Intracellular regulators of neuronal sprouting: calmodulin-binding proteins of nerve growth cones.

The focus of this study is a quantitative biochemical analysis of the calcium-dependent interactions of calmodulin with a nerve growth cone preparation from fetal rat brain (Pfenninger, K. H., L. Ellis, M. P. Johnson, L. B. Freidman, and S. Somlo, 1983, Cell 35:573-584). The presence of calmodulin as an integral component of this preparation is demonstrated, and quantitative binding studies are presented. The binding of 125I-calmodulin to nerve growth cone material is shown to be highly specific, calcium dependent, and saturable at nanomolar calmodulin concentrations. Additionally, the growth cones' binding components appear to be membrane proteins. The individual molecular mass species of growth cone proteins displaying calcium-dependent calmodulin binding are also detailed and presented in comparison with those of synaptosomes. This analysis reveals differences between the calmodulin binding proteins of the growth cone preparation and the synaptosome fraction, suggesting the presence in growth cones of a specialized set of components which may be involved in regulatory mechanisms controlling neuritic sprouting.

Membrane glycoproteins of the nerve growth cone: diversity and growth regulation of oligosaccharides.

A subcellular fraction prepared from fetal rat brain and enriched in growth cone membranes is analyzed for its lectin-binding proteins. Growth-associated glycoproteins are identified by comparing the growth cone glycoproteins with those of synaptosomes. Protein was resolved in one- or two-dimensional gels, electroblotted, and blots probed with radioiodinated concanavalin A, wheat germ agglutinin, and Ricinus communis agglutinins I and II. In one-dimensional gels, each lectin recognizes approximately 20 polypeptides (with substantial overlap) most of which migrate diffusely and have relatively high molecular masses (range 30-200 kD). The seven major Coomassie-staining proteins of the membrane fraction (34-52 kD) are not the major lectin-binding proteins. In two-dimensional gels, the lectin-binding proteins are either streaked across the pH gradient or exist as multiple spots, indicating broad charge heterogeneity. Seven wheat germ agglutinin- and Ricinus communis agglutinin II-binding glycoproteins are present in greater abundance in growth cone fractions compared with synaptosomes. Most notably, an acidic, sialic acid-rich protein (27-30 kD, pI 4.0; termed gp27-30) is most abundant at postnatal day 4, but absent from adult brain. The protein's very acidic isoelectric point is due, at least in part, to its high sialic acid content. Growth regulation of specific protein-linked oligosaccharides suggests that they play a special role in growth cone function. In addition, the great diversity of growth cone glycoproteins from whole brain suggests glycoprotein heterogeneity among growth cones from different neuron types.

Membrane assembly in retinal photoreceptors I. Freeze-fracture analysis of cytoplasmic vesicles in relationship to disc assembly.

To study precursor-product relationships between cytoplasmic membranes of the inner segment of photoreceptors and the continually renewed outer disc membrane, we have compared the density and size distribution of intramembrane particles (IMP) in various membrane compartments of freeze-fractured photoreceptor inner and outer segments. Both rod and cone outer segments of Xenopus laevis are characterized by a relatively uniform distribution of approximately 4,400-4,700 IMP/micron2 in P-face (PF) leaflets of disc membranes. A similar distribution of IMP is found in the outer segment plasma membrane, the ciliary plasma membrane, and in the plasma membrane of the inner segment in the immediate periciliary region. In each case the size distribution of IMP can be characterized as unimodal with a mean diameter of approximately 10 nm. PF leaflets of endoplasmic reticulum, Golgi complex, and vesicles near the cilium have IMP with a size distribution like that in the cilium and outer segment, but with an average density of approximately 2,000/micron2. In contrast, IMP are smaller in average size (approximately 7.5 nm) in PF leaflets of inner segment plasma membrane, exclusive of the periciliary rgion. The similarity of size distribution of IMP in inner segment cytoplasmic membranes and those within the plasmalemma of the cilium and outer segment suggest a precursor-product relationship between the two systems. The structure of the vesicle-rich periciliary region and the segregation of IMP with different size distributions in this region suggest that components destined for incorporation into the outer segment exist as preformed membrane packages (vesicles) which fuse with the inner segment plasma membrane in the periciliary region. Subsequently, membrane components may be transferred to forming discs of the outer segment via the ciliary plasma membrane.

Methods for the freeze-fracturing of nerve tissue cultures and cell monolayers.

Two methods for freeze-cleaving of thin tissue layers are presented. Whereas a simple technique can be employed to fracture continuous, relatively firm tissue layers, a more sophisticated technique employing special carriers is needed to fracture very thin and incomplete layers, e.g., the fiber outgrowth of cultured nerve tissue or sparsely seeded isolated cells. Both methods basically consist of freezing the specimens sandwiched between two small metal carriers which are then fractured apart so that the cleavage plane runs through the tissue. In the resulting replicas of such thin specimens, large membrane areas are exposed, and new information is provided on the topography of membrane properties in entire cells or cell processes. The technique should also be useful for studies on the interactions of cells grown in culture.

Freeze-fracturing of nerve growth cones and young fibers. A study of developing plasma membrane.

Neural and non-neural cellular processes have been studied in organotypic cultures of spinal cord and olfactory bulb by means of the freeze-fracturing technique. Identification of specific cellular elements in replicas has been achieved by comparison with thin-sectioned material in which differences in shape and contents are evident. Freeze-fracturing reveals that neural growth cones may be distinguished from glial pseudopodia by the low number of intramembranous particles within their plasma membrane; the counts of particles within the growth cone membrane average 85/microm(2) (for the inner leaflet) as opposed to hundreds per square micrometer in glial pseudopodia. Whereas the intramembranous particle number in glial pseudopodia is only slightly lower than in their perikaryal plasmalemma, the number of particles in outgrowing axons increases about eightfold from the periphery towards the perikaryon. Furthermore, with prolonged time of growth in culture, the particle density in the young nerve fibers increases by about the same factor. The same phenomenon, i.e. a low intramembranous particle level at earlier stages and an increase in numbers as the nerve fiber matures, is observed in fetal nerve tissue in vivo. These findings suggest that the plasmalemma of the outgrowing nerve, and especially of the growth cone, is immature and that maturation is accompanied by the insertion of intramembranous particles. Furthermore, these data indicate that the chemistry of the growth cone membrane is distinct from that of the neuron soma which may be significant for the mechanisms of guidance and recognition in the growing nerve tip.

Components of the plasma membrane of growing axons. I. Size and distribution of intramembrane particles.

The plasmalemma of mature and growing olfactory axons of the bullfrog has been studied by freeze-fracture. Intramembrane particles (IMPs) of mature olfactory axons are found to be uniformly distributed along the shaft. However, during growth, a decreasing gradient of IMP density is evident along the somatofugal axis. The size histograms of axolemmal IMPs from different segments of growing nerve reveal regional differences in the particle composition. The distribution of each individual size class of particles along the growing nerve forms a decreasing gradient in the somatofugal direction; the slope of these gradients varies directly with particle diameter. These size-dependent density gradients are consistent with a process of lateral diffusion of membrane components that are inserted proximally into the plasma membrane. The membrane composition of the growth cone, however, appears to be independent of these diffusion gradients; it displays a mosaic pattern of discrete domains of high and low particle densities. The relative IMP profiles of these growth cone regions are similar to one another but contain higher densities of large IMPs than the neighboring axonal shaft. The shifting distributions of intramembrane particles that characterize the sprouting neuron give new insights into cellular processes that may underlie the establishment of the functional polarity of the neuron and into the dynamics of axolemmal maturation.

Membrane biogenesis in the sprouting neuron. I. Selective transfer of newly synthesized phospholipid into the growing neurite.

Our goal was to elucidate the pathway of newly synthesized phospholipid into the growing neurite. This was accomplished in pulse-chase studies with the phospholipid precursor [3H]glycerol, using sprouting explant cultures of rat superior cervical ganglion as an experimental system. After the pulse with the precursor and various chase periods, we separated perikarya and neurites microsurgically and extracted their phospholipids. The phospholipid extract from the perikarya exhibited a steep rise followed by a rapid decline in specific radioactivity. In contrast, an increase in neuritic specific radioactivity of phospholipid was observed only after a lag period of approximately 60 min. Nearly quantitative transfer of newly synthesized phospholipid from the perikarya into the neurites could be demonstrated. Both the decline in perikaryal specific radioactivity and the increase in its neuritic counterpart, i.e., the proximodistal transfer, could be blocked with the microtubule drug colchicine and the metabolic uncoupler, 2,4-dinitrophenol. These observations indicate preferential export of newly synthesized phospholipid from the perikaryon (the major or exclusive site of synthesis) into the growing neurites, most likely by rapid axoplasmic transport of formed elements.

Lectin labeling of sprouting neurons. I. Regional distribution of surface glycoconjugates.

Well-defined ferritin-conjugated lectins were used to map glycoconjugates on the surface of sprouting neurons from rat superior cervical ganglion (SCG) and spinal cord (SC). The cultured neurons were exposed to the markers and processed for electron microscopy, and the number of ferritin particles per unit area of plasmalemma was measured in three different regions: perikaryon, neuritic shaft, and growth cone. Three different binding patterns are observed for different lectin: equal receptor density throughout the plasmalemma of the growing neuron (e.g., Ricinus communis agglutinin I in SCG neurons), gradual decrease (e.g., wheat-germ agglutinin in SCG and SC neurons) and gradual increase (e.g., Ricinus communis agglutinin II in SC neurons) in the density of lectin receptors as one moves from the perikaryon to the growth cone. Furthermore, lectin receptor densities differ in the two types of neurons analyzed. We can conclude that the plasmalemma of the growth cone has biochemical properties different from those of the perikaryon, and that the neuron's structural polarity is expressed in its surface glycoconjugates. This phenomenon may be related to the growth cone's special functional properties and to the process of expansion of the plasma membrane.

Lectin labeling of sprouting neurons. II. Relative movement and appearance of glycoconjugates during plasmalemmal expansion.

To study the dynamics of membrane components during neuritic growth, we carried out a series of pulse-chase experiments with ferritin-conjugated and unconjugated lectins on sympathetic neurons sprouting in vitro. Labeling of aldehyde-prefixed cultures with wheat-germ agglutinin or with the galactose-specific lectin of Ricinus communis is consistently dense near the distal end of the neurites. By contrast, if live cultures are labeled with these lectins and chased for 3-20 min, label-free plasmalemmal areas appear in the most peripheral regions of the growth cone, on filopodia and, furthermore, over vesicle clusters (SPVs). These marker-free areas, however, contain lectin receptors, as can be shown by relabeling the chased cultures with the same lectins after the aldehyde fixation. In a further set of experiments, cultures are labeled with a saturating concentration of native lectin, chased, aldehyde-fixed, and then relabeled with the ferritin conjugate of the same lectin. In this case, the surfaces of filopodia and of SPV clusters are selectively labeled with the ferritin conjugate, indicating the insertion of new lectin receptors into the plasma membrane in the growth cone periphery. These results indicate that plasmalemmal expansion in the neuron occurs by a mechanism of polarized growth, possibly involving SPVs as plasmalemmal precursor vesicles.

Regulated plasmalemmal expansion in nerve growth cones.

To study the mechanisms underlying plasmalemmal expansion in the nerve growth cone, a cell-free assay was developed to quantify membrane addition, using ligand binding and sealed growth cone particles isolated by subcellular fractionation from fetal rat brain. Exposed versus total binding sites of 125I-wheat germ agglutinin were measured in the absence or presence of saponin, respectively, after incubation with various agents. Ca2(+)-ionophore A23187 in the presence of Ca2+ increases the number of binding sites (Bmax) but does not change their affinity (KD), indicating that new receptors appear on the plasma membrane. Similarly, membrane depolarization by high K+ or veratridine significantly induces, in a Ca2(+)-dependent manner, the externalization of lectin binding sites from an internal pool. Morphometric analysis of isolated growth cones indicates that A23187 and high K+ treatment cause a significant reduction in a specific cytoplasmic membrane compartment, thus confirming the lectin labeling results and identifying the plasmalemmal precursor. The isolated growth cones take up gamma-amino-butyric acid and serotonin, but show no evidence for Ca2(+)-dependent transmitter release so that transmitter exocytosis is dissociated from plasmalemmal expansion. The data demonstrate that plasmalemmal expansion in the growth cone is a regulated process and identify an internal pool of precursor membrane.

Components of the plasma membrane of growing axons. II. Diffusion of membrane protein complexes.

Intramembrane particles (IMPs) of the plasmalemma of mature, synapsing neurons are evenly distributed along the axon shaft. In contrast, IMPs of growing olfactory axons form density gradients: IMP density decreases with increasing distance from the perikarya, with a slope that depends upon IMP size (Small, R., and K. H. Pfenninger, 1984, J. Cell Biol., 98: 1422-1433). These IMP density gradients resemble Gaussian tails, but they are much more accurately described by the equations formulated for diffusion in a system with a moving boundary (a Stefan Problem), using constants that are dependent upon IMP size. The resulting model predicts a shallow, nearly linear IMP density profile at early stages of growth. Later, this profile becomes gradually transformed into a steep nonlinear gradient as axon elongation proceeds. This prediction is borne out by the experimental evidence. The diffusion coefficients calculated from this model range from 0.5 to 1.8 X 10(-7) cm2/s for IMPs between 14.8 and 3.6 nm, respectively. These diffusion coefficients are linearly dependent upon the inverse IMP diameter in accordance with the Stokes-Einstein relationship. The measured viscosity is approximately 7 centipoise. Our findings indicate (a) that most IMPs in growing axons reach distal locations by lateral diffusion in the plasma membrane, (b) that IMPs--or complexes of integral membrane proteins--can diffuse at considerably higher rates than previously reported for iso-concentration systems, and (c) that the laws of diffusion determined for macroscopic systems are applicable to the submicroscopic membrane system.

Components of the plasma membrane of growing axons. III. Saxitoxin binding to sodium channels.

The density of sodium channels was measured in growing and mature axons of the olfactory nerve of the bullfrog, using as a probe the drug saxitoxin (STX). The toxin binds to control nerves from adult animals in a saturable manner with a dissociation constant of approximately 23 nM at 4 degrees C and a capacity of 72 fmol/mg wet weight, equivalent to about five sites per square micrometer of axolemma. In growing nerves, obtained from adult frogs 4-5 wk following removal of the original nerve, the STX-binding capacity per wet weight of tissue is markedly reduced, to approximately 25% of control values, and appears to decrease in the proximodistal direction. STX-binding data, expressed as STX/mg wet weight, was converted to STX/micron 2 of axolemma using stereologically derived values of membrane area per milligram wet weight of nerve. The axolemmal content (area/mg wet weight) of all regions of growing nerve is substantially decreased compared to controls, but increases in the proximodistal direction by 60%. These changes in axolemmal area result in calculated STX receptor densities (per unit axolemmal area) which, in distal regions, are approximately at the level of the mature nerve and, in proximal regions, are actually increased above controls by 50 to 70%. Upon comparing the axolemmal density of intramembrane particles, reported in the companion paper, with the calculated density of STX receptors in both mature and growing nerves, we find a correlation between STX receptors and intramembrane particles with diameters of 11.5-14.0 nm. The growing axon's gradient of sodium channels and the shift from this gradient to a uniform distribution in the mature axon suggest (a) that sodium channels are inserted into the perikaryal plasmalemma and diffuse from there into the growing axolemma, and (b) that the axolemma undergoes functional maturation during growth.

Nerve growth cones isolated from fetal rat brain. IV. Preparation of a membrane subfraction and identification of a membrane glycoprotein expressed on sprouting neurons.

This study describes the preparation of a membrane subfraction from isolated nerve growth cone particles (GCPs) (see Pfenninger, K. H., L. Ellis, M. P. Johnson, L. B. Friedman, and S. Somlo, 1983, Cell, 35:573-584) and the identification in this fraction of a glycoprotein expressed during neurite growth. While approximately 40 major polypeptides are visible in Coomassie Blue-stained SDS polyacrylamide gels of pelleted (partially disrupted) GCPs, a salt-washed membrane fraction prepared from lysed, detergent-permeabilized GCPs contains only 14% of this protein and has an unusually simple polypeptide pattern of seven major bands. Monoclonal antibodies have been generated to GCP membranes isolated from fetal rat brain. These antibodies have been screened differentially with synaptosomes from adult rat brain in order to identify those which recognize antigens expressed selectively during neurite growth. One such antibody (termed 5B4) recognizes a developmentally regulated membrane glycoprotein that is enriched in GCP membranes and expressed in fetal neurons sprouting in vitro. The 5B4 antigen in fetal brain migrates in SDS polyacrylamide gels as a diffuse band of approximately 185-255 kD, is rich in sialic acid, and consists of a small family of isoelectric variants. Freezing-thawing and neuraminidase digestion result in the cleavage of the native antigen into two new species migrating diffusely around 200 and 160 kD. Prolonged neuraminidase digestion sharpens these bands at about 180 and 135 kD, respectively. In the mature brain, antibody 5B4 recognizes a sparse polypeptide migrating at approximately 140 kD. As shown in the following paper (Wallis, I., L. Ellis, K. Suh, and K. H. Pfenninger, 1985, J. Cell Biol., 101:1990-1998), the fetal antigen is specifically associated with regions of neuronal sprouting and, therefore, can be used as a molecular marker of neurite growth.

Immunolocalization of a neuronal growth-dependent membrane glycoprotein.

Monoclonal antibody (mAb) 5B4 recognizes in the rat a large, developmentally regulated membrane glycoprotein. The larger form of this antigen (185-255 kD) occurs in the developing nervous system and is present in membranes of nerve growth cones, as determined by analysis of a growth cone particle fraction. An immunochemical characterization of this antigen and of a smaller form (140 kD), sparsely present in the mature nervous system, has been described (Ellis, L., I. Wallis, E. Abreu, and K. H. Pfenninger, 1985, J. Cell. Biol., 101:1977-1989). The present paper reports on the localization by immunofluorescence of 5B4 antigen in cultured cortical neurons, developing spinal cord, and the mature olfactory system. In culture, mAb 5B4 stains only neurons; it is sparsely present in neurons at the onset of sprouting while, during sprouting, it appears to be concentrated at the growth cone and in regions of the perikaryon. In the developing spinal cord, 5B4 labeling is faintly detectable on embryonic day 11 but is intense on fetal day 13. At this stage, the fluorescence is observed in regions of the cord where axonal growth is occurring, while areas composed of dividing or migrating neural cells are nonfluorescent. With maturation of the spinal cord, this basic pattern of fluorescence persists initially, but the staining intensity decreases dramatically. In the adult, faint fluorescence is detectable only in gray matter, presumably indicating the presence of the 140 kD rather than the fetal antigen. The only known structure of the adult mammalian nervous system where axonal growth normally occurs is the olfactory nerve. mAb 5B4 intensely stains a variable proportion of olfactory axons in the mucosa as well as in the olfactory bulb. Based on both immunochemical and immunofluorescence data, the 5B4 antigen of 185-255 kD is associated specifically with growing neurons, i.e., neurons that are generating neurites.

Thrombin-induced growth cone collapse: involvement of phospholipase A(2) and eicosanoid generation.

The studies presented here explore intracellular signals resulting from the action of repellents on growth cones. Growth cone challenge with thrombin or thrombin receptor-activating peptide (TRAP) triggers collapse via a receptor-mediated process. The results indicate that this involves activation of cytosolic phospholipase A(2) (PLA(2)) and eicosanoid synthesis. The collapse response to repellents targets at least two functional units of the growth cone, the actin cytoskeleton and substratum adhesion sites. We show in a cell-free assay that thrombin and TRAP cause the detachment of isolated growth cones from laminin. Biochemical analyses of isolated growth cones reveal that thrombin and TRAP stimulate cytosolic PLA(2) but not phospholipase C. In addition, thrombin stimulates synthesis of 12- and 15-hydroxyeicosatetraenoic acid (HETE) from the released arachidonic acid via a lipoxygenase (LO) pathway. A selective LO inhibitor blocks 12/15-HETE synthesis in growth cones and inhibits thrombin-induced growth cone collapse. Exogenously applied 12(S)-HETE mimics the thrombin effect and induces growth cone collapse in culture. These observations indicate that thrombin-induced growth cone collapse occurs by a mechanism that involves the activation of cytosolic PLA(2) and the generation of 12/15-HETE.

Growth-regulated proteins and neuronal plasticity. A commentary.

Growth-regulated proteins (GRPs) of the neuron are synthesized during outgrowth and regeneration at an increased rate and enriched in nerve growth cones. Therefore, they can be used to some degree as markers of neurite growth. However, these proteins are not unique to the growing neuron, and their properties are not known sufficiently to assign them a functional and/or causal role in the mechanisms of outgrowth. During synaptogenesis, GRPs decrease in abundance, and growth cone functions of motility and organelle assembly are being replaced by junctional contact and transmitter release. However, there is a stage during which growth cone and synaptic properties overlap to some degree. We propose that it is this overlap and its continuation that allow for synaptic plasticity in developing and adult nervous systems. We also propose a hypothesis involving (a) trophic factor(s) that might explain the regulation of synaptic sizes and collateral sprouting. Some GRPs, especially GAP43/B50/pp46/F1, are more prominent in adult brain regions of high plasticity, and they undergo change, such as phosphorylation, during long-term potentiation (LTP). Without precise functional knowledge of GRPs, it is impossible to use changes in such proteins to explain the plasticity mechanism. However, changes in these "growth markers" are likely to be an indication of sprouting activity, which would explain well the various phenomena associated with plasticity and learning in the adult. Thus, plasticity and memory may be viewed as a continuation of the developmental process into adulthood.

5B4-CAM expression parallels neurite outgrowth and synaptogenesis in the developing rat brain.

In order to study whether 5B4-CAM expression parallels neurite outgrowth and synaptogenesis, a monoclonal antibody, 5B4, was used, which recognizes both fetal (185-250 kD) and adult (140 kD, 180 kD) forms of the neural cell adhesion molecule (N-CAM), to identify and localize the antigen in rat tissue during developmental ages P1 through P31 and in adults between P60 and 2 years of age. A ubiquitous pattern of intense immunolabelling was detected during the earliest stages of development. 5B4-CAM expression paralleled process outgrowth and the early stages of synaptogenesis in the cerebral cortex, hippocampal formation, and cerebellum. In the adult, immunoreactivity was generally less intense, but the cerebral cortex and hippocampal and cerebellar molecular layers, all areas implicated in learning-associated plasticity, retained substantial immunoreactivity. The inner one-third of the dentate gyrus molecular layer, an area implicated in axonal sprouting and reactive synaptogenesis, was particularly intensely labelled. Evidence from this work suggests that 5B4-CAM expression may be useful in monitoring neurite outgrowth and the early stages of synapse formation during development and possibly axonal sprouting and reactive synaptogenesis in the adult.

Estrogen and insulin synergism in neurite growth enhancement in vitro: mediation of steroid effects by interactions with growth factors?

Addition of estradiol to organotypic cultures of the fetal murine hypothalamus, preoptic area and cerebral cortex has been shown to elicit a striking enhancement of neurite growth which appears restricted to estrogen receptor-containing explant regions. The mechanisms underlying this response are unknown. An important question is whether the neurite enhancement which follows exposure to estradiol is due directly to the interaction of estrogen with the cell that was stimulated (the receptor-containing cell) or whether intermediate steps involving the possible interaction of estrogen and the endogenous polypeptide neurite-promoting growth factors or their receptors may play an important role. Recent findings in the cultures suggest that the effect of estrogen on neurite growth may involve synergistic interactions between estradiol and insulin-related peptides and may be important in regulating estrogen-responsive neurite growth in the central nervous system. Concurrent addition of estradiol and high levels of insulin (10 micrograms/ml or 50 micrograms/ml) to cultures of the olfactory bulb, hypothalamus, preoptic area and cerebral cortex of the fetal rat and mouse results in a dramatic acceleration and increase of neurite outgrowth which appears localized to estrogen receptor-containing explant regions. The supraphysiological concentrations of insulin required to elicit this response suggest that the factor(s) involved is unlikely to be insulin per se. Insulin may activate the receptor of different but closely related molecules such as the insulin-like growth factors (IGF)-I or -II to which it exhibits a relatively low affinity. Interactions between hormones and endogenous growth factors have been implicated in the modulation or mediation of an increasing number of endocrine-dependent, differentiative processes in vivo and in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Two types of presynaptic configurations in insect central synapses: an ultrastructural analysis.

Two structurally distinct types of synapses have been identified in the cockroach metathoracic ganglion. The two synaptic types are distinguished on the basis of (a) the shape and position of the presynaptic density seen by serial thin sectioning and (b) the arrangement and location of vesicle attachment sites (VAS) on the presynaptic membrane obtained from replicas of aldehyde-fixed, freeze-fractured neuropile. Bar-type synapses in thin sections possess a long presynaptic density located in a trough or groove opposite the extracellular space between two contiguous postsynaptic processes. In freeze-fracture, this trough is flanked by two rows of vesicle attachment sites. The second synaptic conformation consists of rows of discrete dense projections located on the convexities of the presynaptic membrane, i.e., directly opposite a single postsynaptic process. This conformation has been correlated with groups of VAS linearly arranged also on the convexities of the presynaptic membrane. These structurally different synapses may represent functionally different contacts within the insect ganglion.

Sites of plasmalemmal expansion in growth cones.

Studies on plasmalemmal expansion in isolated nerve growth cones identified large, clear vesicles characteristically found in growth cones as the plasmalemmal precursor. The present article examines these plasmalemmal precursor vesicles (PPVs) in greater detail in the intact cell. (a) Pulse-chase experiments with the phospholipid precursor, [3H]glycerol, followed by radioautographic analysis show that PPVs in distal neurites and growth cones are labeled prior to equilibration of the label with the plasmalemma. (b) Pulse-chase experiments with lectin-ferritin conjugates demonstrate that PPVs are not endocytotic, that they contain lectin receptors, and that, during growth, patches of lectin receptors appear on the plasmalemma covering PPV clusters. (c) Freeze-fracture studies show that this plasmalemma shares with PPVs a paucity of intramembrane particles. (d) Lectin labeling experiments and freeze-fracture analysis demonstrate, furthermore, that the plasmalemma forms a network of invaginations at the base of PPV clusters. (e) Correlative studies indicate that the refractive 'vacuoles' seen in growth cones by phase-contrast light microscopy correspond to the PPV clusters seen at the ultrastructural level. These results confirm the identity of the plasmalemmal precursor in the intact cell and demonstrate that PPV clusters form distinct, dynamic organelles specialized for plasmalemmal expansion in the growth cone.