Brain Research, 119 (1977) 57-71
57
© Elsevier/North-HollandBiomedicalPress, Amsterdam- Printed in The Netherlands
THE LOCALIZATION OF CONCANAVALIN A BINDING SITES DURING PHOTORECEPTOR SYNAPTOGENESIS IN THE CHICK RETINA
BARBARA J. McLAUGHLIN* and JOHN G. WOOD Department of Anatomy, University of Tennessee, Center for the Health Sciences, Memphis, Tenn. 38163 (U.S.A.)
(Accepted April 27th, 1976)
SUMMARY Carbohydrate containing macromolecules were localized on developing photoreceptor pedicles and processes in the outer plexiform layer (OPL) of the retina in chick embryos and hatchlings by a concanavalin A (Con A)-peroxidase labeling technique. Con A binding sites were visualized along the membrane surfaces of undifferentiated photoreceptor synaptic bases at early embryonic times and later were localized along differentiating synaptic pedicles and at pre- and postsynaptic membranes of early forming ribbon synapses. Membrane surfaces of developing neurites and glia in this layer were labeled also as were the internal membrane surfaces of coated vesicles within developing receptor terminals and neurites. In the hatchling, Con A binding sites were restricted to non-synaptic membrane regions of receptor pedicles and some neuronal and glial membranes. Con A staining in the inner plexiform layer was similar to that in the OPL with membrane labeling on developing synapses, neurites and glia during embryonic development and labeling restricted to nonsynaptic membrane regions in the hatchling. Con A staining in the other layers of the retina was localized on endoplasmic reticulum, nuclear and plasma membranes of all cell types. Cell junctional complexes between pigmented eipthelial cells and the zonulae occludentes of the outer limiting membrane did not label with Con A at the developmental ages sampled in this study. These findings suggest that Con A binding sites are accessible on developing synaptic membranes but become either altered or masked after synaptic junction maturation.
INTRODUCTION An important area in developmental neurobiology is the understanding of under* To whomcorrespondenceshould be addressed.
58 lying mechanisms which account for the specificity of neuronal connections. Because nerve endings are rich in glycoproteins8 and glycoprotein synthesis is greater during neuronal differentiation2,13, it has been speculated that polysaccharides on neuronal surfaces play a major role in interneuronal recognitionL Further support for this suggestion has come from several studies showing that the sialoglycoprotein level increases two-fold during the period of neuronal development4°, and that the size of oligosaccharide chains of brain glycoproteins rapidly increases during development2% Another more recent study has shown that the rapid accumulation of transported glycoproteins is significantly higher when synapses are being formed than after synaptic maturation zl. While it has been known for some time that synaptic junctions contain polysaccharides7,zs,31 as well as protein 6, more specific information about the actual sugar residues present and their location has come from the use of ferritin or peroxidaselabeled lectins 4,27, which localize specific sugar residues in the polysaccharide chain (cf. refs. 26 and 36). Two of these lectins, concanavalin A (Con A), which binds to glycosyl or mannosyl residues, and ricin, which binds to galactosyl residues, have been shown to bind to the synaptic cleft region and the external surface of the postsynaptic membrane of synaptosome preparations 5,11,23. In contrast to these observations of lectin binding on isolated synaptic junctions, we have found that Con A binding sites at intact synaptic junctions are accessible to the label only after removal of the surrounding polypeptides with trypsin digestion44. These observations are of interest in regard to Moscona's (see ref. 25 for refs.) findings on dissociated embryonic neural retina cells, showing that EGTA dispersed cells from young embryos agglutinated readily with Con A, while cells from older embryos did not, except after trypsin dissociation. If the Con A receptors on the surface of immature neurons are at first accessible and become masked or inaccessible later in development, then it is possible that Con A binding sites on the immature synaptic membrane (which is an extension of the neuronal cell surface) are also exposed in a similar topographical fashion during synaptic junction formation. The present study explores this possibility by following the localization of Con A binding sites at developing synaptic junctions in the outer plexiform layer of the chick retina. Evidence will be presented that the distribution of Con A labeling along the photoreceptor synaptic pedicles changes during embryonic development and after functional differentiation of the photoreceptors. These findings will be discussed in regard to current concepts about the role of cell surface carbohydrates in cell recognition and the possible implications of this role in mechanisms of synaptic specificity. MATERIALS AND METHODS White Leghorn chick embryos (obtained from a local hatchery) were kept in a forced draft incubator at 37 °C and staged according to Hamburger and Hamilton14 prior to fixation. Embryos between the ages of 13 and 21 days and 2-7-day-old hatchlings were used in this study. The animals were perfused initially through the heart and then through the eye with a fixative containing 2 ~ glutaraldehyde (70 ~o
59 Ladd)-2 ~ paraformaldehyde--0.5 ~o acrolein-0.5 ~o DMSO in 0.08 M phosphate or cacodylate buffer with 0.001 ~ CaCI2 added 3s. The lower temporal retinal quadrant was dissected free and stored in the same fixative overnight at 4 °C. The central portion of the quadrant a° was sectioned into 50 #m slices with a Sorvall TC-2 tissue sectioner and the slices were collected in 0.08 M phosphate buffered saline (PBS). The best retinal slices were selected under the dissecting microscope for cytochemical staining. The method of Con A-peroxidase labeling of carbohydrates used in this study was essentially as described by Bernhard and Avrameas4. All steps were performed with constant agitation at room temperature. Concanavalin A (Calbiochem., Grade A, San Diego, Calif.), horseradish peroxidase (Sigma Chemical Co., Type VI, St. Louis, Mo.), glucose oxidase (Sigma Type II, a-methyl-D-mannoside, Sigma No. M-3752), and 3,Y-diaminobenzidine (Sigma No. D-8126) were all used in the cytochemical procedures. The retinal slices were washed in PBS for 15 rain and then incubated in 0.5-1 mg/ml of Con A in PBS for 40 min. They were washed in 3-5 changes of PBS for 3 h and incubated in 0.1 mg/ml horseradish peroxidase for 40 min followed by another 3 h washing in PBS. The slices were treated with either 20 #g glucose oxidase, 90 mg glucose and 50 mg of diaminobenzidine (DAB) in 100 ml PBS for 5-15 min or with 2/~1 of 30 ~ H202, and 30 mg DAB in 50 ml PBS for 5-15 min. The slices were washed another 30 min in PBS and were hand cut into smaller pieces and osmicated (2 OsO4), dehydrated and embedded in Epon-Araldite for electron microscopy. Some tissue was block stained in 2 ~ uranyl acetate prior to dehydration17. Control slices were incubated in Con A (0.5-1 mg/ml) in the presence of 0.2 M a-methyl-D-mannoside, an inhibitor of specific Con A binding to sugars36, and the tissue processed as described. Thin sections were cut on a diamond knife and stained with either uranyl acetate and lead citrate 4z or lead citrate only, prior to examination in a Philips EM 301 electron microscope. Some sections were left unstained prior to examination. RESULTS
Con .4 labeling in outer plexiform layer (OPL) The photoreceptor synaptic bases begin to differentiate between 13 and 15 embryonic days2a and filopodial-like protrusions of the receptor cell surface protrude into the OPL at this time (Figs. 1-3). Con A-peroxidase labeling is present on the external membrane surfaces of most of the neuronal and glial profiles in the OPL and along the plasma membrane of the receptor synaptic bases and their filopodial protrusions as well as on adjacent dendritic membranes. The label is particularly concentrated at regions along the receptor membrane and two presumed postsynaptic neurites which are aligned in an apparent dyad-like arrangement on either side of a filopodial protrusion (Figs. 2, 3). These regions, which are characterized by the presence of a filamentous-like undercoating on the cytoplasmic side of the receptor membrane, are thought to represent the first morphogenic stages of photoreceptor synaptogenesis in the chick retina24. Coated vesicles (Figs. 1, 5, 6) are seen throughout
Fig. 1. Con A labeling on nuclear membrane and undifferentiated synaptic base membrane of a photoreceptor cell (R). Several labeled filopodial protrusions (arrows) of the receptor protrude between neurites(*) aligned along the base in dyad-like apposition. Con A staining is also present on membrane surfaces of glial and neuronal profiles in the outer plexiform layer (OPL) and several coated vesicles (arrowheads) are labeled with Con A. 14 embryonic days. Uranyl acetate (UA) and lead (Pb) stained. , 47,500. Fig. 2. Higher magnification of a receptor protrusion in Fig. 1. Con A labeling is particularly concentrated in regions (*) where two neurites are aligned in dyad-like apposition with the receptor membrane. An undercoating is present (arrows) on the cytoplasmic face of the receptor membrane in these regions. 14 embryonic days. UA and Pb stained. >," 90,000. Fig. 3. Another receptor protrusion from Fig. 1 which protrudes between two neurites (*) in dyad-like apposition. Con A labeling appears to be concentrated between the receptor membrane and one of the neurites and an undercoating is present on the receptor membrane in this region (arrows). 14 embryonic days. U A and Pb stained. +i 90,000.
61
Fig. 4. Photoreceptor cells (R) whose synaptic bases have elongated into the outer plexiform layer (OPL) and differentiated into synaptic pedicles (ped). Con A binding sites are localized along the membrane surfaces of each receptor pedicle in both non-synaptic and synaptic (arrows) regions and on the membrane surfaces of profiles in the OPL. The nuclear and plasma membranes of the receptor cells are labeled also. 18 embryonic days. UA and Pb stained. × 25,300. embryonic development in developing neurites and receptor filopodia and are labeled with Con A-peroxidase along their inner vesicular membrane surfaces. In many cases, the labeled coated vesicle and the Con A-labeled plasma membrane of the neurite or receptor cell are joined together (Fig. 6). Between 15 and 21 days, the receptor synaptic bases elongate and expand into conical-shaped pedicles and the protrusions differentiate more fully to form character-
62
Fig. 5. Con A labeling on membrane surfaces of a receptor pedicle (ped) and on profiles in the OPL. The label is particularly concentrated in regions of dyad apposition (*) between the pedicle and potential postsynaptic neurites and at a developing ribbon synapse (arrow) in which a synaptic ribbon (sr) is located nearby. Labeled coated vesicles (arrowheads) are present in the pedicle cytoplasm. 16 embryonic days. UA and Pb stained. × 55,000.
istic ribbon synaptic terminals, in which a synaptic ribbon and its associated vesicles and trough-like density (arciform density of Ladman 18) form a synaptic ridge between two or three apposed neurites in dyad or triad relationship (Fig. 4). The Con A label continues to be present on the external membrane surfaces of the synaptic pedicle and along the synaptic ridges at ribbon synapses as well as on adjacent dendritic membranes (Figs. 4-6). Labeled coated vesicles are observed within the synaptic pedicle (Fig. 5). The numerous synaptic vesicles in the vicinity of coated vesicles are not labeled. Labeling on neuronal and glial plasma membranes in the OPL remains the same as described for earlier embryonic periods, and labeled profiles of coated vesicles are seen in the developing neurites (Fig. 6). In the 2-day hatchling, the expanded receptor pedicles are no longer observed in the same plane of section with their cell bodies, but appear instead as isolated synaptic bodies in the OPL beneath the layer o f receptor cell bodies (Figs. 7, 8). The dyad and
63 triad postsynaptic processes are deeply invaginated within the vitreal borders of the synaptic pedicles. Con A labeling of the OPL is restricted to plasma membrane surfaces of neurites (Figs. 7, 8) surrounding the synaptic pedicles and along the outer or non-synaptic border of the pedicle membrane (Fig. 8). Some labeling is observed occasionally on neuronal profiles invaginated within the synaptic pedicle which are not in any apparent synaptic apposition. Coated vesicles are less numerous in neuronal processes and receptor pedicles at this time, but are labeled along their inner membrane surfaces as described earlier.
Fig. 6. Con A staining of pedicle (ped) membrane in both non-synaptic and at synaptic (arrows) regions in which synaptic ribbons (sr) are present. Staining is present also on profiles in the OPL and within a coated vesicle (arrowhead) which is joined to the labeled plasma membrane. 18 embryonic days. U A and Pb stained, x 68,000.
64
Con A labeling in other retinal layers In addition to plasma membrane labeling, Con A-peroxidase labels the rough and smooth endoplasmic reticulum, the Golgi apparatus and nuclear membranes of all cell types in the retina and pigmented epithelium throughout embryonic development and after hatching (Figs. 9, 10). The zonulae occludentes 37, which form the outer limiting membrane (OLM) of the retina (Fig. 10), are never labeled with Con A at any developmental time, even though there is plasma membrane labeling on the apposed cells above and below the junctional regions. Con A labeling in the inner plexiform layer (IPL) is similar to that in the OPL. During embryonic development the plasma membrane surfaces of developing neurites and glial processes are labeled as are the immature synaptic junction membranes (Fig. 11). After hatching the Con A labeling is restricted to membrane surfaces of profiles not in any apparent synaptic apposition (Fig. 12). No Con A labeling is observed in the control retinas at any developmental time.
Fig. 7. Two mature synaptic pedicles (ped) below photoreceptor cell bodies (R). Con A binding sites are localized on membrane surfaces of someprofiles in the OPL not in synaptic apposition with the pedicles and on the receptor nuclear membranes. 2-day hatchling. UA and Pb stained. × 22,750.
65
Fig. 8. Mature synaptic pedicle (ped) in the OPL in which Con A labeling is localized only on nonsynaptic regions of the pedicle and on membrane surfaces of profiles surrounding the pedicle. 18 embryonic days. UA and Pb stained. × 62,500.
DISCUSSION Studies have shown that the chick eye and contralateral optic lobe respond to photic stimulation by the eighteenth embryonic day 29 but that the synapses in the visual system reach physiological maturity during postnatal life35, 48. The ability of developing photoreceptor synaptic membranes in the embryonic retina to label with Con A and their failure to label after synaptic maturation in the hatchling suggests that lectin binding sites are accessible on immature synaptic membranes but become inaccessible or masked after junctional maturity. While we cannot exclude the possi-
66 bility that failure to label the mature synapse may be due to restricted penetration ot reagents, these developmental changes in lectin labeling may reflect changes in the maturation of cell surface glycoproteins during synaptogenesis. The accessibility of Con A binding sites on immature synaptic membranes may indicate that mannose or glucose residues are relatively exposed or terminal in the oligosaccharide chain, whereas their inaccessibility to labeling after synaptic membrane maturation may indicate that other sugars have been added to the chain thus masking the mannose or glucose residues from the label. Alternatively, these sugars which are present on the presynaptic membrane may become inaccessible to the label by virtue of their being bound to receptors on the postsynaptic membrane, or vice versa. Recent findings 2z in developing brain suggest that glycoproteins undergo a rapid increase in the size of
Fig. 9. Con A-labeled bipolar cells (Bi) in which nuclear membranes, Golgi apparatus and endoplasmic reticulum membranes are stained. Very little labeling is present on plasma membranes. 21 embryonic days. UA and Pb stained. × 17,100.
67 their oligosaccharide chains which may be attributable to the addition of terminal sialic acid residues (see also refs. 10 and 40). Cell surface macromolecules have been implicated in a variety of cellular interactions during morphogenesis and it has been suggested that protein--carbohydrate complexes or 'cell ligands' function as tissue-specific recognition sites on reaggregating cell surfaces 19,2°,25. One of the major carbohydrates in the tissue-specific aggregation factors from embryonic chick neural retina cells is mannose 25 and it is tempting to speculate whether the mannosyl residues which are accessible to Con A during synaptogenesis are also involved in mechanisms underlying interneuronal recognition between potential synaptic membranes in the chick retina. In the OPL, Con A binding sites on immature receptor synaptic bases appear to be concentrated in the regions of
Fig, 10. Photoreceptor cells (R) with Con A binding sites localized on nuclear and plasma membranes of the receptors and their inner segments (IS) and the Mtiller cells (Mu). NO labeling is present in the i'egi0n of zonulae occludentes (arrowheads). 18 embryonic days. UA and Pb stained, x 27,300.
68
i Fig. I1. Con A labeling on developing synaptic membranes (arrows) in the inner plexiform layer (IPL). Label is present also on other non-synaptic membrane surfaces. 14 embryonic days. UA and Pb stained. × 102,000. Fig. 12. Con A labeling on non-synaptic membranes of profiles in the IPL but absence of label at synaptic junction. 18 embryonic days. UA and Pb stained. × 68,000.
membrane alignment between potential dyad synaptic profiles and the pedicle. Such regions of membrane alignment are thought to represent the first stages of synaptic junction differentiation between these components 24 and it would be interesting to know if the exposed Con A receptors play a role in mediating such an initial interaction or specific recognition between these membranes. The fact that other nonsynaptic membrane surfaces in the OPL label also with Con A during the synaptogenic period suggests that there may be other factors besides the mere presence of certain sugars involved in these mechanisms, such as a critical number of Con A binding sites concentrated at synaptic junctional regions as apposed to non-junctional membrane regions as Vaughn et al. 41 have suggested. It is interesting in this regard that the nonsynaptic cell junctions between pigmented epithelial cells and the zonulae occludentes of the O L M do not label with Con A even though p l a s m a membranes adjacent to the junctions are clearly labeled. These junctions are alreadydifferentiated by the earliest embryonic age sampled in this study, which suggests t h a t m a t u r e cell junctions either do not contain Con A receptors or that, like mature synaptic junctions, the binding sites are inaccessible or masked after junction formation. Coated vesicles have been observed to occur in great numbers in developing neurites during synaptogenesis 1,34,39 and it has been suggested that they contribute to the formation of synaptic densities by fusing with the postsynaptic membrane and allowing the coat material to become part of the postsynaptic density 1,3z,a4,a9. It is of
69 interest that the coated vesicles observed in the developing OPL are labeled with Con A at the same time developing synaptic membranes are labeled. This suggests that coated vesicle and immature synaptic membranes share similar cytochemical components during the synaptogenic period which may be related to the formation of synaptic densities or some other aspect of junction formation and neurite growth, such as transport of glycoproteins to the junctional regions and growing ends of neurites 21. Coated vesicles label with Con A throughout development and in the hatchling and these organelles have been implicated in uptake of exogenous protein by growing neurites in the developing cerebellum 12 and in cultureg, 33. During development coated vesicles could also be a source of glycoproteins at the growing tips or at developing junctions by way of vesicle membrane fusion and hence they may play a direct ro!e in mechanisms underlying interneuronal recognition and synaptic specificity. ACKNOWLEDGEMENTS The authors gratefully acknowledge the expert technical assistance of Ms. Lou Boykins. Supported by USPHS R R 05423, USPHS 12590 and 5T01-GM 00202. J G W is an Alfred P. Sloan Research Fellow.
REFERENCES 1 Altman, J., Coated vesicles and synaptogenesis. A developmental study in the cerebellar cortex of the rat, Brain Research, 30 (1971) 311-322. 2 Barondes, S. H., Two sites of synthesis of macromolecules in neurons, In CellularDynamics of the Neuron, Syrup. Int. Soc. Cell Biol., 8 (1969) 351-364. 3 Barondes, S. H., Brain glycomacromoleculesand interneuronal recognition. In F. O. Schmitt (Ed.), The Neurosciences, Second Study Program, Rockefeller Univ. Press, New York, 1970, pp. 747-760. 4 Bernhard, W. and Avrameas, S., Ultrastructural visualization of cellular carbohydrate components by means of concanavalin A, Exp. Cell Res., 64 (1971) 232-236. 5 Bittiger, H. and Schnebli, H. P., Binding of concanavalin A and ricin to synaptic junctions of rat brain, Nature (Lond.), 249 (1974) 370-371. 6 Bloom, F. E. and Aghajanian, G. K., Fine structural and cytochemicalanalysis of the staining of synaptic junctions with phosphotungstic acid, J. Ultrastruct. Res., 22 (1968) 361-375. 7 Bondareff, W., An intercellular substance in rat cerebral cortex: submicroscopic distribution of ruthenium red, Anat. Rec., 157 (1967) 527-536. 8 Brunngraber, E. G., Dekirmenjian, H. and Brown, B. D., The distribution of protein-bound N-acetylneuraminic acid in subcellular fractions of rat brain, Biochem. J., 103 (1967) 73-78. 9 Bunge, M. B., Uptake of peroxidase by growth cones of cultured neurons, Anat. Rec., 175 (1973) 280 (abstract). 10 Ceccarini, C., Muramatsu, T., Tsang, J. and Atkinson, D. H., Growth-dependent alterations in oligomannosyl cores of glycopeptides, Proc. nat. Acad. Sci. (Wash.), 72 (1975) 3139-3143. 11 Cotman, C. W. and Taylor, C., Localization and characterization of concanavalin A receptors in the synaptic cleft, J. Cell Biol., 62 (1974) 236--242. 12 Del Cerro, M., Uptake of tracer proteins in the developingcerebellum, particularly by the growth cones and blood vessels, Jr. comp. Neurol., 157 (1974) 245-280. 13 Dutton, G. R. and Barondes, S. H., Glycoprotein metabolism in developing mouse brain, J. Neurochem., 17 (1970) 913-920. 14 Hamburger, V. and Hamilton, H. L., A series of normal stages in the development of the chick embryo, J. Morph., 88 (1951) 49-92.
70 15 Heuser, J, E. and Reese, T. S., Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction, J. Cell Biol., 57 (1973) 315-344. 16 Hudspeth, A. J. and Yee, A. G., The intercellular junctional complexes of retinal pigment epithelia, Invest. OphthaL, 12 (1973)354-365. 17 Karnovsky, M. J., Ultrastructural basis of capillary permeability studied with peroxidase as a tracer, J. Cell Biol., 35 (1967) 213-236. 18 Ladman, A. J., The fine structure of the rod-bipolar cell synapse in the retina of the albino rat, J. biophys, biochem. Cytol., 4 (1958) 459-465. 19 Lilien, J. E., Specific enhancement of cell aggregation in vitro, Develop. Biol., 17 (1968) 657-678. 20 Lilien, J. E. and Moscona, A. A., Cell aggregation: its enhancement by a supernatant from cultures of homologous cells, Science, 157 (1967) 70-72. 21 Marchisio, P. C., Gremo, F. and Sj6strand, J., Axonal transport in embryonic neurons. The possibility of a proximo-distal axolemmal transfer of glycoproteins, Brain Research, 85 (1975) 281-285. 22 Margolis, R. K. and Gomez, Z., Structural changes in brain glycoproteins during development, Brain Research, 74 (1974) 370-372. 23 Matus, A., DePetris, S. and Raft, M. C., Mobility of concanavalin A receptors in myelin and synaptic membranes, Nature (Lond.), 244 (1973) 278-279. 24 McLaughlin, B. J,, Photoreceptor synaptogenesis in the chick retina, Anat. Rec., 184 (1976) 476 (abstract). 25 Moscona, A. A., Surface specification of embryonic cells: lectin receptors, cell recognition, and specific cell ligands. In A. A. Moscona (Ed.), The Cell Surface in Development, Wiley, New York, 1974, pp. 67-100. 26 Nicolson, G. L., Interactions of lectins with animal cell surfaces, Int. Rev. Cytol., 39 (1974) 89-190. 27 Nicolson, G. L. and Singer, S. J., Ferritin-conjugated plant agglutinins as specific saccharide stains for electron microscopy: application to saccharides bound to cell membranes, Proc. nat. Acad. Sci. (Wash.), 68 (1971) 942-945. 28 Pappas, G. D. and Purpura, D. P., Distribution of colloidal particles in extracellular space and synaptic cleft substance of mammalian cerebral cortex, Nature (Lond.), 210 (1966) 1391-1392. 29 Peters, J. J., Vonderahe, A. R. and Powers, T. H., Electrical studies of functional development of the eye and optic lobes in the chick embryo, J. exp. ZooL, 139 (1958) 459-468. 30 Piddington, R. and Moscona, A. A., Correspondence between glutamine synthetase activity and differentiation in the embryonic retina in situ and in culture, J. Cell Biol., 27 (1965) 247-252. 31 Rambourg, A. and Leblond, C. P., Electron microscope observations on the carbohydrate-rich coat present at the surface of cells in the rat, J. Cell Biol., 32 (1969) 27-54. 32 Rees, R. P., Morphological changes occurring in the neuritic growth cone and target neuron from the time of initial contact to the completion of a synaptic profile, Anat. Rec., 181 (1975) 458 (abstract). 33 Rees, R. P. and Bunge, M. B., Origin of coated vesicles in neuronal perikarya - - a study of peroxidase uptake by cultured neurons with or without neurites, J. Cell Biol., 67 (1975) 357 (abstract). 34 Rees, R. P., Bunge, M. B. and Bunge, R. P., Morphological changes in the neuritic growth cone and target neuron during synaptic junction development in culture, J. Cell Biol., 68 (1976) 240-263. 35 Sedla~,ek, J., Development of optic evoked potentials in chick embryos, Physiol. bohemoslov., 16 (1967) 531-537. 36 Sharon, N. and Lis, H., Lectins: cell agglutinating and sugar-specific proteins, Science, 177 (1972) 949-959. 37 Sheffield, J. B. and Fischman, D. A., Intercellular junctions in the developing neural retina of the chick embryo, Z. Zellforsch., 104 (1970) 405-418. 38 Skoff, R. P. and Hamburger, V., Fine structure of dendritic and axonal growth cones in embryonic chick spinal cord, J. comp. NeuroL, 153 (1974) 107-148. 39 Stelzner, D. J., Martin, A. H. and Scott, G. L., Early stages of synaptogenesis in the cervical spinal cord of the chick embryo, Z. Zellforsch., 138 (1973) 475-488. 40 Van den Eijnden, D. H., Van Dijk, W. and Roukema, P. A., Sialoglycoprotein synthesis in developing rat brain, Neurobiology, 5 (1975) 221-227. 41 Vaughn, J. E., Henrickson, C. K. and Wood, J. G., Surface specializations of neurites in embryonic mouse spinal cord, Brain Research, 110 (1976) 431-445. 42 Venable, J. H. and Coggeshall, R., A simplified lead citrate stain for use in electron microscopy, J. Cell Biol., 25 (1965) 407-408.
71 43 Witkovsky, P., An ontogenetic study of retinal function in the chick, Vision Res., 3 (1963) 341-355. 44 Wood, J. G. and McLaughlin, B. J., Some cytochemical properties of lectin binding sites in rat brain, J. Cell Biol., 67 (1975) 461 (abstract). 45 Wood, J. G., McLaughlin, B. J. and Barber, R. P., The visualization of concanavalin A binding sites in Purkinje cell somata and dendrites of rat cerebellum, J. Cell Biol., 63 (1974) 541-549.
57
© Elsevier/North-HollandBiomedicalPress, Amsterdam- Printed in The Netherlands
THE LOCALIZATION OF CONCANAVALIN A BINDING SITES DURING PHOTORECEPTOR SYNAPTOGENESIS IN THE CHICK RETINA
BARBARA J. McLAUGHLIN* and JOHN G. WOOD Department of Anatomy, University of Tennessee, Center for the Health Sciences, Memphis, Tenn. 38163 (U.S.A.)
(Accepted April 27th, 1976)
SUMMARY Carbohydrate containing macromolecules were localized on developing photoreceptor pedicles and processes in the outer plexiform layer (OPL) of the retina in chick embryos and hatchlings by a concanavalin A (Con A)-peroxidase labeling technique. Con A binding sites were visualized along the membrane surfaces of undifferentiated photoreceptor synaptic bases at early embryonic times and later were localized along differentiating synaptic pedicles and at pre- and postsynaptic membranes of early forming ribbon synapses. Membrane surfaces of developing neurites and glia in this layer were labeled also as were the internal membrane surfaces of coated vesicles within developing receptor terminals and neurites. In the hatchling, Con A binding sites were restricted to non-synaptic membrane regions of receptor pedicles and some neuronal and glial membranes. Con A staining in the inner plexiform layer was similar to that in the OPL with membrane labeling on developing synapses, neurites and glia during embryonic development and labeling restricted to nonsynaptic membrane regions in the hatchling. Con A staining in the other layers of the retina was localized on endoplasmic reticulum, nuclear and plasma membranes of all cell types. Cell junctional complexes between pigmented eipthelial cells and the zonulae occludentes of the outer limiting membrane did not label with Con A at the developmental ages sampled in this study. These findings suggest that Con A binding sites are accessible on developing synaptic membranes but become either altered or masked after synaptic junction maturation.
INTRODUCTION An important area in developmental neurobiology is the understanding of under* To whomcorrespondenceshould be addressed.
58 lying mechanisms which account for the specificity of neuronal connections. Because nerve endings are rich in glycoproteins8 and glycoprotein synthesis is greater during neuronal differentiation2,13, it has been speculated that polysaccharides on neuronal surfaces play a major role in interneuronal recognitionL Further support for this suggestion has come from several studies showing that the sialoglycoprotein level increases two-fold during the period of neuronal development4°, and that the size of oligosaccharide chains of brain glycoproteins rapidly increases during development2% Another more recent study has shown that the rapid accumulation of transported glycoproteins is significantly higher when synapses are being formed than after synaptic maturation zl. While it has been known for some time that synaptic junctions contain polysaccharides7,zs,31 as well as protein 6, more specific information about the actual sugar residues present and their location has come from the use of ferritin or peroxidaselabeled lectins 4,27, which localize specific sugar residues in the polysaccharide chain (cf. refs. 26 and 36). Two of these lectins, concanavalin A (Con A), which binds to glycosyl or mannosyl residues, and ricin, which binds to galactosyl residues, have been shown to bind to the synaptic cleft region and the external surface of the postsynaptic membrane of synaptosome preparations 5,11,23. In contrast to these observations of lectin binding on isolated synaptic junctions, we have found that Con A binding sites at intact synaptic junctions are accessible to the label only after removal of the surrounding polypeptides with trypsin digestion44. These observations are of interest in regard to Moscona's (see ref. 25 for refs.) findings on dissociated embryonic neural retina cells, showing that EGTA dispersed cells from young embryos agglutinated readily with Con A, while cells from older embryos did not, except after trypsin dissociation. If the Con A receptors on the surface of immature neurons are at first accessible and become masked or inaccessible later in development, then it is possible that Con A binding sites on the immature synaptic membrane (which is an extension of the neuronal cell surface) are also exposed in a similar topographical fashion during synaptic junction formation. The present study explores this possibility by following the localization of Con A binding sites at developing synaptic junctions in the outer plexiform layer of the chick retina. Evidence will be presented that the distribution of Con A labeling along the photoreceptor synaptic pedicles changes during embryonic development and after functional differentiation of the photoreceptors. These findings will be discussed in regard to current concepts about the role of cell surface carbohydrates in cell recognition and the possible implications of this role in mechanisms of synaptic specificity. MATERIALS AND METHODS White Leghorn chick embryos (obtained from a local hatchery) were kept in a forced draft incubator at 37 °C and staged according to Hamburger and Hamilton14 prior to fixation. Embryos between the ages of 13 and 21 days and 2-7-day-old hatchlings were used in this study. The animals were perfused initially through the heart and then through the eye with a fixative containing 2 ~ glutaraldehyde (70 ~o
59 Ladd)-2 ~ paraformaldehyde--0.5 ~o acrolein-0.5 ~o DMSO in 0.08 M phosphate or cacodylate buffer with 0.001 ~ CaCI2 added 3s. The lower temporal retinal quadrant was dissected free and stored in the same fixative overnight at 4 °C. The central portion of the quadrant a° was sectioned into 50 #m slices with a Sorvall TC-2 tissue sectioner and the slices were collected in 0.08 M phosphate buffered saline (PBS). The best retinal slices were selected under the dissecting microscope for cytochemical staining. The method of Con A-peroxidase labeling of carbohydrates used in this study was essentially as described by Bernhard and Avrameas4. All steps were performed with constant agitation at room temperature. Concanavalin A (Calbiochem., Grade A, San Diego, Calif.), horseradish peroxidase (Sigma Chemical Co., Type VI, St. Louis, Mo.), glucose oxidase (Sigma Type II, a-methyl-D-mannoside, Sigma No. M-3752), and 3,Y-diaminobenzidine (Sigma No. D-8126) were all used in the cytochemical procedures. The retinal slices were washed in PBS for 15 rain and then incubated in 0.5-1 mg/ml of Con A in PBS for 40 min. They were washed in 3-5 changes of PBS for 3 h and incubated in 0.1 mg/ml horseradish peroxidase for 40 min followed by another 3 h washing in PBS. The slices were treated with either 20 #g glucose oxidase, 90 mg glucose and 50 mg of diaminobenzidine (DAB) in 100 ml PBS for 5-15 min or with 2/~1 of 30 ~ H202, and 30 mg DAB in 50 ml PBS for 5-15 min. The slices were washed another 30 min in PBS and were hand cut into smaller pieces and osmicated (2 OsO4), dehydrated and embedded in Epon-Araldite for electron microscopy. Some tissue was block stained in 2 ~ uranyl acetate prior to dehydration17. Control slices were incubated in Con A (0.5-1 mg/ml) in the presence of 0.2 M a-methyl-D-mannoside, an inhibitor of specific Con A binding to sugars36, and the tissue processed as described. Thin sections were cut on a diamond knife and stained with either uranyl acetate and lead citrate 4z or lead citrate only, prior to examination in a Philips EM 301 electron microscope. Some sections were left unstained prior to examination. RESULTS
Con .4 labeling in outer plexiform layer (OPL) The photoreceptor synaptic bases begin to differentiate between 13 and 15 embryonic days2a and filopodial-like protrusions of the receptor cell surface protrude into the OPL at this time (Figs. 1-3). Con A-peroxidase labeling is present on the external membrane surfaces of most of the neuronal and glial profiles in the OPL and along the plasma membrane of the receptor synaptic bases and their filopodial protrusions as well as on adjacent dendritic membranes. The label is particularly concentrated at regions along the receptor membrane and two presumed postsynaptic neurites which are aligned in an apparent dyad-like arrangement on either side of a filopodial protrusion (Figs. 2, 3). These regions, which are characterized by the presence of a filamentous-like undercoating on the cytoplasmic side of the receptor membrane, are thought to represent the first morphogenic stages of photoreceptor synaptogenesis in the chick retina24. Coated vesicles (Figs. 1, 5, 6) are seen throughout
Fig. 1. Con A labeling on nuclear membrane and undifferentiated synaptic base membrane of a photoreceptor cell (R). Several labeled filopodial protrusions (arrows) of the receptor protrude between neurites(*) aligned along the base in dyad-like apposition. Con A staining is also present on membrane surfaces of glial and neuronal profiles in the outer plexiform layer (OPL) and several coated vesicles (arrowheads) are labeled with Con A. 14 embryonic days. Uranyl acetate (UA) and lead (Pb) stained. , 47,500. Fig. 2. Higher magnification of a receptor protrusion in Fig. 1. Con A labeling is particularly concentrated in regions (*) where two neurites are aligned in dyad-like apposition with the receptor membrane. An undercoating is present (arrows) on the cytoplasmic face of the receptor membrane in these regions. 14 embryonic days. UA and Pb stained. >," 90,000. Fig. 3. Another receptor protrusion from Fig. 1 which protrudes between two neurites (*) in dyad-like apposition. Con A labeling appears to be concentrated between the receptor membrane and one of the neurites and an undercoating is present on the receptor membrane in this region (arrows). 14 embryonic days. U A and Pb stained. +i 90,000.
61
Fig. 4. Photoreceptor cells (R) whose synaptic bases have elongated into the outer plexiform layer (OPL) and differentiated into synaptic pedicles (ped). Con A binding sites are localized along the membrane surfaces of each receptor pedicle in both non-synaptic and synaptic (arrows) regions and on the membrane surfaces of profiles in the OPL. The nuclear and plasma membranes of the receptor cells are labeled also. 18 embryonic days. UA and Pb stained. × 25,300. embryonic development in developing neurites and receptor filopodia and are labeled with Con A-peroxidase along their inner vesicular membrane surfaces. In many cases, the labeled coated vesicle and the Con A-labeled plasma membrane of the neurite or receptor cell are joined together (Fig. 6). Between 15 and 21 days, the receptor synaptic bases elongate and expand into conical-shaped pedicles and the protrusions differentiate more fully to form character-
62
Fig. 5. Con A labeling on membrane surfaces of a receptor pedicle (ped) and on profiles in the OPL. The label is particularly concentrated in regions of dyad apposition (*) between the pedicle and potential postsynaptic neurites and at a developing ribbon synapse (arrow) in which a synaptic ribbon (sr) is located nearby. Labeled coated vesicles (arrowheads) are present in the pedicle cytoplasm. 16 embryonic days. UA and Pb stained. × 55,000.
istic ribbon synaptic terminals, in which a synaptic ribbon and its associated vesicles and trough-like density (arciform density of Ladman 18) form a synaptic ridge between two or three apposed neurites in dyad or triad relationship (Fig. 4). The Con A label continues to be present on the external membrane surfaces of the synaptic pedicle and along the synaptic ridges at ribbon synapses as well as on adjacent dendritic membranes (Figs. 4-6). Labeled coated vesicles are observed within the synaptic pedicle (Fig. 5). The numerous synaptic vesicles in the vicinity of coated vesicles are not labeled. Labeling on neuronal and glial plasma membranes in the OPL remains the same as described for earlier embryonic periods, and labeled profiles of coated vesicles are seen in the developing neurites (Fig. 6). In the 2-day hatchling, the expanded receptor pedicles are no longer observed in the same plane of section with their cell bodies, but appear instead as isolated synaptic bodies in the OPL beneath the layer o f receptor cell bodies (Figs. 7, 8). The dyad and
63 triad postsynaptic processes are deeply invaginated within the vitreal borders of the synaptic pedicles. Con A labeling of the OPL is restricted to plasma membrane surfaces of neurites (Figs. 7, 8) surrounding the synaptic pedicles and along the outer or non-synaptic border of the pedicle membrane (Fig. 8). Some labeling is observed occasionally on neuronal profiles invaginated within the synaptic pedicle which are not in any apparent synaptic apposition. Coated vesicles are less numerous in neuronal processes and receptor pedicles at this time, but are labeled along their inner membrane surfaces as described earlier.
Fig. 6. Con A staining of pedicle (ped) membrane in both non-synaptic and at synaptic (arrows) regions in which synaptic ribbons (sr) are present. Staining is present also on profiles in the OPL and within a coated vesicle (arrowhead) which is joined to the labeled plasma membrane. 18 embryonic days. U A and Pb stained, x 68,000.
64
Con A labeling in other retinal layers In addition to plasma membrane labeling, Con A-peroxidase labels the rough and smooth endoplasmic reticulum, the Golgi apparatus and nuclear membranes of all cell types in the retina and pigmented epithelium throughout embryonic development and after hatching (Figs. 9, 10). The zonulae occludentes 37, which form the outer limiting membrane (OLM) of the retina (Fig. 10), are never labeled with Con A at any developmental time, even though there is plasma membrane labeling on the apposed cells above and below the junctional regions. Con A labeling in the inner plexiform layer (IPL) is similar to that in the OPL. During embryonic development the plasma membrane surfaces of developing neurites and glial processes are labeled as are the immature synaptic junction membranes (Fig. 11). After hatching the Con A labeling is restricted to membrane surfaces of profiles not in any apparent synaptic apposition (Fig. 12). No Con A labeling is observed in the control retinas at any developmental time.
Fig. 7. Two mature synaptic pedicles (ped) below photoreceptor cell bodies (R). Con A binding sites are localized on membrane surfaces of someprofiles in the OPL not in synaptic apposition with the pedicles and on the receptor nuclear membranes. 2-day hatchling. UA and Pb stained. × 22,750.
65
Fig. 8. Mature synaptic pedicle (ped) in the OPL in which Con A labeling is localized only on nonsynaptic regions of the pedicle and on membrane surfaces of profiles surrounding the pedicle. 18 embryonic days. UA and Pb stained. × 62,500.
DISCUSSION Studies have shown that the chick eye and contralateral optic lobe respond to photic stimulation by the eighteenth embryonic day 29 but that the synapses in the visual system reach physiological maturity during postnatal life35, 48. The ability of developing photoreceptor synaptic membranes in the embryonic retina to label with Con A and their failure to label after synaptic maturation in the hatchling suggests that lectin binding sites are accessible on immature synaptic membranes but become inaccessible or masked after junctional maturity. While we cannot exclude the possi-
66 bility that failure to label the mature synapse may be due to restricted penetration ot reagents, these developmental changes in lectin labeling may reflect changes in the maturation of cell surface glycoproteins during synaptogenesis. The accessibility of Con A binding sites on immature synaptic membranes may indicate that mannose or glucose residues are relatively exposed or terminal in the oligosaccharide chain, whereas their inaccessibility to labeling after synaptic membrane maturation may indicate that other sugars have been added to the chain thus masking the mannose or glucose residues from the label. Alternatively, these sugars which are present on the presynaptic membrane may become inaccessible to the label by virtue of their being bound to receptors on the postsynaptic membrane, or vice versa. Recent findings 2z in developing brain suggest that glycoproteins undergo a rapid increase in the size of
Fig. 9. Con A-labeled bipolar cells (Bi) in which nuclear membranes, Golgi apparatus and endoplasmic reticulum membranes are stained. Very little labeling is present on plasma membranes. 21 embryonic days. UA and Pb stained. × 17,100.
67 their oligosaccharide chains which may be attributable to the addition of terminal sialic acid residues (see also refs. 10 and 40). Cell surface macromolecules have been implicated in a variety of cellular interactions during morphogenesis and it has been suggested that protein--carbohydrate complexes or 'cell ligands' function as tissue-specific recognition sites on reaggregating cell surfaces 19,2°,25. One of the major carbohydrates in the tissue-specific aggregation factors from embryonic chick neural retina cells is mannose 25 and it is tempting to speculate whether the mannosyl residues which are accessible to Con A during synaptogenesis are also involved in mechanisms underlying interneuronal recognition between potential synaptic membranes in the chick retina. In the OPL, Con A binding sites on immature receptor synaptic bases appear to be concentrated in the regions of
Fig, 10. Photoreceptor cells (R) with Con A binding sites localized on nuclear and plasma membranes of the receptors and their inner segments (IS) and the Mtiller cells (Mu). NO labeling is present in the i'egi0n of zonulae occludentes (arrowheads). 18 embryonic days. UA and Pb stained, x 27,300.
68
i Fig. I1. Con A labeling on developing synaptic membranes (arrows) in the inner plexiform layer (IPL). Label is present also on other non-synaptic membrane surfaces. 14 embryonic days. UA and Pb stained. × 102,000. Fig. 12. Con A labeling on non-synaptic membranes of profiles in the IPL but absence of label at synaptic junction. 18 embryonic days. UA and Pb stained. × 68,000.
membrane alignment between potential dyad synaptic profiles and the pedicle. Such regions of membrane alignment are thought to represent the first stages of synaptic junction differentiation between these components 24 and it would be interesting to know if the exposed Con A receptors play a role in mediating such an initial interaction or specific recognition between these membranes. The fact that other nonsynaptic membrane surfaces in the OPL label also with Con A during the synaptogenic period suggests that there may be other factors besides the mere presence of certain sugars involved in these mechanisms, such as a critical number of Con A binding sites concentrated at synaptic junctional regions as apposed to non-junctional membrane regions as Vaughn et al. 41 have suggested. It is interesting in this regard that the nonsynaptic cell junctions between pigmented epithelial cells and the zonulae occludentes of the O L M do not label with Con A even though p l a s m a membranes adjacent to the junctions are clearly labeled. These junctions are alreadydifferentiated by the earliest embryonic age sampled in this study, which suggests t h a t m a t u r e cell junctions either do not contain Con A receptors or that, like mature synaptic junctions, the binding sites are inaccessible or masked after junction formation. Coated vesicles have been observed to occur in great numbers in developing neurites during synaptogenesis 1,34,39 and it has been suggested that they contribute to the formation of synaptic densities by fusing with the postsynaptic membrane and allowing the coat material to become part of the postsynaptic density 1,3z,a4,a9. It is of
69 interest that the coated vesicles observed in the developing OPL are labeled with Con A at the same time developing synaptic membranes are labeled. This suggests that coated vesicle and immature synaptic membranes share similar cytochemical components during the synaptogenic period which may be related to the formation of synaptic densities or some other aspect of junction formation and neurite growth, such as transport of glycoproteins to the junctional regions and growing ends of neurites 21. Coated vesicles label with Con A throughout development and in the hatchling and these organelles have been implicated in uptake of exogenous protein by growing neurites in the developing cerebellum 12 and in cultureg, 33. During development coated vesicles could also be a source of glycoproteins at the growing tips or at developing junctions by way of vesicle membrane fusion and hence they may play a direct ro!e in mechanisms underlying interneuronal recognition and synaptic specificity. ACKNOWLEDGEMENTS The authors gratefully acknowledge the expert technical assistance of Ms. Lou Boykins. Supported by USPHS R R 05423, USPHS 12590 and 5T01-GM 00202. J G W is an Alfred P. Sloan Research Fellow.
REFERENCES 1 Altman, J., Coated vesicles and synaptogenesis. A developmental study in the cerebellar cortex of the rat, Brain Research, 30 (1971) 311-322. 2 Barondes, S. H., Two sites of synthesis of macromolecules in neurons, In CellularDynamics of the Neuron, Syrup. Int. Soc. Cell Biol., 8 (1969) 351-364. 3 Barondes, S. H., Brain glycomacromoleculesand interneuronal recognition. In F. O. Schmitt (Ed.), The Neurosciences, Second Study Program, Rockefeller Univ. Press, New York, 1970, pp. 747-760. 4 Bernhard, W. and Avrameas, S., Ultrastructural visualization of cellular carbohydrate components by means of concanavalin A, Exp. Cell Res., 64 (1971) 232-236. 5 Bittiger, H. and Schnebli, H. P., Binding of concanavalin A and ricin to synaptic junctions of rat brain, Nature (Lond.), 249 (1974) 370-371. 6 Bloom, F. E. and Aghajanian, G. K., Fine structural and cytochemicalanalysis of the staining of synaptic junctions with phosphotungstic acid, J. Ultrastruct. Res., 22 (1968) 361-375. 7 Bondareff, W., An intercellular substance in rat cerebral cortex: submicroscopic distribution of ruthenium red, Anat. Rec., 157 (1967) 527-536. 8 Brunngraber, E. G., Dekirmenjian, H. and Brown, B. D., The distribution of protein-bound N-acetylneuraminic acid in subcellular fractions of rat brain, Biochem. J., 103 (1967) 73-78. 9 Bunge, M. B., Uptake of peroxidase by growth cones of cultured neurons, Anat. Rec., 175 (1973) 280 (abstract). 10 Ceccarini, C., Muramatsu, T., Tsang, J. and Atkinson, D. H., Growth-dependent alterations in oligomannosyl cores of glycopeptides, Proc. nat. Acad. Sci. (Wash.), 72 (1975) 3139-3143. 11 Cotman, C. W. and Taylor, C., Localization and characterization of concanavalin A receptors in the synaptic cleft, J. Cell Biol., 62 (1974) 236--242. 12 Del Cerro, M., Uptake of tracer proteins in the developingcerebellum, particularly by the growth cones and blood vessels, Jr. comp. Neurol., 157 (1974) 245-280. 13 Dutton, G. R. and Barondes, S. H., Glycoprotein metabolism in developing mouse brain, J. Neurochem., 17 (1970) 913-920. 14 Hamburger, V. and Hamilton, H. L., A series of normal stages in the development of the chick embryo, J. Morph., 88 (1951) 49-92.
70 15 Heuser, J, E. and Reese, T. S., Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction, J. Cell Biol., 57 (1973) 315-344. 16 Hudspeth, A. J. and Yee, A. G., The intercellular junctional complexes of retinal pigment epithelia, Invest. OphthaL, 12 (1973)354-365. 17 Karnovsky, M. J., Ultrastructural basis of capillary permeability studied with peroxidase as a tracer, J. Cell Biol., 35 (1967) 213-236. 18 Ladman, A. J., The fine structure of the rod-bipolar cell synapse in the retina of the albino rat, J. biophys, biochem. Cytol., 4 (1958) 459-465. 19 Lilien, J. E., Specific enhancement of cell aggregation in vitro, Develop. Biol., 17 (1968) 657-678. 20 Lilien, J. E. and Moscona, A. A., Cell aggregation: its enhancement by a supernatant from cultures of homologous cells, Science, 157 (1967) 70-72. 21 Marchisio, P. C., Gremo, F. and Sj6strand, J., Axonal transport in embryonic neurons. The possibility of a proximo-distal axolemmal transfer of glycoproteins, Brain Research, 85 (1975) 281-285. 22 Margolis, R. K. and Gomez, Z., Structural changes in brain glycoproteins during development, Brain Research, 74 (1974) 370-372. 23 Matus, A., DePetris, S. and Raft, M. C., Mobility of concanavalin A receptors in myelin and synaptic membranes, Nature (Lond.), 244 (1973) 278-279. 24 McLaughlin, B. J,, Photoreceptor synaptogenesis in the chick retina, Anat. Rec., 184 (1976) 476 (abstract). 25 Moscona, A. A., Surface specification of embryonic cells: lectin receptors, cell recognition, and specific cell ligands. In A. A. Moscona (Ed.), The Cell Surface in Development, Wiley, New York, 1974, pp. 67-100. 26 Nicolson, G. L., Interactions of lectins with animal cell surfaces, Int. Rev. Cytol., 39 (1974) 89-190. 27 Nicolson, G. L. and Singer, S. J., Ferritin-conjugated plant agglutinins as specific saccharide stains for electron microscopy: application to saccharides bound to cell membranes, Proc. nat. Acad. Sci. (Wash.), 68 (1971) 942-945. 28 Pappas, G. D. and Purpura, D. P., Distribution of colloidal particles in extracellular space and synaptic cleft substance of mammalian cerebral cortex, Nature (Lond.), 210 (1966) 1391-1392. 29 Peters, J. J., Vonderahe, A. R. and Powers, T. H., Electrical studies of functional development of the eye and optic lobes in the chick embryo, J. exp. ZooL, 139 (1958) 459-468. 30 Piddington, R. and Moscona, A. A., Correspondence between glutamine synthetase activity and differentiation in the embryonic retina in situ and in culture, J. Cell Biol., 27 (1965) 247-252. 31 Rambourg, A. and Leblond, C. P., Electron microscope observations on the carbohydrate-rich coat present at the surface of cells in the rat, J. Cell Biol., 32 (1969) 27-54. 32 Rees, R. P., Morphological changes occurring in the neuritic growth cone and target neuron from the time of initial contact to the completion of a synaptic profile, Anat. Rec., 181 (1975) 458 (abstract). 33 Rees, R. P. and Bunge, M. B., Origin of coated vesicles in neuronal perikarya - - a study of peroxidase uptake by cultured neurons with or without neurites, J. Cell Biol., 67 (1975) 357 (abstract). 34 Rees, R. P., Bunge, M. B. and Bunge, R. P., Morphological changes in the neuritic growth cone and target neuron during synaptic junction development in culture, J. Cell Biol., 68 (1976) 240-263. 35 Sedla~,ek, J., Development of optic evoked potentials in chick embryos, Physiol. bohemoslov., 16 (1967) 531-537. 36 Sharon, N. and Lis, H., Lectins: cell agglutinating and sugar-specific proteins, Science, 177 (1972) 949-959. 37 Sheffield, J. B. and Fischman, D. A., Intercellular junctions in the developing neural retina of the chick embryo, Z. Zellforsch., 104 (1970) 405-418. 38 Skoff, R. P. and Hamburger, V., Fine structure of dendritic and axonal growth cones in embryonic chick spinal cord, J. comp. NeuroL, 153 (1974) 107-148. 39 Stelzner, D. J., Martin, A. H. and Scott, G. L., Early stages of synaptogenesis in the cervical spinal cord of the chick embryo, Z. Zellforsch., 138 (1973) 475-488. 40 Van den Eijnden, D. H., Van Dijk, W. and Roukema, P. A., Sialoglycoprotein synthesis in developing rat brain, Neurobiology, 5 (1975) 221-227. 41 Vaughn, J. E., Henrickson, C. K. and Wood, J. G., Surface specializations of neurites in embryonic mouse spinal cord, Brain Research, 110 (1976) 431-445. 42 Venable, J. H. and Coggeshall, R., A simplified lead citrate stain for use in electron microscopy, J. Cell Biol., 25 (1965) 407-408.
71 43 Witkovsky, P., An ontogenetic study of retinal function in the chick, Vision Res., 3 (1963) 341-355. 44 Wood, J. G. and McLaughlin, B. J., Some cytochemical properties of lectin binding sites in rat brain, J. Cell Biol., 67 (1975) 461 (abstract). 45 Wood, J. G., McLaughlin, B. J. and Barber, R. P., The visualization of concanavalin A binding sites in Purkinje cell somata and dendrites of rat cerebellum, J. Cell Biol., 63 (1974) 541-549.
