Melina E. Hale

A topographic map of recruitment in spinal cord

Animals move over a range of speeds by using rhythmic networks of neurons located in the spinal cord. Here we use electrophysiology and in vivo imaging in larval zebrafish (Danio rerio) to reveal a systematic relationship between the location of a spinal neuron and the minimal swimming frequency at which the neuron is active. Ventral motor neurons and excitatory interneurons are rhythmically active at the lowest swimming frequencies, with increasingly more dorsal excitatory neurons engaged as swimming frequency rises. Inhibitory interneurons follow the opposite pattern. These inverted patterns of recruitment are independent of cell soma size among interneurons, but may be partly explained by concomitant dorso-ventral gradients in input resistance. Laser ablations of ventral, but not dorsal, excitatory interneurons perturb slow movements, supporting a behavioural role for the topography. Our results reveal an unexpected pattern of organization within zebrafish spinal cord that underlies the production of movements of varying speeds.

A confocal study of spinal interneurons in living larval zebrafish.

We used confocal microscopy to examine the morphology of spinal interneurons in living larval zebrafish with the aim of providing a morphological foundation for generating functional hypotheses. Interneurons were retrogradely labeled by injections of fluorescent dextrans into the spinal cord, and the three‐dimensional morphology of living cells was reconstructed from confocal optical sections through the transparent fish. At least eight types of interneurons are present in the spinal cord of larval zebrafish; four of these are described here for the first time. The newly discovered cell types include classes of commissural neurons with axons that ascend, descend, and bifurcate in the contralateral spinal cord. Our reexamination of previously described cell types revealed functionally relevant features of their morphology, such as undescribed commissural axons, as well as the relationships between the trajectories of the axons of interneurons and the descending Mauthner axons. In addition to describing neurons, we surveyed their morphology at multiple positions along the spinal cord and found longitudinal changes in their distribution and sizes. For example, some cell types increase in size from rostral to caudal, whereas others decrease. Our observations lead to predictions of the roles of some of these interneurons in motor circuits. These predictions can be tested with the combination of functional imaging, single‐cell ablation, and genetic approaches that make zebrafish a powerful model system for studying neuronal circuits. J. Comp. Neurol. 437:1–16, 2001.

EVOLUTION OF BEHAVIOR AND NEURAL CONTROL OF THE FAST-START ESCAPE RESPONSE

Abstract The fast‐start startle behavior is the primary mechanism of rapid escape in fishes and is a model system for examining neural circuit design and musculoskeletal function. To develop a dataset for evolutionary analysis of the startle response, the kinematics and muscle activity patterns of the fast‐start were analyzed for four fish species at key branches in the phylogeny of vertebrates. Three of these species (Polypterus palmas, Lepisosteus osseus, and Amia calva) represent the base of the actinopterygian radiation. A fourth species (Oncorhynchus mykiss) provided data for a species in the central region of the teleost phylogeny. Using these data, we explored the evolution of this behavior within the phylogeny of vertebrates. To test the hypothesis that startle features are evolutionarily conservative, the variability of motor patterns and kinematics in fast‐starts was described. Results show that the evolution of the startle behavior in fishes, and more broadly among vertebrates, is not conservative. The fast‐start has undergone substantial change in suites of kinematics and electromyogram features, including the presence of either a one‐ or a two‐stage kinematic response and change in the extent of bilateral muscle activity. Comparative methods were used to test the evolutionary hypothesis that changes in motor control are correlated with key differences in the kinematics and behavior of the fast‐start. Significant evolutionary correlations were found between several motor pattern and behavioral characters. These results suggest that the startle neural circuit itself is not conservative. By tracing the evolution of motor pattern and kinematics on a phylogeny, it is shown that major changes in the neural circuit of the startle behavior occur at several levels in the phylogeny of vertebrates.

Grading movement strength by changes in firing intensity versus recruitment of spinal interneurons.

Animals can produce movements of widely varying speed and strength by changing the recruitment of motoneurons according to the well-known size principle. Much less is known about patterns of recruitment in the spinal interneurons that control motoneurons because of the difficulties of monitoring activity simultaneously in multiple interneurons of an identified class. Here we use electrophysiology in combination with in vivo calcium imaging of groups of identified excitatory spinal interneurons in larval zebrafish to explore how they are recruited during different forms of the escape response that fish use to avoid predators. Our evidence indicates that escape movements are graded largely by differences in the level of activity within an active pool of interneurons rather than by the recruitment of an inactive subset.

Structure, function, and neural control of pectoral fins in fishes

Fin-based propulsion systems perform well for both high-speed cruising and high maneuverability in fishes, making them good models for propulsors of autonomous underwater vehicles. Labriform locomotion in fishes is actuated by oscillation of the paired pectoral fins. Here, we present recent research on fin structure, fin motion, and neural control in fishes to outline important future directions for this field and to assist engineers in attempting biomimicry of maneuverable fin-based locomotion in shallow surge zones. Three areas of structure and function are discussed in this review: 1) the anatomical structure of the fin blade, skeleton, and muscles that drive fin motion; 2) the rowing and flapping motions that fins undergo for propulsion in fishes; and 3) the neuroanatomy, neural circuitry, and electrical muscle activity that are characteristic of pectoral fins. Research on fin biomechanics, muscle physiology and neural control is important to the comparative biology of locomotion in fishes and application of fin function for aid in aquatic vehicle design. Recommendations are made regarding fin propulsor designs based on the fin shape, activation pattern, and motion. Research on neural control of fins is a key piece in the puzzle for a complete understanding of comparative fin function and may provide important principles for engineers designing control systems for fin-like propulsors.

Duplication events and the evolution of segmental identity.

Summary Duplication of genes, genomes, or morphological structures (or some combination of these) has long been thought to facilitate evolutionary change. Here we focus on studies of the teleost fishes to consider the conceptual similarities in the evolutionary potential of these three different kinds of duplication events. We review recent data that have confirmed the occurrence of a whole‐genome duplication event in the ray‐finned fish lineage, and discuss whether this event may have fuelled the radiation of teleost fishes. We then consider the fates of individual duplicated genes, from both a theoretical and an experimental viewpoint, focusing on our studies of teleost Hox genes and their functions in patterning the segmented hindbrain. Finally, we consider the duplication of morphological structures, once again drawing on our experimental studies of the hindbrain, which have revealed that experimentally induced duplicated neurons can produce functionally redundant neural circuits. We posit that the availability of duplicated material, independent of its nature, can lead to functional redundancy, which in turn enables evolutionary change.

Behavioral evidence for the evolution of walking and bounding before terrestriality in sarcopterygian fishes

Tetrapods evolved from sarcopterygian fishes in the Devonian and were the first vertebrates to colonize land. The locomotor component of this transition can be divided into four major events: terrestriality, the origins of digited limbs, solid substrate-based locomotion, and alternating gaits that use pelvic appendages as major propulsors. As the sister group to tetrapods, lungfish are a morphologically and phylogenetically relevant sarcopterygian taxon for understanding the order in which these events occurred. We found that a species of African lungfish (Protopterus annectens) uses a range of pelvic fin-driven, tetrapod-like gaits, including walking and bounding, in an aquatic environment, despite having a derived limb endoskeleton and primitively small, muscularly supported pelvis. Surprisingly, given these morphological traits, P. annectens also lifts its body clear of the substrate using its pelvic fins, an ability thought to be a tetrapod innovation. Our findings suggest that some fundamental features of tetrapod locomotion, including pelvic limb gait patterns and substrate association, probably arose in sarcopterygians before the origin of digited limbs or terrestriality. It follows that the attribution of some of the nondigited Devonian fossil trackways to limbed tetrapods may need to be revisited.

Swimming of larval zebrafish: fin-axis coordination and implications for function and neural control

SUMMARY Adult actinopterygian fishes typically perform steady forward swimming using either their pectoral fins or their body axis as the primary propulsor. In most species, when axial undulation is employed for swimming, the pectoral fins are tucked (i.e. adducted) against the body; conversely, when pectoral fins are beating, the body axis is held straight. In contrast to adults, larval fishes can combine their pectoral fin and body-axis movements during locomotion; however, little is known about how these locomotor modes are coordinated. With this study we provide a detailed analysis of the coordinated fin and axial movements during slow and fast swimming by examining forward locomotion in larval zebrafish (Danio rerio L.). In addition, we describe the musculature that powers pectoral fin movement in larval zebrafish and discuss its functional implications. As larvae, zebrafish alternate their pectoral fins during slow swimming (0.011±0.001 mm ms–1) in conjunction with axial undulations of the same frequency (18–28 Hz). During fast swimming (0.109±0.030 mm ms–1; 36–67 Hz), the fins are tucked against the body and propulsion occurs by axial undulation alone. We show that during swimming, larval fishes can use a similar limb–axis coordination pattern to that of walking and running salamanders. We suggest that the fin–axis coordination observed in larval zebrafish may be attributed to a primitive neural circuit and that early terrestrial vertebrates may have gained the ability to coordinate limbs and lateral bending by retaining a larval central pattern generator for limb–axis coordination in the adult life history stage.

Repression of the hindbrain developmental program by Cdx factors is required for the specification of the vertebrate spinal cord

The spinal cord is a unique vertebrate feature that originates, together with the hindbrain, from the caudal neural plate. Whereas the hindbrain subdivides into rhombomeres, the spinal cord remains unsegmented. We have identified Cdx transcription factors as key determinants of the spinal cord region in zebrafish. Loss of Cdx1a and Cdx4 functions causes posterior expansion of the hindbrain at the expense of the unsegmented spinal cord. By contrast, cdx4 overexpression in the hindbrain impairs rhombomere segmentation and patterning and induces the expression of spinal cord-specific genes. Using cell transplantation, we demonstrate that Cdx factors function directly within the neural ectoderm to specify spinal cord. Overexpression of 5′ Hox genes fails to rescue hindbrain and spinal cord defects associated with cdx1a/cdx4 loss-of-function, suggesting a Hox-independent mechanism of spinal cord specification. In the absence of Cdx function, the caudal neural plate retains hindbrain characteristics and remains responsive to surrounding signals, particularly retinoic acid, in a manner similar to the native hindbrain. We propose that by preventing the posterior-most region of the neural plate from following a hindbrain developmental program, Cdx factors help determine the size of the prospective hindbrain and spinal cord territories.

Strikes and startles of northern pike (Esox lucius): a comparison of muscle activity and kinematics between S-start behaviors.

SUMMARY S-starts are a major class of fast-start behaviors that serve diverse locomotor functions in fishes, playing roles in both feeding strike and escape startle events. While movement patterns are similar during strike and startle, their motor control mechanisms have not been compared. To investigate heterogeneity in S-start responses and to test the hypothesis that S-starts are generated by the same patterns of muscle activity regardless of the behavioral context in which they function, we examined kinematic and muscle activity patterns of northern pike (Esox lucius) performing feeding and escape S-starts. Movements were recorded with high-speed video (250 Hz). Muscle activity was recorded from seven electrodes, one in the left adductor mandibulae and bilaterally in the anterior, midbody and posterior epaxial white muscle. Although S-shaped movements are produced in both feeding and escape, kinematics and electromyogram (EMG) patterns differ. Stage 1 (pre-propulsive movement) is significantly slower and more variable during feeding strikes and involves caudal bending with less rostral movement than recorded for startle behaviors. Correspondingly, there is strong caudal muscle activity prior to rostral activity during strikes, whereas in startles caudal muscle activity had near simultaneous onset with contralateral rostral activity. Onset of jaw muscle activity occurred significantly after the onset of axial muscle activity during feeding strikes. By contrast, during startles, jaw activity onset was nearly simultaneous with the onset of axial muscle activity. Stage 2 kinematics generally did not differ between the strike and startle; however, EMGs indicate that stage 2 movements are generated by different patterns of muscle activity for the two behaviors. Although strikes and startles are similar in their propulsive performance, they appear to be initiated and driven by fundamentally different motor control mechanisms. We suggest that S-start startle behavior is mediated by a simple system of descending reticulospinal input to spinal neurons while the S-start strike involves a more complex neural circuit, allowing greater modulation of stage 1 movements while maintaining high stage 2 performance.