THE JOURNAL OF COMPARATIVE NEUROLOGY 306:95-116 (1991)

Outgrowth of the Pyramidal Tract in the Rat Cervical Spinal Cord: Growth Cone Ultrastructure and Guidance T.G.M.F. GORGELS Department of Anatomy and Embryology, University of Nijmegen, 6500 HB, Nijmegen, The Netherlands

ABSTRACT In order to examine the mode of outgrowth of the pyramidal tract in the rat, the ultrastructure of its pathway in the dorsal funiculus of the spinal cord was analysed. The analysis was performed by means of serial sections of the third cervical segment before and during the arrival of pyramidal tract axons, and focussed on the morphology and microenvironment of the growth cones. Growth cones appear as elongated terminal enlargements without side branches. Two zones could be discerned: the distal, usually lamellipodial fine granular zone, containing no organelles, except for an occasional clear vesicle; and the proximal organelle-rich zone, which contains various organelles, such as agranular reticulum and vesicular structures. In addition, the proximal organelle-rich zone contains round or elliptic structures, limited by two concentric membranes, that enclose reticular and vesicular elements. The electron density of these structures varied from as low as the surrounding growth cone matrix to as dark as lysosomal structures, suggesting their involvement in turnover processes. At embryonic day 20, the most ventral part of the dorsal funiculus, where the first pyramidal tract axons are due to arrive within two days, is populated by axons that are relatively small compared to those in the rest of the dorsal funiculus. At birth, the arrival of the first pyramidal tract axons is marked by the presence of numerous large growth cone profiles in between small axons in the most ventral part of the dorsal funiculus; no circumscript bundle separated from the ascending sensory fiber tracts is present yet. The growth cones descend, club-shaped and 1to 2 pm in diameter, without lamellipodia or filopodia. Within the same area a second growth cone type is present, which contains dense-core vesicles and has spread-out lamellipodia. Most of these growth cones are ascending and they probably belong to primary afferent or propriospinal fibers. At postnatal day 2, the pyramidal tract can be readily delineated from the adjacent fasciculus cuneatus where myelination has already started, but no glial boundary is present. The abundant growth cones are 1-2 pm wide and extend single unbranched lamellipodia, up to 15 pm long, which often enfold parallel axons or other growth cones. At postnatal day 4,growth cones are scarce in the tract. They measure 1pm or less in diameter and each extends a single, straight lamellipodium or filopodium over 1to 7 pm in the caudal direction. At all ages examined, most of the surface of the growth cones is in apposition to longitudinally oriented axons and growth cones. The caudally outgrowing growth cones also make contact with glial elements. These are predominantly oriented perpendicularly to the longitudinal axis and are not joined together into continuous arrays. Furthermore, growth cones form synaptic contacts with dendrites that extend into the dorsal funiculus from the adjacent spinal grey. These observations indicate that the morphology of growth cones changes during the formation of the pyramidal tract in the cervical spinal cord. It is proposed that the outgrowing axons are probably guided into their pathway by selective fasciculation on their precursors, and by local differences in oligodendrocyte and axon maturation within the dorsal funiculus. Furthermore, it is suggested that the leading growth cones of the pyramidal tract grow into the most immature region of the ascending sensory fiber tracts without actively fasciculating on the axons of these tracts. Key words: corticospinaltract, axon fasciculation, axon loss, cell adhesion molecules, lysosomes

Accepted November 21,1990. Q

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The pyramidal tract (PT) represents a major descending fiber system in the mammalian central nervous system (CNS), originating from the neocortex and projecting into the brainstem and the spinal cord (Wise et al., '79; Armand, '82; Kuypers, '82). The PT is characterized by relatively late development. In the rat, the timing of its outgrowth has been determined with normal axonal staining, anterograde fiber degeneration, and anterograde tract tracing using horseradish peroxidase (HRP) or wheat germ agglutinin-conjugated HRP (DeMyer, '67; Donatelle, '77; Schreyer and Jones, '82; De Kort et al., '85; Gribnau et al., '86). These studies have shown that the first corticospinal axons arrive around birth in the cervical spinal cord, where they grow into the dorsal funiculus (DF), which already contains the fasciculi cuneatus and gracilis (Altman and Bayer, '84). The formation of the PT in the most ventral part of the DF comes about by the successive arrival of new fibers over a period of at least one week (Schreyer and Jones, '82; Gribnau et al., '86; Gorgels et al., '89a). Several mechanisms have been proposed for the guidance of outgrowing axons and for the formation of axon tracts. These mechanisms focus on interactionsbetween the growth cones, the motile enlargements at the tips of the outgrowing axons, and their local environment. The growth cones may be guided mechanically by boundaries or channels, formed by glial cells (Singer et al., '79; Poston et al., '88). Since growth cones in vitro grow along substrates to which they adhere best (Letourneau, '75), they might also follow a pathway in vivo, determined by the distribution of adhesion molecules in the extracellular matrix and at neuronal and non-neuronal cell surfaces in the developing CNS (Katz et al., '80; Letourneau, '82; Edelman, '83; Dodd and Jessel, '88). Recent in vitro studies demonstrated that during axon outgrowth, inhibitory factors may also play a role in defining the pathways followed (Patterson, '88; Schwab and Caroni, '88). Apart from these contact-mediated guidance cues, in vitro experiments have indicated that gradients of diffusible substances (Gundersen and Barrett, '80; Lumsden and Davies, '83; Heffner et al., '90) and weak electrical fields (Hinkle et al., '81; Pate1 et al., '85) can direct axon outgrowth. The morphology and ultrastructural characteristics of growth cones have been described in detail (Skoff and Hamburger, '74; Landis, '83; De Kort et al., '85). In vitro studies have demonstrated that the shape of growth cones is influenced by the geometry and chemical properties of the substrate (Letourneau, '82; Harris et al., '851, by contact with other cells (Kapfhammer and Raper, '871, and by focal application of neurotransmitters (Mattson, '89). In vivo, the morphology of growth cones in developing nerve tracts varies with their position along the pathway, which in part may reflect differences in the local microenvironment of the growth cones (Tosney and Landmesser, '85; Bovolenta and Mason, '87; Nordlander, '87; Kalil, '88). In the present study, the outgrowth and guidance of the PT in the rat spinal cord is studied by examining the ultrastructure of the developing PT at the level of the third cervical segment, just caudal to the pyramidal decussation. The ventral part of the dorsal funiculus, where the PT in the rat is situated (cf. Kuypers, '81; Schreyer and Jones, '82) is analysed by serial section electron microscopy over the period from embryonic day 20, two days before the arrival of the first PT axons, t o postnatal day 4, when most of the PT axons have arrived at this level of the tract (Schreyer and Jones '82; Gribnau et al., '86; Gorgels et al.,

T.G.M.F. GORGELS '89a). The analysis was performed on normal, unlabeled material in order to ensure optimal preservation of ultrastructural details, and focussed on the precise shapes, contents and cellular relationships of the growth cones that successively pass this segment.

MATERIALS AND METHODS The animals used in this study were Wistar albino rats; animals were taken on the 20th day of gestation (embryonic day 20 = E20), and 0, 2, and 4 days postnatally (PO, P2, P4). Embryonic age was calculated by regarding the day upon which spermatozoa were observed in vaginal smears taken from the dams as embryonic day 1. In our colony, bred at the central animal laboratory of the University of Nijmegen, litters were usually born on the 22nd day of gestation (E22 = PO). Animals designated as PO were sacrificed within 2 hours of birth. Embryos were delivered by Caesarian section and fixed immediately by transcardial perfusion with 2% glutaraldehyde, 2% formaldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, containing 2 mM CaCl,. Postnatal animals were anesthetized by intraperitoneal injection with sodium pentobarbital (40 mg per kg body weight), prior to perfusion. After perfusion fixation, the cervical spinal cord was removed and immersed in the fixative overnight at 4°C. The next day, the third cervical segment (C,) was transversely resected from the cord, rinsed in buffer, and postfixed in 1% OsO,, 0.05 M K,Fe(CN), in buffer (modified from Karnovsky, '71) for 2 hours and, after rinsing in doubledistilled water, stained en bloc for 2 hours in 0.5% aqueous uranyl acetate. Subsequently, the tissue was dehydrated in ethanol and embedded in Epon. Series of ultrathin sections (80-90 nm thick) were cut and collected on single slot Formvar-coated copper grids, contrasted with uranyl acetate and lead citrate, and examined in a Philips EM 300 electron microscope at 60 kV. Serial section electron microscopy was employed to allow for identification of the neuronal and glial elements and to reconstruct the 3-D shape of growth cones in the developing PT. In total 6 series of 200-300 transverse sections and 8 series of 50-200 horizontal sections were prepared. Growth cones, which were at each age randomly selected in various parts of the tract, were traced in the series of transverse sections. At least every fifth section was photographed at a primary magnification of 3,600-5,700 X , and printed at an enlargement of 4 x higher. In total 51 sets of micrographs, containing parts of several hundred growth cones, were obtained. Graphical reconstructions were made of the growth cones that terminated in the series of sections: the outlines of these growth cones were traced onto transparent paper, together with the outlines of at least 5 cross-sectioned axons that served as points of reference (Stevens et al., '80). The successive transparencies were then superimposed and aligned by manoeuvring the points of reference to the "best fit" position (Hinds and Hinds, '74). A 3-D drawing of the growth cone was made by successively placing the aligned growth cone profiles at appropriate distances on a z-axis oriented at an angle of 30" to the x-axis of the individual drawings. Measurements of the size of growth cones and axons were made using a Kontron videoplan measuring system, calibrating the magnification with a diffraction grating replica (2,160 lines per mm). For elliptic to round structures the minimum diameter was measured.

GROWTH CONES IN THE RAT CERVICAL PYRAMIDAL TRACT

RESULTS Ultrastructural features of PT growth cones Before describing the variation in morphology of growth cones encountered in the development of the PT, it is necessary to give a detailed description of the general ultrastructural characteristics of PT growth cones, in order to set up a frame of reference on growth cone morphology and terminology. This description is mainly based upon data from the cervical PT of the 2-day-old rat, since at this stage growth cones are abundant and well elaborated. In horizontal sections growth cones appear as elongated terminal enlargements, oriented longitudinally in the tract. On the basis of an uneven distribution of organelles, two subdivisions can be distinguished in the longitudinal direction. In the distal part of the growth cone, a fine granular cytoplasmicmatrix is present without organelles, except for an occasional clear vesicle (Fig. 1).Small indentations into this part of the growth cone, caused by neighbouring axons, are frequently observed (Fig. 1). The distal, fine granular part of the growth cone has in transverse sections an irregular contour and forms an undulating, lamellipodial extension of the growth cone. In contrast, the proximal part of the growth cone contains a great variety of organelles; an agranular reticulum is the most prominent content of these (Fig. 1).The agranular reticulum consists of predominantly longitudinally oriented, presumably interconnected strands of membranes. In transverse sections these have the appearance of small strands, triangles, or dots composed of 2 membranes. In contrast to perinuclear agranular reticulum (e.g., Figs. 7, 8, 10) agranular reticulum in growth cones has no lumen or only a very narrow lumen with an electron-dense content (Fig. 2E). In transverse sections larger, more-or-less straight profiles of 2 tightly packed membranes were also observed which sometimes spanned nearly the full width of the growth cone (Figs. ID, 2D,F, 7A, 15). In serial sections, these membranous profiles appeared to be sections of discs of two tightly packed membranes. Apart from the meshwork of agranular reticulum, the proximal organelle-rich region of the growth cone contains vesicles of various sizes and contents, including clusters of clear vesicles of approximately 45 nm (Fig. 1).Occasionally, the fusion or formation of a coated vesicle was discerned at the growth cone membrane (Fig. 1D). Furthermore, the proximal organelle-rich region contains microtubules, but neurofilaments were not observed (Fig. 1A). Mitochondria are also present, although not in great abundance. Ribosomes and glycogen granules were not observed. In the proximal organelle-rich growth cone region, round or elliptic structures were frequently observed which appeared to be portions of growth cone cytoplasm limited by 2 concentric membranes and containing several strands of

Fig. 1. Electron micrographs of a horizontal section of the PT at the third cervical spinal cord segment in a 2-day-old rat, illustrating the general features of PT growth cones. A A growth cone profile (clear stars) oriented rostrocaudally in the tract. B-D Details of the proximal organelle-rich part of the growth cone, which contains strands of agranular reticulum, and various other organelles, including microtubules (arrowhead in A), a cluster of small clear vesicles (small arrow in B), a coated vesicle (small arrow in D), a membranous disc (arrowhead in D) and a so called growth cone parcel (black star in C). E: Detail of the distal part, the lamellipodium with a fine filamentous ground substance. Bars represent 1km (A) and 0.25 krn (B-E).

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agranular reticulum and occasionally some vesicles (Figs. l C , 2). Serial sectioning showed that these structures, which will be referred to as growth cone parcels, are in general completely enclosed by the two enveloping membranes. These two membranes are often tightly packed together, with no interstitial space (Fig. 2E). Occasionally, a growth cone parcel was observed in close apposition to a membranous disc (Fig. 2F). The electron density of the matrix within growth cone parcels is variable: Often the density is similar to the density of the surrounding growth cone cytoplasm, but also more electron-dense growth cone parcels are found. The darkest growth cone parcels with a mottled, electron-dense matrix and strands or whorls of membranes (Figs. 2A,B) resemble lysosomal structures (Peters et al., '76). In addition, growth cones contain electron-dense bodies, presumably lysosomes, with a homogeneously dense granular matrix (Fig. 2). Some gradual proximo-distal differences were noticed in the distribution of organelles in the organelle-rich growth cone region: Microtubules become rare in more distal parts and stop close to the transition into the fine granular, lamellipodial zone. In contrast, agranular reticulum prevails in the distal part of the organelle-rich growth cone region, where it forms a dense meshwork, and gradually decreases in the proximal part, where in transverse sections, only individual, small strands or dots are observed. Electron-dense bodies are most frequently found in the proximal part of the organelle-rich region. The shape of the organelle-rich region of PT growth cones is, except for the distal part close to the lamellipodium, more-or-less tubular, measuring generally 1-2 pm in diameter, which is considerably larger than the surrounding axon shafts of on average 0.3 +m. In general, no sharp transition of the growth cone into the axon shaft was observed: In serial sections, the growth cone proximally decreases gradually in diameter. The borderline between the axon shaft and the growth cone cannot be indicated precisely, since local constrictions and swellings occur and axon shafts often contain, apart from microtubules, also strands of agranular reticulum and vesicles, while neurofilaments are usually absent. This means that the length of the growth cone cannot be determined precisely.

Embryonic day 20: ultrastructure of the dorsal funiculus prior to arrival of PT axons At E20, the ascending sensory fiber tracts are present in the DF. In the most ventral part of the DF, where the PT is due to arrive within 2 days, mainly small unmyelinated axons are present, which are not organized into fascicles (Fig. 3). These axons measure 0.2-0.4 pm in diameter and contain microtubuli and only seldomly neurofilaments. More dorsally in the DF, the axons are on average considerably larger and frequently contain neurofilaments. There is a gradual transition from the ventral part of the DF to more dorsal regions without a distinct glial boundary or differ-

ences in glial organization. Longitudinally oriented growth cones were observed in both ventral and dorsal regions of the DF. Apart from the general growth cone organelles as described above, these growth cone profiles contain characteristically dense-core vesicles (Fig. 3B). The diameter of the organelle-rich region of these growth cones rarely exceeds 1 pm (Fig. 3C).

Postnatal day 0: Two types of growth cones In the 6 animals examined a t this age, a considerable variation in ultrastructural appearance of the most ventral part of the DF was found. In 2 animals, a distinct PT was readily observed with a substantial number of axons, whereas in 1animal no indication of the arrival of PT axons was observed. In the remaining 3 animals there were several indications of the arrival of some P T axons, but a distinct bundle had not yet been formed. The latter situation in particular is most suitable for the analysis of the outgrowth of the first P T axons, and the following description is based upon observations made on 2 of these animals. The most striking difference in ultrastructural appearance of the DF at PO compared with E20 is the presence of numerous large growth cone profiles in between small unmyelinated axons in the ventralmost part of the DF. The area rich in growth cones is limited to the ventralmost part of the DF, but is not distinctly separated from the rest of the DF. Neither a clear glial boundary nor a distinct bundle is present. There is a gradual decrease in growth cone density in dorsal and lateral direction. The examination of serial sections and reconstruction of growth cones revealed the presence of two types of growth cones in the most ventral part of the DF (Fig. 4).Most of the growth cone profiles belong to a type indicated as type A. These growth cones terminate in the caudal direction and thus belong to descending axons. They contain a loose meshwork of agranular reticulum. Other organelles are relatively scarce, although the full complement of general growth cone elements as described above is present. In transverse sections, the profiles are generally round to elliptic and measure 1 to 2 pm in diameter. In the distal, i.e., caudal, direction the meshwork of agranular reticulum gradually becomes more dense. Type A growth cones end rather abruptly in distal direction, where a fine granular zone, devoid of organelles, is virtually absent: Agranular reticulum extends into one or two sections (0.1-0.2 pm) from the leading edge of the growth cone (Fig. 5). Only one of the type A growth cones traced in the serial sections (Fig. 4,No A l ) terminated in a very small (0.3 pm long) filopodial protrusion (Fig. 7). Clear vesicles are observed frequently close to the leading edge of the growth cone. The generally blunt ending, tubular form and large diameter gives type A growth cones the overall appearance of club-shaped, bulbous endings. Most of the type A growth cones were longer than the 25 pm covered by the series of 300 transverse sections used for reconstruction. In horizontal sections, profiles of type A

Fig. 2. Growth cone profiles (clear star in A) in horizontal (A) and transverse sections (B-F) of the cervical PT in a 2-day-old rat, illustrating the morphology of growth cone parcels (straight arrows in A-F), electron-dense bodies (white asterisks in B,F), and membranous discs (bent arrows in D,F). The electron density of the matrix within growth cone parcels often may be similar to that of the surrounding growth cone matrix (B,D-F), but also darker (A,B). Growth cone

parcels are surrounded by two generally tightly packed membranes (El. D,F: membranous discs (bent arrows), visible as more or less straight profiles of two tightly packed membranes that span nearly the full width of the growth cone profiles. Figure F shows the apposition of a growth cone parcel to a disc. Bars represent 0.5 1J-m(A-D) and 0.25 1J-m (E,F).

GROWTH CONES IN THE RAT CERVICAL PYRAMIDAL TRACT

Figure 2

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Fig. 3. Ultrastructure of the most ventral part of the dorsal funiculus in transverse sections of the third cervical segment at E20, prior to the arrival of the PT (pyramidal tract).A shows the presence of unmyelinated axons without apparent fascicular organization. B: A growth cone profile in the DF (dorsal funiculus), identified by its

relatively large size and the presence of agranular reticulum and vesicles. C : A growth cone profile at the transition between the proximal organelle-rich growth cone region and the distal lamellipodial region. Note the presence of dense-core vesicles in these growth cones (B,C). Bars represent 1pm (A) and 0.25 pm (B,C).

growth cones of 30-40 pm length can be readily observed. In tracings of about 100 growth cones over the length of the series of transverse sections (25 pm), lateral protrusions were never observed. Twice, a growth cone bifurcated with both parts extending in parallel in the longitudinal direction. Apart from the club-shaped type A growth cones just described, another, less abundant type of growth cone, indicated as type B, was present in the ventralmost part of the DF (Fig. 4).Six out of 7 randomly selected growth cones of this type, that were traced in the serial sections, terminated in the rostral, ascending direction, whereas one was oriented in the caudal direction. The proximal organellerich region of type B growth cones contains all the general growth cone organelles, as described above. In addition, type B growth cones contain dense-core vesicles, which is a major difference with type A growth cones. The dense-core vesicles are present in the organelle-rich growth cone region as well as in the proximal part of the lamellipodia (Fig. 6). They are also present within growth cone parcels. The diameter of the proximal organelle-rich region of type B growth cones is relatively small and increases steadily in distal direction up to approximately 1 km at the transition to the fine granular zone. The fine granular zone consists of a single or, more often, multiple lamellipodia, that may break up distally in filopodia (Fig. 4,No. B2). The lamellipodia and filopodia extend over 8-15 km in longitudinal

direction, spanning 3-7 km in the lateral direction. Thus type B growth cones are more-or-less funnel-shaped with spread-out lamellipodia, which contrasts sharply with the bulbous, club-shaped type A growth cones, that lack lamellipodia. In addition to these differences between the two types of growth cones, the cytoplasmic matrix in the type B growth cones appears to be slightly more electron-dense. Type B growth cones belong most likely to primary afferent or propriospinal fibers, and not to corticospinal axons as will be discussed later.

Environment of PO growth cones Axons and growth cones. The cellular environment of both types of growth cones in the PT consists of axons and other growth cones, glial elements, and dendrites. Most of the surface of growth cones is in apposition to aligned, parallel oriented axons and growth cones. Serial sections show that this also holds true for the most distal parts of the growth cones. Immediately in front of the growth cones no enlarged extracellular space was observed between the longitudinal axons and growth cones. The aligned axons are small and unmyelinated. They contain a few microtubuli, agranular reticulum and occasional vesicles, but no neurofilaments (Figs. 5, 6). At the contact zone between growth cones and aligned axons distinct membrane specializations or sub-membrane coatings were not observed, but protru-

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Fig. 4. 3-D graphical reconstructions based on every tenth section of the terminal parts of growth cones traced in series of transverse sections at PO. Black profiles represent sections through the organellerich growth cone region whereas sections through the fine granular, lamellipodial region are dotted. Arrow points in caudal direction. Two

distinct types of growth cones were discerned. Type A growth cones terminate all in caudal direction without or with only very small filopodial extensions. Type B growth cones may ascend (No. 1)as well as descend (No. 2) and have long and branching lamellipodia and filopodia. Scale: 1 Fm.

sions and invaginations can occur frequently, most notably of axons into lamellipodia (Figs. 1, 15).A slight tendency of the bulbous type A growth cones to group together was noticed. Several mutual protrusions can be observed between these neighbouring type A growth cones and one growth cone terminated as a small filopodial protrusion into a neighbouring growth cone (Fig. 7). Appositions between type A and type B growth cones were not observed. Glial elements. Both type A and type B growth cones were occasionally found in apposition to glial elements in the tract. The glial processes generally follow a winding path perpendicularly to the longitudinally oriented growth cones, and are not joined together into continuous arrays in the longitudinal direction. The glial profiles generally have an irregular shape and contain occasional profiles of endoplasmic reticulum and microtubules. Some of them have an electron-lucent cytoplasm and contain many glycogen granules (Fig. 12); others have a relatively electron-dense cytoplasm and often contain free ribosomes. Bundles of intermediate filaments, a characteristic of mature astrocytes, were only rarely observed in glial processes. At the

contact zone of growth cones and these immature glial elements, synapse-like specializations and small protrusions of the growth cone into the glial cell were frequently observed. The morphology of these contacts and of the glial elements involved are dealt with in detail in the accompanying paper (Gorgels, '91). Glial elements do not seem to form physical boundaries around the PT, except for the posterior median septum (Fig. 8C), which separates the right and the left PT. Growth cones were occasionally observed along this septum, but close to the septum no increased growth cone density was apparent. Dendrites. Dendrites of neurons located in the spinal grey are present in the most ventral part of the DF, where the caudal outgrowth of the PT takes place (Fig. 8). These processes are predominantly oriented within the transverse plane and extend more-or-less radially from the grey matter into the DF. Microtubuli, mitochondria, granular and agranular reticulum, and free ribosomes are present in the proximal part of these processes, whereas these organelles, apart from microtubules were only scarcely observed in the

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Fig. 5. 3-D graphical reconstruction based on every fifth section of the terminal part of a PO type A growth cone (Fig. 4, No. 61,with electron micrographs illustrating the ultrastructure a t or close to the levels of the profiles indicated in black. Arrow points in caudal direction. Agranular reticulum is the most prominent organelle in this growth

cone. In the micrograph on the left, which is taken at 0.2 pm of the leading edge of the growth cone, the growth cone profile is indicated by arrows. Note the presence of agranular reticulum (arrowhead) in this profile. Scale: 1pm.

distal parts. Along their course they frequently have varicosities (Figs. 9B,C) with a relatively electron-lucent cytoplasm in which a few microtubuli and occasionally agranular reticulum, vesicles and vacuoles were observed. Examination of serial sections revealed that some of these varicosities are terminal, with small filopodial protrusions, and thus probably represent dendritic growth cones (Hinds and Hinds, '72; Skoff and Hamburger, '74; Vaughn et al., '74). The dendrites observed in the ventralmost part of the DF at PO, make several synaptic contacts with distinct pre- and postsynaptic densities. The presynaptic elements involved contain round synaptic vesicles or, very rarely, elliptic vesicles. The presynaptic elements include axons as well as axonal growth cones (Figs. 8, 9). Presynaptic growth cones are usually oriented longitudinally, thus perpendicular to the dendrites (Figs. 9A,B) and included both type A and B

growth cones. In addition, however, 4 growth cones were observed that were oriented along the transversely running dendrite with which they make contact (Figs. 9C,D). These growth cones all contained dense-core vesicles, characteristic of type B growth cones. For two of these growth cones it was established by examining serial sections that they had lamellipodia pointed at or into the grey matter.

Postnatal day 2: large growth cones with a single unbranched lamellipodium In the 2-day-old rat the P T can be discerned as a circumscript, homogeneous bundle in the ventralmost part of the DF, located ventral and medial to the fasciculi cuneatus and gracilis. Although this bundle is not separated from the rest of the DF by glial boundaries, a

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Fig. 6. 3-D representation based on every tenth section of the terminal part of a PO type B growth cone (Fig. 4,Bl), with electron micrographs illustrating the ultrastructure at or close to the levels of the profiles indicated in black. Arrow points in caudal direction. This

growth cone terminates in rostra1 direction with two perpendicularly oriented lamellipodia. Dense-core vesicles are present in this growth cone. Scale: 1pm.

borderline can be readily drawn between the PT and the ascending sensory fiber tracts (Fig. 10): The former contains numerous growth cones and small immature axon shafts, whereas the latter contains many larger axons with neurofilaments. In the fasciculus cuneatus myelinating oligodendrocyte processes are abundantly present (Fig. 10). All growth cones that were traced in the series of sections in various parts of the tract terminate in the caudal direction and thus belong to descending axons. As described above, PT growth cones at P2 terminate in a fine granular, lamellipodial zone. This fine granular zone varies greatly in shape and size, but consists basically of a single unbranched lamellipodium which is directed caudally (Fig. 11).The length of this lamellipodium varies from 1 to 15 pm. In transverse sections these lamellipodia can span up to 10 pm, but most typically they tend to form S shapes or more complex forms in the transverse direction, since they fold around contiguous axons and growth cones (Fig. 11, Nos. 6-9). Distally, the lamellipodium may terminate in 1 or 2 small filopodial protrusions. The diameter of the proximal organelle-rich region varies along the length of the growth cone. Generally, this part of the growth cone is a 1-2 pm wide tubular varicosity that

flattens in distal direction to form the lamellipodium. Most growth cones are longer than the 25 pm length of the series of transverse sections. Lateral branching of the growth cone was not observed. Occasionally, the growth cone is split into two parts that are both directed caudally (Fig. 11, Nos. 5, 6, 12). At the site of bifurcation a glial process was observed laying across the growth cone (Fig. 12).In comparison to the club-shaped PO type A growth cones, the P2 growth cones appear to contain a higher density of organelles. In contrast with PO type B growth cones, P2 growth cones do not contain dense-core vesicles.

Environment of P2 growth cones At P2, growth cones appear to be randomly distributed within the transverse plane of the tract and are as at PO predominantly located in between parallel axons and other growth cones (Fig. 10). The large lamellipodia of P2 growth cones contact many axons and growth cones and frequently fold around them over distances of up to 10 pm (Figs. 11, 13). This contrasts markedly with the large spread-out lamellipodia of PO type B growth cones that do not display this behavior.

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Fig. 7. A illustrates the ultrastructure of the ventralmost part of the dorsal funiculus at PO. Several large profiles of type A growth cones with agranular reticulum, discs and mitochondria are present between small axon shafts. In addition, a lamellipodium of an ascending type B growth cone is present (arrow). The type A growth cone indicated by a clear star (see Fig. 4 No. A l ) was graphically reconstructed as shown in

the drawing which represents every tenth section. The micrographs A-C were taken at the levels of this growth cone as indicated in the drawing. The growth cone terminates in the caudal direction in a small protrusion (arrows in C,D)into a neighbouring growth cone (black stars in A-D). The electron micrograph in D, taken between B and C , shows this protrusion more clearly. Scale: 1 bm.

The relations between growth cones and glial elements are essentially the same as described for PO. Synaptic contacts between growth cones and dendrites appear to be less numerous than at PO, and were only observed between longitudinal growth cones and perpendicularly oriented dendrites. Axons and glial processes were frequently observed lying along the scarce dendrites in the tract.

cones, P4 growth cones are small and have a simple shape. Nevertheless, all the growth cone organelles described above are present in these growth cones (Fig. 15).

Postnatal day 4: small, simple growth cones At P4, growth cone profiles are much less numerous in the tract. In general, they terminate in a fine granular zone which extends in the caudal direction as a single, unbranched, straight lamellipodium or filopodium of 1-7 pm length (Fig. 14). The breadth of the lamellipodia of P4 growth cones can measure 2 pm but generally is much smaller. The diameter of the core region of P4 growth cones does not exceed 1 pm. Thus, in comparison to P2 growth

Environment of P4 growth cones At P4, the growth cones are distributed throughout all parts of the tract with no apparent distribution pattern in the transverse plane. As at PO and at P2, the growth cones are predominantly situated in between axom and several invaginations between axons and growth cones, most frequently into the lamellipodial parts are present (Fig. 15). Lamellipodia of P4 growth cones were not observed to enfold axons. The relations with glia are essentially the same as described for PO and P2. Dendrites are scarce in the tract and at P4 synaptic contacts between P4 growth cones and dendrites were not observed. Axons and glial processes were frequently observed lying along these dendrites.

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Fig, 8. Axodendritic synapses in the DF at PO. A The most ventral part of the DF, the adjacent spinal grey matter and the posterior median septum (PMS).A dendrite (clear star) of a neuron (N) located in the spinal grey, extends into the DF. B,C: Details of the sites indicated by arrows in A, either in the same section (C) or in a nearby section (B),

showing axodendritic synapses (arrowheads). These synapses are made by an axon oriented parallel to the dendrite (B) and by an axon oriented perpendicular to the dendrite (0.In B, dense-core vesicles are present in the presynaptic element (bent arrow). Bars represent 2 p,m (A) and 0.5 W r n (B,C).

Degenerating growth cones

only four of such growth cones encountered during the analysis were traced in the series of sections. One changed gradually into a growth cone with a normal appearance, whereas the other was continuous with a very small, relatively electron-dense axon shaft. Vacuoles, electrondense bodies and a disorganization of the membrane were observed in this growth cone (Fig. 16A-C), which suggest that the growth cone was degenerating. These growth cone profiles were not apposed to glial cells.

During the analysis of growth cone profiles, abnormal ultrastructural features were occasionallyobserved, suggesting degeneration. Most of such growth cone profiles display ruptures of the plasma membrane, disorganization of the meshwork of agranular reticulum, and an accumulation of vesicular structures, including electron-dense bodies. These growth cones generally were in apposition to, or sometimes even surrounded by, electron-lucent glial processes, which possessed intermediate filaments and therefore probably represent astrocytic processes (Fig. 16D). In addition, large profiles (2-15 km in diameter) with densely packed mitochondria and electron-dense bodies were present which display growth cone characteristics, such -as strands of agranular reticulum as well as growth cone parcels, and frequently also glial characteristics such as intermediate filaments and glycogen granules (Fig. 16E). Such profiles were observed most frequently at P4, only rarely at P2, but not at PO. Finally, a few growth cone profiles were observed with an abnormally electron-dense cytoplasm. Two of the

DISCUSSION Ultrastructural features of PT growth cones The PT growth cones examined in serial sections for the present study are elongated, virtually unbranched, terminal enlargements, oriented longitudinally in the dorsal funiculus. On the basis of an uneven distribution of organelles, the growth cone could be divided into 2 zones: A distal fine granular zone, which contains a fine filamentous matrix with no organelles except for a few small clear

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Fig. 9. Synaptic contacts of growth cones on dendrites at PO, in transverse (A,C,D) and horizontal (B) sections. A,B: Synaptic contacts of longitudinally oriented growth cones (clear stars) with perpendicular dendrites (triangle in B). Note the varicosity in the dendritic process in B. C: Two profiles of the same growth cone (clear stars) oriented within the tranverse plane and pointing toward the grey matter. The growth

T.G.M.F. GORGELS

cone is situated along a presumably dendritic process (triangles) with varicose swellings, with which it makes synaptic contact (large arrow). D: The borderzone of the DF (top) and the grey matter (bottom). A growth cone (clear star) oriented into the grey matter makes synaptic contact on a dendritic process (triangle). The growth cones in C and D contain dense-core vesicles (small arrows). Bars represent 1 km.

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Fig. 10. Electron micrograph of the border between the fasciculus cuneatus and the pyramidal tract in a transverse section of the 2-day-old rat. The borderline (dashed line) runs approximately along the diagonal and separates the pyramidal tract containing numerous

growth cone profiles and small immature axons, from the fasciculus cuneatus, which contains many larger axons, including myelinated axons, and many oligodendrocyte processes. Bar represents 1Fm.

vesicles, and a proximal organelle-rich zone, which contains a great diversity of organelles. Although the ultrastructure of PT growth cones corresponds well on major points to previous ultrastructural descriptions of growth cones in situ (Skoff and Hamburger, '74; Landis, '83;Williams et al., '861, there are nonetheless some remarkable differences, such as the absence of neurofilaments in PT growth cones, as noted before by De Kort et

al. ('85). Another remarkable feature of the growth cones examined in the present study, is the abundant presence of peculiar structures, designated as growth cone parcels, which contain agranular reticulum and vesicles, surrounded by 2 generally tightly packed membranes. These organelles probably occur in other growth cones as well, since a great variety of vesicular and lysosomal structures is usually present in growth cones (Landis, '83). Lampert

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1

n

3

9

Fig. 11. 3-Dgraphical reconstructions based on every tenth section of the terminal parts of growth cones traced in series of transverse sections of a 2-day-old rat. Black profiles represent sections through the organelle-rich region of growth cones, whereas sections through the fine granular, lamellipodial region are dotted. Arrow points in caudal

direction. The growth cones extend a single lamellipodium of highly variable length, width, and shape. The larger lamellipodia tend to fold, forming S shapes or more complex forms in the transverse direction, while enfolding longitudinally oriented axons and growth cones (Nos. 7-9). Scale: 1 km.

('67)has described similar portions of axoplasm enclosed by 2 or 3 membranes in the tip of regenerating axons. In the present study, fixation with K,Fe (CN), reduced OsO, was employed which is known to enhance preservation of membranes and membrane contrast (Langford and Coggeshall, '80). This fixation method may render these organelles more conspicuous, since their enclosing layer of typically 2, tightly packed, membranes stands out clearly. The electron density of the growth cone parcels is variable and may be as dark as lysosomal structures, which suggests

that they are involved in the turnover of growth cone material or of endocytosed material. In addition, growth cones in the PT contain discs of 2 tightly packed membranes, which often span nearly the full width of the growth cone. Similar structures were described by Cheng and Reese ('85) in growth cones of quick-frozen and then freeze-substituted chick optic tectum, where stacked and single discs appear to replace the abundant agranular reticulum found in conventionally, chemically fixed tissue. In the present study, both membranous discs

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Fig. 12. 3-D representation based on every fifth section of the terminal part of a P2 growth cone (Fig. 11, No. 5), with electron micrographs illustrating the ultrastructure at or close to the levels of the profiles indicated in black. Arrow points in caudal direction. There,

where a glial process (G) lays across the growth cone, the growth cone bifurcates with both parts (clear stars) extending in the caudal direction. Scale: 1 pm.

and abundant strands of agranular reticulum were observed within the same growth cones after chemical h a tion, suggesting that these different forms of organization of membrane structures coexist in PT growth cones. Cheng and Reese ('87) suggested that the discs are involved in the recycling of the plasmalemma. The resemblance of the layer of 2 membranes that constitute the discs and surround the growth cone parcels, may indicate that both organelles are interrelated, and play a role in the recycling of growth cone components.

tures, including electron-dense bodies. They resemble growth cones in the cat optic nerve interpreted as necrotic by Williams et al. ('86). The suggestion that these PT growth cones are actually degenerating is supported by the observat,ion that they are frequently in apposition to or even surrounded by glial processes. The large profiles which contain, in addition to electron-dense bodies, growth cone parcels, and strands of agranular reticulum, also abundant mitochondria, some intermediate filaments and glycogen granules might well represent glial processes that have taken up growth cone material. Glial cells have been implicated in phagocytosis and removal of degenerating axons in various nerve tracts (Peters et al., '76; Chu-Wang and Oppenheim, '781, including the developing P T (Das and Hine, '72; Reh and Kalil, '82; Gorgels, '90). Consequently, these observations suggest that the massive axon loss in the development of the PT, which is apparent from the decreas-

Degenerating growth cones A small fraction of the growth cones exhibited a set of ultrastructural features which suggests that they are degenerating. Most of these growth cones have ruptures of the plasma membrane, a disorganization of the meshwork of agranular reticulum, and accumulations of vesicular struc-

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JFig. 13. 3-D graphical reconstruction based on every tenth section of part of a lamellipodium of a growth cone at P2. The lamellipodium enfolds 3 longitudinally oriented axons, as illustrated in the micrograph. Scale: 1 wm. Arrow points in caudal direction.

Fig. 14. 3-D graphical reconstructions based on every tenth section of the terminal parts of growth cones traced in series of transverse sections in a 4-day-old rat. Black profiles represent sections through the organelle-rich region of growth cones, whereas sections through the

fine granular, lamellipodial region are dotted. Arrow points in caudal direction. All growth cones are longitudinally oriented in the tract and terminate in the caudal direction (arrow). Scale: 1 pm.

ing numbers of axons in the cervical PT from P4 onwards (Gorgels et al., '89a), already starts with the elimination of growth cones during the outgrowth of the tract. Growth cone degeneration was most frequently observed at P4, which may suggest that the survival chances of growth cones become smaller during ongoing formation of the pyramidal tract.

the majority of growth cones has grown past this level (Schreyer and Jones, '82; Gribnau et al., '86; Gorgels et al., '89a), two major findings were made: First, the morphology of growth cones differs at different stages. Second, two different types of growth cones are simultaneously present at PO. Differences in growth cone morphology can be attributed to intrinsic factors, such as origin and age of the parent neuron, as well as to extrinsic factors, i.e., factors in the microenvironment of the growth cone (Letourneau, '79; Argiro et al., '84; Tosney and Landmesser, '85; Bovolenta and Mason, '87; Nordlander, '87; Kalil, '88). At PO, two

Differences in growth cone morphology Examining the morphology of growth cones in the PT pathway at the level of the third cervical segment over the period from PO, when the first PT axons arrive, to P4 when

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Fig. 15. 3-D graphical reconstruction based on every fifth section of a P4 growth cone (Fig. 14,No. 21, illustrating the ultrastructure at or close to the levels of the profiles indicated in black. Note the invagination of a n axon into the lamellipodium (arrow). Scale: 1km. Arrow points in caudal direction.

discrete types of growth cones were observed within the same environment: Funnel-shaped growth cones with densecore vesicles and spread-out lamellipodia, and large clubshaped growth cones without lamellipodia. These differences probably relate to a different origin of these growth cones. The lamellipodial growth cones are predominantly ascending. With respect to their shape and the presence of dense-core vesicles they resemble the growth cones present

in the ascending sensory fiber tracts at E20, before the arrival of PT axons. Therefore, most probably these growth cones belong to primary afferent or, possibly, propriospinal axons (cf. Patterson et al., '89). The club-shaped, descending PO growth cones and the other types of exclusively descending growth cones at P2 and at P4 most probably represent corticospinal, PT growth cones. The timing of their massive appearance in the ventral part of the DF,

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Fig. 16. Electron micrographs of putative signs of degenerating growth cones in the PT at PO (A-C), at P2 (D), and at P4 (El. A-C: Serial sections of a growth cone with an abnormally electron-dense cytoplasmic matrix. The presence of electron-dense bodies and many vacuoles as well as the irregular outline (B) may indicate that the growth cone is degenerating. This growth cone was continuous with a relatively small and electron-dense axon shaft (C, arrow). D: A growth

cone profile is completely surrounded by an astrocytic process, identified by the presence of a small group of intermediate filaments (arrow). In the growth cone profile electron-dense bodies are present and the organization of the agranular reticulum appears to be distorted. E: A large profile, containing electron-dense bodies, abundant mitochondria, agranular reticulum, and a growth cone parcel. Bars represent 0.5 bm (A-D) and 1.0 km (E).

where the descending corticospinal fibers in the rat are located (cf. Kuypers, '81), agrees well with the arrival of corticospinal fibers as determined in tracer studies (Schreyer and Jones, '82; Gribnau et al., '86). The age-related differences between the PT growth cones, appear to be gradual in nature when the variability at each age is taken into account, but still a different type can be discerned at each age: at PO, growth cones are club-shaped without lamellipodia; at P2, they have a single unbranched lamellipodium; at P4, they are comparatively small and simple. This change in morphology of growth cones during the formation of the PT might result in part from differences in location of the parent neurons in the cortex. In the development of the corticospinal projection a topographic organization emerges in the distribution of neurons projecting to cervical and lumbar regions of the spinal cord, involving in its extreme form the elimination of transient PT axon

collaterals originating from ectopic cortical areas, like the occipital cortex (Stanfield et al., '82). This collateral elimination is caused by factors within the cortex (O'Leary and Stanfield, '89). Tracer studies (Joosten et al., '87; Schreyer and Jones, '88; Uozumi et al., '88) did not establish firmly whether a differential timing exists in the outgrowth of axons projecting to either the cervical or the lumbar spinal cord, but probably a large overlap exists (Joosten et al., '87). However, the transient projections from ectopic cortical areas seem to lag behind in their outgrowth: Projections from the occipital cortex do not arrive until P2 at upper cervical levels (Stanfield and O'Leary, '85; Joosten et al., '87). Therefore, the smaller size of P4 growth cones and the apparent increase in signs of degenerating growth cones at P4, might to some extent relate to a different cortical origin of these growth cones. In vitro studies have shown that the elaboration of lamellipodia and the size of the growth cone are positively

GROWTH CONES IN THE RAT CERVICAL PYRAMIDAL TRACT correlated with growth speed and negatively correlated with neuronal age (Letourneau, '82; Argiro et al., '84). In the present study the differences in neuronal age amount only to a few days. Still, the smaller size of P4 growth cones in comparison with the P2 growth cones might reflect differences in neuronal age or growth speed. The shape of the PT growth cones that are the first to arrive in the spinal cord, at PO, however, cannot be explained on the basis of these intrinsic factors. Despite the lack of lamellipodia, these growth cones probably do move rather fast, since the extension of the corticospinal projection spreads swiftly in the caudal direction through the spinal cord (Schreyer and Jones, '82; Gribnau et al., '86). In addition to these intrinsic factors like cortical location, age of the parent neuron, and growth speed, extrinsic factors may also account for the age-related differences in PT growth cone morphology, since in vitro studies have indicated that the local environment influences growth cone morphology substantially (Letourneau, '82; Harris et al., '85; Kapfhammer and Raper, '87; Mattson, '89). Conversely, the change in morphology of growth cones during the formation of the PT, may reflect changes in the local environment, such as a change in adhesive properties of the substrate elements: In vitro, growth cones elaborate larnellipodia upon contact with adhesive substrates (Letourneau, '79) and when offered a choice among various substrates they grow on the most adherent substrate (Letourneau, '75). The configuration of the growth cones and their relations with the surrounding elements might therefore give an indication of the local substrate properties and the preferences of PT growth cones. The substrate elements in the tract consist of axons and growth cones, glial elements, and dendrites. The following may be noticed on the significance of the relations between outgrowing P T axons and these elements.

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Fig. 17. Artist's impression of a growth cone enfolding 3 axons (see Fig. 13), illustrating the fasciculative outgrowth of trailing PT growth cones. Caudal is to the bottom.

The first PT growth cones encounter axons of a different Most of the surface area of P T growth cones, including origin in the most ventral part of the DF. The origin of this their most distal parts, is in apposition to aligned small population of axons is not known with certainty. The axons and other growth cones. Therefore, it appears that apparent continuity with the ascending sensory fiber tracts the longitudinally oriented axon shafts in the DF represent and the presence of ascending growth cones which resemble the growth cones in the sensory tracts at E20, suggest that a preferred substrate for P T growth cones. A distinction has to be made between the axonal sub- these axons pertain to the sensory tracts. Within the DF, strate of the first PT growth cones and that of subsequently the ventralmost axons seem to be the most immature ones, arriving PT growth cones. The latter growth cones grow judging from their relatively small diameter and the abinto the most ventral part of the DF, where the axon shafts sence of neurofilaments. Consequently, it seems that the of previously arrived PT axons are already present. The first P T axons preferentially grow in the region of the most observation that these later-arriving growth cones in the immature axons of the sensory tracts. In this environment, P T elaborate lamellipodia that are for the largest part PT growth cones display a slight tendency to group together contiguous with axom and other growth cones, and often and do not extend lamellipodia, in contrast to the growth even fold around them, suggests that these growth cones cones of presumably primary afferent or propriospinal selectively fasciculate on their P T precursors (Fig. 17). axons, which have large lamellipodia. This suggests that Such a fasciculative outgrowth of the PT has been previ- although the most immature region of the DF represents a ously suggested by Schreyer and Jones ('83) on the basis of permissive environment for P T outgrowth, the PT growth studies of the outgrowth of corticospinal axons after lesion- cones do not fasciculate with these axons of noncortical ing the pathway. Furthermore, immunocytochemical stud- origin. The nonfasciculative outgrowth of the leading PT ies on the expression of the cell adhesion molecule L1, that growth cones might facilitate the proper segregation of the is involved in the extension of growth cones along axons and different fiber tracts in the DF when subsequently PT in the bundling of axons (Rathjen and Schachner, '84; axons arrive, that selectively fasciculate on their PT precurChang et al., '87; Rathjen et al., '87), give support to this sors. view: The highest levels of L1 immunoreactivity in the DF Relations of growth cones with glial cells of the early postnatal rat are found in the area where the Growth cones were only occasionally observed in apposioutgrowth of the P T takes place (Joosten and Gribnau, tion to glial elements. The dispersed glial elements are '89a).

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predominantly oriented perpendicularly to the longitudinal axis of the spinal cord (Joosten and Gribnau, '89b). They are not joined together into continuous arrays in the longitudinal direction within or around the PT except for the glial processes in the posterior median septum which separates the left and the right PT. Since along this septum no increased growth cone density was observed (Gorgels et al., '89a), the septum does not represent a preferred substrate for the caudal outgrowth of the PT, although it may serve as a barrier to prevent PT axons from decussating (Joosten and Gribnau, '89b). Consequently, PT growth cones do not grow along glial cells but instead grow predominantly in between or along axons, and make only "en passant" contacts with perpendicularly oriented glial elements. These observations on the relations between P T growth cones and glial processes are not in line with a previous ultrastructural study on the outgrowth of the corticospinal tract by Schreyer and Jones ('82). These authors suggested that the first corticospinal axons grow out as tight fascicles of small axons through a dense fabric of predominantly longitudinally oriented glial processes. However, in a recent immunocytochemical study using vimentin and glial fibrillary acidic protein as glial markers, Joosten and Gribnau ('89b) could not visualize such longitudinally oriented glial fibers. De Kort et al. ('85)suggested on the basis of serial section electron microscopy that the structures tentatively identified as glia by Schreyer and Jones ('82) in fact represent varicose axons, a view which is supported by the localization of the neuron-specific phosphoprotein B-50 in virtually all longitudinally oriented processes in the developing PT (Gorgels et al., '89b). Although glial cells do not form a continuous adhesive substrate pathway for the caudal outgrowth of PT axons, glial cells probably still play an important role in PT outgrowth. During the outgrowth of the PT, growth cones grow in a region which contains immature glial cells, while in the adjacent fasciculus cuneatus myelination already has started and mature, myelinating oligodendrocyte processes are abundantly present. In vitro studies have demonstrated that mature oligodendrocytes and CNS myelin represent, in contrast to immature oligodendrocytes, a nonpermissive substrate for axon outgrowth (Schwab and Caroni, '881, probably due to inhibitory proteins at the surface of mature oligodendrocytes and myelin (Caroni and Schwab, '88). These inhibitory surface proteins appear in CNS nerve tracts just before the onset of myelination (Caroni and Schwab, '89). In the cervical spinal cord, maturation of oligodendrocytes and the onset of myelination occur in the fasciculus cuneatus just after birth (Matthews and Duncan, '71; Schwab and Schnell, '891, which may canalize the outgrowth of PT fibers into the most ventral part of the DF and contribute to the separation of these fiber tracts. Interestingly, PT growth cones show junctional specializations consisting of synapse-like specializations and invaginations, at the site of contact with immature glial cells, which may suggest that outgrowing PT axons themselves exert influence on the timing of glial maturation and appearance of inhibitory substrate properties in the developing PT (Gorgels, '91).

Relations of growth cones with dendrites Neurons of the spinal grey matter extend dendrites into the most ventral part of the DF, during the outgrowth of the PT. At PO and at P2, several growth cones may establish synaptic contacts with these dendrites. Most

synapses are made by longitudinal growth cones, thus oriented perpendicularly to the dendrites, but occasionally also by growth cones that were pointing toward or into the grey matter and were oriented along the dendrite. Similar structural relationships have been described in the developing lateral marginal layer in the mouse embryonic spinal cord, where the synapses have been postulated to influence both dendrite morphology and axon collateralization (Vaughn et al., '74; Vaughn and Sims, '78). The former effect is supported by in vitro studies of neurotransmitter influence on dendrite morphology (Mattson, '89). Glutamate selectively prunes dendrites of hippocampal neurons in culture (Mattson et al., '88). Since glutamate is a putative neurotransmitter of the PT (Giuffrida and Rustioni, '891, the synapses of the growth cones in the developing DF possibly subserve a similar function. On the other hand, the lengthy appositions between growth cones or axons along dendrites within the transverse plane indicates that dendrites might guide or even induce the outgrowth of axons or axoncollaterals into the spinal grey. However, since such parallel appositions were only observed for the growth cones of presumably primary afferent or propriospinal axons, and only at PO when outgrowth of PT axons into the spinal grey does not yet occur (Schreyer and Jones, '82; Gribnau et al., '86), the present study does not provide morphological indications that PT outgrowth into the grey matter occurs by contact with dendrites.

ACKNOWLEDGMENTS The author is greatly endebted to H.T.H. Van Aanholt and M.M.A. Helsen for excellent technical assistance, T. Hafmans for skilled photographic assistance, J. Russon for preparing the line drawing, and Drs. A.A.M. Gribnau, J. Meek, A.B. Oestreicher, D. Iles, the late Dr. E.J.M. De Kort, and Professor Dr. R. Nieuwenhuys for valuable suggestions. This study was supported by a grant of the Foundation for Medical and Health Research (MEDIGON), which is subsidized by the Netherlands Organization for Scientific Research (NWO).

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Outgrowth of the pyramidal tract in the rat cervical spinal cord: growth cone ultrastructure and guidance.

In order to examine the mode of outgrowth of the pyramidal tract in the rat, the ultrastructure of its pathway in the dorsal funiculus of the spinal c...
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