David L. McLean

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.

Ontogeny and Innervation Patterns of Dopaminergic, Noradrenergic, and Serotonergic Neurons in Larval Zebrafish

We report the development of aminergic neurons from 0–10 days postfertilization (dpf) in zebrafish (Danio rerio). This study was prompted by the lack of information regarding patterns of spinal aminergic innervation at early stages, when the fish are accessible to optical, genetic, and electrophysiological approaches toward understanding neural circuit function. Our findings suggest that aminergic populations with descending processes are among the first to appear during development. Descending aminergic fibers, revealed by antibodies to tyrosine hydroxylase (TH) and serotonin (5‐hydroxytryptamine; 5‐HT), innervate primarily the ventral (TH, 5‐HT), but also the dorsal (5‐HT) aspects of the spinal cord by 4 dpf, with the extent of innervation not changing markedly up to 10 dpf. By tracking the spatiotemporal expression of TH, 5‐HT, and dopamine beta hydroxylase reactivity, we determined that these fibers likely originate from neurons in the posterior tuberculum (dopamine), the raphe region (5‐HT) and, possibly, the locus coeruleus (noradrenaline). In addition, spinal neurons positive for 5‐HT emerge between 1–2 dpf, with processes that appeared to descend along the ventrolateral cord for only 1–2 muscle segments. Their overall morphology distinguished these cells from previously described “VeMe” (ventromedial) interneurons, which are also located ventromedially, but have long, multisegmental descending processes. We confirmed the distinction between spinal serotonergic and VeMe interneurons using fish genetically labeled with green fluorescent protein. Our results suggest that the major aminergic systems described in adults are in place shortly after hatching, at a time when zebrafish are accessible to a battery of techniques to test neuronal function during behavior. J. Comp. Neurol. 480:38–56, 2004.

Relationship of tyrosine hydroxylase and serotonin immunoreactivity to sensorimotor circuitry in larval zebrafish.

Our previous study tracked the ontogeny of aminergic systems in zebrafish (Danio rerio). Here we use tyrosine hydroxylase (TH) and serotonin (5‐hydroxytryptamine; 5‐HT) immunoreactivity, in conjunction with retrograde and genetic labeling techniques, to provide a more refined examination of the potential synaptic contacts of aminergic systems. Our focus was on different levels of the sensorimotor circuit for escape, from sensory inputs, through identified descending pathways, to motor output. We observed 5‐HT reactivity in close proximity to the collaterals of the Rohon‐Beard sensory neurons in spinal cord. In the brainstem we found TH and 5‐HT reactivity closely apposed to the dendritic processes of the nucleus of the medial longitudinal fascicle (nMLF), in addition to the ventral dendrites of the Mauthner neuron and its serial homologs MiD2cm and MiD3cm. Only TH reactivity was observed near the lateral dendrites of the Mauthner cell. TH and 5‐HT reactivity were also positioned near the outputs of reticulospinal cells in spinal cord. Finally, both TH and 5‐HT reactivity were detected close to the dendritic processes of primary and secondary spinal motor neurons. We also confirmed, using dual TH and 5‐HT staining and retrograde labeling, that the sources of spinal aminergic reactivity include the posterior tuberculum (dopamine) and inferior raphe region (5‐HT). Our data indicate that aminergic systems may interact at all levels of the sensorimotor pathways involved in escape. The identification of some of these likely sites of aminergic action will allow for directed studies of their functional roles using the powerful combination of techniques available in zebrafish. J. Comp. Neurol. 480:57–71, 2004.

Spinal Interneurons Differentiate Sequentially from Those Driving the Fastest Swimming Movements in Larval Zebrafish to Those Driving the Slowest Ones

Studies of neuronal networks have revealed few general principles that link patterns of development with later functional roles. While investigating the neural control of movements, we recently discovered a topographic map in the spinal cord of larval zebrafish that relates the position of motoneurons and interneurons to their order of recruitment during swimming. Here, we show that the map reflects an orderly pattern of differentiation of neurons driving different movements. First, we use high-speed filming to show that large-amplitude swimming movements with bending along much of the body appear first, with smaller, regional swimming movements emerging later. Next, using whole-cell patch recordings, we demonstrate that the excitatory circuits that drive large-amplitude, fast swimming movements at larval stages are present and functional early on in embryos. Finally, we systematically assess the orderly emergence of spinal circuits according to swimming speed using transgenic fish expressing the photoconvertible protein Kaede to track neuronal differentiation in vivo. We conclude that a simple principle governs the development of spinal networks in which the neurons driving the fastest, most powerful swimming in larvae develop first with ones that drive increasingly weaker and slower larval movements layered on over time. Because the neurons are arranged by time of differentiation in the spinal cord, the result is a topographic map that represents the speed/strength of movements at which neurons are recruited and the temporal emergence of networks. This pattern may represent a general feature of neuronal network development throughout the brain and spinal cord.

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.

Some principles of organization of spinal neurons underlying locomotion in zebrafish and their implications

Recent studies of the spinal motor system of zebrafish, along with work in other species, are leading to some principles that appear to underlie the organization and recruitment of motor networks in cord: (1) broad neuronal classes defined by a set of transcription factors, key morphological features, and transmitter phenotypes arise in an orderly way from different dorso‐ventral zones in spinal cord; (2) motor behaviors and both motoneurons and interneurons differentiate in order from gross, often faster, movements and the neurons driving them to progressively slower movements and their underlying neurons; (3) recruitment order of motoneurons and interneurons is based upon time of differentiation; (4) different locomotor speeds involve some shifts in the set of active interneurons. Here we review these principles and some of their implications for other parts of the brain, other vertebrates, and limbed locomotion.

Using Imaging and Genetics in Zebrafish to Study Developing Spinal Circuits In Vivo

Imaging and molecular approaches are perfectly suited to young, transparent zebrafish (Danio rerio), where they have allowed novel functional studies of neural circuits and their links to behavior. Here, we review cutting‐edge optical and genetic techniques used to dissect neural circuits in vivo and discuss their application to future studies of developing spinal circuits using living zebrafish. We anticipate that these experiments will reveal general principles governing the assembly of neural circuits that control movements.

Metamodulation of a Spinal Locomotor Network by Nitric Oxide

Flexibility in the output of spinal networks can be accomplished by the actions of neuromodulators; however, little is known about how the process of neuromodulation itself may be modulated. Here we investigate the potential “meta”-modulatory hierarchy between nitric oxide (NO) and noradrenaline (NA) in Xenopus laevis tadpoles. NO and NA have similar effects on fictive swimming; both potentiate glycinergic inhibition to slow swimming frequency and GABAergic inhibition to reduce episode durations. In addition, both modulators have direct effects on the membrane properties of motor neurons. Here we report that antagonism of noradrenergic pathways with phentolamine dramatically influences the effect of the NO donor S-nitroso-N-acetylpenicillamine (SNAP) on swimming frequency, but not its effect on episode durations. In contrast, scavenging extracellular NO with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) does not influence any of the effects of NA on fictive swimming. These data place NO above NA in the metamodulatory hierarchy, strongly suggesting that NO works via a noradrenergic pathway to control glycine release but directly promotes GABA release. We confirmed this possibility using intracellular recordings from motor neurons. In support of a natural role for NO in the Xenopus locomotor network, PTIO not only antagonized all of the effects of SNAP on swimming but also, when applied on its own, modulated both swimming frequency and episode durations in addition to the underlying glycinergic and GABAergic pathways. Collectively, our results illustrate that NO and NA have parallel effects on motor neuron membrane properties and GABAergic inhibition, but that NO serially metamodulates glycinergic inhibition via NA.

The development of neuromodulatory systems and the maturation of motor patterns in amphibian tadpoles

The relative simplicity of the amphibian tadpole nervous system has been utilised as a model for the mechanisms underlying the generation and development of vertebrate locomotion. In this paper, we review evidence on the role of descending brainstem projections in the maturation and intrinsic modulation of tadpole spinal motor networks. Three transmitter systems that have been investigated utilise the biogenic amines serotonin (5HT) and noradrenaline (NA) and the inhibitory amino acid gamma-aminobutyric acid (GABA). The distribution, development and spinal targets of these systems will be reviewed. More recent data on the role of nitric oxide (NO) will also be discussed. This ubiquitous gaseous signalling molecule is known to play a crucial role in the developing nervous system, but until recently, had not been directly implicated in the brain regions involved in motor control. NO appears to be produced by three homologous brainstem clusters in the developing motor networks of two closely related amphibian species, Xenopus laevis and Rana temporaria but, surprisingly, it plays contrasting roles in these species. Given the presumed co-localisation and interaction of nitric oxide with conventional neurotransmitters, we discuss the potential relationship of nitrergic neurons with 5HT, NA and GABA in these amphibian models.

Modular Organization of Axial Microcircuits in Zebrafish

Spinal Circuit Complexity in Fish Rapid coordination of opposing muscle groups helps zebrafish zip through water. Bagnall and McLean (p. 197) now describe the neuronal circuits that stabilize swimming fish in their three-dimensional environment. By studying the self-righting behavior of larval zebrafish immobilized in agar, the authors identified parallel excitatory and inhibitory circuits driving dorsal and ventral hemisegments that could be activated independently. Larval zebrafish show more refined musculature control than expected. Locomotion requires precise control of spinal networks. In tetrapods and bipeds, dynamic regulation of locomotion is simplified by the modular organization of spinal limb circuits, but it is not known whether their predecessors, fish axial circuits, are similarly organized. Here, we demonstrate that the larval zebrafish spinal cord contains distinct, parallel microcircuits for independent control of dorsal and ventral musculature on each side of the body. During normal swimming, dorsal and ventral microcircuits are equally active, but, during postural correction, fish differentially engage these microcircuits to generate torque for self-righting. These findings reveal greater complexity in the axial spinal networks responsible for swimming than previously recognized and suggest an early template of modular organization for more-complex locomotor circuits in later vertebrates.