j o u r n a l o f N e u r o c y t o l o g y 7, 3 3 7 - 3 6 3 ( 1 9 7 8 )

Axonal growth cones and developing axonal collaterals form synaptic junctions in embryonic mouse spinal cord JAMES

E.

VAUGHN

and

TERRY

J.

SIMS

Division of Neurosciences, City of Hope National Medical Center, 1500 East Duarte _Road, Duarte, California 91010, U.S.A. Received 11 O c t o b e r 1977; revised and a c c e p t e d 9 D e c e m b e r 1977

Summary Axonal growth cones and developing axonal collaterals have been studied in electron microscopic and Golgi preparations of embryonic mouse cervical spinal cord. These structures are first observed on embryonic day 11 (Ell), and by E12 both axonal growth cones and developing collaterals are observed to be the presynaptic elements of developing synaptic junctions. These relatively undifferentiated portions of axons comprise nearly one-half of the presynaptic components of the synaptic contacts observed on E12, and they continue to constitute about this same proportion during the rest of the embryonic period examined (El 3--16). These early synaptic interactions of developing axonal collaterals may be involved in establishing the basic ramification patterns of mature axons by determining which developing collaterals will continue to grow and differentiate. Coated vesicles are observed fused with axonal collateral plasma membranes at the perimeters of developing synaptic contacts. Occasionally thin extensions of postsynaptic surfaces protrude into these coated vesicles, and similar relationships are also observed between presynaptic surfaces and coated vesicles fused with the membranes of postsynaptic elements. Such coated vesicle/ synaptic surface relationships may represent an early stage in the endoeytosis of synaptic membranes by both the pre- and postsynaptic elements of developing synaptic contacts. This might represent a mechanism for the exchange of 'molecular information' between the components of protosynaptic contacts, and the results of such an exchange could be involved in determining whether protosynaptic contacts will differentiate into synaptic junctions. Developing axonal collaterals exhibit a preferential growth toward the sources of potential postsynaptic elements, and this growth implies that the axons present in the embryonic marginal zone are actively searching for appropriate postsynaptic processes rather than passively awaiting their arrival. Many of the collaterals weave around numerous intervening processes in order to reach their postsynaptic destinations, and unusual subaxolemmal lamellae are commonly observed where collaterals make acute curvatures around neurites which block their direct access to postsynaptic processes. The location of these lamellae suggests that they could be involved in producing changes in the direction of growth as developing axonal collaterals search for synaptic partnerships. Main axonal shafts commonly exhibit synaptic contacts adjacent to where they give rise to developing collaterals, and this observation hints that the formation of synapses by axonal shafts might stimulate collateral sprouting. However, other observations (see text) indicate that the formation of synaptie contacts by axonal shafts is not an obligatory prelude to collateral development. Therefore synaptic interactions of main axonal shafts do not appear to be a 9 1978 Chapman and Hall Ltd. Printed in Great Britain

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requirement for the primary induction of collateral development,but they may facilitate collateral growth by providinga confirmation that axons are located in appropriate synaptogenicfields. Introduction

Studies of the developing central nervous system have provided evidence that dendritic growth cones and dendritic growth cone filopodia are postsynaptic components of early forming synaptic junctions (Bodian, 1966; Del Cerro and Snider, 1968; Hinds and Hinds, 1972, 1976; Skoff and Hamburger, 1974; Vaughn et al.,. 1974; Fox et al., 1976). In addition, many of the above investigations show that axonal growth cones may also participate in the early formation of synaptic contacts, and this has been confirmed by serial section analysis (Hinds and Hinds, 1976). Dendritic growth cones and filopodia appear to comprise a substantial proportion of the total postsynaptic population during the early stages of synaptogenesis (Vaughn et al., 1974), but there is no information available concerning the frequency with which axonal growth cones appear as the presynaptic components of developing synaptic junctions. In addition, previous studies have not provided a detailed ultrastructural description of the formation of central axonal collaterals during early synaptogenic periods. Hayes and Roberts' (1974) schematic diagram of relationships between growing axons and dendrites in embryonic toad spinal cord shows that varicosities of main axonal shafts form all of the early synaptic contacts and that the early marginal zone axons in this species are devoid of developing collaterals. Their diagram also suggests to us that the early-arriving axons of the embryonic toad marginal zone are relatively passive elements of synaptogenesis in the sense that they course straight through their synaptogenic fields awaiting the arrival of potential postsynaptic elements. In contrast to embryonic toad spinal cord, developing axonal collaterals appear to be rather common during the early stage of synaptogenesis in embryonic mouse spinal cord, and these collaterals form synaptic contacts with postsynaptic profiles which are located some distance from the parent axonal shafts (Vaughn et al., 1977). This implies that early marginal zone axons may be engaged in an active search for potential postsynaptic elements. The main objectives of the present report are: (1) to provide a detailed description of the development of axon collaterals during the early synaptogenic period in embryonic mouse spinal cord; (2) to document the participation of developing axonal collaterals in the early formation of synaptic junctions; and (3) to provide information pertaining to the relative frequency with which the undifferentiated, and presumably growing, portions of axons are the presynaptic components of developing synaptic junctions. Materials and methods Mice of the inbred strain C57BL/6J were obtained from the Jackson Laboratory, Bar Harbor, Maine, and maintained on a reversedday-night cycle. Femaleswere placed in breeding cages with

A x o n a l g r o w t h cones and developing axonal collaterals f o r m synapses

3 39

males for 4 h during the middle of the dark period. The females were then removed from the breeding cages and examined for vaginal plugs. 24 h after the detection of vaginal plugs was designated as embryonic day 1 (El). The embryos used in this study were delivered by Caesarian section at approximately 2.00 p.m. on embryonic days, 11 through 16. The younger specimens (El I and 12) were fixed by immersion in glutaraldehyde-paraformaldehyde-acrolein mixtures, and the older (E13--16) specimens were perfused with a similar tri-aldehyde fixative (see Henrikson and Vaughn, 1974 for details of tissue preservation). Cervical spinal cord segments 5 and 6 were prepared for routine and serial section electron microscopic examinations by the procedures described elsewhere (Henrikson and Vaughn, 1974; Vaughn et al., 1974, 1975, 1977). Additional specimens were obtained for Golgi studies by removing the cervical spinal column from unfixed, E12--16 embryos with the spinal cord intact within the vertebral canal. These specimens were impregnated by a rapid Golgi procedure (Palay and Chan-Palay, 1974), and embedded in celloidin following ethanolic dehydration. The embedded specimens were sectioned in either the transverse, horizontal, or sagittal plane on a Sorvall TC-2 tissue sectioner at thicknesses ranging between 100 and 200/lm depending upon specimen age and the plane of section. The cartilaginous vertebrae in celloidin embedded material sectioned easily and prevented the crust of silver deposition that formed upon the outer surfaces of the specimens from obscuring the marginal zone of the embryonic spinal cords. In addition, the vertebrae served as locational and orientational guides for sections cut in the horizontal and sagittal planes. Golgi-impregnated marginal zone axons from at least 10 different spinal cords for each embryonic age were either drawn with the aid of a Leitz optical microscope equipped with a drawing tube or were photographed using a Leitz Orthoplan photomicroscope. The procedures used to determine the relative proportions of synaptic contacts formed by different kinds of presynaptic profiles in this study were the same as those used previously to determine the relative proportions of developing synapses formed upon postsynaptic dendritic growth cones and differentiated dendrites (Vaughn et al., 1974). The sampled area consisted of the portion of the lateral marginal zone that is contiguous with the lateral edge of the lateral motor column and extended 50-75/~m laterally from the most lateral motor somata. 10-20 non-overlapping electron micrographs of this area were taken for each specimen using a primary magnification (x 8000) that was insufficient for identification of synaptic contacts directly on the viewing screen. The electron micrographic negatives were printed at an enlargement of x 2.5, and synaptic contacts could be clearly identified on the resulting prints. Specimens from at least two animals for each embryonic day were analysed in the quantitative part of this investigation. Observations All o f the s t r u c t u r e s described in this r e p o r t have been observed w i t h i n either the lateral or ventral marginal zones in the vicinity o f the lateral and medial m o t o r c o l u m n s respectively. As has b e e n described in detail elsewhere (Vaughn et al., 1977), these regions c o n t a i n the earliest s y n a p t i c c o n t a c t s which have been observed in e m b r y o n i c m o u s e spinal cords. These early s y n a p t i c fields are c o m p o s e d o f longitudinal axons which are divided into large fascicles b y radial t r a b e c u l a e o f cellular processes which e m a n a t e f r o m the m o t o r columns. T h e cellular processes include m o t o r n e u r o n a l dendrites, d e n d r i t i c g r o w t h cones, d e n d r i t i c g r o w t h cone f i l o p o d i a (e.g., V a u g h n et al., 1974) and radial glial processes w h i c h e x p a n d t o f o r m the glia limitans at the peripheral margin o f the spinal cord ( H e n r i k s o n and Vaughn, 1974).

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Golgi impregnations The earliest successful Golgi impregnations of marginal zone axons have been obtained on E12, and this coincides with the earliest Golgi impregnations of interneurons within the intermediate zone. The axons of these interneurons enter the marginal zone perpendicular to the longitudinal axis of the spinal cord, but they turn to a longitudinal course shortly after leaving the intermediate zone (Fig. 1). Only a few marginal zone axons are impregnated on E12, and they display a thin, uniform calibre. Impregnated axons seldom exceed two spinal segments in length on E12, and they rarely exhibit varicosities or collateral processes. The number of Golgi-impregnated axons in both the lateral and ventral marginal zones substantially increases on E13. This increase of marginal zone axons coincides with an increase in the number of impregnated interneurons within the intermediate zone. In addition to their numerical increase, both the calibre and the length of marginal zone axons increase on E13. Short collateral processes can also be observed emanating from the main axonal shafts of E13 specimens, and these collaterals frequently arise from varicosities of the main shafts (Fig. 1). The short collaterals are observed to terminate in growth cone swellings (Fig. 1) which are smaller than those of main axonal shafts (Figs. 1, 2, 3). A large majority of the E13 collaterals project perpendicularly from the axonal shafts in the direction of the adjacent motor column. Some marginal zone axonal shafts which span the entire length of the cervical spinal cord are impregnated on days E14-16. A marked increase in the number of impregnated short axons also occurs during this time, and the terminals of short axons often have the appearance of growth cones. Collaterals arising from long axons penetrate the motor columns, and the number of collaterals arising from a single long axon appears to average only one or two per spinal segment. Short

Figs. 1--4. Drawings of Golgi-impregnated marginal zone axons in the developing cervical spinal

cord. Fig. 1. Camera lucida drawings illustrate the progressive growth of marginal zone axon collaterals on embryonic days 12 through 15. All drawings were made from horizontal sections with the exception of the top axon in the E13 group (see below), and they are all positioned as if the motor column (not shown) were located above each drawing. Examples were selected to represent the most differentiated marginal zone axons observed on each embryonic day, The marginal zone axon impregnated on E l 2 (top single axon) has no detectable collaterals, and the main shaft terminates in a growth cone (arrow) that displays filopodial extensions, On E13, short collateral processes (small arrows) are observed, and they are directed predominately toward the motor column. The large arrow on the uppermost axon drawn from a sagittal, section at El3 designates a portion of a primary axon within the intermediate zone that branches into ascending and descending axonal shafts when it reaches the marginal zone, On E14 and E15, the collateral processes (short arrows) increase in length and they often end in growth cone-like swellings (arrowheads). x 1000. Figs. 2--4. Golgi drawings made from a through-focus series of photographic enlargements. Figs. 2 and 3 are examples of terminal growth cones (arrows) on the main shafts of E l 4 marginal zone axons. In Fig. 3 a presumptive developing collateral (arrowhead) arises from the main shaft just proximal to the terminal growth cone of the axonal shaft. Fig. 4 shows two collateral growth cones (arrows) branching from the main shaft of a marginal axon on E15. x 3500.

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EI~

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axons, on the other hand, give rise to numerous collaterals which exhibit irregular profiles (Fig. 4), and which often give rise to secondary branches within the marginal zone and the adjacent intermediate zone. The results obtained from our Golgi impregnations are in basic agreement with those reported for comparable stages of chick embryonic spinal cord (Ram6n y Cajal, 1960). They also essentially agree with our electron microscopic findings (see below) except for the fact that axonal collaterals can be detected in electron microscopic preparations two days earlier than in Golgi impregnations. This difference is probably due to the apparent inability of Golgi methods to impregnate neurons at very early stages of their differentiation (see Ram6n y Cajal, 1960, p. 217; Sims and Vaughn, 1977). Thus the neurons whose axons give rise to the collaterals observed in electron microscopic preparations of E11 and E 12 specimens are probably not sufficiently differentiated in other respects to be impregnated b y the Golgi procedure used in our study.

Electron microscopy Many of the early-forming synaptic junctions of the marginal zone are observed upon dendritic profiles located in the radial trabeculae described above. Dendritic growth cones and dendritic growth cone filopodia are more common postsynaptic elements than differentiated dendritic profiles during the early stage (El 1 - 1 4 ) of synaptogenesis (Table 1; Vaughn et al., 1974). In addition, some of the presynaptic components of the early-forming synaptic contacts have the characteristics of axonal growth cones. For example, the presynaptic profiles shown in Figs. 5 and 6 (also see Fig. 5 in Vaughn et al., 1974) contain growth cone vesicles, cisternae of smooth endoplasmic reticulum and display numerous microfilamentous components within

All of the electron micrographs shown in Figs. 5--32 are from transversely-sectioned, cervical (C5 and 6) spinal cords. Figs. 5 and 6. Axonal growth cones form synaptic contacts in the lateral marginal zone of E14 spinal cord. The growth cone in Fig. 5 contains numerous growth cone vesicles (GV) but no microtubules. A portion of the axonal growth cone is the presynaptic component of a developing synaptic contact (arrows). Two axonal growth cone-like profiles in Fig. 6 form developing synaptic contacts (arrows) with a dendritic growth cone filopodium (D). One of the presynaptic elements contains several growth cone vesicles (GV), while the other displays prominent smooth reticulum (SR). Notice that the main axonal shafts in Figs. 5 and 6 exhibit numerous transversely-sectioned microtubules (arrowheads), and some main shafts also contain a few neurofilaments (f). Fig. 5, x 44 000; Fig. 6, x 43 600. Fig. 7. Lateral marginal zone of an El4 spinal cord. A developing collateral protrusion from an axonal shaft (*) contains growth cone vesicles (GV) and forms a synaptic contact (arrow) with a dendritic profile (D). x 44 600. Fig. 8. Lateral marginal zone of an El3 spinal cord. A short, developing axonal collateral arises from the main axonal shaft (*) adjacent to the area where the shaft forms a synaptic contact (arrows). The collateral is packed with longitudinally arranged microfilaments, and it also contains several synaptic vesicles (arrowheads). A cistern of smooth reticulum (SR) is located within the main axonal shaft near the origin of the collateral, x 33 000.

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their cytoplasm. The cytoplasm of these profiles contains fewer microtubules and mitochondria than more differentiated axons. Growth cone-like structures are also observed as simple collateral protrusions which branch from main axonal shafts at approximately right angles. These collateral protrusions contain growth cone vesicles, and occasionally their more distal portions are the presynaptic elements of developing synaptic contacts (Fig. 7). Somewhat more differentiated axonal collaterals frequently arise at sites where the main axonal shafts form synaptic junctions, and they contain numerous microfilaments arranged parallel to the long axis of the developing collateral (Fig. 8). The distal tips of shorter collaterals are observed to be presynaptic to adjacent neurites (Fig. 9), and longer collaterals display presynaptic sites along their lengths at variable distances from the parent axonal shafts (Figs. 11,13 and 15). In some cases, developing axonal collaterals form synaptic contacts with postsynaptic elements that are immediately adjacent to the parent axonal shafts (Fig. 12), while in other cases developing collaterals weave around intervening neurites in order to reach postsynaptic processes (Figs. 13, 19, 20, 22 and 26). Nonsynaptic portions of developing collateral profiles commonly contain numerous, scattered vesicles which are the same size and shape as synaptic vesicles (Fig. 11, 14-16). Most of the longer profiles of developing axonal collaterals also display well-organized linear arrays of microfilaments (Figs. 8, 14 and 16), but these arrays are replaced by longitudinally-oriented microtubules in a few collaterals which appear to be more differentiated (Fig. 17). Although microfilaments and synaptic vesicles are the principal organelles of developing axonal collaterals, a few profiles of coated vesicles (Fig. 15), small cisternae of smooth reticulum as well as growth cone vesicles (Fig. 21) can also be observed in collaterals. However, coated vesicles and well-developed cisternae of smooth reticulum are found much more frequently where developing collaterals arise from axonal shafts (Figs. 11, 14, 17 and 18) than within the collaterals proper. Most of the coated vesicles associated with developing axonal collaterals measure between 80 and 100 nm in diameter. Certain developing axonal collaterals contain rather unusual lamellar structures

Figs. 9 and 10. Developing axonal collaterals in the lateral marginal zone of El3 spinal cord. In Fig. 9, a short collateral arises from a main axonal shaft identified by numerous transversely-sectioned microtubules (arrowheads). The collateral contains synaptic vesicles aggregated at the site where it forms a developing synaptic contact (arrows) with a dendritic growth cone (D). The long, irregular process located immediately to the right of the short collateral is also a developing collateral whose origin from an axonal shaft is not shown in the figure. The long collateral contains scattered synaptic vesicles (SV). Both of the axonal collaterals in Fig. 9 are directed medially through the lateral marginal zone toward the lateral motor column. A main axonal shaft (*) in Fig. 10 gives rise to two developing collaterals. The shorter collateral forms a punctum adhaerens (opposed arrows) with a different main axonal shaft. The longer collateral forms a punctum adhaercns with a dendritic growth cone filopodium (D), and a synaptic vesicle (single arrow) is closely associated with this junction. The longer collateral is directed medially toward the lateral motor column. Fig. 9, x 58 500; Fig. 10, x 35 000.

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(Figs. 19 and 20) w h i c h are l o c a t e d a b o u t 10 n m b e n e a t h - and precisely parallel to -- the inner leaflet o f the a x o l e m m a . T h e s e s u b a x o l e m m a l lamellae are a p p r o x i m a t e l y 4 n m t h i c k and are c o n t i n u o u s f o r at least 3 to 4 serial sections ( a b o u t 0 . 2 5 - 0 . 3 3/am). T h e s u b a x o l e m m a l lamellae o f a x o n a l collaterals are o b s e r v e d w h e r e t h e collaterals c i r c u m v e n t o t h e r processes w h i c h b l o c k their direct access to p o s t s y n a p t i c e l e m e n t s , and the lamellae are invariably f o u n d on the s a m e side as the d i r e c t i o n o f collateral c u r v a t u r e (Figs. 19 and 20). A t such sites t h e a x o n a l m e m b r a n e s a p p e a r to b e v e r y rigid. Similar s t r u c t u r e s are also l o c a t e d i m m e d i a t e l y s u b j a c e n t to a c u t e c u r v a t u r e s of d e n d r i t i c and s o m a l p l a s m a m e m b r a n e s . M o s t of the d e v e l o p i n g a x o n a l collaterals w i t h i n t h e m o r e distal p o r t i o n s o f t h e marginal z o n e a p p e a r t o be d i r e c t e d t o w a r d the radial t r a b e c u l a e w h i c h e m a n a t e f r o m the m o t o r columns. O n c e t h e y reach the t r a b e c u l a e , t h e y c o n t i n u e on a course

Figs. 11-13. Developing axonal collaterals in El3 (Figs. 11 and 12) and E14 (Fig. 13) spinal cords. The slender collateral in Fig. 11 forms a synaptic contact (arrows) with a dendritic growth cone (D) near its origin from the main axonal shaft (*). A cistern of smooth reticulum (SR) is located in the main shaft near the presynaptic site. A mixture, of synaptic and growth cone vesicles are aggregated in the tip of the collateral near the right hand edge of Fig. 11. The tip of this collateral exhibits a focal surface specialization (see Vaughn et al., 1976) similar to the one shown at higher magnification on the tip of a different axonaI collateral in the inset to Fig. 11 (arrowheads). The axonal collateral in Fig. 11 is directed medially toward the lateral motor column. In Fig. 12, an axonal collateral forms developing synaptic contacts (arrows.) with a dendrite (D) that is contiguous with the collateral's parent axonal shaft (*), while the axonal collateral in Fig. 13 projects from its shaft (*) and traverses several layers of marginal zone axons before it forms a synapse (arrows) with a dendritic profile (D). The collateral in Fig. 13 is directed toward the medial motor column. Fig. 11, x 69 700; inset, x 105 300; Fig. 12, x 25 200; Fig. 13, x 45 300. Fig. 14. Portion of a radial trabecula in the lateral marginal zone of an El4 spinal cord. A developing axonal collateral projects from its axonal shaft (*) and courses medially along a radial glial process (GP) toward the lateral motor column. The origin of the collateral contains three coated vesicles (short arrows) which appear to be fused to a common region of the axolemma. Another coated vesicle is located between these three coated vesicles and the adjacent mitochondrial profile, while a fifth coated vesicle (arrowhead) is present somewhat nearer the centre of the main axonal shaft. The collateral contains numerous synaptic vesicles (SV). Another axonal shaft (**) located in the lower right hand corner of Fig. 14 gives rise to two, short collateral projections (curved arrows). One collateral forms a synapse (double arrows) with a dendritic growth cone filopodium (D), while the other displays a coated vesicle (CV) in apparent fusion with its surface, x 26 200. Figs. 15-17. Developing axonal collaterals in the marginal zone of E13 spinal cord. The collateral in Fig. 15 forms a synaptic contact (arrow) with a dendritic growth cone (D) and contains synaptic vesicles (SV) along its entire length. A coated vesicle (CV) is located just to the right of the presynaptic site. Numerous synaptic vesicles (SV) and longitudinally-arranged microfilaments characterize the developing collateral in Fig. 16. This collateral forms a punctum adhaerens (opposed arrowheads) with a dendritic growth cone (D). The axonal collateral in Fig. 17 appears to be more differentiated than those in Figs. 14--16 because it contains microtubules (arrows) instead of microfilaments. A long cistern of smooth reticulum (SR) is located where the collateral arises from the main shaft(*). Fig. 15, x 35 700; Fig. 16, x 38 400; Fig. 17, x 26 800.

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toward the m o t o r column and commonly form one or more synaptic contacts with dendritic profiles encountered along the way. Developing axonal collaterals located in the more internal portions of the marginal zone also appear to follow trabeculae toward the motor columns, but some axons that are very closely adjacent to the m o t o r somata frequently give rise to collaterals that take a direct, nontrabecular course toward the motor columns. These collaterals are c o m m o n l y observed to form axosomatic synaptic contacts (Figs. 21, 22 and 26). However, they also form axodendritic synapses, and occasionally an individual collateral forms both axosomatic and axodendritic synapses (for example, collateral 3 in Figs. 21 and 26). Some of the axonal shafts which course longitudinally along the interface between t h e marginal zone and the motor somata display a complex collateralization. For instance, one of the axonal shafts located in a series of sections partially shown in Figs. 2 1 - 2 9 gives rise to three collateral projections (1, la and lb) within a 2.6/.tm interval. One of these collaterals (la) projects to an adjacent m o t o r soma and forms what appears to be an axosomatic synaptic contact (Fig. 26). Another collateral (1) takes a course that approximately parallels the plane of the marginal zone interface with the m o t o r somata and forms two axodendritic synaptic contacts (Figs. 28 and 29). One of these synaptic contacts is formed with what appears to be a dendritic growth cone filopodium contained in an invagination of the presynaptic collateral (Fig. 28). Collateral 1 contains several coated vesicles near the place where it begins to be invaginated by the dendritic growth cone filopodium (not shown). In addition, a coated vesicle is fused with the plasma membrane of the collateral where the membrane is indented by the filopodial tip (Figs. 21 and 24). Another relationship of a presynaptic coated vesicle to a developing synapse is shown in Figs. 21, 22 and 25. The site of an axosomatic synapse formed b y collateral 2 (Fig. 22) is contiguous with a region that displays a minute extension of the adjacent somal surface into a large coated vesicle (approximately 100 nm in diameter) that is fused with the surface of the developing axonal collateral (Figs. 21 and 25). Similar relationships also exist between presynaptic surface membranes and postsynaptic coated vesicles (Figs. 3 0 - 3 2 ) .

Fig. 18. Lateral marginal zone of El3 spinal cord. The large, main axonal shaft located in the upper right of the figure contains numerous, transversely-sectioned microtubules (arrowheads), a long cistern of smooth reticulum (SR) and a mitochondriaI profile (M). The shaft gives rise to two short collateral profiles (curved arrows) which contain microfilamentous cytoplasmic material that extends into the main shaft for only a short distance. The smaller main axonal shaft (*) at the left of the Fig. gives rise to a single collateral that extends to the bottom left hand corner. The smaller shaft also displays a cistern of smooth reticulum (SR). x 30 600. Figs, 19 and 20. Subaxolemmai lamellae within developing axonai collaterals of E13 spinal cord. Collaterals in each figure curve around intervening axonal shafts (A) and form developing synaptic contacts (arrows). Subaxolemmal lamellae (arrow heads) are located along the region of axolemmal curvature. The postsynaptic element in Fig. 20 is a dendritic protrusion from the soma of a developing motor neuron (MN). The collateral in Fig. 20 is in continuity with its axonal shaft (*) of origin. Fig. 19, x 60 300; Fig. 20, x 54 800.

A x o n a l g r o w t h c o n e s and d e v e l o p i n g a x o n a l collaterals f o r m s y n a p s e s

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Quantitative study T a b l e 1 s h o w s the relative p r o p o r t i o n s o f s y n a p t i c c o n t a c t s t h a t are f o r m e d b y a x o n a l g r o w t h c o n e s and d e v e l o p i n g a x o n a l collaterals (i.e., ' d e v e l o p i n g ' a x o n s ) as c o m p a r e d w i t h t h o s e f o r m e d b y m a i n a x o n a l shafts (i.e., ' d i f f e r e n t i a t e d ' axons). In o r d e r to be j u d g e d as ' d e v e l o p i n g ' axons, profiles h a d to display the characteristics o f the a x o n a l g r o w t h c o n e s or d e v e l o p i n g a x o n a l collaterals d e s c r i b e d a b o v e (Figs. 5--7, 9, 12, 13 and 15) and n o t c o n t a i n m i c r o t u b u l e s a n d / o r n e u r o f i l a m e n t s . Profiles o f ' d i f f e r e n t i a t e d ' a x o n s w e r e classified as such b e c a u s e t h e y c o n t a i n e d m i c r o t u b u l e s , m i t o c h o n d r i a and, occasionally, a f e w n e u r o f i l a m e n t s (Figs. 8, 10 and 11). T h e m a i n criteria f o r i d e n t i f y i n g s y n a p t i c c o n t a c t s w e r e an aggregation o f s y n a p t i c vesicles at p r e s y n a p t i c m e m b r a n e s and a r i g o r o u s l y parallel a p p o s i t i o n o f pre- and p o s t s y n a p t i c m e m b r a n e s . A n a d d i t i o n a l criterion was the p r e s e n c e o f p a r a m e m b r a n o u s d e n s e m a t e r i a l at the sites o f s y n a p t i c c o n t a c t . T h e relative p r o p o r t i o n s o f a x o d e n d r i t i c s y n a p t i c c o n t a c t s f o r m e d b y d e v e l o p i n g and d i f f e r e n t i a t e d a x o n a l profiles are a p p r o x i m a t e l y equal in all o f the e m b r y o n i c

Figs. 21--29. Serial section analysis of developing axonal collaterals adjacent to a motor neuronal soma (MN) in the lateral motor column of E13 spinal cord. The collaterals of three different axonal shafts are shown in this series, and isolated profiles of each of these three collateral systems that are continuous within the series are designated by common numerals. For example, in Fig. 21, a main axonal shaft (1, left centre of micrograph), and an isolated portion of one of its collaterals (1, upper right of micrograph) are continuous in Fig. 23 (curved arrows). Isolated collateral 1 begins to be indented by what appears to be a dendritic growth cone filopodium (D) in Fig. 21, and as shown at higher magnification in Fig. 24, a coated vesicle (CV) is fused with the portion of isolated collateral l's plasma membrane that is associated with this indentation. Somewhat farther into the series, this collateral (1) forms a developing synaptic contact (Fig. 28, arrow) with the indenting dendritic profile (D), and the dendritic profile then decreases markedly in size until it becomes an extremely thin stalk (Figs. 26 and 29, arrow heads) surrounded by the collateral's (1) plasma membrane. Collateral 1 also forms a more conventional developing synaptic contact (arrow) with another dendritic profile in Fig. 29. A second collateral branch (la) of axonal shaft 1 begins to appear in Figs. 21 and 22, and it forms a punctum adhaerens (Fig. 27, opposed arrow heads) with the base of a filopodial extension from the motor soma. Farther into the series, this collateral (la) makes what appears to be a grazed synaptic contact (Fig. 26, arrow) with the motor soma as well as another punctum adhaerens (Fig. 26, opposed arrow heads). A third collateral branch (lb) of axonal shaft 1 extends obliquely away from the motor soma (Figs. 21, 22 and 27) and expands (Fig. 26, lb) into a swelling that is filled with growth cone vesicles near the end of the series (not shown). A different main axonal shaft (2) in Fig. 21 projects a collateral (2) to the motor soma, and the axolemma at the tip of collateral 2 is fused with a coated vesicle (CV) that surrounds what appears to be a thin extension of the motor somal surface. This relationship is shown at higher magnification in Fig. 25 where the thin somal extension is designated by an arrow. This coated vesicle/somal surface relationship of collateral 2 is located at the perimeter of the developing synaptic contact (Fig. 22, arrows) between collateral 2 and the motor soma. A third axonat collateral (3) forms what may be a developing synapse (Fig. 21, arrow) with the motor soma and, elsewhere in the series, it forms a developing synaptic contact (Fig. 26, arrow) with a dendritic profile (D). A portion of collateral 3 in Fig. 21 contains numerous growth cone vesicles (GV). Fig. 21, x 24 200; Fig. 22, x 24 200; Fig. 23, x 30 600; Fig. 24, x 58 500; Fig. 25, x 55 600; Fig. 26, x 24 900; Fig. 27, x 22 300; Fig. 28, x 55 600; Fig. 29, x 26 800.

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Figs. 30--32. Serial sections of possible dendritic endocytosis of a presynaptic membrane. A main axonal shaft (A1) forms a developing synaptic contact (arrow) with a dendritic growth cone (D) in Fig. 30. Note the grazed coat material (arrowhead) within the dendritic profile (D) in Fig. 30 is a portion of the coated vesicle (CV) shown in Fig. 31. This coated vesicle is fused with the perimeter of the dendrite's postsynaptic membrane, and it is invaginated by a thin surface extension (Fig. 31, arrows) from the presynaptic element A 1 . Neither the coated vesicle nor the synaptic relationship betweeen A1 and the dendritic profile is present in Fig. 32 (separated from Fig. 31 by one ultrathin section, or approximately 80 nm). However, a less specialized interdigitation (Fig. 32; arrowhead) between the dendritic growth cone (D) and a different axon (A2) does occur in Fig. 32, and another large coated vesicle (CV) appears to be fused with the dendritic profile's plasma membrane. Figs. 30-32 are from the lateral marginal zone of El4 spinal cord. x 34 900. specimens. This relationship is very different f r o m t h a t observed for the proportions of synapses f o r m e d u p o n developing dendrites (i.e., dendritic growth cones and dendritic growth cone filopodia) and differentiated dendrites (Vaughn et al., 1974). Developing dendritic profiles constitute substantially larger proportions of synaptic populations t h a n do differentiated dendritic profiles during the early period of synaptogenesis, b u t this gradually changes with developmental age so t h a t b y days E15 and 16 differentiated dendritic profiles are p o s t s y n a p t i c sites m u c h more f r e q u e n t l y t h a n developing dendrites (Table 1).

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Table 1. Mean percentages* of developing and differentiated pre- and postsynaptic elements in the lateral marginal zone of embryonic mouse spinal cord.

Age

El2 El3 El4 E15 El6

Developing Axons I

Differentiated Axons 2

S.E.M. *

43.32 45.00

56.68 55.00 54.02 54.11 41.67

2.14 7.88 10.27 7.96 9.85

45.98 45.89

58,33

Developing Dendrites 3

Differentiated Dendrites 4

64.17

35.83 24.44 36.61

75.56

63.39 48.41 28.79

S.E.M. *

51.59

0.51 12.37 0.89 13.12

71.21

7.58

*Data are the means of percentages obtained from two animals for each embryonic day except E13 where percentages obtained from three animals were averaged to obtain the reported mean. S.E.M., standard error of the mean. 1 Axonal growth cones and developing axonal collaterals (see text). 2Main axonal shafts (see text). 3Motor dendritic growth cones and growth cone filopodia (see Vaughn et al., 1974). 4Motor dendritic profiles with numerous microtubules (see Vaughn et al., 1974).

A few synaptic contacts have also been observed in the marginal zone of E l l spinal cord (Vaughn et al., 1977), but they are sufficiently rare that they are not detected by our quantitative sampling procedure. For this reason the synaptic contacts observed at E l l have not been included in the data shown in Table 1. Nevertheless, it is relevant to point out that both synaptic contacts observed in the extremely thin E l l lateral marginal zone (Vaughn et al., 1977, Figs. 2 and 3) occur between main axonal shafts and a differentiated primary dendrite of a motor neuron. The presynaptic elements of all four synaptic contacts observed in the E l l ventral marginal zone are also main axonal shafts, and two of the postsynaptic components of these synaptic contacts are differentiated dendritic profiles while the remaining two postsynaptic elements appear to be dendritic growth cones (Vaughn et al., 1977, Figs. 4 and 5). Although a few axonal growth cones and developing axonal collaterals are present in the marginal zone of E11 specimens, they have not been observed forming synaptic contacts. Discussion

Development of axonal collaterals The development of the axonal collaterals observed in this study apparently begins with the formation of short, cytoplasmic protrusions from main axonal shafts. Since these protrusions contain the vesicular and microfilamentous components which have been used to identify growth cones in previous electron microscopic studies of developing C.N.S. (reviewed by Hinds and Hinds, 1972; Bunge, 1973), they would appear to be similar to the growth cones associated with other growing neurites. This interpretation is supported by the observation that Golgi-impregnations reveal short, developing axonal collaterals which terminate as growth cone swellings that are

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smaller than those located at the tips of main axonat shafts (Ramdn y Cajal, 1960; and present observations). The short, collateral growth cones contain synaptic vesicles and are the presynaptic elements of developing synaptic contacts. A subsequent step in collateral development appears to be the formation of longer projections which are characterized by linear arrays of microfilaments and scattered synaptic vesicles. Some of the longer collateral projections are sheet-like structures, while some of the shorter ones are finger-like, filopodial extensions (also see Hinds and Hinds, 1972; Roberts, 1976). Both types of collateral projections are observed to be the presynaptic elements of developing synaptic junctions, and they commonly form synaptic contacts at several different sites along their lengths. The microfilamentous arrays within axonal collaterals appear to be replaced with linearly arranged microtubules in the later stages of development, and mitochondrial profiles also become progressively more common as the collaterals become more differentiated. Role of the smooth reticulum The smooth reticulum appears to be involved in the development of axonal collaterals because its cisternae are much more prominent within main axonal shafts at the sites where collaterals arise than they are along the main shafts generally. Similar concentrations of the smooth reticulum are also commonly seen in main axonal shafts at sites of synaptic junctions. If the smooth reticulum is involved in axoplasmic transport as has been suggested by other investigators (for example, Byers, 1974; Droz et al., 1975 ; Gray, 1976), it is plausible that sites of collateral and synaptic development might display a specialized development of this organelle as a reflection of an increased distribution of the raw materials necessary for further growth and differentiation. Roles o f coated vesicles Possible functions of the coated vesicles observed in developing axonal collaterals are conceptually more complex than the one suggested above for the smooth reticulum. The results of numerous previous investigations indicate that coated vesicles commonly occur within developing postsynaptic elements, and it has been suggested that these organelles may be involved in the insertion of specific molecular arrays into differentiating postsynaptic membranes (e.g. Altman, 1971 ; Stelzner et al., 1973; Rees et al., 1976; McLaughlin and Wood, 1977). Since the present investigation shows that coated vesicles are also commonly found in presynaptic elements where they fuse with developing presynaptic membranes, it is possible that coated vesicles may be involved in the insertion of specific molecules into presynaptic, as well as postsynaptic, membranes. However, our observation that some presynaptic coated vesicles are invaginated by minute extensions of postsynaptic membranes suggests that certain presynaptic coated vesicles may be engulfing small portions of postsynaptic elements. This interpretation is consistent with the evidence that large diameter (80-100 nm) coated vesicles generally appear to be involved in the

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endocytosis or ingestion of extracellular material (for reviews see Peters et al., 1976; Rees et al., 1976). Since we have also observed an apparent endocytosis of presynaptic membranes by postsynaptic coated vesicles, it is intriguing to imagine that a microphagocytosis of pre- and/or postsynaptic membranes might be a mechanism for the exchange of molecular information between neurons engaged in the formation of synaptic relationships. Such exchanges might result in alterations of the synthesis of new membrane components (see Roberts and Matthysse, 1970) on the part of contacting cells which, in turn, might lead to the differentiation of low affinity protosynaptic contacts into synaptic junctions. It is possible to suppose that one population of coated vesicles might mediate the addition of new components into the membranes of developing synaptic junctions while another class of coated vesicles might be involved in an intercellular exchange of membrane material. The former possibility implies a centrifugal movement of coated vesicles from the Golgi apparatus to the synaptogenic sites, while the latter would involve a centripetal flow of coated vesicles from the synaptogenic sites to the neuronal somata. Although there are indications of such a bidirectional flow of coated vesicles (reviewed by Rees et al., 1976), more experimental information must be obtained if the functions of coated vesicles in neuronal development are to be completely understood. A case in point is Privat's (1974) often ignored suggestion that coated vesicles may be associated with the destruction of transitory intercellular contacts rather than the construction of permanent synaptic junctions. Privat based this on coated vesicles, invaginated by portions of junctional membranes, similar to those described here, but he has only described such relationships at puncta adhaerentia in contrast to our observations that they are located at the perimeters of clearly identifiable, developing synaptic contacts. Since we have observed these membrane/coated vesicle relationships associated with 'normal-appearing' synaptic contacts, we favour a view that they are involved in constructive processes, but on the basis of our observations alone we cannot rule out the possibility that they could represent initial steps in the direct destruction of inappropriate synaptic contacts (Privat, 1974). However, the occurrence of similar membrane protrusions into coated vesicles in other developing systems would seem to support a constructive, rather than destructive, function for these relationships at developing synaptic junctions. For example, Harris and Hopkins (1977) show that similar 'ball and socket' junctions form between neuroblastoma cells (origin of the 'balls') and muscle cells (sites of the 'socket', coated vesicles) which form synapse-like contacts in vitro, but that 'ball and socket' junctions are not observed between mouse L cells (fibroblastic cell line) and muscle cells which do not form synapse-like contacts. In addition, James and Tresman (1969) observed ball and socket-like junctions between axons ('ball') and muscle cells ('socket') in primary cultures. They described this relationship as 'axon pinocytosis', and suggested that it might represent a mechanism for the uptake of axonal material by muscle cells and/or an initial step in the adherence of axons and muscle cells. Similar examples of axon pinocytosis were also observed by Bird and James (1973) at synapses developing in vitro between

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previously dissociated chick spinal cord neurons, and thes< investigators suggested that 'complex (i.e. coated) vesicles may be involved in processes other than those concerning the transport or release of transmitters, or simple pinocytosis of substances in the nutrient medium.' Furthermore, Henkart (1975) has stated that coated vesicles 'pinch-off' small amounts of adjacent neurons and that the frequency with which this occurs is dependent upon extracellular Ca 2§ concentration. Henkart (1975) proposes that these coated vesicle relationships could represent a morphological basis for the transfer of macromolecules between adjacent neurons and alludes to the possibility that this might 'represent a transfer of information' between functionally interconnected elements of particular neuronal circuits. It is also germane to draw attention to the similarity between the 'balls' of ball and socket junctions and the 'dense membrane knobs' observed by Roberts and Hayes (1974) in embryonic toad nervous system. These knobs are apparently formed by an 'outpushing' of the plasma membrane, and they are generally directed into wide extracellular spaces. Roberts and Hayes (1974) suggest that one possible function of the knobs might be to 'serve as points of adhesion for growing processes.' It is tempting to speculate further along this line that such unarticulated membrane knobs (Roberts and Hayes, 1974) might represent an initial step in the formation of ball and socket junctions. Based upon the findings discussed above, it is reasonable to think that coated vesicles may mediate several different functions all of which are important to the formation and maintenance of appropriate synaptic connectivity patterns in the central and peripheral nervous systems. However, it should be obvious that this concept presently rests almost entirely upon descriptive, circumstantial evidence, and that a firm experimental foundation remains to be established for the roles played by coated vesicles in the formation and/or destruction of synaptic as well as other types of intercellular junctions.

Relation of synapses to collateral sprouting Since coated vesicles are frequently observed in presynaptic elements at developing synaptic contacts, the clusters of coated vesicles located at the origin of developing collaterals from axonal shafts may indicate that these portions of the collaterals are forming synaptic contacts just out of the plane of section. Such a possibility is supported by the fact that developing synaptic junctions are commonly located at the sites of collateral origin from main axonal shafts. This observation suggests that the early synaptic contacts formed by main axonal shafts may stimulate collateral sprouting near the developing synaptic contacts. However, other observations suggest that the formation of synaptic contacts by main axonal shafts is not a mandatory prelude to collateral development. For example, collaterals are observed emanating from main axonal shafts located in the distal marginal zone well beyond the extent of dendritic growth. Thus no appropriate postsynaptic elements are in the area for the formation of synaptic contacts with axons that have already begun to develop collateral branches. Furthermore, we have not observed synaptic contacts at or near the origin of some of the collateral branches which have been examined in

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359

serial sections. Therefore the formation of synaptic contacts by axonal shafts would not seem to be an absolute requirement for the primary induction of collateral sprouting, and it appears that the capacity to form axonal collaterals may be an endogenous propertT of neurons which, as in the case of dendrogenesis (Rakic, 1974; Bradley and Berry, 1976; Privat and Drian, 1976; Sotelo and Arsenio-Nunes, 1976; Vaughn et al., 1977), does not require synaptic contact for its expression. Nevertheless, the formation of synaptic contacts could facilitate or hasten collateral development by signalling axonal shafts that they are located in a synaptogenic field. A similar facilitation of dendritic growth by early forming axosomatic synapses has been suggested previously (for example, Bradley and Berry, 1976; Vaughn et al., 1977).

Bases and implications o f preferential collateral growth A large majority of developing axonal collaterals in both Golgi and electron microscopic preparations exhibit a preferential direction of growth toward the locations of potential postsynaptic elements. Collaterals from distal axons are almost invariably directed toward the trabeculae which contain the dendrites growing from the somata of the medial and lateral motor columns. Furthermore collaterals originating from main axonal shafts that are located adjacent to these trabeculae are most frequently observed to be directed centrally toward the source of the dendritic growth, while collaterals from axons located in the marginal zone immediately adjacent to the motor columns are often directed toward the motor somata. Partial explanations for this apparent 'directed growth' of axonal collaterals may be that collaterals growing toward the motor columns are likely to form synaptic contacts, and that once they have formed such contacts they are no longer transitory structures which only probe short distances away from the main axonal shaft before being withdrawn. Conversely collateral growth directed away from the source of postsynaptic elements would not form synaptic contacts, and thus it would not be maintained sufficiently long to be observed frequently. However, this possibility is not adequate to explain the orientation of the distal collaterals which have a very low probability of forming synaptic contacts because they have not reached the more proximal, dendrite-rich areas of the developing marginal zone. Despite this fact, such collaterals appear to be directed preferentially toward the encroaching dendritic growth, and perhaps this phenomenon hints at the existence of a positive dendrotropism that is independent of physical contact for its attractive, or 'alluring' (Ram6n y Cajal, 1959), effect upon collateral growth (see Jacobson, 1970 for a review of chemotaxic concepts). Regardless of what might be the basis for the apparent preferential growth of axonal collaterals toward potential postsynaptic elements, this growth per se suggests that the axons present in the embryonic marginal zone are actively searching for postsynaptic elements rather than passively awaiting their arrival. Striking reflections of such an active search are provided by those collaterals which bend around intervening profiles in order to establish synaptic contacts with

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postsynaptic elements located some distances away from the parent axonal shafts. The ability of collaterals to grow around processes which are blocking their direct access to postsynaptic elements may involve the subaxolemmat lamellar structures described earlier in this report. These structures might serve to modify the properties of the collateral membrane and/or juxtamembranous cytoplasm in such a way as to allow for the acute curvatures of collaterals around intervening processes. There are several hypothetical ways in which a subaxolemmal lamella might cause a change in the direction of a collateral's growth (for example, by effecting an asymmetric insertion of membrane vesicles into the tips of growing filopodia; c.f., Bray, 1973). However, it is sufficient for the present to suggest that current ideas concerning mechanisms of neurite growth should encompass the probabilities that axonal collaterals developing in vivo can execute rather marked changes in their direction of growth, and that this ability may play an integral role in their active search for potential postsynaptic elements.

Significance o f 'developing axons and dendrites'as synaptic elements Main axonal shafts form the extremely small number of synaptic contacts first observed in E l l specimens of developing mouse spinal cord. However, axonal growth cones and developing axonal collaterals are present in the marginal zone of E l l specimens, and they are substantially involved in synaptogenesis by the next embryonic day (E12). Indeed developing axonal collaterals and axonal growth cones ('developing axons' as defined above) comprise nearly one-half of the presynaptic components of the synaptic contacts observed in the quantitative study of El2 specimens, and they continue to constitute about this same proportion throughout the remainder of the embryonic days examined. This rather constant proportion of developing presynaptic elements observed during the E12-E16 period contrasts with the progressively~ decreasing proportion of dendritic growth cones (i.e., 'developing dendrites') which are observed to be postsynaptic elements during this same developmental period (Table 1 and Vaughn et al., 1974). This difference is probably due to the fact that the marginal zone axons course longitudinally within the sampled portions of the marginal zone, while the motor neuronal dendrites are directed radially through the sample space (Materials and methods, and Vaughn et al., 1974). Thus axonal growth cones and the ramifications of developing axonal collaterals would be likely to remain within the sampled portions of the marginal zone over the embryonic period examined, while motor neuronal dendrites would grow progressively through and eventually leave the.sample space. The most striking observation, however, is not that there are differences in the proportions of the synaptic contacts formed by the relatively undifferentiated, and presumably growing, portions of axons and dendrites, but that these portions of both axons and dendrites constitute such a large percentage of the pre- and postsynaptic elements observed during the E12--16 period. It has been suggested previously (Vaughn et al., 1974) that the formation of synaptic contacts on motor dendritic growth cone filopodia may play a role in determining the dendritic

Axonal growth cones and developing axonal collaterals form synapses

3 61

branching patterns ultimately exhibited by differentiated motor neurons. The central idea behind this suggestion is that synaptically-contacted portions of growing dendrites have a high probability of continued differentiation while noncontacted dendritic growth is more likely to be retracted. Observations made in the present study suggest that this same idea may be extended to include a determination of the ramification patterns of axonal collaterals. Thus, growing collaterals which form synaptic contacts would be more likely to persist than those that do not, and this would result in the establishment of the basic ramification patterns of mature axonal collaterals. However, presently available information indicates that the formation of synaptic junctions is not an obligatory prelude either to dendrogenesis or to the initial development of collateral sprouts from main axonal shafts. Therefore it would appear that early synaptic interactions are not critically involved in a primary induction of neurite growth (Rakic, 1974; Bradley and Berry, 1976; Privat and Drian, 1976; Sotelo and Arsenio-Nunes, 1976; Vaughn at al., 1977), but that they may play a significant role in directing the formation of the 'characteristic' dendritic and axonal branching patterns exhibited by different neuronal classes (Vaughn et al., 1974; present report; Berry and Bradley, 1976a,b). Acknowledgement We would like to thank Mariko Nakashima, Christine S. Vaughn, Donna Dreier, Lynn Anderson, Robert P. Barber and Dr Dee Ann Matthews for their assistance in this investigation. This work was supported b y U.S.P.H.S. Grant NS 09578 from the National Institute of Neurological and Communicative Disorders and Stroke. References ALTMAN, J. (1971) Coated vesicles and synaptogenesis. A developmental study in the cerebellar cortex of the rat. Brain Research 30, 311-22. BERRY, M. and B R A D L E Y , P. (1976a) The application of network analysis to the study of branching patterns of large dendritic fields. Brain Research 109, 111-32. B E R R Y , M. and BRADLEY, P. (1976b) The growth of the dendritic trees of Purkinje ceils in the cerebellum of the rat. Brain Research 112, 1-35. BIRD, M. M. and JAMES, D. W. (1973) The development of synapses in vitro between previously dissociated chick spinal cord neurons. Zeitschrift fi~r Zeliforschung und mikroskopische Anatomie 140, 203--16. BODIAN, D. (1966) Development of fine structure of spinal cord in monkey fetuses. I. The motoneuron neuropil at the time of onset of reflex activity. Bulletin of the Johns Hopkins Hospital 119, 129-49. BRADLEY, P. and BERRY, M. (1976) The effects of reduced climbing and parallel fibre input on Purkin.je cell dendritic growth. Brai~* Research 109, 133-51. BRAY, D. (1973) Model for membrane movements in the neural growth cone. Nature 244, 93--96. BUNGE, M. B. (1973) Fine structure of nerve fibers and growth cones of isolated sympathetic neurons in culture. Journal of Cell Biology 56, 713-35.

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