Calcium signals monitored from leopard frog optic tectum after the optic nerve has been selectively loaded with calcium sensitive dye.

We loaded adult leopard frog optic nerves with the calcium-sensitive dye Calcium Green-1 3000 mw dextran conjugate. The dye was transported to the optic tectum in approximately 6 days and selectively labeled optic nerve terminals as seen with confocal microscopy. Viewed with an intensified CCD system, electrical stimulation of the optic nerve in vitro increases Calcium Green-1 fluorescence significantly. With increasing number of pulses in pulse trains there was increased presynaptic facilitation as measured by increased fluorescence. Addition of nicotine to the bathing solution increased baseline fluorescence. These results suggest that Calcium Green-1 dextran conjugate can be actively transported in adult nerve fibers over a significant distance and is retained in presynaptic terminals in a form that allows monitoring of presynaptic calcium levels.

Lithium perturbation and goosecoid expression identify a dorsal specification pathway in the pregastrula zebrafish.

The zebrafish dorsoventral axis can first be distinguished at gastrulation, upon formation of the embryonic shield, the site of the organizer. We have asked whether the shield is specified before gastrulation. First, we show that brief exposure of premidblastula embryos to lithium, which is known to shut down the phospho-inositol signaling pathway, produces excessive shield formation and extreme hyper-dorsal development. Second, we show that the zebrafish goosecoid homeobox gene is activated at or just after the midblastula in a localized domain of cells that subsequently populate the most anterior region of the incipient shield and axial hypoblast, goosecoid expression is elevated and radialized by early lithium treatment, suggesting that goosecoid plays a role in establishing the organizer and shield. Our results demonstrate that the zebrafish dorsal axis is signaled by a pathway initiated in the cleavage-stage embryo. Furthermore, they provide novel insights into anterior morphogenesis.

Cell-cell interactions during the migration of an identified commissural growth cone in the embryonic grasshopper.

One of the fascicles of the posterior commissure of the embryonic grasshopper is pioneered by an individually identifiable neuron named Q1. Q1 initially grows along a longitudinal pathway established by another pioneer neuron, MP1, and then crosses to the midline, where it meets and fasciculates with the axon of the contralateral Q1. The Q1 growth cone follows the contralateral Q1 axon to the contralateral longitudinal pathway, where it then fasciculates with axons of the MP1/dMP2 fascicle. In this work, we have identified a small set of early neurons that Q1 could use as guidance cues while negotiating its way along a specific and stereotyped pathway to the midline. Furthermore, we have observed characteristic morphological changes in the Q1 growth cone that could indicate responses to changing adhesivity in the substrates it contacts. We have also quantified the pattern of dye coupling between neurons in this system. Most of the neurons to which Q1 becomes coupled retain a strong, consistent pattern of dye coupling that shows no recognizable variation at times when growth cones are making pathway decisions. However, we have found one clear instance of transient, site-specific dye coupling between the Q1 growth cone and the ipsilateral MP1 soma. The timing and pattern of dye coupling in this system suggest that dye coupling may play a role in synchronizing the initiation of axon outgrowth among a small population of neurons. Although dye coupling may not play a direct role in neuronal pathfinding, it may exert a profound indirect influence on neuronal interactions by regulating the timing of axon outgrowth.

Growth cone dynamics during the migration of an identified commissural growth cone.

We have used time-lapse video microscopy to study the behavior of a neuron, Q1, that pioneers the posterior commissure of the embryonic grasshopper. Our goal is to use time-lapse video as a tool to acquire a precise picture of normal development over time, and thereby identify stereotypic activities that might indicate important interactions necessary for proper formation of the commissure. We have identified specific and reproducible behaviors that suggest the presence of underlying cellular interactions that may play a role in pathfinding. In particular, the Q1 growth cone undergoes several morphological changes as it contacts the midline. As a commissural neuron, the midline may be a target in its outgrowth; Q1's typical response upon contacting the midline with its filopodia, however, is a rapid retraction. This inhibitory reaction can be overridden by contact with filopodia of its contralateral homolog. Q1's growth cone can translocate across the midline at an accelerated rate by a process resembling "filopodial dilation" (O'Connor et al., 1990) once the two Q1 growth cones meet. Ablation of the contralateral Q1 blocks Q1's advance across the midline. We have also analyzed in detail the behavior of individual filopodia to identify behavioral differences that could indicate differences in substrate adhesivity. Except for instances of filopodial dilation seen only at the midline, we found no significant asymmetries in rates of filopodial extension and retraction, or in the survival times of individual filopodia. We suggest that either the adhesive signal used by Q1 is relatively weak, requiring the integration of many adhesive interactions by many filopodia to be resolved, or the guidance cues may not be adhesive in nature.

NeuroVideo: a program for capturing and processing time-lapse video.

We have developed a program for the Macintosh computer to control a Panasonic Optical Memory Disk Recorder (OMDR) in order to generate time-lapse video recordings of growing neurons. The software, in addition to regulating the timing of a recording in a flexible way, can also digitize and pre-process images before writing them out to the optical disk. NeuroVideo includes a complete set of functions to enhance images, and provides both an easy-to-use graphical interface and a simple but powerful text-based scripting language.

Primary neurons that express the L2/HNK-1 carbohydrate during early development in the zebrafish.

In zebrafish, many nerve pathways in both the CNS and periphery are pioneered by a small and relatively simple set of 'primary' neurons that arise in the early embryo. We now have used monoclonal antibodies to show that, as they develop, primary neurons of several functional classes express on their surfaces the L2/HNK-1 tetrasaccharide that is associated with a variety of cell surface adhesion molecules. We have studied the early labeling patterns of these neurons, as well as some non-neural cells, and found that the time of onset and intensity of immunolabeling vary specifically according to cell type. The first neuronal expression is by Rohon-Beard and trigeminal ganglion neurons, both of which are primary sensory neurons that mediate touch sensitivity. These cells express the epitope very strongly on their growth cones and axons, permitting study of their development unobscured by labeling in other cells. Both types initiate axogenesis at the same early time, and appear to be the first neurons in the embryo to do so. Their peripheral neurites display similar branching patterns and have similar distinctive growth cone morphologies. Their central axons grow at the same rate along the same longitudinal fiber pathway, but in opposite directions, and where they meet they appear to fasciculate with one another. The similarities suggest that Rohon-Beard and trigeminal ganglion neurons, despite their different positions, share a common program of early development. Immunolabeling is also specifically present on a region of the brain surface where the newly arriving trigeminal sensory axons will enter the brain. Further, the trigeminal expression of the antigen persists in growth cones during the time that they contact an individually identified central target neuron, the Mauthner cell, which also expresses the epitope. These findings provide descriptive evidence for possible roles of L2/HNK-1 immunoreactive molecules in axonal growth and synaptogenesis.

Development and axonal outgrowth of identified motoneurons in the zebrafish.

We have observed the development of live, fluorescently labeled motoneurons in the spinal cord of embryonic and larval zebrafish. There are 2 classes of motoneurons: primary and secondary. On each side of each spinal segment there are 3 individually identifiable primary motoneurons, named CaP, MiP, and RoP. The motoneurons of the embryo and larva are similar in morphology and projection pattern to those of the adult. During initial development, axons of primary motoneurons make cell-specific, divergent pathway choices and grow without error to targets appropriate for their adult functions. We observed no period of cell death, and except for one consistently observed case, there was no remodeling of peripheral arbors. We have observed a consistent temporal sequence of axonal outgrowth within each spinal segment. The CaP motor axon is the first to leave the spinal cord, followed by the axons of the other primary motoneurons. The Mauthner growth cone enters the spinal cord after all the primary motoneurons of the trunk spinal cord have begun axonal outgrowth. Secondary motor growth cones appear only after the Mauthner growth cone has passed by. Our results suggest that this stereotyped temporal sequence of axonal outgrowth may play a role in defining the contacts between the Mauthner axon and the motoneurons; the behavior of growth cones in the periphery suggests that interactions with the environment, not timing, may determine path-finding and peripheral connectivity of the motoneurons.

Pathway selection by growth cones of identified motoneurones in live zebra fish embryos.

How is the adult pattern of connections between motoneurones and the muscles that they innervate established during vertebrate development? Populations of motoneurones are thought to follow one of two patterns of development: (1) motor axons initially follow stereotyped pathways and project to appropriate regions of the developing muscle or (2) motor axons initially project to some regions that are incorrect, the inappropriate projections being eliminated subsequently. Here we observed individually identified motoneurones in live zebra fish embryos as they formed growth cones and as their growth cones navigated towards their targets. We report that from axogenesis, each motor axon followed a stereotyped pathway and projected only to the specific region of the muscle appropriate for its adult function. In addition, the peripheral arbor established by each motoneurone was restricted to a stereotyped region of its own segment and did not overlap with the peripheral arbor of the other motoneurones in that segment. We conclude that the highly stereotyped pattern of innervation seen in the adult is due to initial selection of the appropriate pathway, rather than elimination of incorrect projections.

Spinal motoneurons of the larval zebrafish.

Application of horseradish peroxidase to lesions of the muscles and the central nervous system of larval zebrafish Brachydanio rerio was used to identify several types of neurons present in the spinal cord. The spinal cord was found to contain three distinct motoneuronal types: primary and secondary motoneurons that innervate the axial muscles, and pectoral fin motoneurons that innervate the muscles of the pectoral girdle. The cell types are similar to those described in larvae of other anamniote vertebrates. The axial muscles of a given hemisegment are innervated by two or three primary motoneurons and a larger number of secondary motoneurons in the corresponding spinal segment, whereas fin muscles are innervated by a pool of motoneurons spanning several spinal segments.

CHROMPAC III: an improved package for microcomputer-assisted analysis of karyotypes.

An improved package of BASIC computer programs designed to facilitate complete analysis of karyotypes is described. The package's many functions include facilities for measurement of chromosomes, analysis of data, pairing of homologues, designation of sex chromosomes and supernumeraries , storage and recall of data, and generation of idiograms and karyograms .