neurons. Because the total number
of neurons appears to be relatively small, functionally
signi cant numbers of them (in principle, any desired subset)
can be speci cally targeted and laser-ablated. It should be
possible, therefore, to optically dissect the larva s descending
control system to elucidate the cellular control of swimming,
turning and other locomotor behaviors. Because these same
neurons are present and identi able in both adult zebra sh (Lee
and Eaton, 1991) and adult gold sh ( Carassius auratus) (Lee
et al., 1993), they may form the core of an adult teleost
locomotor control system (Prasada Rao et al., 1987).
Fishes, as a group, exhibit a great diversity of swimming
styles (Wardle et al., 1995; Van Raamsdonk et al., 1998).
Single species can exhibit a variety of swimming patterns, as
occurs in bluegill sun sh ( Lepomis macrochirus) in which
three distinct patterns were observed at successively greater
swimming speeds (Jayne and Lauder, 1994). Because of their
small size, larval swimming has not been examined in as great
detail. The early development of motor behaviors has recently
been characterized in zebra sh embryos (Saint-Amant and
Drapeau, 1998). In several larval shes, including plaice
(Pleuronectes platessa), herring (Clupea harengus) and
chinook salmon (Oncorhynchus tshawytscha), detailed
kinematics have been reported for burst swimming (Batty and
Blaxter, 1992; Hale, 1996). In larval and juvenile zebra sh,
ontogenetic changes in swimming speed and acceleration have
been reported along with duration and distance covered during
bouts of routine swimming (Fuiman and Webb, 1988), while
a more recent study characterized hydrodynamic ow patterns
around larval and adult zebra sh (M ller et al., 2000). In no
case, however, has a larval sh at a particular developmental
stage been reported to exhibit several distinct patterns of
swimming analogous to those observed in adult sh.
Less is known about turning behavior in shes. While
escape-related turning behaviors have been studied extensively
(see, for example, Kimmel et al., 1974; Foreman and Eaton,
1993), and kinematic data are available on the S-starts used in
predation (Domenici and Blake, 1997; Spierts and Van
Leeuwen, 1999), other more routine turning behaviors used in
navigation, foraging or related behaviors have been less studied
(McClellan and Hagevik, 1997). Fuiman and Webb (1988)
reported that in zebra sh larvae the proportion of swimming
bouts that begin with large-angle turns increases with the length
of the larva, but the frame rate of the video recordings used at
that time limited the kinematic analyses that could be
performed. Regarding prey capture by larval zebra sh, there
are, to our knowledge, no published high-speed kinematic
studies, although prey capture by other larval sh has been
shown to involve both ram- and suction-feeding strategies
(Drost and Van den Boogaart, 1986; Coughlin, 1994).
Zebra sh have attracted intense interest as a model
vertebrate organism, and many central nervous system and
behavioral mutants have recently been generated (see, for
example, Brockerhoff et al., 1995; Nicolson et al., 1998). Our
objective was to generate a catalogue of larval zebra sh
locomotor behaviors using high-speed digital imaging. While
high-speed imaging is known to be essential for examining fast
behaviors such as the escape response (Eaton et al., 1977;
Harper and Blake, 1989), precise kinematic analysis of even
the slower swimming and turning behaviors described here
required high-speed imaging. Our speci c goal in describing
the locomotor repertoire was to establish the range of behaviors
in which de cits might ultimately be produced by laser-
ablation experiments. We report here variations in swimming
and turning behaviors that have implications for the neural
control of locomotion.
Materials and methods
Animals
Fertilized eggs were collected from a breeding laboratory
population of zebra sh ( Brachydanio rerio), transferred to
10 % Hanks solution and maintained at approximately 25 ¡C
(Wester eld, 1995). After hatching, the larvae were kept under
the same conditions for the duration of the study. Maintaining
zygotes and larvae at this relatively low temperature slightly
retards both their rate of growth and depletion of the larval yolk
sac. This allows a somewhat longer period to study locomotor
behaviors before feeding the larvae becomes necessary. Larvae
were not fed prior to the evaluation of locomotor or feeding
behaviors. Behavioral observations were performed on sh
between 6 and 9 days post-fertilization. Unfed larvae continue
to grow during this period: the mean total length of the sh at
6 days post-fertilization was 3.68–0.14 mm (N=8), while 9-
day-old sh measured 3.93–0.13 mm (means – S.E.M., N=8).
Experimental protocols
To observe swimming and turning behaviors, larvae were
individually transferred to small plastic Petri dish lids (4 cm
diameter) containing 10 % Hanks solution. To observe
S. A. BUDICK AND D. M. O MALLEY