The Journal of Neuroscience, June 1991, 1 I(6): 1617-l 626 The Distribution of Synapsin I and Synaptophysin in Hippocampal Neurons Developing in Culture Tara L. Fletcher,’ Patricia Cameron,2 Pietro De Camilli,2 and Gary Banker’ ‘Department of Anatomy, Cell Biology, and Neurobiology, Albany Medical College, Albany, New York 12208 and *Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510 As a first step toward elucidating mechanisms involved in synaptic target. Thus, in cultured hippocampal neurons, the the sorting of synaptic vesicle proteins in neurons, we have localization of synaptic vesicles in presynaptic specializa- used immunofluorescence microscopy to determine the dis- tions is the result of sorting mechanisms intrinsic to individ- tribution of two synaptic vesicle proteins, synapsin I and ual neurons as well as to mechanisms mediated by cell-cell synaptophysin, in hippocampal neurons developing in cul- contact. ture. In mature cultures, synapsin I and synaptophysin im- munoreactivity was concentrated in puncta that were re- The mechanismsthat lead to the formation and maintenance stricted to sites where axons contacted neuronal cell bodies of synapsesare of critical importance to an understanding of or dendrites. Electron-microscopic immunocytochemistry the functional development of the nervous system. The avail- demonstrated that these puncta corresponded to vesicle- ability of antibodies that recognize specific synaptic vesicle pro- filled axonal varicosities that were exclusively presynaptic. teins has made it possible to begin to examine some of the At early stages of development, before cell-cell contact, cellular events associatedwith the formation of presynaptic spe- both synapsin I and synaptophysin were preferentially lo- cializations. For example, it has been possibleto identify syn- calized in axons, where they were particularly concentrated aptic vesicle antigensin axons and growth conesprior to contact in the distal axon and growth cone. In axons that did not with target cells (Mason, 1986; Sarthy and Bacon, 1985; Chun contact other cells, immunostaining for these two proteins and Shatz, 1988) and, subsequently,to follow the accumulation had a granular appearance, which persisted for at least 7 d, of these proteins in varicosities at synaptic sites, for example, but focal accumulations of vesicles comparable to those at sites opposite ACh receptor clusters when neurites contact seen at sites of synaptic contact were not observed. When myotubes (Bixby and Reichardt, 1985; Lupa and Hall, 1989). neurons contacted one another, numerous puncta of syn- The experiments described in this report use this approach apsin I and synaptophysin formed within the first week in to examine changesin the distribution of two synaptic vesicle culture. Double-label immunofluorescence demonstrated that proteins, synapsin I and synaptophysin (or p38), during the the two vesicle antigens were closely codistributed through- development of CNS neurons in culture. Synapsin I is the col- out these stages of development. lective name for two very similar neuron-specific polypeptides These observations demonstrate that synaptic vesicle with molecular weights of 86,000 (Ia) and 80,000 (Ib) Da (Ueda proteins assume a polarized distribution within nerve cells and Greengard, 1977), which result from the alternative splicing beginning early in development, as soon as the axon can be of the transcript of a single gene (Sudhof et al., 1989). It is a identified. In contrast, differences in microtubule polarity ori- peripheral membrane protein that is associatedwith the cyto- entation that distinguish mature axons and dendrites, and plasmic face of virtually all small synaptic vesiclesand is there- that have been proposed to account for the selective sorting fore widely distributed in presynaptic specializationsthroughout of some materials in nerve cells, first appear at a subsequent the CNS and PNS (De Camilli et al., 1979, 1983a,b; Navone stage of development. The selective distribution of synaptic et al., 1984). Synapsin I also binds to cytoskeletal proteins, vesicle proteins to the axon occurs in isolated cells, inde- including F-actin. It is thought to act asa linking protein between pendent of interactions with other cells. In contrast,‘the for- small synaptic vesiclesand the actin-basedcytoskeleton of nerve mation of large clusters of vesicles typical of presynaptic terminals, thereby mediating the clustering of small synaptic specializations requires contact with an appropriate post- vesiclesat releasesites (Bainesand Bennett, 1985, 1986; Gold- em-inget al., 1986; Bahler and Greengard, 1987; Petrucci and Morrow, 1987). Synapsin I is a prominent substratefor CAMP- Received July 3, 1990; revised Dec. 18, 1990; accepted Jan. 9, 199 1. dependent and calcium/calmodulin-dependent protein kinases, This research was supported by NIH Grants NS 17 112 to G.B. and MH45 19 1 and its state of phosphorylation is thought to regulate neuro- to P.D., who was also the recipient of an award from the Muscular Dystrophy Association. We wish to thank Dr. Reinhard Jahn for his generous Sift of syn- transmitter releaseby controlling availability of synaptic vesi- aptophysin antisera, Dr. Jeffrey Deitch for his substantial contributions to the clesfor exocytosis (Nestler and Greengard, 1982; Huttner et al., electron-microscopic immunocytochemistry, and Colleen Fitzgerald and Cindy 1983; Llinas et al., 1985; Schiebler et al., 1986). Synaptophysin Tmutman for their expert technical assistance. This paper was submitted by T.L.F. in partial fulfillment of the requirements for the degree of Doctor of Philosophy, is an integral membraneglycoprotein, with a molecular weight Albany Medical Center Graduate School of Health Sciences, Albany, NY. of 38,000 Da and containing four membrane-spanningdomains. Correspondence should be addressed to Dr. Gary Banker, Department of Neu- roscience, University of Virginia School of Medicine, Medical Center Box 230, It is a major component of small synaptic vesicles in neurons Charlottesville, VA 22908. and of a population of small vesicles in neuroendocrine cells Copyright 0 199 1 Society for Neuroscience 0270-6474/9 l/l 11617-10$03.00/O (Jahn et al., 1985; Wiedenmann and Franke, 1985; Navone et 1616 Fletcher et al. l Synapsin I and Synaptophysin in Cultured Neurons al., 1986; Johnston et al., 1989). It is a calcium-binding protein, and for this reason it is thought to be involved in calcium- 2 4 9 15 dependent exocytosis (Rehm et al., 1986). Analysis of its quaternary structure and functional properties suggests that syn- aptophysin may be a voltage-sensitive, hexameric channel pro- tein (Thomas et al., 1988). a In the present study, we have investigated the distribution of synapsin I and synaptophysin during the development of hip- pocampal neurons in culture. These cultures have proven to be a useful model system for studying several aspects of neuronal development, including the growth of axons and dendrites and the development of the molecular differences that make these b two classes of processes functionally distinct (Caceres et al., 1986; Baas et al., 1988; Dotti et al., 1988; Goslin et al., 1988, 1990). In addition, the axons and dendrites that develop in such cultures form synapses with one another and exhibit a normal Figure 1. Immunoblots of cultured hippocampal cells stained with antisera directed against synapsin I (a) or synaptophysin (b). Equal synaptic polarity: axons are predominantly presynaptic, and volumes of hippocampal cell culture extracts (corresponding to equal dendrites, postsynaptic (Bartlett and Banker, 1984b). Moreover, numbers of cells) were loaded onto each lane. Numbers at the top of each the neuronal types present in these cultures normally form syn- lane indicate the number of days in vitro. Synapsin I was not resolved apses with one another in situ, and the properties of such syn- as a doublet by the minigel system used. apseshave been extensively studied. Pyramidal neurons, the most abundant cells inthe hippocampus, make excitatory con- sample was loaded onto a 7.5% (synapsin I) or 10% (synaptophysin) nections with one another via recurrent collaterals, Schaffer col- discontinuous polyacrylamide gel and electrophoresed according to the laterals, and commissuralprojections (reviewed by Swanson et method of Laemmli (1970) using a minigel system (Biorad). The gel al., 1987). Interneurons, including GABAergic cells that make was then electroblotted onto nitrocellulose using a semidry transfer inhibitory connectionswith pyramidal neuronsin situ, have also apparatus (Biorad) for 35 min at 1 A in a buffer containing 25 mM Tris, been identified in these cultures (C. Dotti, unpublished obser- 192 mM glycine, 20% methanol, and 0.025% SDS. Blots were processed according to Towbin et al. (1979) using the following buffer for quench- vations). The synapsesthat form betweenhippocampal neurons ing nonspecific protein binding sites, antibody dilutions, and washes: in culture are physiologically functional (C. Allen, unpublished 5% dry milk, 20 mM Tris (pH, 7.5), 150 mM NaCI, and 0.1% Tween observations) and, when examined by electron microscopy, are 20. Immunolabeling was performed with polyclonal rabbit sera directed comparable in structure to synapsesin situ (Bartlett and Banker, against synapsin I and against synaptophysin (gift of R. Jahn) followed by incubation with lZ51 protein A (Amersham). These antibodies have 1984b). These features suggestthat hippocampal