Neuronal Form in the Central Nervous System of the Tadpole Larva of the Ascidian Ciona intestinalis.
S. STANLEY MACISSAC [+]
Y. KATSUYAMA 
Y. OKAMURA [++]
I. A. MEINERTZHAGEN ,[ss]
The dorsal tubular central nervous system (CNS) of the ascidian tadpole larva is a diagnostic feature by which the chordate affinities of this group, as a whole, are recognized. We have used two methods to identify larval neurons of Ciona intestinalis. The first is serial electron microscopy (EM), as part of a dedicated study of the visceral ganglion , and the second is the transient transfection of neural plate progeny with green fluorescent protein (GFP) , to visualize the soma and its neurites of individual neurons in whole-mounted larvae of C. intestinalis. Our observations reveal that ascidian larval neurons are simple in form, with a single axonal neurite arising from a soma that is either monopolar or has only very few, relatively simple neurites arising from it, as part of a presumed dendritic arbor. Somata in the visceral ganglion giving rise to axons descending in the caudal nerve cord are presumed to be those of motor neurons.
Regardless of whether ancestral tunicates more closely resembled the adult than its larva [3, 4], or vice versa , urochordates form a sister group to chordates (e.g., ref. 6). Yet, possibly because of their small size and transient life, the neurobiology of ascidian larvae has not received sufficient attention, even though they may be better known than other urochordates . This is not only a taxonomic omission but is also an opportunity missed, given that these simple nervous systems contain relatively few cells, only about 340 in the larva of C. intestinalis, an estimated 100 of which are neurons in that species . The criteria by which cells are recognized are, however, weak. Although cell maps exist for the entire larval CNS [8, 9], few studies identify these cells in any way. Markers identify a number of cell types , and the number of these has increased in recent years, but reports on the morphology of individual neurons are either lacking or are limited to partial studies on the receptor cells of the sensory vesicle [11, 12]. Of particular interest are the motor neuron outputs of the CNS, because these generate undulations of the tail, rhythmical and otherwise , that underlie the larva's swimming behavior. The motor neurons have been widely presumed to lie in the visceral ganglion, the middle division of the CNS, between the rostral swelling of the sensory vesicle and the caudal nerve cord [8, 14].
To visualize neurons in their entirety, in whole-mounted larvae, we used a modification of the GFP transfection method of Corbo et al. . Alternatively, neurons in the visceral ganglion were reconstructed in three dimensions from serial-section EM. For GFP observations, using fluorescence microscopy we first selected those with intense GFP fluorescence from among several hundred transfected, fixed larvae. About 70% of the larvae had such a positive signal, and we then observed about 50 of these by confocal microscopy. Those that were finally selected had either a single neuronal transfection or transfected neurons that were well separated, so that we could observe the entire morphology of the individual neurons, without the possibility of confusion with other GFP profiles. Some larvae had multiple transfections, but the number of cells simultaneously transfected was always restricted and never included all the neurons of a region in the CNS, possibly because of the low concentration of DNA we used.
Various forms of cell outline were visualized after GFP transfection (Fig. lA-D). Confirming the neuron-specific promoter for the GEP, the cytological profiles were identified as those of neurons on the basis of the defining feature of a long slender neurite, the presumed axon. The terminals of these were rarely conspicuous. In addition, we saw few presumed dendritic neurites arising from the soma. Most somata therefore appeared to be monopolar, with a shape approximating a simple prolate spheroid, although two neurons were bipolar (data not shown).
Soma size in GFP transfected neurons varied between about 5.5 and 8 [micro]m for the long axis and 2.5 and 5 [micro]m for the minor axis (Fig. 1A-D); motor neurons reconstructed in three dimensions by computer were somewhat larger than this, with minor axis diameters about 5 [micro]m (Fig. lE). Neuron size in the ascidian larva is thus quite small, similar to the sizes of motor neurons in, for example, the salp ganglion  or the arthropod brain. For the latter, an average density of 4.6 X [10.sup.6] per cubic millimeter in the housefly Musca domestica  corresponds to a mean cell diameter of 7.5 [micro]m; and neuronal diameters in the Drosophila optic lobe fall between 3.6 and 5.0 [micro]m for lamina monopolar cells and 2.8 and 4.2 [micro]m for medulla cells (measurements from ref. 17). In the vertebrate brain, the density of the cerebellar granule cell layer is, for reference, 3-7 X [10.sup.6] . The small size of ascidian larval neurons is compatible with the restricted extent of their arborizatio ns, which are among the smallest examples of deuterostome neuron.
Different forms of neurons were identified with respect to the location of their somata and the direction of their axons (Fig. 1). Most neurons were descending with axons passing either from the sensory vesicle to the visceral ganglion region or from the visceral ganglion to the caudal nerve cord. For some reason, transfection of cells in the anterior region of the sensory vesicle occurred much less frequently than in either the posterior region of the sensory vesicle or the smaller visceral ganglion, even though there are many more cells in the anterior sensory vesicle.
To confirm that the morphological picture of ascidian neurons was correct, we compared images of GFP transfected neurons with three-dimensional reconstructions of motor neurons obtained from serial EM (Fig. lE). The two neurons illustrated are one of five bilaterally symmetrical pairs of motor neurons in the visceral ganglion that have been reconstructed from one individual (1). Observations from two other serially sectioned larvae confirm the number of cells and their general morphology but were not analyzed in detail (Stanley MacIsaac, unpub.). The EM series contained images from only every third section, insufficient for fully tracing the finest neurites but sufficient to have seen coarse basal dendrites; the absence of the latter supports the picture, of simple somata lacking an extensive dendritic arbor, seen from GFP transfection. Neuromuscular terminals traced out to sites of presynaptic contact with the tail muscle do, however, indicate that these cells have bulbous terminals (Fig. lE), in at least t wo sites each, that were not clearly seen after GFP transfection (Fig. lA). Thus, GFP expression in a neuron may not always reveal the axon terminals, perhaps because there is insufficient GFP protein transported down the axon. On the other hand, the trajectory of the axon is clearly revealed.
Compared with the typical multipolar neuron of the vertebrate CNS, the form of neurons in the ascidian larval nervous system seems to be remarkably simple. All examples of neurons have somata with few slender neurites, or sometimes none, and a single axon with simple terminals. Although it is possible that GEP protein does not transport completely into the finest dendrites, serial EM of the motor neurons bears out the general impression that most neurons lack well-developed dendrites. In addition to monopolar neurons, at least two transfected cells each had two neurites, an axon and perhaps a stout dendrite, and thus appeared bipolar.
In the general level of their complexity, the transfected neurons resemble motor neurons in the salp ganglion  or in the amphioxus larva . By comparison, neurons in another deuterostome group, echinoderms [20, 21], seem morphologically more like vertebrate neurons. Thus neuron form does not relate closely to phylogenetic position. It does, however, correlate in general with neuron number, the simplest morphological types of neuron correlating with the least populous nervous systems. Indeed, one view of the dendritic tree is that it provides a structure to segregate different inputs to different dendritic limbs , the size and complexity of neuronal branching in a parasympathetic neuron, for example, increasing with the body size of a particular species [23, 24] and with the number of inputs on the neuron .
One distinction between vertebrate and invertebrate nervous systems lies in the placement of the soma with respect to its neurites. The monopolar cells of invertebrates can be seen as the product of segregating somata into a cortex of cell bodies surrounding the neuropile formed by their neurites. Because the neuropile region is very thin in the ascidian larval CNS [1, 26], in practice this distinction is hard to make in our case. A corollary of the arrangement in invertebrates is that the axonal and dendritic regions of the neuron are not strictly segregated at different regions of the neuronal arbor . In contrast, inputs in vertebrate neurons generally are exclusively received over the soma and its dendritic tree, quite separate from the output region at the axon terminal [28, 29]. Our evidence does not yet allow us to examine whether this distinction is upheld by all neurons in the ascidian larval CNS. Available evidence does, however, indicate the presence of synaptic inputs at the soma [1, 11, 12] a nd outputs at neuromuscular terminals [1, 30]. Our GFP evidence also does not allow us to determine whether axons arise from the apical or basal region of the soma , with respect to the neural canal, because this feature is not resolved in wholemount preparations.
The cells of the visceral ganglion are the subject of a recent study . Further documentation of the cells in the nervous system of the ascidian tadpole larva will, however, require a dedicated study of the sensory vesicle, where at least 60% of the cells in the CNS reside . Application of the GEP method we report here is a first step towards a systematic cataloging of cell types, and their correlation with cell maps derived either from histological methods  or from nuclear stains  will be a first step toward the analysis of the neural circuits these form and the behavior for which these are a substrate. Likewise, if expression turns on sufficiently early in the embryo and persists sufficiently long in the larva, the GEP method may be a way to examine the growth of neurites and their possible later regression during larval metamorphosis. We currently lack evidence on these points.
We thank Ms. Alison Cole for sharing unpublished work on BOBO-3 staining, Ms. Grazyna Tokarczyk for help with confocal imaging of larvae, and Ms. Jane Anne Home for support with computer 3-D reconstructions. Supported by NSERC grant 0000065 (to I.A.M.).
(1.) Neuroscience Institute, Life Sciences Centre, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4J1; and (2.) Department of Developmental and Cell Biology, Developmental Biology Center, BioScience II, University of California Irvine, Irvine, California 92697
(*.) Present address: Howard Hughes Medical Institute, Department of Neurobiology and Behavior, State University of New York, Stony Brook, NY 11794-5230.
(+.) Present address: 405 Penn Circle, Apt. H, Allentown, PA 18102.
(++.) Present address: Biomolecular Engineering Dept. (Ion Channel Group), National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Higashi 1-1, Tsukuba-shi, Ibaraki 305-8566, Japan.
(ss.) To whom correspondence should be addressed. E-mail: IAM@IS.DAL.CA.
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|Author:||OKADA, T.; MACISSAC, S. STANLEY; KATSUYAMA, Y.; OKAMURA, Y.; MEINERTZHAGEN, I. A.|
|Publication:||The Biological Bulletin|
|Date:||Jun 1, 2001|
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