Joseph R. Fetcho

Laser Ablations Reveal Functional Relationships of Segmental Hindbrain Neurons in Zebrafish

Segmentation of the vertebrate brain is most obvious in the hindbrain, where successive segments contain repeated neuronal types. One such set of three repeated reticulospinal neurons--the Mauthner cell, MiD2cm, and MiD3cm--is thought to produce different forms of the escape response that fish use to avoid predators. We used laser ablations in larval zebrafish to test the hypothesis that these segmental hindbrain cells form a functional group. Killing all three cells eliminated short-latency, high-performance escape responses to both head- and tail-directed stimuli. Killing just the Mauthner cell affected escapes from tail-directed but not from head-directed stimuli. These results reveal the contributions of one set of reticulospinal neurons to behavior and support the idea that serially repeated hindbrain neurons form functional groups.

Imaging the Functional Organization of Zebrafish Hindbrain Segments during Escape Behaviors

Although vertebrate hindbrains are segmented structures, the functional significance of the segmentation is unknown. In zebrafish, the hindbrain segments contain serially repeated classes of individually identifiable neurons. We took advantage of the transparency of larval zebrafish and used confocal calcium imaging in the intact fish to study the activity of one set of individually identified, serially homologous reticulospinal cells (the Mauthner cell, MID2cm, and MID3cm) during behavior. Behavioral studies predicted that differential activity in this set of serially homologous neurons might serve to control the directionality of the escape behavior that fish use to avoid predators. We found that the serially homologous cells are indeed activated during escapes and that the combination of cells activated depends upon the location of the sensory stimulus used to elicit the escape. The patterns of activation we observed were exactly those predicted by behavioral studies. The data suggest that duplication of ancestral hindbrain segments, and subsequent functional diversification, resulted in sets of related neurons whose activity patterns create behavioral variability.

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.

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.

Distribution of prospective glutamatergic, glycinergic, and GABAergic neurons in embryonic and larval zebrafish.

Zebrafish are an excellent model for studies of the functional organization of neuronal circuits, but little is known regarding the transmitter phenotypes of the neurons in their nervous system. We examined the distribution in spinal cord and hindbrain of neurons expressing markers of transmitter phenotype, including the vesicular glutamate transporter (VGLUT) genes for glutamatergic neurons, the neuronal glycine transporter (GLYT2) for glycinergic neurons, and glutamic acid decarboxylase (GAD65/67) for GABAergic neurons. All three markers were expressed in a large domain in the dorsal two‐thirds of spinal cord, with additional, more ventral expression domains for VGLUT2 and GAD/GABA. In the large dorsal domain, dual in situ staining showed that GLYT2‐positive cells were intermingled with VGLUT2 cells, with no dual‐stained neurons. Many of the neurons in the dorsal expression domain that were positive for GABA markers at embryonic stages were also positive for GLYT2, suggesting that the cells might use both GABA and glycine, at least early in their development. The intermingling of neurons expressing inhibitory and excitatory markers in spinal cord contrasted markedly with the organization in hindbrain, where neurons expressing a particular marker were clustered together to form stripes that were visible running from rostral to caudal in horizontal sections and from dorsomedial to ventrolateral in cross sections. Dual labeling showed that the stripes of neurons labeled with one transmitter marker alternated with stripes of cells labeled for the other transmitter phenotypes. The differences in the distribution of excitatory and inhibitory neurons in spinal cord versus hindbrain may be tied to differences in their patterns of development and functional organization. J. Comp. Neurol. 480:1–18, 2004.

Engrailed-1 Expression Marks a Primitive Class of Inhibitory Spinal Interneuron

Studies in chicks and mice have suggested that transcription factors mark functional subtypes of interneurons in the developing spinal cord. We used genetic, morphological, and physiological studies to test this proposed association in zebrafish. We found that Engrailed-1 expression uniquely marks a class of ascending interneurons, called circumferential ascending (CiA) interneurons, with ipsilateral axonal projections in both motor and sensory regions of spinal cord. These cells express the glycine transporter 2 gene and are the only known ipsilateral interneurons positive for this marker of inhibitory transmission. Patch recordings show that the CiA neurons are rhythmically active during swimming. Pairwise recordings from the CiA interneurons and postsynaptic cells reveal that the Engrailed-1 neurons produce monosynaptic, strychnine-sensitive inhibition of dorsal sensory interneurons and also inhibit more ventral neurons, including motoneurons and descending interneurons. We conclude that Engrailed-1 expression marks a class of inhibitory interneuron that seems to provide all of the ipsilateral glycinergic inhibition in the spinal cord of embryonic and larval fish. Individual Engrailed-1-positive cells are multifunctional, playing roles in both sensory gating and motor pattern generation. This primitive cell type may have given rise to several, more specialized glycinergic inhibitory interneurons in birds and mammals. Our data support the view that the subdivision of spinal cord into different regions by transcription factors defines a primitive functional organization of spinal interneurons that formed a developmental and evolutionary foundation on which more complex systems were built.

Spinal Network of the Mauthner Cell (Part 1 of 2)

Most swimming vertebrates, particularly fishes and amphibians, avoid predators by producing an escape behavior initiated by a single action potential in one of a pair of cells, the Mauthner cells, loc

A structural and functional ground plan for neurons in the hindbrain of zebrafish

The vertebrate hindbrain contains various sensory-motor networks controlling movements of the eyes, jaw, head, and body. Here we show that stripes of neurons with shared neurotransmitter phenotype that extend throughout the hindbrain of young zebrafish reflect a broad underlying structural and functional patterning. The neurotransmitter stripes contain cell types with shared gross morphologies and transcription factor markers. Neurons within a stripe are stacked systematically by extent and location of axonal projections, input resistance, and age, and are recruited along the axis of the stripe during behavior. The implication of this pattern is that the many networks in hindbrain are constructed from a series of neuronal components organized into stripes that are ordered from top to bottom according to a neurons age, structural and functional properties, and behavioral roles. This simple organization probably forms a foundation for the construction of the networks underlying the many behaviors produced by the hindbrain.

The spinal motor system in early vertebrates and some of its evolutionary changes.

Recent studies of the spinal motor systems of vertebrates allow us to begin to infer the organization of the motor apparatus of primitive vertebrates. This paper attempts to define some of the features of the motor system of early vertebrates based on studies of the motor systems in anamniotes and in Branchiostoma. It also deals with some changes in the primitive motor system during evolution. The primitive motor system consisted of myomeric axial muscles, with a functional subdivision of the musculature into non-spiking slow muscle fibers segregated in the myomeres from spiking fast ones. These fibers were innervated by two major classes of motoneurons in the cord-large motoneurons innervating faster fibers and small motoneurons innervating slow fibers. There was not a simple isomorphic mapping of the position of motoneurons in the motor column onto the location of the muscle fibers they innervated in the myomeres. Early vertebrates used these axial muscles to bend the body, and the different types of muscle fibers and motoneurons reflect the ability to produce slow swimming movements as well as very rapid bending associated with fast swimming or escapes. The premotor network producing bending was most likely a circuit composed of a class of descending interneurons (DIs) that provided excitation of ipsilateral motoneurons and other interneurons, and inhibitory commissural interneurons (CIs) that blocked contralateral activity and played an important role in generating the rhythmic alternating bending during swimming. This DI/CI network was retained in living anamniotes. At least two major descending systems linked the sensory systems in the head to these premotor networks in the spinal cord. The ability to turn on swimming by activation of DI/CI premotor networks in the cord resided at least in part in a midbrain locomotor region (MLR) that influenced spinal networks via projections to the reticular formation. Reticulospinal neurons were important not only for initiation of rhythmic swimming but also in the production of turning movements. The reticulospinal cells involved in turns produced their effects in part via monosynaptic connections with motor neurons and premotor interneurons, including some involved in rhythmic swimming. A prominent and powerful Mauthner cell was most likely present and important for rapid escape or startle movements. Some features of this primitive motor apparatus were conserved during the evolution of vertebrate motor systems, and others changed substantially. Many features of the early motor system were retained in living anamniotes; major changes occur among amniotes.(ABSTRACT TRUNCATED AT 400 WORDS)