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Previous Article | Next Article
Volume 16, Number 18, Issue of September 15, 1996 pp. 5749-5761
Copyright ©1996 Society for NeuroscienceDevelopment of an Identified Spinal Commissural Interneuron Population in an Amniote: Neurons of the Avian Hofmann Nuclei
Anne Lill Eide1 and Joel C. Glover2 1 Department of Physiology, and 2 Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, 0317 Oslo, Norway
ABSTRACT
The commissural interneurons of the Hofmann nuclei (HN) of the avian spinal cord (The axonal projections of the Hofmann nuclei in the spinal cord of the late stage chicken embryo, Anat Embryol (Berl), A. L. Eide, 1996
Key words: birthdating; spinal interneuron; neuronal migration; axon outgrowth; collateral sprouting; spinal cord; chicken embryo, Vol 193, pp 543-557) provide a unique opportunity to describe the development of an identified spinal commissural axon projection and its terminal collaterals in an amniote vertebrate. Here, we use the lipophilic tracer DiI to label these and other commissural projections anterogradely and retrogradely from the time the HN neurons are born. [3H]thymidine birthdating shows that the final mitoses of HN neurons occur at stages 21-24 [developmental day (d) 4]. By direct comparison, this follows the generation of motoneurons and of large, dorsally located commissural interneurons. The first HN neurons reach the ventrolateral margin of the spinal cord by d6 by a radial migration through the ventral horn. Radial migration occurs after the extension of HN axons across the midline. Thus, HN neurons are determined to be commissural interneurons before attaining their definitive locations. The HN neurons subsequently aggregate into segmentally iterated clusters at the ventrolateral margin of the spinal cord by d8. Also by d8 their longitudinal axons attain mature extent in the ventral funiculus of the contralateral side and begin to sprout collaterals. The collaterals are directed predominantly toward the medial aspect of the ventral horn at all stages, forming by d12 a dense thicket of terminals that thins out over several segments to each side of the HN of origin. The initial direction of collateral outgrowth is largely appropriate for the mature termination pattern of the HN. Terminal arbors, however, are less focused at early developmental stages than at later stages.
INTRODUCTION
Spinal interneurons are responsible in large part for the integrated function of the spinal cord. Many project intersegmentally to link different axial levels of the cord, and many of these decussate to effect bilateral coordination. These interneuronal connections engender many complex motor patterns with a spinal autonomy (Fukson et al., 1980Despite the cardinal role of spinal interneurons in organizing spinal function, relatively little is known about their connectivity, especially in amniotes. This necessarily hampers the study of the development of spinal interneurons, a subject that has received increasing attention in recent years. Existing descriptions of the development of spinal interneurons in amniotes are fragmentary, based mostly on retrograde axonal tracing or immunohistochemistry for cytoskeletal or membrane proteins (for review, see Yaginuma et al., 1994
We have recently shown that the paragriseal Hofmann nuclei (HN) of the avian spinal cord contain commissural interneurons whose spatial isolation makes them especially amenable to anatomical and physiological analysis (Eide, 1996
Here we examine the development of the HN neurons in the chicken embryo with particular reference to their birth, migration, axonal projections, and termination patterns.
MATERIALS AND METHODS
White Italian chicken embryos were incubated in a forced-draft incubator at 37°C until the desired stage, at which time they were either used for neuronal birthdating or killed for axonal tracing. Staging was according to Hamburger and Hamilton (1951)The HN are located between two consecutive ventral roots. Their segmental location is designated with reference to the roots. For example, HN-LS5/6 refers to the HN located between the roots of lumbosacral segments (LS) 5 and 6.
Birthdating by autoradiography. Thirty-two embryos at successive stages of development from stage 15 to stage 30 (d2-d6) were exposed by opening a window in the egg. Methyl-[3H]thymidine (20-45 µCi; Amersham-TRK686, specific activity 83 Ci/mmol) in a volume of 40-90 µl of saline was applied to each embryo through the window with a microliter syringe.
At d14-d16, by which time the HN are easily identified, the embryos were removed from the egg, anesthetized by cooling in oxygenated PBS, pH 7.4, and killed by decapitation. The preparations were fixed overnight in 4% formaldehyde in 0.1 phosphate buffer. A hemisection of the cord including HN-LS5/6 was cut out for further analysis. The tissue was equilibrated in 15% sucrose in phosphate buffer, mounted in Tissue-Tek O.C.T. compound (Chemi-Teknik, Oslo, Norway), frozen at
20°, sectioned transversely at 10 µm on a cryostat, and mounted onto gelatin-coated glass slides. The sections were treated with 1 ethanolamine for 10 min to eliminate free aldehyde groups that can react with film emulsion and increase background. They were then dehydrated and rehydrated through a series of ethanol (50%-70%-96%-96%-70%-H2O) and air dried. Kodak NTB2 emulsion (diluted 1:1 in distilled water) was applied by the method of Kopriwa and LeBlond (1962)
This application method has been shown to give cumulative labeling, as the [3H]thymidine evidently remains available throughout the duration of neurogenesis, although this is dependent on the stage of application (Langman and Haden, 1970
The assessment of birthdate using cumulative labeling is based on the presumption that any neuron that is not labeled has been born before the application of [3H]thymidine. The generation of a neuron population is thus chronicled by determining the stage(s) at which labeling ceases.
The sections were examined in the light microscope. Only cells that contained a distinct nucleus in the plane of section were scored. Labeled cells contained grains of developed photoemulsion over the nucleus and were easy to identify. In each preparation, the proportion of labeled to unlabeled HN neurons was established. Motoneurons and large interneurons in the dorsal horn were also scored to correlate the birthdates of the different populations.
DiI tracing. A total of 65 embryos at successive developmental stages from d3 to d14 were killed for axonal tracing experiments (Table 1). The spinal cord from thoracic segment (Th) 4 to LS14 was dissected out in cold oxygenated PBS, the ventral and dorsal cartilage (or bone at late stages) was removed, and the dura was slit open. The preparations were fixed in cold 4% formaldehyde in phosphate buffer and kept refrigerated in this solution for 1 d to several months before tracer was applied. Variation in time of storage before tracer application did not influence the results.
Table 1. Number of preparations used for DiI-labeling experiments at each development stage
Day of development Number d3 1 d4 5 d5 10 d6 6 d7 13 d8 7 d9 2 d10 7 d11 1 d12 6 d13 0 d14 7 Total 65 The lipophilic tracer 1,1
-dioctadecyl-3,3,3
Because DiI diffuses throughout the neuronal membrane, both anterograde and retrograde labeling inevitably occur in all neurons that intersect the application site. For anterograde labeling of the HN neurons, DiI was generally applied to HN-LS5/6 or, before the overt appearance of the HN, to the spinal cord at the same level. For retrograde labeling of the HN neurons, DiI was applied unilaterally to the spinal cord either in the middle lumbosacral region or rostral to the lumbosacral region. The various application paradigms and representative examples of anterograde labeling are illustrated in Figure 1.
Fig. 1. The different DiI application paradigms. A-D illustrate schematically the sites of DiI application (indicated by arrowheads and/or dots). E and F show representative examples of anterograde labeling from a single HN at d8. A, Anterograde tracing of the entire commissural projection from the level corresponding to a single HN in preparations from d4 to d8. DiI is applied to one side of the spinal cord between two ventral roots. The spinal cord is split along the midline except for between the two roots. This restricts the anterograde labeling to those axons crossing within this region. It also eliminates retrograde labeling of commissural interneurons on the contralateral side at all other levels. B, Retrograde tracing of commissural interneurons in preparations from d4 to d14. DiI is applied to the ventral and ventrolateral funiculi on one side midway between two ventral roots. The spinal cord is split along the midline at the same level. This eliminates anterograde labeling of commissural axons originating from the injected side. It also restricts retrograde labeling to commissural axons originating from the contralateral side of more rostral and caudal levels. This reveals the distribution of the commissural interneurons, including HN neurons, on the contralateral side, the longitudinal axons of which traverse the injected level. C, Selective anterograde tracing of the HN projection. DiI is applied selectively to one HN. The spinal cord is split along the midline except for the region in which the axons from the injected HN cross. This eliminates retrograde labeling of contralateral commissural interneurons rostral and caudal to the split and allows the HN axons and collaterals to be viewed in isolation. D, Retrograde tracing of the entire HN population. DiI is applied to one side of the spinal cord over many segments. This reveals the longitudinal disposition of the HN somata during the formation of the HN. E, Ventral view of a whole-mount spinal cord at d8 in which DiI has been applied to a single HN. The HN axons are visible along their commissural and longitudinal trajectories. F, Transverse section through a similar preparation showing the HN axon trajectory directed radially toward the ventral pole of the central canal and then crossing in the ventral commissure. Note that labeling extends all the way to the ventricular surface. This represents the ventricular attachments of radial glia that extend to the HN, not ventricular attachments of the HN neurons themselves (see text). Scale bars: 500 µm in E and 200 µm in F.
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Labeled preparations were first examined in whole mount. They were then rinsed in phosphate buffer overnight, embedded in 17% gelatin in phosphate buffer, and sectioned transversely at 50-100 µm on a Vibratome (Campden Instruments LTD, 752 Vibroslice). To maintain the integrity of the labeling, it was necessary to keep the sections wet at all times; even a few minutes of drying disrupted the stain. To attach the wet sections to a glass slide for microscopic examination, the gelatin edges of the sections were melted onto the slide with a hot needle. The sections were covered with 4% formaldehyde, coverslipped, and could then be stored in slide jars containing 1-2% formaldehyde at 4°C for many months. Contamination of the surface of the section occurred over time, presumably because labeled and unlabeled membranes are disrupted and come into contact there. Thus, microscopic examination of the sections was performed during the first 1-3 d after sectioning.
Fluorescence microscopy. Whole-mount preparations and transverse sections were examined under a Leitz Ortholux II epifluorescence microscope outfitted with a rhodamine filter.
RESULTS
This study covers a developmental period characterized by tremendous growth and morphometric change in the spinal cord. Figure 2 provides an anatomical framework for the comparison of results obtained at different stages. The HN can be discerned as protrusions along the lateral edge of the lumbar spinal cord from d8. This is the earliest time at which the HN neurons can be anterogradely labeled selectively.
Fig. 2. The development of the HN in the context of the morphometric changes occurring in the spinal cord during the developmental period studied. For each developmental stage, we show: (1) a drawing of a ventral view of the lumbar region of the spinal cord (the series of drawings is at the same scale to illustrate relative size); (2) a photomicrograph of a ventral view of one side of the spinal cord showing the disposition of the HN (first distinctly visible from d8); and (3) a photomicrograph of a transverse section through the lumbar spinal cord at the level of an HN (arrows). Scale bars: 2 mm in all drawings, 500 µm in the left photomicrographs, and 200 µm in the right photomicrographs.
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Birthdating
The mitoses that generate the HN neurons occur between stages 21 and 24. In embryos exposed to [3H]thymidine before stage 21, all HN neurons were labeled, whereas in those exposed after stage 24 (d4), none was labeled. Two preparations at stage 23 contained, respectively, 80 and 50% labeled HN neurons.Two other neuron types could be identified readily in transverse sections and compared with the HN neurons with respect to time of generation: namely, the motoneurons of the lateral motor column and large commissural interneurons located between laminae II and III and at the boundary between the intermediate zone and the dorsal horn (Fig. 3) (Eide and Glover, 1996
Fig. 3. [3H]thymidine birthdating of HN neurons and other neuron populations. A, Autoradiographic transverse section from the spinal cord of a d15 embryo labeled with [3H]thymidine at stage 23 (early d4). Circles enclose the regions shown in B, C, and F. B, Intermingled labeled and unlabeled HN neurons; the latter were born before [3H]thymidine application. C, All motoneurons in the lateral motor column are unlabeled, demonstrating that this neuron population is born before the HN cells. D, Retrograde labeling of a large dorsal commissural interneuron at d7. E, Retrograde labeling of a large dorsal commissural interneuron at d15. F, Large dorsal interneurons of the same size, shape, and location as in E are unlabeled (arrow), demonstrating that this neuron population is born before the HN neurons. Scale bars: 500 µm in A, 50 µm in B, C, E, and F, and 200 µm in D.
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HN neuron migration and aggregation
When DiI was injected into the ventral funiculus on one side of the lumbar spinal cord, interneurons projecting ipsilaterally were labeled retrogradely on the same side, and commissural interneurons were labeled retrogradely on the opposite side. The pattern of commissural interneuron labeling changed over time and will be discussed in more detail below. Here we draw attention to a feature of the labeling that bears specifically on the migration of the HN neurons.In the immediate vicinity of a unilateral DiI injection, all cells, including neurons and radial glia, were labeled simply because of nonspecific spread. But at levels rostral or caudal to the injected region on the same side, or at any level on the contralateral side, only interneurons whose axons projected to or through the injected region became labeled. This is shown in Figure 4A, in a section taken caudal to the injected region on d7. The labeling of commissural interneurons has been limited to those crossing rostral and caudal to the injected region by the midline split, as shown in Figure 4D. The domains of the lateral motor columns are outlined by labeled interneurons and their dendrites (both sides), as well as by labeled ventral axon tracts (injected side). The labeled processes radiating through the lateral motor column toward the lateral margin of the spinal cord on the side contralateral to the injection are the retrogradely labeled axons of the HN neurons, which were consistently seen only on the contralateral side (Fig. 4A). Elongated somata with radial orientation and devoid of dendrites were clearly associated with these axons on d6 (Fig. 4B,C), but no longer by d8. Our interpretation is that the HN somata have already extended a commissural axon by this stage of their development, but are still in the process of migrating laterally by nuclear translocation. The temporal sequence of events thus appears to be as follows: HN neurons, after undergoing their terminal mitoses in the ventral ventricular zone by d4, extend migratory leading processes through the lateral motor column to the pial surface, parallel to radial glia, and begin to migrate by nuclear translocation. By d6, while still migrating radially, they have extended commissural axons across the midline. By d8, their radial migration brings them to their definitive locations at the ventrolateral margin of the spinal cord.
Fig. 4. Lateral migration of HN neurons. Transverse sections through the ventral spinal cord showing the distribution of retrogradely labeled commissural interneurons at d7 (A) and d6 (B, C) after DiI application to the right side as shown in D. A, The labeling pattern is different on the injected (right) and contralateral (left) sides immediately caudal to the level of injection. Radial processes extending through the lateral motor column are present only on the side contralateral to the injection and represent the retrogradely labeled axons of the HN neurons. B and C show examples of somata (presumed HN somata) associated with these axons. D summarizes schematically the distribution of axons and cell bodies labeled by this DiI application procedure. The small black ellipses represent interneurons, HN neurons on the left side and an ipsilaterally projecting interneuron on the right, the latter lying lateral to longitudinal axons. Hatching indicates the DiI application site, and arrowheads indicate the location of the sections shown in A-C. Scale bars, 100 µm.
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In addition to the radially oriented leading processes of the HN neurons, radial glia (which are not labeled in Fig. 4) extend through the lateral motor column. At d8, selective application of DiI to the HN typically labeled not only the HN commissural axons but also fibers that extended to the ventricular surface (Fig. 1F). These ventricular attachments were not labeled when the HN axons were traced retrogradely from the contralateral side. Thus, they cannot be ventricular attachments of the HN neurons themselves. Rather, they must be the ventricular attachments of radial glia cells that extend from the ventricular surface to the HN. Evidently these cells are eliminated or retract from the ventricular surface after d8, because application of DiI to the HN at later stages did not label any ventricular attachments.
After their radial migration, the HN underwent a longitudinal aggregation. At d7, the HN neuron population appeared as a fairly continuous column along the ventrolateral margin (Fig. 5). By d8, the column was clearly segmented into clusters that gradually coalesced to form the definitive protruding HN (Fig. 5). Support for a segmental aggregation of an initially unsegmented column of HN neurons comes from the relationship between soma and axon distributions in late stage embryos, as described by Eide (1996)
Fig. 5. The longitudinal aggregation of HN neurons. Ventral views of the spinal cord at the indicated developmental stages. All HN neurons have been retrogradely labeled as shown in Figure 1D. Scale bars, 500 µm.
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The commissural interneuron population as a whole
The first commissural axons in the lumbar spinal cord of the chicken embryo cross the midline at about d3, and then turn either rostrally or caudally in the contralateral ventral funiculus (Yaginuma et al., 1991
Fig. 6. Retrograde labeling of the entire commissural interneuron population located between LS5 and LS6 at the indicated developmental stages (DiI application as in Fig. 1B). Each row of plates shows the pattern of labeling at the same indicated stage, with distance rostrad increasing from left to right over a distance of about two to three segments. Scale bars, 100 µm.
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Development of longitudinal projections
The extension of longitudinal axons after bifurcation of the commissural axon population was examined using three different experimental approaches. In the first, used from d4 to d7.5, we labeled anterogradely all commissural axons crossing at a specific level by applying DiI to the ventral region of one side of the spinal cord (Fig. 7). The ventral commissure was split rostral and caudal to the DiI application site to restrict labeling to the commissural axons crossing at the level of application (Fig. 1A). In this case, it was impossible to distinguish labeled HN axons from other labeled axons. In the second, used from d5.5 to d8, we labeled retrogradely all commissural axons that had turned or bifurcated to extend through a given spinal level by applying DiI to the ventral funiculus on one side of the spinal cord (Fig. 1B). In this case, from d7 onward, we could determine selectively the longitudinal extent of the HN axons by virtue of the retrograde labeling of their distinctly localized and easily identified somata (see Fig. 5). In the third, used from d8 on, we selectively labeled the axons of the HN neurons anterogradely by applying DiI to the HN itself (Fig. 1C). Projection distances were quantitated in at least three preparations from each developmental day, starting at d4 (stage 23) (Fig. 8).
Fig. 7. The progressive longitudinal growth of axonal projections from commissural interneurons. Ventral views of the spinal cord at the indicated developmental stages. At d8, a single HN is labeled selectively; at earlier stages, the entire commissural axon population is labeled. Scale bars: 100 µm (d4.5), 500 µm (all subsequent stages).
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Fig. 8. The progressive longitudinal growth of axonal projections from the entire commissural interneuron population located between LS5 and LS6 (points and solid lines) or from HN-LS5/6 (dashed lines). Axon extent is expressed in terms of segmental reach (A) and absolute length (B) at the indicated developmental stages. Points represent maximum extents observed in single preparations, whereas the solid lines represent averages of these. Points are only shown for the early stages when maximum extents are more variable. For the entire commissural axon population, average extents are derived from three, seven, and four preparations at d4, d5, and d6, respectively. For the HN commissural axon population, average extents are derived from three preparations at each of d8 and d14.
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By d4, a few commissural axons extended directly across the ventral midline, some of them then turned either rostrally or caudally or bifurcated near the ventrolateral margin and extended longitudinally up to a distance of about one segment in one or both directions (Figs. 7, 8). By d5, the number of commissural axons had increased dramatically and had spurted 4 to 7 segments, or ~1.5 mm, in each direction (Figs. 7, 8). By d6, the axons had extended about another millimeter in each direction, although this was equivalent to only about two additional segments because of the concomitant longitudinal growth of the spinal cord (Figs. 7, 8). Throughout this period, the rostral and caudal projection distances were roughly symmetrical about the segment of origin.
At d7 and later, we could not determine the full longitudinal extent of the entire commissural axon population because the DiI labeling faded out before reaching the axon termini. This means that some commissural axons must extend many segments in each direction already at an early stage of spinal cord development. By contrast, we could determine the full reach of the HN axons at all stages because these were always strongly labeled throughout their extent.
At d8, the earliest time at which the HN axon population could be labeled selectively (Figs. 7, 8), the HN axons had extended about four segments in each direction. This is approximately equivalent to the mature HN projection (Eide, 1996
Development of collateral branches
When we labeled anterogradely all commissural axons crossing at a given level, the process of collateral sprouting could be followed in transverse sections rostral and caudal to the DiI application (Fig. 9). The first collaterals appeared at d7, i.e., approximately 3 d after the first commissural axons had begun to extend longitudinally in the ventral funiculus. Initially, the collaterals were straight unbranched neurites oriented at various angles into the gray matter in the transverse plane. Later, the angle made by the collaterals in the transverse plane generally depended on the mediolateral positions of the parent longitudinal axons; collaterals originating from more medial axons projected more dorsad, whereas those originating from more lateral axons projected more mediad (not shown). The collaterals were thus oriented approximately parallel to radial glia. By d9, the collaterals had initiated secondary branching (not shown).
Fig. 9. Early development of axon collaterals from the entire commissural interneuron population located between LS5 and LS6. Transverse sections through the contralateral side of the spinal cord. The commissural axon population has bifurcated to extend longitudinally in the ventral and ventrolateral white matter at d5 (A) and d6 (B), but collaterals first appear at d7 (C, arrow). By d8 (D, E), only a few unbranched collaterals have been elaborated. All sections are from the region just rostral to the DiI application site (DiI applied as in Fig. 1A). Scale bars, 100 µm.
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When we first selectively labeled the HN axons anterogradely at d8, only a very few collaterals were present, and these were slightly less mature than the collaterals labeled from the entire commissural projection at d8. This indicates that the HN axons lag the earliest commissural axons slightly in the establishment of collaterals. Moreover, it shows that the entire process of HN collateral sprouting can be followed by selectively labeling the definitive HN, starting at d8.
The first HN collateral sprouts appeared along much of the longitudinal extent covered by the HN axons on d8 (Fig. 10). With further development, collaterals gradually increased in number, most rapidly at proximal positions. Secondary branching was initiated between d10 and d12 (Fig. 10). The collaterals gradually became more elaborate, and by d14 the HN collateral arbors contacted much of the ventromedial gray matter (Fig. 10). Most collaterals were oriented toward the medial region of the ventral horn throughout this process, a feature especially prominent in collaterals arising distally, where the parent longitudinal axons have acquired more lateral positions. The termination pattern at d14 is similar to, but less focused than, the heavy termination in lamina VIII seen at d18 (Eide, 1996
Fig. 10. The development of axon collaterals from a single HN-LS5/6 at successively greater distances rostral to the HN (DiI application as in Fig. 1C). Each row of plates shows the pattern of labeling at the same indicated stage, with distance rostrad increasing from left to right over a distance of about four segments. Note the change in the mediolateral location of the longitudinal axons with increasing distance and the corresponding change in angle of collateral ingrowth. Scale bars, 100 µm.
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DISCUSSION
We have described the development of commissural neurons in the avian spinal cord, with special emphasis on the Hofmann nuclei major located at the ventrolateral margin of the spinal cord. The HN neurons exhibit many of the general properties of commissural spinal interneurons, and thus provide a good model for the study of commissural interneuron development in amniotes (Eide, 1996Birth and radial migration
The HN neurons are born between stages 21 and 24, starting about a half day after the very first interneurons are born in the lumbar spinal cord (Hollyday and Hamburger, 1977By analogy to the mammalian cortex (McConnell, 1992
Although we have not witnessed the migration of HN neurons from their birth sites in the ventricular zone, their radial trajectories in the ventral neural tube suggest that they are born in the ventral part of the ventricular zone (see Fig. 2). A ventral origin of the HN neurons stands in contrast to the apparent dorsal origin of at least some other commissural interneurons (Oppenheim et al., 1988
Somatic motoneurons also originate from the ventral ventricular zone and migrate laterally in advance of the HN neurons, establishing the lateral motor column through which the HN neurons migrate. Evidently, motoneuron and HN neuron lineages have diverged before the birth of the motoneurons. After labeling motoneuron progenitors with a retroviral lineage tracer at stages 11-18 (d2-d3), Leber et al. (1990)
Commissural axon outgrowth
The radially migrating HN neurons can first be labeled retrogradely from the contralateral side starting at approximately d6. Other commissural interneuron types located medial and dorsal to the motoneuron column evidently are the source of the first commissural axons we have observed with anterograde labeling. Oppenheim et al. (1988)The HN neurons can be labeled retrogradely from the contralateral side of the spinal cord before reaching their definitive positions. Thus, they appear to be determined as commissural neurons before or during migration and not by virtue of their eventual location at the ventrolateral margin of the neural tube. In this regard, the HN neurons are similar to spinal motoneurons. Before reaching their definitive migratory positions, motoneurons express specific combinations of transcription factors of the LIM homeobox gene family (Tsuchida et al., 1994
In general, commissural axon outgrowth in the spinal cord follows a circumferential trajectory that is believed to be steered by contact guidance cues, followed by attraction to and interaction with the floor plate (Colamarino and Tessier-Levigne, 1995). The site of axonogenesis for interneurons located dorsal to the motor column is from the leading (pial) process of the commissural interneuron, because contact with the circumferential pathway is made near the pial surface (see Fig. 6). But ventrally, where the HN neurons are generated, the circumferential pathway lies not at the pial surface, but rather medial to the lateral motor column. Because HN neurons eventually migrate past this point, their axons could derive either from the leading (pial) process or the trailing (ventricular) process, depending on the timing of axonogenesis and migration. This illustrates a potential diversity in the way commissural interneurons interact with their pathways.
Termination pattern
To our knowledge, this is the first description of the development of termination pattern by spinal interneurons in an amniote. Several features warrant mention. (1) Collateral sprouting does not begin until the full segmental range of the longitudinal axons is established. Such delayed sprouting has been observed in other axon systems (O'Leary et al., 1990
FOOTNOTES
Received March 21, 1996; revised June 27, 1996; accepted July 2, 1996.
This work was supported by grants from the Norwegian Medical Research Council and the Nansen Foundation.
Correspondence should be addressed to Anne Lill Eide, Department of Physiology, Institute of Basic Medical Sciences, P.B. 1103 Blindern, University of Oslo, 0317 Oslo, Norway.
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