Synapsins in the vertebrate retina: Absence from ribbon synapses and heterogeneous distribution among conventional synapses

Neuron, Vol. 5, 19-33,July,1990,Copyright© 1990by Cell Press

Synapsins in the Vertebrate Retina: Absence from Ribbon Synapses and Heterogeneous Distribution Among Conventional Synapses James W. Mandell* Ellen Townes-Anderson* Andrew J. Czernik, t Richard Cameron, t§ Paul Greengard,t and Pietro De Camillit * Department of Physiology Cornell University Medical College New York, New York 10021 t Laboratory of Molecular and Cellular Neuroscience The Rockefeller University New York, New York 10021 Department of Cell Biology Yale University School of Medicine New Haven, Connecticut 06510

Summary The vertebrate retina contains two ultrastructurally dip tinct types of vesicle-containing synapses,conventional synapses, made predominantly by amacrine cells, and ribbon synapses, formed by photoreceptor and bipolar cells. To identify molecular differences between these synapsetypes, we have compared the distribution of the synapsins, a family of nerve terminal phosphoproteins, with that of synaptophysin (p38) and SV2, two intrinsic membrane proteins of synaptic vesicles. We report an absence of synapsin I and II immunoreactivity from all ribbon-containing nerve terminals. These include terminals of rod cells in developing and adult rat retina, rod and cone cells in monkey and salamander retinas, and rat bipolar cells. Furthermore, we show that synapsins I and II are differentially distributed among conventional synapses of amacrine cells. The absence of the synapsins from ribbon synapses suggests that vesicle clustering and mobilization in these terminals differ from that in conventional synapses. Introduction Synapsin is the collective name for a family of four closely related synaptic vesicle-associated phosphoproteins, synapsins la, Ib, lla, and llb, that are thought to play a key role in the regulation of neurotransmitter release (De Camilli and Greengard, 1986). They arise from the differential splicing of transcripts from two genes (Siidhof et al., 1989). Synapsin I binds both to the cytoplasmic surface of synaptic vesicles and to actin and may be responsible for the clustering of small synaptic vesicles at presynaptic sites (Huttner et al., 1983; Biihler and Greengard, 1987; Landis et al., 1988; Hirokowa et al., 1989). The binding affinities of the molecule for both actin and the vesicle surface are regulated by phosphorylation (Huttner et al., 1983; B~ihler and Greengard, 1987; Schiebler et al., 1986; §Present address: Section of Neuroanatomy, Yale University School of Medicine, New Haven, Connecticut 06510.

B~ihler et al., 1989; Benfenati et al., 1989a, 1989b), suggesting that synapsin I may regulate availability of synaptic vesicles for exocytosis at active zones. Synapsin II (formerly referred to as protein III) has been less thoroughly studied. However, the similarity of its primary structure to synapsin I (S~idhof et al., 1989) and its similar association with synaptic vesicles (Browning et al., 1987) suggest that it has a related function. Previous studies have shown that synapsin I is present in virtually all synapses. More recently, the availability of anti-peptide antibodies specific for each of the four synapsins has enabled their immunocytochemical localization. These studies have confirmed the widespread distribution of the synapsins, but have shown that the relative expression of the four isoforms differs among nerve terminals and that some nerve terminals may lack some of the isoforms (SCidhof et al., 1989). A possible exception to the widespread distribution of synapsin I was revealed in a previous study, which suggested an absence of synapsin I in retinal photoreceptor terminals (De Camilli et al., 1983). This finding was of interest because terminals of photoreceptors have specific structural and functional features. They contain dense bars or ribbons anchored to the presynaptic membrane and covered with a layer of synaptic vesicles (Sj6strand, 1953). Ribbon synapses are also found in a variety of other sensory receptor cells, including pineal photoreceptors (Hopsu and Arstila, 1964), hair cells of the cochlea (Smith and Sj0strand, 1961) and vestibular organs (Flock, 1964), teleost lateral line, and electroreceptor cells (Wachtel and Szamier, 1966; Szabo, 1974), as well as in a nonsensory neuron, the retinal bipolar cell (Kidd, 1962). From a functional standpoint, photoreceptors and all other ribbon-containing cells are distinct from most neurons in that they do not utilize regenerative action potentials. Transmitter is released from these cells in response to small, graded potential changes. Ribbons have been proposed to shuttle synaptic vesicles to exocytotic sites (Bunt, 1971; Gray and Pease,1971). Changes in ribbon morphology have been correlated with changes in synaptic efficacy (Fields and Ellisman, 1985). Establishing whether the absence of all of the synapsins is a general feature of ribbon terminals would contribute to the elucidation of molecular differences between the exocytotic machinery of ribbon and conventional synapses. In this studywe have used an immunocytochemical approach to determine whether ribbon-containing terminals of the retina contain any of the synapsins. In the retina, ribbon synapses are formed by photoreceptors in the outer plexiform layer (OPL) and by bipolar cells in the inner plexiform layer (IPL). The majority of conventional synapses are formed by amacrine cells, a heterogeneous population of interneurons with terminals located in the inner plexiform

Neuron 20

Table 1. Specifications of Primary Antibodies Antibody G-35/36 14 G-115 19.31 G-306 G-278 G-281 G-211 G-62163 C7.1 10H3

Type Rb, AP Rb Rb, AP MAb,s Rb, AP Rb, AP Rb, AP Rb, AP Rb MAb, a MAb, s

Antigen(s) Recognized Synapsin la, Ib, Ila, lib Synapsin la, Ib Synapsin,la, Iba Synapsin lla, llb Synapsin laa Synapsin Iba Synapsin llaa Synapsin Ilba Synaptophysin Synaptophysin SV2

Concentrations/Dilutions for Immunofluoresence:Blotting 25 gg/ml; 2.5 p.g/ml 11100; 111000 25 lig/ml; 2.5 lig/ml 11100; 111000 25 p.glml; 2.5 llglml 25 llglml; 2.5 llglml 25 p,glml; 2.5 llg/ml 25 p.g/ml; 2.5 p.g/ml 1/100; 111000 112000;1140,000 1110; 11100

References Browning et al., 1987 De Camilli et al., 1983 S(idhof et al., 1989 Browning et al., 1987 S(idhof et al., 1989 Siidhof et al., 1989 S(idhof et al., 1989 SCidhof et al., 1989 Jahn et al., 1985 Jahn et al., 1985 Buckley and Kelly, 1985

Rb, rabbit polyclonal; AP, affinity-purified; MAb, mouse monoclonal; s, culture supernatant; a, ascites fluid. a Anti-peptide antibodies.

layer. As a control for our studies, we have used antibodies directed against two intrinsic membrane proteins of small synaptic vesicles, synaptophysin (p38) and S~12.These proteins have previously been shown to be present in all small synaptic vesicles irrespective of the type of neurotransmitter that they store (Buckley and Kelly, 1985; Jahn et al:, 1985; Wiedenmann and Franke, 1985; Navone et al., 1986; Floor and Feist, 1989). We report that in contrast to synaptophysin and SV2, which are present in all synapses of the retina, none of the synapsin isoforms is present atdetectable concentration in ribbon-containing nerve terminals. Moreover, our results have revealed a heterogeneous distribution of the four synapsins among conventional nerve terminals in retina.

apsin llb antibody also labeled unidentified bands of higher molecular weights in both retina and cortex. The anti-synaptophysin monoclonal antibody ((]7.1) labeled identical 38 kd bands in both tissues (Figure IC, lane p38). Anti-SV2 revealed a difference between retina and cortex: 80 kd bands were present in both tissues, but an additional 70 kd band was detected in retina (Figure lC, lane SV2). The basis for this difference is unknown, but previous work has demonstrated tissue-specific variations in the apparent molecular weight of the SV2 antigen (Buckley and Kelly, 1985). Control lanes, labeled with Sal-1, an irrelevant mouse monoclonal antibody (Figure IC, lane CTLI), or with normal rabbit serum (Figure IC, lane CTL2) showed no detectable labeling.

Results

The Synapsins Are Absent from Ribbon Synapses in Adult Retinas Immunolabeling of frozen sections of rat retina with antibodies directed against synaptophysin and SV2 produced identical staining patterns. Intense immunofluorescence was seen in both the outer and inner plexiform layers. This pattern differed from that produced by antibodies directed against the synapsins, as demonstrated by both conventional and confocal fluorescence microscopy. Anti-synapsin antibodies left the OPL almost completely unstained. In addition, some synaptophysin- and SV2-immunoreactive structures in the IPL did not contain immunoreactive synapsin. These differences are consistent with an absence of the synapsins from ribbon synapses, as discussed below.

Characterization of Rat Retinal Synaptic Vesicle Proteins by Immunoblotting The antibodies used in this study have been characterized previously and are listed in Table 1. To determine whether the antibodies recognized molecular species in retina similar to those present in other CNS regions, immunoblot analysis was simultaneously performed on homogenates of rat retina and cerebral cortex. Anti-synapsin 1/11 (G-35/36) recognized four bands of identical electrophoretic mobilities (86, 80, 78, and 55 kd) in both retina and cortex (Figure 1A, lane I/ll). These bands represent synapsin la, Ib, Ila, and lib, respectively. However, the intensity of the lib band relative to the other synapsins appeared substantially lower in retina than in cortex. The anti-synapsin I antiserum labeled an identical 86180 kd doublet in retina and cortex (Figure 1A, lane la,b), as did an anti-peptide antibody directed against the synapsin I doublet (Figure 1A, lane la,b*). Anti-peptide antibodies, specific for each of the four synapsins, also revealed bands of identical molecular weight in retina and cortex (Figure 1B, lanes la, Ib, lla, and llb). Again, synapsin llb in the retina was barely detectable in comparison with the other synapsins. The anti-syn-

Rod Photoreceptors Immunolabeling for synaptophysin (Figure 2B) and SV2 (data not shown) revealed brightly fluorescent, teardrop-shaped terminals in the OPL. Many of these large terminals were continuous with thin connecting fibers coursing toward the photoreceptor cell bodies in the outer nuclear layer (ONL). The shape and position of these terminals unambiguously identify them as photoreceptor synaptic terminals. Furthermore, because the rat has essentially an all rod retina, they are

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rod synaptic terminals, also known as spherules. In contrast to synaptophysin or SV2 immunolabeling, no synapsin 1/11labeling was detectable in large rod terminals, as seen by conventional double-label immunofluorescence (Figures 2C and 2D). In some sections, however, slight synapsin immunc~reactivity was detectable in the OPL as sparsely distributed puncta. These synapsin-immunoreactive puncta were much smaller in diameter (<1 I~m) than the large SV2- or synaptophysin-immunoreactive photoreceptor terminals. Furthermore, they occupied a position closer to the inner nuclear layer (INL) than the large photoreceptor terminals. A panel of anti-peptide antibodies directed against each of the four synapsins (la, Ib, Ila, and lib) provided further evidence that all synapsins are absent from photoreceptor synaptic terminals. None of the four antibodies showed any significant labeling in the large OPL rod terminals (see Figure 8). Additional proof that the photoreceptor terminals are devoid of synapsin I immunoreactivity was obtained with double immunofluorescence labeling of enzymatically isolated photoreceptors. Many of these cells retained intact synaptic terminals after isolation (Figure 3). Previous work has demonstrated that the spherules of isolated rods are filled with synaptic vesicles and retain functional ribbon complexes (Townes-Anderson et al., 1988). Rod photoreceptors were identified by their characteristic morphology and by labeling with a monoclonal antibody against a rod-specific opsin (MAb 4D2; data not shown). Spherules showed prominent immunoreactivity for SV2 and synaptophysin but not for synapsin I. Finally, to verify the localization of synaptophysin in photoreceptor terminals, ultrathin cryosections of rat retina were labeled with a polyclonal antiserum against synaptophysin followed by protein A-colloidal gold (Figures 4A and 4B). Gold particles were seen on most small, clear vesicles within photoreceptor spherules. Vesicles bound to ribbons were also labeled. Nonspecific labeling was minimal, as seen, for example, by the absence of gold particles on mitochondria. Cone Photoreceptors To d e t e r m i n e w h e t h e r c o n e as well as rod photore-

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Figure 1. Immunoblot Analysis of Synaptic Vesicle Proteins in Rat Retina (A and B) Anti-synapsinantibodies detect speciesidentical molecular weight (la, Ib, Ila, and lib; 86, 80, 74, 55 kd, respectively) in retina (r) and cerebralcortex(c). However,synapsinlib (55 kd) is barelydetectablein retina.Anti-synapsin lib antibodies label bands of higher molecularweight in addition to the 55 kd protein in both tissues. (C) Anti-SV2 revealsa difference between retina and cortex: 80 kd bands are present in both tissues, but an additional 70 kd band is detected in retina. Identical 38 kd bands are detected by anti-synaptophysinin retina and cortex. No bands are recognized by an irrelevantmonoclonal antibody (CTL1)or by normal rabbit serum (CTL2).Total homogenatesof rat retina (40 p.g protein; r) or cerebralcortex(10 p.gprotein; c) were separatedon an 8% SDS,acrylamidegel and transferredto nitrocellulose.Anti-

ceptors lack synapsin immunoreactivity, retinas of two species known to contain abundant cones were investigated. The tiger salamander (Ambystoma tigrinum) retina contains approximately 50% cones. Doublelabel immunofluorescence of frozen sections of salamander retina (Figure 5A) with antibodies to SV2 and to synapsin 1/11revealed intense SV2 immunoreactivity in large OPL rod and cone terminals as well as in puncta throughout the IPL In contrast, synapsin 1/11 immunoreactivity was detectable only in the I PL. Sire-

bodies used: 1/11,G-35/36; la,b, 14;la,b*, G-115; Ila,b, MAb 19.31; la, G-306; Ib, G-278; Ila, G-281; liD, G-211; SV2, MAb 10H3; p38 (synaptophysin),MAb C7.1;CTL1, MAb Sal-1;CTL2, normal rabbit serum. Molecular weight standards: 116,84, 48, and 36 kd.

Neuron 22

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Figure 2. Double-Label Immunofluorescence of Rat Retina (A-D) The synapsins are absent from ribbon-containing synaptic terminals of rod photoreceptors. (A) Phase-contrast micrograph of rat retina frozen section. GCL, ganglion cell layer; IPI_, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OS, outer segments of rod photoreceptors. (B) Bright anti-synaptophysin labeling (rhodamine) is present in the

S~napsins in Retina

ilar results were obtained by double-labeling a monkey retina (Figure 5B) with anti-synaptophysin and anti-synapsin 1/11.Cell isolation experiments confirmed that the SV2- and synaptophysin-immunoreactive terminals in the salamander OPI. were derived from both cones and rods (Mandell et al., 1988, Soc. Neurosci., abstract). It appears that both cone and rod photoreceptor terminals contain immunoreactive synaptophysin and SV2 but lack synapsins I and II.

Bipolar Cells

Figure 1 Double-Label Immunofluorescence of Isolated Rod Photoreceptors Enzymatically isolated rod cells retain characteristic morphology, including outer and inner segment and attached spherule (top panels; arrows). Synaptic terminals (arrows) show intense SV2 and synaptophysin immunoreactivity (middle panels) but no detectable synapsin I (bottom panels).Antibodies used: synaptophysin (p38), MAb C7.1;SV2,MAb 10H3; synapsin I, 14.Bar, 20 p.m.

A second major difference between synaptophysin or SV2 and synapsin immunolabeling was found in the rat IPL. This was most clearly revealed by confocal fluorescence microscopy, which provides thin optical sections of labeled structures, eliminating fluorescence above or below the focal plane. Synaptophysin or SV2 immunolabeling revealed large, 4-8 I~m diameter, bulbous structures deep in the IPL (Figure 6B). These structures were not labeled with the antisynapsin 1/11 antibody, leaving "holes" in the synapsin-immunostained IPL. Strikingly, these holes were rimmed by punctate synapsin immunoreactivity (Figure 6C). Electron microscopic analysis of the rat IPL revealed ribbon-containing terminals of bipolar cells surrounded by conventional synaptic terminals (Figure 6A; Sosula and Glow, 1970). A large synapsin-negative bipolar terminal surrounded by smaller synapsin-positive boutons would be consistent with the rimming pattern of synapsin immunoreactivity seen with confocal microscopy. To provide unambiguous identification of bipolar cell terminals, cell isolation experiments were performed. Bipolar cells, identified by their characteristic morphology, retained large, bulbous terminals after enzymatic dissociation. Double-label immunofluorescence showed that these terminals contained SV2 and synaptophysin but lacked synapsin I immunoreactivity (Figures 6D-61). SV2 and synaptophysin immunoreactivity associated with these bulbous terminals could not be attributed to attached conventional nerve terminals because previous electron microscopic studies demonstrated that synaptic terminals from a variety of retinal neurons are free from adhering presynaptic boutons under the isolation conditions used in the present study CTownes-Anderson and Vogt, 1989). Thus, the punctate synapsin immunoreactivity seen rimming the profiles of bipolar cell terminals in intact sections of retina is presumed to be associated with amacrine cell synaptic boutons, which are removed by papain treatment.

OPL, where large rod photoreceptor terminals can be seen. The IPL is also positive for synaptophysin immunolabeling. (C) In contrast, synapsin 1/11immunoreactivity (fluorescein) is absent from the OPL but present in the IPL. Note that synapsin I/ll immunoreactivity is concentrated within five IPL sublayers. (D) A double-exposureusing both fluorescein and rhodamine filter sets revealsthe colocalization of synapsin I/ll and synaptophysin in the IPL as yellow fluorescence and the exclusive localization of synaptophysin in the OPL as red fluorescence. Antibodies used: synaptophysin, MAb C7.1;synapsin 1/11,G-35/36. (E-G) Members of the synapsin family show differential localization within the IPL. (E) Synapsin I is distributed throughout the IPL. (F) Synapsin II, however,is localized within three discrete sublayersof the IPL. Note again that the OPL is devoid of immunolabeling for both synapsins. (G) A double exposuredemonstratesthe spatial relationship between synapsin I and II immunoreactive layers.Antibodies used: synapsin la,b, G-115; synapsin Ila,b, MAb 19.31. Bars, 20 p.m.

Figure 4. Immunocytochemical Localization of Synaptophysin to Synaptic Vesicles of Photoreceptor Terminals Immunogold labeling of ultrathin frozen sections of rat retina. (A) A photoreceptor terminal in the OPL contains abundant gold particles over small clear vesicles but almost none over a mitochondrion (m). Bar, 03 p.m. (B) Heavy labeling is seen on synaptic vesicles surrounding a synaptic ribbon (rb) as well as vesicles away from the ribbon. Antibody used: synaptophysin, G-62/63. Bar, 0.25 Izm.

Figure 5. Double-Label Immunofluorescence of Tiger Salamander and Monkey Retinas Cryostat sections of tiger salamander (A) and monkey (B) retinas were doubie-labelecl for synapsin 1/11and either SV2 or synaptophysin. Rod and cone synaptic terminals in the OPL of both species are brightly immunoreactive for SV2 or synaptophysin. In contrast, synapsin 1/11immunoreactivity is virtually absent from terminals in the OPL. The IPL stains brightly for SV2 or synaptophysin as well as synapsin 1/11.Antibodies used: synaptophysin, MAb C7.1; SV2, MAb 10H3; synapsin 1/11,G-35/36. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. Bars, 50 Izm.

2~napsinsin Retina

Synapsin I Is Expressed in the Somata of Some Developing Retinal Neurons, but Not in Developing Photoreceptors In adult neurons synapsin I is detectable by immunocytochemistry only in nerve terminals. In contrast, in at least some developing neurons, it can also be detected in cell bodies (De Camilli et al., 1980, Soc. Cell Biol., abstract). Since mature photoreceptors are de. void of synapsin I immunoreactivity, it was of interest to determine whether, at any developmental stage, synapsin I was present in the cell bodies of photoreceptors. For this purpose the distribution of synapsin I immunoreactivity was examined in retinas of developing rats between I and 19 days postnatal. At day 1, before the establishment of the prominent laminar organization of the retina, only a few cells were immunoreactive. Examination of 5-day-old (data not shown) and 7-day-olcl (Figure 7B) retinas revealed three populations of cell bodies positive for synapsin I immunoreactivity. The most abundant and intensely stained somata were those located in the region of the INL directly adjacent to the forming IPL. These cells were identified as amacrine cells based on their position and the emergence of a single process from their vitreal pole. Their terminals in the IPL were also brightly immunoreactive. Intense immunoreactivity was also observed in a small population of cell bodies located in the outermost portion of the INL and in their processes coursing through the OPL. Based on their position and shape, these cells were identified as horizontal cells. Finally, a few immunoreactive cell bodies were detectable in the forming ganglion cell layer. These were either ganglion cells or displaced areacrine cells. By day 19, only a few amacrine cell bodies were immunoreactive, as seen in the adult. At all developmental stages examined, cell bodies and terminals of developing photoreceptor cells were devoid of synapsin I immunoreactivity. That some of the cells in the ONL are developing photoreceptors is supported by high levels of opsin expression at similar developmental stages (Hicks and Barnstable, 1987). To confirm that the lack of synapsin I immunoreactivity in the forming photoreceptor terminals was not merely due to the absence of recognizable terminals at this developmental stage, synaptophysin (Figure 7C, p38) and SV2 (data not shown) immunofluorescence was performed on a day 7 retina. Synaptophysin and SV2 immunoreactivity was present in large terminals in the forming OPL as well as in puncta within the IPL. The large OPL terminals were continuous with thin connecting fibers coursing toward the developing photoreceptor somata. In addition, their size, shape, and position were consistent with previous electron microscopic descriptions of vesicle-filled photoreceptor terminals in early postnatal rat retina (VVeidman and Kuwabara, 1969). Double-labeling experiments (data not shown) demonstrated that these large synaptophysin-immunoreactive terminals were separate structures from the synapsin-stained processes of presumptive horizontal cells. Thus, neither photorecep-

tor cell bodies nor terminals contained detectable synapsin immunoreactivity during postnatal development.

Heterogeneous Distribution of Synapsins la, Ib, Ila, and lib among Conventional Retinal Synapses Examination of the IPL labeled with the anti-peptide synapsin antibodies revealed a further heterogeneity of synapsin expression in the rat retina. Whereas synapsin la and Ib immunoreactivity was distributed relatively homogeneously across the IPL (Figure 8, la and Ib), synapsin Ila and lib immunoreactivity was concentrated in three sublayers of the IPL (Figure 8, Ila and lib). Two of these were narrow sublayers located at 25% and 55% of the IPL width (measured from the INL border). The wider layer spanned from 70% to 100% of the IPL width (i.e., to the border of the ganglion cell layer). Though the patterns of synapsin Ila and lib labeling were identical, the intensity of labeling was far less for synapsin lib than for synapsin Ila and was undetectable in some experiments. This observation was consistent with the low levels of synapsin lib detectable by immunoblot. The labeling patterns within the IPL and their relative intensities were unchanged over a wide range of antibody concentrations. Immunofluorescence was abolished by preincubation of the antisera with the corresponding free peptide (right-hand panels in Figure/3) but was unaffected by preincubation with unmatched synapsin peptides (data not shown). Furthermore, immunolabeling identical to that revealed with anti-synapsin la and Ib antibodies was seen with an anti-peptide antibody recognizing both synapsin la and Ib (Figure 2E). Similarly, the same pattern of immunolabeling seen with synapsin Ila and lib antibodies was revealed with a monoclonal antibody to both synapsin Ila and lib (Figure 2F). The spatial relationship between synapsin I- and synapsin II-immunoreactive layers was demonstrated by double-exposure micrographs of a doublelabeling experiment (Figure 2G). Results of representative immunofluorescence experiments are presented semi-quantitatively in the form of averaged densitometric scans of stained rat retina sections (Figure 9). For comparison, the number of small synaptic vesicles (SSVs) per unit area across the width of the plexiform layers was determined by quantitation of vesicular profiles in electron micrographs. SV2 and synaptophysin immunofluorescence both produced sharp peaks in the OPL that were absent from sections labeled with any of the synapsin antibodies. SV2 and synaptophysin immunofluorescence also produced remarkably similar densitometric profiles in the IPL. The peak intensities were located near the border of the IPL and the ganglion cell layer. These peaks were probably due to the abundance of synaptophysin- and SV2-immunoreactive bipolar cell terminals in the deep IPL. The densitometric profiles of SV2 and synaptophysin immunofluorescence were quite similar to the plot of SSVs per unit area. This result is consistent with the finding

Neuron 26

S}~napsinsin Retina

Figure Z Immunofluorescence Localization of Synapsin I and Synaptophysin in the Developing Rat Retina (A) Phase-contrast micrograph of a postnatal day 7 rat retina. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. (B) Synapsin I immunofluorescence reveals brightly immunoreactive perikarya in the inner retina (arrows). In addition, a population of presumptive horizontal cells exhibits high levels of synapsin I immunoreactivity in cell bodies and in their processes within the OPL (arrowheads). (C) Synaptophysin immunolabeling of an adjacent section revealsterminals of developing photoreceptors in the forming OPL (arrows) that are unlabeled with anti-synapsin I. Antibodies used: synaptophysin, G-62/63; synapsin I, 14. Bar, 20 p.m.

that these two proteins are c o m m o n to virtually all SSVs. In contrast, anti-synapsin 1/11 staining consistently produced five peaks in the IPL, a feature never observed in the densitometric profiles of synaptophysin or SV2 immunostaining. Synapsin la and Ib immunoreactivities had a relatively even profile across the w i d t h of the IPL w i t h a trough of lower intensity between 40% and 70% of the IPL width. In contrast, both synapsin Ila and lib immunoreactivities had three major peaks at 25%, 55%, and 70%-100% of the IPL w i d t h . In addition, two minor peaks were detectable at 5% and 40% of the IPL width. Thus, the sublayering of the IPL revealed by the synapsin 1/11 antibody was largely due to the laminar distribution of synapsin Ila and, to a lesser extent, synapsin lib.

Discussion

Lack of Detectable Synapsin Immunoreactivity in Photoreceptors and Bipolar Cells The synapsins are thought to play a key role in the exocytotic release of neurotransmitter from conventional nerve terminals (De Camilli and Greengard, 1986). We have shown that ribbon-containing nerve terminals of the retina lack immunoreactivity for all of the synapsin isoforms, suggesting that the synapsins are not required for exocytosis at these synapses. The rat retina does contain proteins identical to brain synapsins, as characterized by immunoblotting. The studies presented here strongly suggest their localization to conventional synaptic terminals in the IPL.

Figure 6. SV2, Synaptophysin, and Synapsin Immunolabeling of Rat Bipolar Cell Terminals (A) An electron micrograph of the rat IPL showing large, vesicle-filled bipolar cell terminals (identifiable by the presence of ribbons, rb) surrounded by smaller, conventional synaptic terminals (asterisks). The majority of these large bipolar cell terminals were located in the deep (vitreal) portion of the IPL, near the ganglion cell layer. The terminal shown was located 1.8 I~m from a ganglion cell body. (B) With confocal microscopy, large, bulbous synaptophysin-immunoreactive terminals (arrows) are evident along the IPUGCL border. (C) Synapsin 1/11immunofluorescence of an adjacent section shows no labeling in the bulbous structures but a punctate rimming of synapsin-negative holes (arrows). (D-I) Double-label immunofluorescence of isolated bipolar cells demonstrates SV2 and synaptophysin immunoreactivity in their terminals (arrows), but no synapsin I immunoreactivity. Antibodies used: synaptophysin, MAb CZ1; SV2, MAb 10H3; synapsin 1/11,G-35/36; synapsin I, 14. Bars, 1 lam (A); 5 p.m (C); 20 lain (D); 10 p.m (G).

Neuron

28

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Figure 8. Differential Distribution of the Four Synapsins in Rat Retina (la and Ib) Both synapsin la and Ib immunofluorescence is present throughout the IPL. (lla and lib) In contrast, both synapsin Ila and lib immunofluorescence is concentrated in three sublayers of the IPL. Synapsin lib immunofluorescence is barely detectable above background, but is identical in pattern to that of synapsin Ila. Right-hand panelsdemonstrate the specificity of immunolabeling. Incubation of antibodies with corresponding peptides completely abolished immunofluomscence. Antibodies used: la, G-306; Ib, G-278; Ila, G-281; lib, G-211. Bar, 25 p.m.

A negative immunocytochemical result does not prove the absence of a molecule from a tissue or cell type. Theoretical and practical aspects of detection methods set a lower limit on the amount of antigen that can be detected, tack of an immunocytochemical signal can be the result of inaccessibility of antigen to antibody. In the present study, the latter is unlikely since terminals in the IPL were labeled by all synapsin antibodies tested and other synapti~ vesicle-associated antigens (SV2 and synaptophysin) in photoreceptor and bipolar terminals were accessible to antibody labeling both in tissue sections and in isolated cells. Whether synapsins I and II are truly absent from ribbon synapses or as present at levels too low for detection cannot be determined. Clearly, however, lack of synapsin immunoreactivity is a special feature of these terminals. In addition to retinal photoreceptors and bipolar cells, ribbon synapses are found in a variety of sensory transducing cells. It is of interest that vestibular hair cells also appear to lack synapsin I immunoreac-

tivity, although they contain synaptophysin immunoreactivity (Favre et al., 1986; Scarfone et al., 1988). It seems likely that other cells forming ribbon synapses are similarly devoid of the synapsins. Absence of the synapsins may be related to structural and functional differences between ribbon and conventional nerve terminals. In the latter, the synapsins are thought to connect small synaptic vesicles with F-actin. The regulation of this association by phosphorylation may play a crucial role in the control of neurotransmitter release. At ribbon synapses, the presynaptic terminal has a very different structural organization. The ribbon appears to be the key structural element enabling accumulation of vesicles and possibly their guidance to release sites. Synaptic vesicles are linked to ribbons by 30-50 nm filaments that are not recognized by antiactin antibodies (Usukura and Yamada, 1987). Cells forming ribbon synapses have other features in common: they respond to stimuli with slow, graded potential changes and do not generate action potentials in vivo. Electrical signals can only reach the site of

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Figure 9. Densitometric Analysis of Synaptic Vesicle Protein Immunofluorescence in Rat Retina Densitometric scans of rat retina sections showing the relative intensity of synaptic vesicle protein immunofluorescence across the synaptic layers (OPL, outer plexiform layer; IPL, inner plexiform layer). For comparison, the areal density of small synaptic vesicles (SSVs/p.m2; determined from electron micrographs) across the synaptic layers is shown. Synaptophysin and SV2 show remarkably similar intensity profiles, with large peaks in the OPL due to staining of rod synaptic terminals. The densitometric profiles of these two integral membrane proteins of synaptic vesicles are very similar to the SSVdensity profile in both the OPL and IPL. In contrast, none of the synapsinsshow significant OPL peaks.Synapsin 1/11has five peaks in the IPL. This sublayering was primarily attributable to the concentration of synapsins Ila and lib within these sublayers, seen as sharp peaks in their scan profiles. Points in the SSV density profile are the mean of 8 measurements + standard error of the mean. Other densitometric measurements are averaged profiles made from representative portions of negatives of immunofluorescence micrographs. Backgroundfluorescence(defined as fluorescence present in inner and outer nuclear layers) has been subtracted. Antibodies used: synaptophysin, MAb C7.1;SV2,MAb 10H3; la, G-306; Ib, G-278; Ila, G-281; lib, G-211.

release by electrotonic spread. Application of cable theory to current flow in a rat rod indicated that a maximal light-induced hyperpolarization in the outer segment would be attenuated to less than 200 I~V at the synaptic termir~al (Hagins et al., 1970). Furthermore, the authors calculated that the hyperpolarization induced by the absorption of a single photon, known to be detectable by human rods (Hecht et al., 1942), would be less than 5 I~V at the ribbon synapse. Such a synapse must be able to modulate its rate of neurotransmitter release in response to extremely small potential changes. This exquisite sensitivity probably requires a mechanism distinct from that present in conventional nerve terminals (i.e., synapsin-containing terminals), which release transmitter in response to the much larger voltage changes brought on by propagated action potentials. Experiments on the squid giant synapse demonstrated that transmitter release at such a "classic" nerve terminal requires a minimal depolarization of about 45 mV (Katz and Miledi, 1967), 4 orders of magnitude larger than that predicted for rod synaptic terminals. The ribbon synapse may provide sensory transducing cells and bipolar cells with an exquisitely sensitive stimulus-secretion coupling mechanism. A difference in sensitivity between ribbon and conventional synapses might arise at a number of loci. One candidate might be the presynaptic Ca2+ channels thought to play a critical role in vesicular neurotransmitter release (Augustine et al., 1988). Recent evidence suggests that the photoreceptor synapse uses a Ca2+ channel distinct from the L-type channel found in most nerve terminals (Kleinschmidt, 1989, Soc. Neurosci., abstract). Other candidate molecules may be proteins of the secretory machinery itself, including synaptic vesicle proteins. Identification of molecules unique to ribbon-containing terminals, including the molecular components of the ribbon itself, will be important steps in the elucidation of the mechanism of transmitter release at ribbon synapses. The presence of a presynaptic compartment with specialized biochemical, structural, and functional properties may be viewed as part of a program of cellspecific gene expression that distinguishes these cells from classic neurons. It is of interest that all of these cells lack the long, highly branched processes typical of most neurons and secrete neurotransmitters either directly from the basal pole or from one or a few short processes originating from this pole. Furthermore, at least photoreceptors and bipolar cells have in common a lack of the high molecular weight microtubuleassociated protein MAP-2 and neurofilament proteins (Dr~iger, 1983; Shaw and Weber, 1984; De Camilli et al., 1984). These two proteins are expressed by virtually all CNS neurons. However, the expression of another microtubule-associated protein, tau, suggests a subdivision of cells that form ribbon synapses: it is found in bipolar cells (as well as amacrine and ganglion cells) but not in photoreceptors (Tucker and Matus, 1988). As our knowledge of neuronal cell lineage and

Neuron 3O

gene expression grows, a molecular taxonomy of neural cells is emerging.

Expression of Synapsin I by Developing Retinal Neurons Identification of populations of cell bodies containing high levels of synapsin I immunoreactivity in the developing rat retina supported the finding that ribbon-containing cells lack synapsin I. As in the adult, developing photoreceptors were devoid of synapsin I immunoreactivity whereas their terminals contained abundant immunoreactive synaptophysin and SV2. A previous study has shown that these developing terminals also contain another integral membrane protein of synaptic vesicles, p65 (Sarthy and Bacon, 1985). In contrast, inner retinal neurons, presumably amacrine and ganglion cells, which form only conventional synapses, expressed abundant synapsin I in both somata and processes. Synaptophysin and SV2 immunoreactivity in these neurons was seen only in processes in the forming IPL. These observations suggest a differential subcellular localization of integral and peripheral membrane synaptic vesicle proteins during development. High levels of synapsin I expression during development may result in accumulation of a large nonvesicular pool. Indeed, in vitro studies of developing neurons show that prior to cell- cell contact, diffuse synapsin I immunostaining is seen throughout the cytoplasm. Upon contact with postsynaptic neurons, however, synapsin I becomes concentrated at presynaptic active zones (1".L. Fletcher, P. Cameron, P. De Camilli, and G. Banker, submitted). In addition to presumptive amacrine and ganglion cells, a population of synapsin I-immunoreactive somata, thought to be horizontal cells, was observed adjacent to the forming OPL. These cells appeared very similar in morphology and position to cells previously identified by their immunoreactivity for glutamate decarboxylase in developing mouse retina (Schnitzer and Rusoff, 1984) and for y-aminobutyric acid, calbindin, and corticotropin releasing factor in the developing rat retina (l"rojanczyk et al., 1988, Soc. Neurosci., abstract; Versaux-Botteri et al., 1989). However, expression of these transmitter traits and synapsin immunoreactivity is transient, making positive identification of cell type difficult. The transient expression of high levels of synapsin I together with a variety of neurotransmitter phenotypes suggests that these substances may be released by a conventional vesicular mechanism during development of the OPL. Although some mature horizontal cells form conventional synapses (Dowling et al., 1966), anatomical and physiological studies have suggested that horizontal cells make predominantly nonvesicular synapses and release neurotransmitter in a Ca2÷-independent manner (Dowling and Boycott, 1966; Schwartz, 1987). The punctate synapsin I immunoreactivity seen in the adult OPL is consistent with the presence of a few conventional, vesicular synapses in horizontal cell terminals. However, some of the OPL puncta are proba-

bly terminals of interplexiform cells, which are known to make conventional, vesicle-containing synapses in the OPL of many vertebrate retinas (Dowling and Ehinger, 1975; Kolb and West, 1977).

Heterogeneous Distribution of Synapsins I and II in the Inner Plexiform Layer We have found a marked difference between the distributions of synapsin I- and synapsin II-immunoreactive terminals in the IPL. Nerve terminals immunoreactive for synapsins la and Ib were distributed across the width of the IPL. In contrast, terminals imrnunoreactive for synapsins Ila and lib were concentrated within three discrete sublayers. Since virtually all conventional synapses in the mammalian IPL are made by amacrine cell processes, we attribute the differential localization of the synapsins to heterogeneity of amacrine cell terminals. Interplexiform cells make conventional synapses in the IPL, but far fewer in number than amacrine cell synapses. In amphibian retinas, single bipolar cells have been shown to make both conventional and ribbon synapses (Wong-Riley, 1974). It would be of interest to determine whether these cells express synapsin and, if so, whether it is exclusively associated with conventional synapses. However, bipolar cells in mammalian retinas form only ribbon synapses (Dowling and Boycott, 1966). Ganglion cell dendrites are thought to be only postsynaptic in the mammalian retina, though conventional synapses made by ganglion cell dendrites have been documented in the catfish retina (Sakai et al., 1986). Thus, the observed differential localization of synapsins I and II in the rat I PL is likely to be due to a widespread expression of the synapsin I gene in all amacrine cells and a selective expression of the synapsin II gene in a subset of amacrine cells. Alternatively, such a distribution could arise from the selective transport of one or more synapsins to individual terminals of single amacrine cells. We consider the first possibility to be a more likely explanation of the observed heterogeneity. Stratification of the IPL has been revealed in many studies of the inner retina. Golgi impregnation studies have revealed precise patterns of lamination by amacrine cell processes (Ram6n y Cajal, 1892). Localization of neurotransmitters in the IPL has demonstrated specific patterns of process lamination for distinct populations of amacrine cells. These data suggest a five-sublayer scheme by which to classify such stratification (Karten and Brecha, 1983; Marc, 1986). Our localization of the synapsins revealed five sublayers, consistent with this scheme. A simple hypothesis to explain the localization of synapsin II would be that it is expressed by amacrine cells of particular neurotransmitter phenotypes. However,the localization of synapsins Ila and lib, primarily in sublayers 2, 4, and 5, is not consistent with the sublayering pattern of any known single neurotransmitter. The positions of the two narrow bands of synapsin II immunoreactivity were found to correspond exactly to those of cho-

~ napsinsin Retina

linergic amacrine cell terminals (sublayers 2 and 4), i d e n t i f i e d by i m m u n o l o c a l i z a t i o n w i t h an a n t i - c h o line acetyltransferase a n t i b o d y (Voigt, 1986; o u r unp u b l i s h e d data), but t h e third, w i d e r layer of synapsin II i m m u n o r e a c t i v i t y d i d not c o i n c i d e w i t h a c h o l i n e acetyltransferase-positive sublayer. Thus, the presence (or absence) of synapsin II does not a p p e a r to be exclusively linked to a single n e u r o t r a n s m i t t e r p h e n o type. It w i l l be of interest to d e t e r m i n e w h e t h e r the presence o r absence of synapsin II correlates w i t h some o t h e r p r o p e r t y of conventional presynaptic terminals, such as the class of n e u r o t r a n s m i t t e r released (e.g., excitatory versus i n h i b i t o r y ) o r the t e m p o r a l pattern of release. In c o n c l u s i o n , we have d e m o n s t r a t e d t h e existence of significant differences in t h e d i s t r i b u t i o n of t h e synapsins a m o n g a variety of retinal synapses. O n the o n e hand, t h e r i b b o n - c o n t a i n i n g t e r m i n a l s of phot o r e c e p t o r s and b i p o l a r cells lack both synapsins I and II. O n t h e o t h e r hand, t e r m i n a l s of a m a c r i n e cells may express o n l y synapsin I, o r in some cases, both synapsins I and II. In contrast to this heterogeneous d i s t r i b u t i o n of the synapsins, intrinsic m e m b r a n e proteins of small synaptic vesicles, i n c l u d i n g SV2 and • synaptophysin (present paper; Buckley and Kelly, 1985; W i e d e n m a n n and Franke, 1985), as well as p65 (Matt h e w et al., 1981), have a m o r e w i d e s p r e a d d i s t r i b u t i o n . This suggests that some b i o c h e m i c a l features are shared by all small synaptic vesicles, i n c l u d i n g the small synaptic vesicles of r i b b o n synapses. It is possible that t h e heterogeneous d i s t r i b u t i o n of t h e synapsins is related t o variations in the regulation of stimulus-secretion c o u p l i n g a m o n g different cell types. Experimental Procedures Antibodies The primary antibodies used in this study have been previously characterized and are listed in Table 1. Rhodamine- and fluorescein-conjugated goat anti-rabbit and goat anti-mouse IgGs were obtained from Organon Teknika/Cappel (Malvern, PA).Alkaline phosphatase-conjugated anti-rabbit and anti-mouse antibodies were from Promega (Madison, WI) or PeI-Freez(Rogers, AR). Colloidal gold-conjugated protein A (5 nm diameter) was from Janssen (Belgium). Tissue Preparation Light-adapted adult female Sprague-Dawley rats were given a lethal dose of Chloropent (Ft. Dodge Labs,Ft. Dodge, IA). The eyes were removed and placed in oxygenated Hank's balanced salt solution (HBSS), and the anterior segments were cut away. For immunocytochemistry, the resulting eyecup was fixed by immersion in 4% formaldehyde in 0.1 M phosphate buffer (PB; pH 7A) at 4°C for 3-12 hr. After 3 rinses in PBS, fixed eyecups were infiltrated with 30% sucrose in PBSat 4°C for at least 12 hr. The eyecups were embedded in a 1:1 mixture of Aquamount (Lerner Labs, New Haven, CT) and O. C. T. (Miles, Elkhart, IN) for 4 hr at 4°C (Jones et al., 1986) and frozen with -40°C freon. Sections (15-20 lain) were cut on a Bright model 5030 cryostat and mounted on gelatin-chromium potassium sulfate-coated slides. Light-adapted tiger salamanders (A. tigrinum; aquatic phase) were killed by decapitation, and the eyeswere placed in amphibian Ringer's solution. After removal of the anterior segment, the eyecup was fixed by immersion in 4% formaldehyde, 0.1 M PB and processed for immunofluorescence as described above.

Monkey (Macaca fasicularis) retina sections were generously provided by Dr. Peter MacLeish, Rockefeller University. Following an acute electrophysiological experiment, a deeply anesthetized animal was perfused with 4% formaldehyde, 0.1 M PB. Following 3 PBS rinses and sucrose infiltration, eyes were embedded in O. C. T., cryosectioned, and mounted as above. Immunoblolfin8 Rat retina and frontal cortex were homogenized in boiling 1% SDS. Protein concentration was measured by the method of Bradford (1976).Tissue homogenates were used immediately or stored at -20°C. Samples (10 p.g of protein per lane for cortex and 40 I~g of protein per lane for retina) were subjected to SDS-PAGE (Laemmli, 1970) and transferred to nitrocellulose (rowbin et al., 1979). Proteins were visualized by staining with Ponceau-S. All incubations were performed at room temperatu re. The blots were blocked with PBS,2.5% nonfat dry milk, 0.1% Tween-20 for 1 hr. Blots were probed with primary antibodies diluted in blocking solution (see Table 1 for dilutions) for 8-12 hr, washed in PBS,0.1%Tween-20,and then incubated with alkaline phosphatase-conjugated secondary antibodies in blocking solution. After 3 washes in PBS,0.1%Tween-20and a rinse in 50 mM Tris (pH 7.4),blots were reacted with Nitro Blue Tetrazolium, 5-bromo-4-chloro-3-indoyl phosphate p-toluidine salt, 10 mM MgCI2 in 10 mM Tris-HCI (pH 9.5). Cell Dissociation The procedure was similar to that previously described for rabbit retina (lownes-Anderson et al., 1988) with the following changes. The retina was removed from the eyecup in HBSS and immediately placed in Ringer's solution with papain as described, but without ascorbic acid and with 7 U/ml papain (Worthington, Malvern, PA). In some experiments, CaCI2was omitted from the enzyme solution. Retinas were incubated in the enzyme solution with agitation at 37°C for 15 min for photoreceptor dissociation and 45 rain for bipolar cell dissociation. After 3 rinses in HBSS + 0.05% BSA, retinas were gently triturated with a wide-bore glass pipette. The resulting cell suspension (100 I~1) was fixed by adding 1 ml of 4% formaldehyde in PBS.After 30 rain in fixative, cells were washed 3 times by centrifugation in 5 mM PB (pH 7.4)and dried onto 1% agarose-coated slides (McGrail and Sweadner, 1986). Immunofluorescence Frozen sections and dried cell suspensions were processed identically for indirect immunofluorescence. Goat serum dilution buffer (GSDB; 450 mM NaCI, 17% goat serum, 0.3% Triton X-100, 20 mM sodium phosphate buffer [pH 7A]) was used in all antibody incubations. Immunolabeling was performed as follows: -Incubation in GSDB for 30 min. -Incubation in primary antibody or normal rabbit serum diluted in GSDB for 3-12 hr at room temperature. -Three 10 rain rinses in wash buffer (450 mM NaCI, 0.3% Triton X-100, 20 mM sodium phosphate buffer [pH Z4]) and one in PBS (30 rain). -Incubation in secondary antibody diluted in GSDB for 2 hr. -Three 10 rain rinses in wash buffer and one in PBS (30 rain) followed by a 15 rain rinse in 5 mM PB (pH 7.4). For double-label experiments, primary antibodies were added to tissue sections or cells simultaneously. Slides were coverslipped with 90% glycerol, 10% PBS, 2.5% (w/v) 1,4 diazobicyclo[2,2,2]octane (Johnson et al., 1982) and examined with a Zeiss ICM 405 photomicroscope equipped for epifluorescence. Black and white photographs were taken with Kodak T-Max 400 film (developed with D-76 diluted 1:1), and color photographs were taken with Kodak Ektachrome 160 film. A Dage/MTI 68 video camera was used to acquire images of black and white negatives. Computerized densitometric scans of digitized images were performed with the JAVAimage analysis program (Jandel Scientific, Corte Madera, CA) using the vertical intensity average function. Confocal fluorescence microscopy was performed with a Sarastro Phoibos 1000, equipped for fluorescein

Neuron 32

excitation. Black and white photos were taken directly from the video monitor. The specificity of the immunofluorescence technique was tested by omitting the primary antibodies or by substituting an irrelevant primary antibody from the same species. In either case, the observed fluorescence was no higher than the tissue autofluorescence. The cross-reactivity of the fluorescent secondary antibodies was tested by applying a rabbit primary antibody followed by a goat anti-mouse secondary, or a mouse primary antibody followed by a goat anti-rabbit secondary. In both cases, no immunofluorescence was observed. For the anti-pep tide antibodies, immunofluorescence was abolished by incubating the diluted primary antibody with the appropriate free peptide at 0.5 mg/ml for 1 hr (see Figure 8); staining was unaffected by incubation with an unrelated synapsin peptide at the same concentration. Electron Microscopy and Electron Microscopic Immunolabeling For conventional electron microscopy, rat retinas were fixed by immersion in 4% formaldehyde, 0.5% glutaraldehyde, 0.1 M PB; postfixed in 1% OsO4, 1.5% potassium ferrocyanide; stained en bloc with 1% uranyl acetate; dehydrated; and embedded in Epon. To quantify the areal density of SSVs across the plexiform layers, ultrathin sections were collected on formvar-coated, onehole grids. A photographic montage measuring 100 x 20 p.m and including both the inner and outer plexiform layers was made and divided into vertical strips 0.7 p.m wide. Profiles of SSVswere counted within strips using a grid overlay. Profiles were counted as SSVs if they showed a complete circular profile and had a diameter of 40-60 nm. Eight separate strips were counted and averaged. For immunocytochemistry, rat retinas were fixed by immersion in 4% formaldehyde, 0.1 M PB and ultrathin cryosectioned and immunolabeled as described previously (Navone et al., 1986). Sections were viewed on a Jeol CXII electron microscope. Acknowledgments We thank Dr. Kathleen Buckley for the gift of the anti-SV2 monoclonal antibody (10H3), Dr. Michael Browning for the synapsin Ila/llb monoclonal antibody (19.31) and synapsin fill antiserum (G-35/36), Dr. Reinhard Jahn for the anti-p38 antiserum (G-62/63) and monoclonal antibody (C7.1), Dr. David Hicks for the antiopsin monoclonal antibody (4D2), and Dr. Peter MacLeish for the Sal-1 monoclonal antibody and the monkey retina sections. We are grateful to Hans Stukenbrok for performing the ultrathin cryosectioning and to Dr. Ross Smith for providing the use of and assistance with the Sarastro confocal microscope at the NYU School of Medicine. This work was supported by NIH grants EY06135(E. T.-A.)and MH39327 (P. G.). P. D. C. is supported by a Muscular Dystrophy Association grant. J. W. M. is the recipient of a Life and Health Insurance Medical Research Fund Scholarship. Received February 14, 1990; revised May 1, 1990.

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3S~napsins in Retina

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