vesicles. As discussed earlier, newly synthesized proteins are often transported to the plasma membrane by the constitutive route. A careful immunoelectron microscopic study by Navone et al. (1986) could be consistent with a constitutive route to the plasma membrane. Antibodies to p38 were easily detectable attached to small electron lucent vesicles using immunogold labeling but were not detected in the membranes of DCSVs. The more sensitive immunoadsorption techniques reveal, however, the presence of three synaptic vesicle antigens on the membranes of DCSVs (Lowe et al. 1988). Two explanations appear possible: a slight leak of constitutively transported membrane proteins into the path to the DCSVS or a concentration of DCSV membrane proteins into small, clear vesicles on internalization. We know internalization can occur, but do not know its significance.

Small synaptic vesicles and the DCSVs have properties in common and others in which they differ. The small resemblance between the membranes of adrenergic vesicles, whether they have protein content or not, has suggested to several investigators that those that lack protein content are generated from the membranes of those that have protein content (Winkler et al. 1987). On the other hand, other authors have stressed the differences in the regulation and the sites of exocytosis of small SVs and DCSVs (Navone et al. 1986; Zhu et al. 1986). Left unresolved, therefore, is whether the function of proteins such as p38 is one of those shared by SSVS and DCSVs, or is exclusive to SSVs.

Models of Neuronal Development

The attractions of models in which DCSVS give rise to SSVs are developmental. If a neuronal precursor is endocrine-like, it will have DCSVs and a route to recycle membrane proteins in endocytotic vesicles back to the Golgi. DCSV membrane proteins accumulate in the Golgi region because the cell has a reserve capacity of membrane packaging material to prevent inappropriate secretion if peptide hormone is rapidly induced. When such a precursor becomes a neuron, the endocytotic vesicles remain at the cell periphery instead of recycling to the Golgi. In this model, the depletion of Golgi stores is due to redistribution, not a turnoff in synthesis. Contact with a synaptic target causes the mechanisms holding the vesicles at the periphery to accumulate at the point of contact.

The conversion of an endocrine cell to a neuron with induction of SSVS can be mimicked in PC12 by treating with NGF. Furthermore, we have recently been able to induce secretory granules in a variant of AtT-20 cells lacking them by exposing the cells to the oncogene H-ras. When we know more about these two developmental transitions in tumor cells in culture, the

next task will be to determine the validity of treating tumor cells as cells arrested in development.

Conclusion

Understanding the postsynaptic side of the synapse has been enormously facilitated by the availability of toxins and drugs. We have had no such advantage in studying neurotransmitter release. At last, however, antibody and DNA reagents to nerve terminal components are available that should lead to rapid advances in our understanding of how nerve terminals form, how they function, and how they might be modified by synaptic activity.

REFERENCES

Buckley, K.M., and Kelly, R.B. Identification of a transmembrane glycoprotein specific for secretory vesicles of neural and endocrine cells. Journal of Cell Biology 100:1284-1294, 1985.

Burgess, T.L.; Craik, C.S.; and Kelly, R.B. The exocrine protein trypsinogen is targeted into the secretory granules of an endocrine cell line: Studies by gene transfer. Journal of Cell Biology 101:639-645, 1985.

Gumbiner, B., and Kelly, R.B. Two distinct intracellular pathways transport secretory and membrane glycoproteins to the surface of pituitary tumor cells. Cell 28:51-59, 1982.

Hooper, J.E.; Carlson, S.S.; Kelly, R.B. Antibodies to synaptic vesicles purified from Narcine electric organ bind a subclass of mammalian nerve terminals. Journal of Cell Biology 87:104-114, 1980.

Kelly, R.B.; Carlson, S.S.; von Wedel, R.J.; Hooper, J.E.; Miljanich, G.P.; and Brasier, A.R. Antibodies to pure cholinergic synaptic vesicles, nerve terminals and their plasmamembranes. In: McKay, R.; Raff, M.C.; and Reichardt, L.F., eds. Monoclonal Antibodies to Neural Antigens. Cold Spring Harbor, NY: Cold Spring Harbor Lab Press, 1981. pp. 153-161.

Lowe, A. W.; Madeddu, L.; and Kelly, R.B. Endocrine secretory granules and neuronal synaptic vesicles have three integral membrane proteins in common. Journal of Cell Biology 106:51-59, 1988.

Matsuuchi, L.; Buckley, K.; Lowe, A.; and Kelly, R.B. Targeting of secretory vesicles to cytoplasmic domains in AtT-20 and PC-12 cells. Journal of Cell Biology 106:239251, 1988.

Matthew, W.D.; Tsavaler, L.; and Reichardt, L.F. Identification of a synaptic vesiclespecific membrane protein with a wide distribution in neuronal and neurosecretory tissue. Journal of Cell Biology 91:257-269, 1981.

Moore, H.-P.H.; Gumbiner, B.; and Kelly, R.B. A subclass of proteins and sulfated macromolecules secreted by AtT-20 cells is sorted with ACTH into dense secretory granules. Journal of Cell Biology 97:810-818, 1983.

Navone, F.; Jahn, R.; Digioia, G.; Stukenbrok, H.; Greengard, P.; and DeCamilli, P.

Protein p38: An integral membrane protein specific for small vesicles of neurons and neuroendocrine cells. Journal of Cell Biology 103:2511-2527, 1986. Orci, L.; Ravazzola, M.; Amherdt, M.; Perrelet, A.; Powell, S.K.; Quinn, D.L.; and Moore, H.-P.H. The trans-most cisternae of the Golgi complex: A compartment for sorting of secretory and plasma membrane proteins. Cell 51:1039-1051, 1987. Schweitzer, E., and Kelly, R.B. Selective packaging of human growth hormone into synaptic vesicles in a rat neuronal (PC-12) cell line. Journal of Cell Biology 101:667676, 1985.

Sudhof, T.; Lottspeich, F.; Greengard, P.; Mehl, E.; and Jahn, R. A synaptic vesicle protein with a novel cytoplasmic domain and four transmembrane regions. Science 238:1142-1144, 1987.

Tooze, J., and Burke, B. Accumulation of adrenocorticotropin secretory granules in the midbody of telophase AtT20 cells: Evidence that secretory granules move anterogradely along microtubules. Journal of Cell Biology 104:1047-1057, 1987. Tooze, J.; Tooze, S.A.; and Fuller, S.D. Sorting of progeny coronavirus from condensed secretory proteins at the exit from the Trans-Golgi network at AtT20 cells. Journal of Cell Biology 105:1215-1226, 1987.

Wiedenmann, B., and Franke, W. Identification and localization of synaptophysin, an integral membrane glycoprotein of Mr 38,000 characteristic of presynaptic vesicles. Cell 41:1017-1028, 1985.

Winkler, H.; Sietzen, M.; and Schober, M. The life cycle of catecholamine-storing vesicles. Annals of the New York Academy of Sciences 493:3-19, 1987.

Zhu, P.D.; Thureson-Klein, A.; and Klein, R.L. Exocytosis from large dense cored vesicles outside the active synaptic zones of terminals within the trigeminal subnucleus caudalis: A possible mechanism for neuropeptide release. Neuroscience 19:43-54, 1986.

Agrin-Like Molecules in Synaptic Basal Lamina and in Motor Neurons

C. Magill-Solc, N.E. Reist, and U.J. McMahan

Stanford University School of Medicine, Department of Neurobiology, Stanford, CA 94305

Structural specializations directly involved in synaptic transmission occur at synapses throughout the nervous system. For example, axon terminals have active zones that play a role in the release of neurotransmitter, and the postsynaptic membrane of target cells has a high concentration of receptors for the transmitter. Over the last two decades, much effort has been directed toward determining how such specializations are formed in the embryo, how they are maintained in the adult, and, in cases where regeneration occurs, how they are reformed. Many of these studies have been conducted on the neuromuscular junction, which is convenient for experimentation both in vivo and in vitro and where the synaptic specializations are well characterized. It is now clear that the formation and maintenance of these specializations is dependent on communication between the motor neuron's axon terminals and muscle fibers. That is, muscle fibers provide molecules that direct the formation of the active zones in axon terminals, which release the transmitter acetylcholine. Axon terminals provide molecules that direct the formation of acetylcholine receptor (AChR) and acetylcholinesterase (AChE) aggregates on the surface of muscle fibers. We have been conducting experiments aimed at identifying and characterizing the molecules involved in this communication.

Our initial studies, made on damaged adult muscles, revealed that molecules stably bound to the portion of the muscle fiber's basal lamina that occupies the synaptic cleft direct the aggregation of AChRs and AChE on the surface of regenerating muscle fibers (Anglister and McMahan 1985; Burden et al. 1979; McMahan and Slater 1984). These findings led to an analysis of basal lamina-containing extracts from the electric organ of Torpedo californica. The electric organ is rich in cholinergic synapses and has been useful for identifying and characterizing other synaptic molecules. Such extracts were found to contain polypeptides that cause the formation of patches on the surface of cultured myotubes at which AChRs and AChE are concentrated

(Godfrey et al. 1984; Nitkin et al. 1987; Wallace 1987; Wallace et al. 1985). The active polypeptides, called agrin, have been purified (Nitkin et al. 1987), and antigenically similar molecules that cause the aggregation of AChRs on cultured myotubes have been extracted from muscle (Godfrey et al. 1984), although in much lower amounts than from the electric organ. Moreover, monoclonal antibodies (mAbs) against agrin stain molecules stably bound to the synaptic basal lamina at the neuromuscular junction (Reist et al. 1987).

Here we review some of these findings and present a brief account of recent studies which indicate that motor neurons in embryos and adults contain agrin-like molecules (Magill et al. 1987; Magill-Solc and McMahan, submitted; Smith et al. 1987). Together, these findings support the hypothesis that agrin or a molecule very similar to it is synthesized by motor neurons and is released from their axon terminals to become incorporated into the synaptic basal lamina where it directs the formation of AChR and AChE aggregates on muscle fibers during development and regeneration.

Anti-Agrin Antibodies Stain the Synaptic
Basal Lamina

We made a library of monoclonal antibodies that immunoprecipitated agrin. Thirteen stained neuromuscular junctions in Torpedo; four also stained neuromuscular junctions in frog and/or chicken (Reist et al. 1987). In all cases, the distribution of stain was the same; it filled the synaptic cleft and junctional folds and lined the surface of the Schwann cells that capped the axon terminals (figure 1a). In chicken and Torpedo, the mAbs stained the basal lamina both in the extrajunctional region of slow muscle fibers and on smooth muscle fibers of blood vessels (Reist et al. 1987). Thus, molecules antigenically related to agrin are not restricted to the neuromuscular junction, but at the junction, agrin-like molecules are highly concentrated in the synaptic cleft, the site where molecules that cause AChR and AChE aggregation on regenerating muscle fibers are known to be.

That the molecules recognized by the mAbs were stably bound to the basal lamina was demonstrated by damaging frog muscles in a way that caused degeneration of all cells in the neuromuscular junction – muscle fibers, axon terminals, and their Schwann cells - while leaving much of the basal lamina intact (Reist et al. 1987). Regeneration was prevented. Three weeks later, when the cellular debris had been phagocytized, the empty basal lamina sheaths were treated with anti-agrin mAbs. As shown in figure 1b, the synaptic region of the muscle fiber's basal lamina and Schwann cell basal lamina stained, and such staining could account for all of that observed at normal neuromuscular junctions (compare with figure la).