THE JOURNAL OF COMPARATIVE NEUROLOGY 306~117-128 (1991)
Junctional Specializations Between Growth Cones and Glia in the Developing Rat Pyramidal Tract: Synapse-Like Contacts and Invaginations T.G.M.F. GORGELS Department of Anatomy and Embryology, University of Nijmegen, 6500 HB, Nijmegen, The Netherlands
ABSTRACT The ultrastructure of contacts between axonal growth cones and glial cells in the developing pyramidal tract was examined by serial sectioning at the third cervical spinal cord segment in 0-, 2-, and 4-day-old rats. Junctional specializations, composed of synapse-like contacts and invaginations, were frequently observed at the contact zone between growth cones and glial elements. The synapse-likecontacts consist of clear, round vesicles of 43 2 6 nm in the presynaptic growth cone, a pre- and a postsynaptic density, separated by a cleft of 12.1 0.9 nm. The invaginations consist of small protrusions of the growth cone into the glial element. The invaginated glial membrane is coated. Within the glial element, close to the invagination, frequently organelles were observed that closely resemble endosomes and prelysosomes. Therefore, it is suggested that the invagination represents a stage in endocytosis or possibly phagocytosis of the protruding part of the growth cone by the glial cell. The junctional specializations are formed by growth cones and, less frequently, by axon shafts. The targets of these specialized contacts are, in general, immature glial cells located within the tract area. Occasionally, however, invaginations were also observed into myelinating oligodendrocytes, suggesting that the population of immature target cells includes oligodendrocyteprecursors. With regard to the functional significance of these temporary growth cone-glial contacts, several possibilities are discussed, including the suggestion that outgrowing pyramidal tract axons provide immature glial cells with chemical messages, which may influence the timing of glial cell maturation in the tract.
*
Key words: axon guidance, glia maturation,endocytosis,oligodendrocyte,synaptogenesis
The formation of the ordered network of connections in the central nervous system (CNS) depends on a delicate interplay of outgrowing neurites and maturing glial cells. The spatial distribution of glial cells in the developing CNS suggests that they are involved in guidance of outgrowing axons and in the formation of axon tracts by forming barriers and channels, or by serving as pathway for axon outgrowth (Singer et al., '79; Poston et al., '88). Both in vivo and in vitro, a preference of growing axons for glial surfaces has been observed (Noble et al., '84; Silver and Rutishauser, '84; Fallon, '85). The outgrowth promoting properties of astrocytes apparently depend on their developmental stage (Smith et al., '86; Kalderon, '88), and oligodendrocytes become upon maturation even inhibitory for axonal outgrowth (Caroni and Schwab, '88, '89; Schwab and Caroni, '88). This developmental change, which may constitute an important factor in CNS failure to regenerate (Schnell and
o 1991 WILEY-LISS, INC.
Schwab, 'go), might influence the guidance of axons during CNS development, since striking regional differences exist in the timing of oligodendrocyte maturation (Schwab and Schnell, '89). Conversely, an influence of axons on glia survival or development has been postulated in order to account for the impaired gliogenesis observed after optic nerve transection (David et al., '84; Valat et al., '88). Furthermore, fiber tracts differ in the timing of the glial maturation (Schwab and Schnell, '891, and the time course of astroglial maturation over the length of axon tracts is correlated with the timing of axon outgrowth (Bovolenta et al., '87; Joosten and Gribnau, '89). These observations suggest, in spite of the fact that a properly timed glial maturation can occur in vitro in absence of axons (Abney et al., '81; Temple and RafF, '85; Zeller et al., '851, that Accepted November 21,1990.
T.G.M.F. GORGELS
118
outgrowing axons influence glial maturation (Hatten and Mason, '86). The developing pyramidal tract (PT) in the rat spinal cord represents an attractive system to analyse the relation between outgrowing axons and maturing glial cells, because of its late development in comparison to neighbouring fiber tracts: In the rat, the first PT axons arrive around birth in the cervical spinal cord, where they grow into the dorsal funiculus, which already contains the fasciculi cuneatus and gracilis (Altman and Bayer, '84).The PT is then formed in the most ventral part of the dorsal funiculus by the successive arrival of new fibers during the first postnatal week (Schreyer and Jones, '82; Gribnau et al., '86; Gorgels et al., '89). Maturation of both astrocytes and oligodendrocytes occurs in the PT several days later than in the adjacent sensory fiber tracts (Valentino et al., '83; Schwab and Schnell, '89). Myelination already starts in the fasciculus cuneatus immediately after birth, whereas the onset of myelination of the PT takes place 10 days later (Matthews and Duncan, '71, Gorgels et al., '89). During the outgrowth of the PT, growth cones contact with immature glial cells in the tract (Joosten and Gribnau, '89; Gorgels, '91). In the present study, these contacts between growth cones and glial cells are examined by serial section electron microscopy, with particular emphasis on the precise ultrastructural characteristics of these contacts and on the ultrastructural characterization and identification of the axonal and glial elements involved.
MATERIALS AND METHODS The material and method used are basically the same as described previously (Gorgels, '91). Briefly, Wistar albino rats, 0,2, and 4 days old (PO,P2, P4), were anaesthetized by intraperitoneal injection with sodium pentobarbital (40 mg per kg body weight) and fixed by transcardial perfusion with 2% paraformaldehyde, 2% glutaraldehyde in 0.1 M sodium cacodylate buffer, containing 2 mM CaCI,, pH 7.4. The isolated third cervical spinal cord segments were immersed overnight in the same fixative, postfixed for 2 hours in 1% OsO,, 0.05 M K,Fe(CN), in buffer (adapted from Karnovsky, '711, stained en bloc for 2 hours in 0.5% aqueous uranyl acetate, and embedded in Epon. Series of (up to 300) transverse ultrathin sections were cut and collected on single slot, formvar coated copper grids. After counterstaining with lead citrate and uranyl acetate, the sections were examined in a Philips EM 300 electron microscope. Magnification was calibrated using a diffraction grating replica (2,160lines per mm).
Fig. 1. A synapse-like contact formed by a growth cone (star) on a glial process in the cervical pyramidal tract of a 2-day-old rat. The synaptic characteristics include the presynaptic vesicles, the pre- and postsynaptic membrane densities, and the synaptic cleft. The postsynaptic element contains a relatively electron-dense cytoplasmic matrix. Bar represents 0.25 pm.
the presynaptic element, a pre- and a postsynaptic density, and a synaptic cleft (Fig. 1). The postsynaptic density generally is quite modest. The synaptic cleft is 12.1 0.9 nm wide (n = 15).Amoderate electron-dense staining was observed within the cleft, and rarely, midway between the presynaptic and the postsynaptic membranes a line of higher electron-density was discerned. In transverse sections, the synapse-like contacts are up to 500 nm long with a mean value of 248 130 nm (n = 24). In the longitudinal direction, they have similar dimensions, since they mostly extend over only 3-4 sections (200-300 nm). The synapselike contact generally has a concave shape, and in an extreme form, the contact is observed at the base of a protrusion into the postsynaptic element (Fig. 3A). Occasionally, however, also slightly convex synapse-like contacts are found (Fig 3Bf. The presynaptic vesicles are round and measure 43 2 6 nm (n = 27) in diameter. The number of RESULTS synaptic vesicles is usually small (but see Fig. 3B). Because Ultrastructure of growth cone-glial contacts of the close resemblance of these growth cone-glial contacts In the neonatal rat (PO-P4), the PT at the level of the to interneuronal synaptic contacts, the constituent elethird cervical segment is mainly composed of longitudinally ments will be referred to as the presynaptic and the oriented small, unmyelinated axons and growth cones. Glial postsynaptic element. processes follow a winding path between the axons and are In addition to the synapse-like specialization, invaginapredominantly oriented perpendicular to the axons and tions of the presynaptic element into the postsynaptic growth cones. Growth cones were occasionally found in element were frequently observed, next to or even within apposition to glial elements, and at the contact zone the region of the synapse-like contact (Figs. 2, 3D, 9). This junctional specializations, consisting of synapse-like con- invagination consisted as a rule of a 100 to 200 nm long, tacts and invaginations, were frequently observed (Figs. 1, more-or-less drop-shaped growth cone protrusion, which 2). The synapse-like contacts, which growth cones make was enveloped by the glial process. The membranes of the with glial cells, contain the 4 defining components of growth cone and the glial cell were not fused but maininterneuronal synapses (Peters et al., '76): clear vesicles in tained a fairly constant distance, roughly similar to the
*
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119
GROWTH CONE-GLIAL CONTACTS IN THE PYRAMIDAL TRACT
Fig. 2. Electron micrographs of 2 adjacent sections of the 0-day-old rat PT, showing a drop-shaped invagination (arrow in B)of the growth
cone membrane into the postsynaptic glial process. The invagination is located in between 2 synapse-like contacts. Bars represent 0.4 pm.
width of the synaptic cleft. In the cleft and in the protrusion, material of a moderate density was observed. The invaginated glial membrane displayed the furry submembrane coating of coated pits and coated vesicles (Peters et al., '76). Although this was the general appearance of the invaginations (Fig. 3D), occasionally the protrusion of the growth cone did not extend far into the coated pit, and rarely, a coated pit was observed at the glial membrane without a protrusion, as was verified by examining the adjacent sections as well (Figs. 3C, 5D, 8C). Within the postsynaptic element, frequently vesicular or tubular structures with a smooth-surface (Figs. 2A, 9) were observed as well as profiles of a larger diameter with an often irregular, tubular outline, containing vesicular and reticular elements embedded in a matrix of variable electron-density (Figs. 3B, 4C,E,F, 9). These structures resemble closely endosomes, prelysosomes, and multivesicular bodies, organelles that are implicated in the process of endocytosis (Geuze et al., '83; Helenius et al., '83; Griffiths, '89). In the approximately 150 invaginations examined in the present study, coated vesicles with a single membrane and electron-lucent content were never observed in the postsynaptic element. Rarely, vesicles with some coating and a moderately electrondense content (Fig. 9) or consisting of two concentric membranes (Fig. 5C) were observed in the postsynaptic element. These vesicles were not in contact with the growth cone, as was ascertained by examining serial sections. The junctional specializations at glial cells were found at all ages examined. The number of synapse-like contacts and invaginations that were counted in single transverse sections of the left PT, decreases, however, from 27 at P2 to 7 at P4. At PO, no counts were made since at this age the PT axons do not yet form a circumscript bundle (Gorgels et al.,
The presynaptic element
'89).
Growth cones in the PT generally consist of a distal, lamellipodial region, devoid of organelles except for an occasional clear vesicle, and a proximal organelle-rich region, which is more-or-less tubular and contains predominantly agranular reticulum and vesicles. The diameter of growth cone profiles generally measures 0.5-2 Fm, whereas the immature axon shafts in the tract measure approximately 0.3 pm (De Kort et al., '85; Gorgels et al., '89; Gorgels, '91). On the basis of this description, the presynaptic element generally was identified as the proximal part of the growth cone. Invaginations were made occasionally by lamellipodia as well (Fig. 4B). Furthermore, invaginations and small synaptic contacts were sometimes made by small axon shafts, which contain a few microtubuli (Figs. 4C,D). Examination of the presynaptic elements of 21 synapse-like contacts that were randomly sampled at PO, P2, and P4, identified 13as the proximal organelle-rich part of a growth cone and 3 as axon shafts. The remaining 5 presynaptic elements probably represent the transition zone of the growth cone into the axon shaft, but might also include lamellipodia. At PO, two different types of growth cones are present in the ventralmost part of the dorsal funiculus: the more frequently occurring, descending PT growth cones, which are club-shaped and do not possess lamellipodia, and in addition, lamellipodial growth cones, which probably belong to cells in the sensory ganglia or in the spinal cord. The latter growth cones typically contain dense-core vesicles (Gorgels, '91). Most of the contacts observed at PO were made by growth cones with characteristics of PT growth cones, i.e., large profiles, containing predominantly agranular reticulum. One of these profiles was traced in the series
120
T.G.M.F. GORGELS
Fig. 3. Variation observed in the morphology of the specialized contacts between growth cones and glial cells in the 2-day-old rat PT. A A small concave synapse-like contact (arrow) at the base of a protrusion of a growth cone into a glial process. B: A slightly convex synapse-like contact with a large accumulation of vesicles. In the postsynaptic element the cap of an invagination is present and also an irregularly formed, tubular, probably prelysosomal organelle (arrow). C: A coated
pit a t a glial membrane without a protrusion of the growth cone membrane, as verified by serial sectioning. D The growth cone membrane extends into the glial process with a fairly constant cleft between the growth cone membrane and the glial membrane. Note the coating of the glial membrane and the moderately electron-dense material in the cleft and in the protrusion. Bars represent 0.25 pm.
of sections towards its ending in descending direction, confirming its similarity to club-shaped descending PT growth cones. Occasionally, however, presynaptic growth cones were observed also, which contain dense-core vesicles (Figs. 4E,F).
somes (Figs. 4B, 5 , 6 ) . Glycogen granules were not observed. These ultrastructural features correspond very well with descriptions of immature glial cells in the rat optic nerve (Vaughn, '69; Skoff et al., '76). The shape of the postsynaptic cells was variable. Examination of the cells in serial sections reveals that thev v a n from uniuolar cells with one broad process (Fig. 5) to multipolar cells possessing several, although not numerous, slender processes (Fig. 6). Especially on the processes of the latter cell subtype specialized contacts are abundant: 8 junctional specializations were counted on one of these processes, which was traced in the series of sections over a length of approximately 15 km. The postsynaptic cells described above represent the most common glial cell type in the cervical P T during the first postnatal days, but other glial elements are present in the neonatal PT as well. The surface of these elements was scanned in order to assess the full spectrum of glial structures involved in the growth cone-glial interaction (Figs. 6-8). Electron-lucent, thin processes with accumulations of glycogen granules are present in the neonatal PT. These processes can often be observed extending from the posterior median septum which separates the right and the left PT. On these processes, presumably of radial glia (cf. Henrikson and Vaughn, '741, no junctional specializations were found. Occasionally, synapse-like contacts were observed on processes, which resemble these processes in shape and in abundance of glycogen granules, but which contain a slightly more electron-densematrix (Fig. 7C).
Junctional Specializations Between Growth Cones and Glia in the Developing Rat Pyramidal Tract: Synapse-Like Contacts and Invaginations T.G.M.F. GORGELS Department of Anatomy and Embryology, University of Nijmegen, 6500 HB, Nijmegen, The Netherlands
ABSTRACT The ultrastructure of contacts between axonal growth cones and glial cells in the developing pyramidal tract was examined by serial sectioning at the third cervical spinal cord segment in 0-, 2-, and 4-day-old rats. Junctional specializations, composed of synapse-like contacts and invaginations, were frequently observed at the contact zone between growth cones and glial elements. The synapse-likecontacts consist of clear, round vesicles of 43 2 6 nm in the presynaptic growth cone, a pre- and a postsynaptic density, separated by a cleft of 12.1 0.9 nm. The invaginations consist of small protrusions of the growth cone into the glial element. The invaginated glial membrane is coated. Within the glial element, close to the invagination, frequently organelles were observed that closely resemble endosomes and prelysosomes. Therefore, it is suggested that the invagination represents a stage in endocytosis or possibly phagocytosis of the protruding part of the growth cone by the glial cell. The junctional specializations are formed by growth cones and, less frequently, by axon shafts. The targets of these specialized contacts are, in general, immature glial cells located within the tract area. Occasionally, however, invaginations were also observed into myelinating oligodendrocytes, suggesting that the population of immature target cells includes oligodendrocyteprecursors. With regard to the functional significance of these temporary growth cone-glial contacts, several possibilities are discussed, including the suggestion that outgrowing pyramidal tract axons provide immature glial cells with chemical messages, which may influence the timing of glial cell maturation in the tract.
*
Key words: axon guidance, glia maturation,endocytosis,oligodendrocyte,synaptogenesis
The formation of the ordered network of connections in the central nervous system (CNS) depends on a delicate interplay of outgrowing neurites and maturing glial cells. The spatial distribution of glial cells in the developing CNS suggests that they are involved in guidance of outgrowing axons and in the formation of axon tracts by forming barriers and channels, or by serving as pathway for axon outgrowth (Singer et al., '79; Poston et al., '88). Both in vivo and in vitro, a preference of growing axons for glial surfaces has been observed (Noble et al., '84; Silver and Rutishauser, '84; Fallon, '85). The outgrowth promoting properties of astrocytes apparently depend on their developmental stage (Smith et al., '86; Kalderon, '88), and oligodendrocytes become upon maturation even inhibitory for axonal outgrowth (Caroni and Schwab, '88, '89; Schwab and Caroni, '88). This developmental change, which may constitute an important factor in CNS failure to regenerate (Schnell and
o 1991 WILEY-LISS, INC.
Schwab, 'go), might influence the guidance of axons during CNS development, since striking regional differences exist in the timing of oligodendrocyte maturation (Schwab and Schnell, '89). Conversely, an influence of axons on glia survival or development has been postulated in order to account for the impaired gliogenesis observed after optic nerve transection (David et al., '84; Valat et al., '88). Furthermore, fiber tracts differ in the timing of the glial maturation (Schwab and Schnell, '891, and the time course of astroglial maturation over the length of axon tracts is correlated with the timing of axon outgrowth (Bovolenta et al., '87; Joosten and Gribnau, '89). These observations suggest, in spite of the fact that a properly timed glial maturation can occur in vitro in absence of axons (Abney et al., '81; Temple and RafF, '85; Zeller et al., '851, that Accepted November 21,1990.
T.G.M.F. GORGELS
118
outgrowing axons influence glial maturation (Hatten and Mason, '86). The developing pyramidal tract (PT) in the rat spinal cord represents an attractive system to analyse the relation between outgrowing axons and maturing glial cells, because of its late development in comparison to neighbouring fiber tracts: In the rat, the first PT axons arrive around birth in the cervical spinal cord, where they grow into the dorsal funiculus, which already contains the fasciculi cuneatus and gracilis (Altman and Bayer, '84).The PT is then formed in the most ventral part of the dorsal funiculus by the successive arrival of new fibers during the first postnatal week (Schreyer and Jones, '82; Gribnau et al., '86; Gorgels et al., '89). Maturation of both astrocytes and oligodendrocytes occurs in the PT several days later than in the adjacent sensory fiber tracts (Valentino et al., '83; Schwab and Schnell, '89). Myelination already starts in the fasciculus cuneatus immediately after birth, whereas the onset of myelination of the PT takes place 10 days later (Matthews and Duncan, '71, Gorgels et al., '89). During the outgrowth of the PT, growth cones contact with immature glial cells in the tract (Joosten and Gribnau, '89; Gorgels, '91). In the present study, these contacts between growth cones and glial cells are examined by serial section electron microscopy, with particular emphasis on the precise ultrastructural characteristics of these contacts and on the ultrastructural characterization and identification of the axonal and glial elements involved.
MATERIALS AND METHODS The material and method used are basically the same as described previously (Gorgels, '91). Briefly, Wistar albino rats, 0,2, and 4 days old (PO,P2, P4), were anaesthetized by intraperitoneal injection with sodium pentobarbital (40 mg per kg body weight) and fixed by transcardial perfusion with 2% paraformaldehyde, 2% glutaraldehyde in 0.1 M sodium cacodylate buffer, containing 2 mM CaCI,, pH 7.4. The isolated third cervical spinal cord segments were immersed overnight in the same fixative, postfixed for 2 hours in 1% OsO,, 0.05 M K,Fe(CN), in buffer (adapted from Karnovsky, '711, stained en bloc for 2 hours in 0.5% aqueous uranyl acetate, and embedded in Epon. Series of (up to 300) transverse ultrathin sections were cut and collected on single slot, formvar coated copper grids. After counterstaining with lead citrate and uranyl acetate, the sections were examined in a Philips EM 300 electron microscope. Magnification was calibrated using a diffraction grating replica (2,160lines per mm).
Fig. 1. A synapse-like contact formed by a growth cone (star) on a glial process in the cervical pyramidal tract of a 2-day-old rat. The synaptic characteristics include the presynaptic vesicles, the pre- and postsynaptic membrane densities, and the synaptic cleft. The postsynaptic element contains a relatively electron-dense cytoplasmic matrix. Bar represents 0.25 pm.
the presynaptic element, a pre- and a postsynaptic density, and a synaptic cleft (Fig. 1). The postsynaptic density generally is quite modest. The synaptic cleft is 12.1 0.9 nm wide (n = 15).Amoderate electron-dense staining was observed within the cleft, and rarely, midway between the presynaptic and the postsynaptic membranes a line of higher electron-density was discerned. In transverse sections, the synapse-like contacts are up to 500 nm long with a mean value of 248 130 nm (n = 24). In the longitudinal direction, they have similar dimensions, since they mostly extend over only 3-4 sections (200-300 nm). The synapselike contact generally has a concave shape, and in an extreme form, the contact is observed at the base of a protrusion into the postsynaptic element (Fig. 3A). Occasionally, however, also slightly convex synapse-like contacts are found (Fig 3Bf. The presynaptic vesicles are round and measure 43 2 6 nm (n = 27) in diameter. The number of RESULTS synaptic vesicles is usually small (but see Fig. 3B). Because Ultrastructure of growth cone-glial contacts of the close resemblance of these growth cone-glial contacts In the neonatal rat (PO-P4), the PT at the level of the to interneuronal synaptic contacts, the constituent elethird cervical segment is mainly composed of longitudinally ments will be referred to as the presynaptic and the oriented small, unmyelinated axons and growth cones. Glial postsynaptic element. processes follow a winding path between the axons and are In addition to the synapse-like specialization, invaginapredominantly oriented perpendicular to the axons and tions of the presynaptic element into the postsynaptic growth cones. Growth cones were occasionally found in element were frequently observed, next to or even within apposition to glial elements, and at the contact zone the region of the synapse-like contact (Figs. 2, 3D, 9). This junctional specializations, consisting of synapse-like con- invagination consisted as a rule of a 100 to 200 nm long, tacts and invaginations, were frequently observed (Figs. 1, more-or-less drop-shaped growth cone protrusion, which 2). The synapse-like contacts, which growth cones make was enveloped by the glial process. The membranes of the with glial cells, contain the 4 defining components of growth cone and the glial cell were not fused but maininterneuronal synapses (Peters et al., '76): clear vesicles in tained a fairly constant distance, roughly similar to the
*
*
119
GROWTH CONE-GLIAL CONTACTS IN THE PYRAMIDAL TRACT
Fig. 2. Electron micrographs of 2 adjacent sections of the 0-day-old rat PT, showing a drop-shaped invagination (arrow in B)of the growth
cone membrane into the postsynaptic glial process. The invagination is located in between 2 synapse-like contacts. Bars represent 0.4 pm.
width of the synaptic cleft. In the cleft and in the protrusion, material of a moderate density was observed. The invaginated glial membrane displayed the furry submembrane coating of coated pits and coated vesicles (Peters et al., '76). Although this was the general appearance of the invaginations (Fig. 3D), occasionally the protrusion of the growth cone did not extend far into the coated pit, and rarely, a coated pit was observed at the glial membrane without a protrusion, as was verified by examining the adjacent sections as well (Figs. 3C, 5D, 8C). Within the postsynaptic element, frequently vesicular or tubular structures with a smooth-surface (Figs. 2A, 9) were observed as well as profiles of a larger diameter with an often irregular, tubular outline, containing vesicular and reticular elements embedded in a matrix of variable electron-density (Figs. 3B, 4C,E,F, 9). These structures resemble closely endosomes, prelysosomes, and multivesicular bodies, organelles that are implicated in the process of endocytosis (Geuze et al., '83; Helenius et al., '83; Griffiths, '89). In the approximately 150 invaginations examined in the present study, coated vesicles with a single membrane and electron-lucent content were never observed in the postsynaptic element. Rarely, vesicles with some coating and a moderately electrondense content (Fig. 9) or consisting of two concentric membranes (Fig. 5C) were observed in the postsynaptic element. These vesicles were not in contact with the growth cone, as was ascertained by examining serial sections. The junctional specializations at glial cells were found at all ages examined. The number of synapse-like contacts and invaginations that were counted in single transverse sections of the left PT, decreases, however, from 27 at P2 to 7 at P4. At PO, no counts were made since at this age the PT axons do not yet form a circumscript bundle (Gorgels et al.,
The presynaptic element
'89).
Growth cones in the PT generally consist of a distal, lamellipodial region, devoid of organelles except for an occasional clear vesicle, and a proximal organelle-rich region, which is more-or-less tubular and contains predominantly agranular reticulum and vesicles. The diameter of growth cone profiles generally measures 0.5-2 Fm, whereas the immature axon shafts in the tract measure approximately 0.3 pm (De Kort et al., '85; Gorgels et al., '89; Gorgels, '91). On the basis of this description, the presynaptic element generally was identified as the proximal part of the growth cone. Invaginations were made occasionally by lamellipodia as well (Fig. 4B). Furthermore, invaginations and small synaptic contacts were sometimes made by small axon shafts, which contain a few microtubuli (Figs. 4C,D). Examination of the presynaptic elements of 21 synapse-like contacts that were randomly sampled at PO, P2, and P4, identified 13as the proximal organelle-rich part of a growth cone and 3 as axon shafts. The remaining 5 presynaptic elements probably represent the transition zone of the growth cone into the axon shaft, but might also include lamellipodia. At PO, two different types of growth cones are present in the ventralmost part of the dorsal funiculus: the more frequently occurring, descending PT growth cones, which are club-shaped and do not possess lamellipodia, and in addition, lamellipodial growth cones, which probably belong to cells in the sensory ganglia or in the spinal cord. The latter growth cones typically contain dense-core vesicles (Gorgels, '91). Most of the contacts observed at PO were made by growth cones with characteristics of PT growth cones, i.e., large profiles, containing predominantly agranular reticulum. One of these profiles was traced in the series
120
T.G.M.F. GORGELS
Fig. 3. Variation observed in the morphology of the specialized contacts between growth cones and glial cells in the 2-day-old rat PT. A A small concave synapse-like contact (arrow) at the base of a protrusion of a growth cone into a glial process. B: A slightly convex synapse-like contact with a large accumulation of vesicles. In the postsynaptic element the cap of an invagination is present and also an irregularly formed, tubular, probably prelysosomal organelle (arrow). C: A coated
pit a t a glial membrane without a protrusion of the growth cone membrane, as verified by serial sectioning. D The growth cone membrane extends into the glial process with a fairly constant cleft between the growth cone membrane and the glial membrane. Note the coating of the glial membrane and the moderately electron-dense material in the cleft and in the protrusion. Bars represent 0.25 pm.
of sections towards its ending in descending direction, confirming its similarity to club-shaped descending PT growth cones. Occasionally, however, presynaptic growth cones were observed also, which contain dense-core vesicles (Figs. 4E,F).
somes (Figs. 4B, 5 , 6 ) . Glycogen granules were not observed. These ultrastructural features correspond very well with descriptions of immature glial cells in the rat optic nerve (Vaughn, '69; Skoff et al., '76). The shape of the postsynaptic cells was variable. Examination of the cells in serial sections reveals that thev v a n from uniuolar cells with one broad process (Fig. 5) to multipolar cells possessing several, although not numerous, slender processes (Fig. 6). Especially on the processes of the latter cell subtype specialized contacts are abundant: 8 junctional specializations were counted on one of these processes, which was traced in the series of sections over a length of approximately 15 km. The postsynaptic cells described above represent the most common glial cell type in the cervical P T during the first postnatal days, but other glial elements are present in the neonatal PT as well. The surface of these elements was scanned in order to assess the full spectrum of glial structures involved in the growth cone-glial interaction (Figs. 6-8). Electron-lucent, thin processes with accumulations of glycogen granules are present in the neonatal PT. These processes can often be observed extending from the posterior median septum which separates the right and the left PT. On these processes, presumably of radial glia (cf. Henrikson and Vaughn, '741, no junctional specializations were found. Occasionally, synapse-like contacts were observed on processes, which resemble these processes in shape and in abundance of glycogen granules, but which contain a slightly more electron-densematrix (Fig. 7C).
