Synaptic vesicle alterations in rod photoreceptors of synaptophysin-deficient mice

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Neuroscience Vol. 107, No. 1, pp. 127^142, 2001 ß 2001 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522 / 01 $20.00+0.00

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SYNAPTIC VESICLE ALTERATIONS IN ROD PHOTORECEPTORS OF SYNAPTOPHYSIN-DEFICIENT MICE I. SPIWOKS-BECKER,a L. VOLLRATH,a M. W. SEELIGER,b G. JAISSLE,b L. G. ESHKINDc and R. E. LEUBEa * a b

c

Department of Anatomy, Johannes Gutenberg University, Becherweg 13, 55128 Mainz, Germany

Retinal Electrodiagnostics Research Group, Department of Pathophysiology of Vision and Neuroophthalmology, University Eye Hospital, SchleichstraMe 12^16, 72076 Tu«bingen, Germany

Laboratory of Mouse Genetics, Institute of Toxicology, Johannes Gutenberg University, Obere Zahlbacher StraMe 67, 55131 Mainz, Germany

AbstractöThe abundance of the integral membrane protein synaptophysin in synaptic vesicles and its multiple possible functional contributions to transmitter exocytosis and synaptic vesicle formation stand in sharp contrast to the observed lack of defects in synaptophysin knockout mice. Assuming that de¢ciencies are compensated by the often coexpressed synaptophysin isoform synaptoporin, we now show that retinal rod photoreceptors, which do not synthesize synaptoporin either in wild-type or in knockout mice, are a¡ected by the loss of synaptophysin. Multiple pale-appearing photoreceptors, as seen by electron microscopy, possess reduced cytoplasmic electron density, swollen mitochondria, an enlarged cell surface area, and, most importantly, a signi¢cantly reduced number of synaptic vesicles with an unusually bright interior. Quanti¢cation of the number of synaptic vesicles per unit area, not only in these, but also in all other rod terminals of knockout animals, reveals a considerable reduction in vesicles that is even more pronounced during the dark period, i.e., at times of highest synaptic activity. Moreover, activity-dependent reduction in synaptic vesicle diameter, typically occurring in wild-type mice, is not detected in knockout animals. The large number of clathrin-coated pits and vesicles in dark-adapted synaptophysin knockout mice is taken as an indication of compensatory usage of synaptophysin-independent pathway(s), and, conversely, in view of the overall reduction in the number of synaptic vesicles, as an indication for the presence of another synaptophysin-dependent synaptic vesicle recycling pathway. Our results provide in vivo evidence for the importance of the integral membrane protein synaptophysin for synaptic vesicle recycling and formation. ß 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: retina, ribbon synapse, knockout mouse, synaptic vesicle protein, vesicle tra¤c, ERG.

Johnston and Su«dhof, 1990). Multiple phosphorylation sites are present in its cytoplasmic carboxy-terminus and are subject to serine and tyrosine phosphorylation by enzymes that co-distribute with synaptophysin in the same vesicles (Pang et al., 1988; Barnekow et al., 1990; Rubenstein et al., 1993; Janz et al., 1999). Each of the intravesicular loop domains contains a pair of cysteines that form intraloop disul¢de bonds (Johnston and Su«dhof, 1990). An N-glycosylation site is located in the ¢rst loop (Leube et al., 1989). Synaptophysin is a member of a small polypeptide family including synaptoporin (also referred to as synaptophysin II), which is often coexpressed with synaptophysin in SVs (Knaus et al., 1990; Fykse et al., 1993), pantophysin, which is present in ubiquitous transport vesicles (Leube, 1994; Haass et al., 1996; Windo¡er et al., 1999), and mitsugumin29, which has been detected in the triad junction of skeletal muscle and parts of the tubule system in kidney (Shimuta et al., 1998; Takeshima et al., 1998). The precise function(s) of synaptophysin are still largely unknown, despite numerous e¡orts to clarify its role. One major area of research has concentrated on the contribution of synaptophysin to the regulation of exocytosis in neurons and neuroendocrine cells. Originally, it

Synaptophysin is a homomultimeric integral membrane protein that is highly enriched in the `classic' electrontranslucent, 30^80-nm-diameter synaptic vesicles (SVs) of neurons and is also a major constituent of synaptic-like microvesicles of neuroendocrine cells (e.g., see Wiedenmann and Franke, 1985; Jahn et al., 1985). Detection of synaptophysin is a reliable indicator for the presence of these microvesicles, which occur exclusively in neuronal and neuroendocrine cells, and is widely used in tissue and tumor diagnosis (for reviews, see Bu¡a et al., 1988; Wiedenmann and Huttner, 1989). Synaptophysin is embedded in the membrane by means of four transmembrane regions, its amino- and carboxy-termini protruding into the cytoplasm (Leube et al., 1987;

*Corresponding author. Tel.: +49-6131-3922731; fax: +49-61313924615. E-mail address: [email protected] (R. E. Leube). Abbreviations : DA, dark-adapted ; ERG, electroretinogram; IPL, inner plexiform layer; LA, light-adapted ; OPL, outer plexiform layer; PBS, phosphate-bu¡ered saline; SCAMPs, secretory carrier-associated membrane proteins ; SR, synaptic ribbon; SV, synaptic vesicle. 127

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was proposed that the synaptophysin homomultimer forms a fusion pore through which neurotransmitter is released by coupling to a cognate plasma membrane receptor (Thomas et al., 1988). This hypothesis, however, has been challenged (Su«dhof and Jahn, 1991), and the putative receptor has not been found. The participation of synaptophysin in regulated secretion has also been suggested based on studies in which alterations in synaptophysin expression have been shown to a¡ect calcium-dependent exocytosis, although the reasons for its di¡ering e¡ects in di¡erent systems are not understood (Alder et al., 1992a,b, 1995; Sugita et al., 1999). The linkage of synaptophysin to the vesicular soluble N-ethylmaleimide-sensitive fusion factor attachment protein receptor synaptobrevin II may be one way in which synaptophysin regulates secretion of SV contents (Calakos and Scheller, 1994; Edelmann et al., 1995; Washbourne et al., 1995). Furthermore, synaptophysin may a¡ect transmitter release by the formation of a calcium-sensitive complex with myosin V, which is important for the translocation of SVs to presynaptic docking sites (Prekeris and Terrian, 1997). Another idea that has been pursued over many years is that synaptophysin is important for SV formation by speci¢c functional contributions to vesicle recycling and/or by `creating' its own type of vesicle. It has been demonstrated, in non-neuroendocrine cells lacking SVs and synaptic-like microvesicles, that cDNA-encoded synaptophysin is predominantly targeted to the constitutive endosomal recycling system, indicating a close relationship between this pathway and specialized SV recycling (Johnston et al., 1989; Cameron et al., 1991; Linstedt and Kelly, 1991; Leube et al., 1994). The recent ¢nding of a calcium-dependent association of synaptophysin with dynamin I at sites of SV fusion provides evidence for molecular interactions of synaptophysin in this process (Daly et al., 2000). We have also observed that a certain proportion of synaptophysin segregates from the pre-existing constitutive recycling vesicles in transgenic non-neuroendocrine cells and accumulates in distinct microvesicles, observations that have led us to propose that synaptophysin itself participates in vesicle formation (Leube et al., 1989, 1994; Leimer et al., 1996). In support of this, Thiele et al. (2000) have recently reported that synaptophysin binds speci¢cally to cholesterol and that the depletion of cholesterol impairs synaptic-like microvesicle formation in neuroendocrine PC12 cells. The signi¢cant reduction in the number of SVs occurring during high frequency stimulation of giant squid terminals injected with inhibitory synaptophysin peptides can be taken as another indication for the morphogenetic function of synaptophysin (Daly et al., 2000). To test the various hypotheses for synaptophysin function, we and others have generated synaptophysin knockout mice (Eshkind and Leube, 1995; McMahon et al., 1996). Remarkably, these animals are healthy and show no apparent defect, except for a slight decrease in synaptobrevin II expression (McMahon et al., 1996), thereby demonstrating that synaptophysin is not essential in itself in mice. The reason for this seeming paradox between the multiple functions expected from several

experimental observations and the lack of defects in knockout animals is not known, although it is possible that synaptophysin isoforms take over some of the functions of synaptophysin. Furthermore, secretory carrier-associated membrane proteins (SCAMPs) and synaptogyrins that share the same tetraspan membrane topology with synaptophysin and that are also expressed in cytoplasmic microvesicles including SVs (Brand et al., 1991; Brand and Castle, 1993; Stenius et al., 1995; Singleton et al., 1997; Janz and Su«dhof, 1998) may correct the loss of synaptophysin. The observation that a double knockout of synaptophysin and synaptogyrin I results in a phenotype with a reduction of short- and long-term synaptic plasticity in hippocampal neurons supports this notion (Janz et al., 1999). However, given the abundance of synaptophysin in SVs (Schlaf et al., 1996), it is hard to believe that any compensatory mechanism would be complete. Based on careful analyses, we now report activity-dependent alterations of SV number and morphology in rod photoreceptors of synaptophysin-de¢cient mice, i.e., in neurons lacking detectable levels of synaptoporin. EXPERIMENTAL PROCEDURES

Animals All animal experiments were carried out in accordance with guidelines laid down in the European Communities Directive of 24 November 1986 (86/609/EEC), and all e¡orts were made to minimize animal su¡ering and to use only the number of animals necessary to produce reliable scienti¢c data. The synaptophysin knockout mice were kept and bred in our institute and C57BL/6 mice were obtained from Harlan Winkelmann, Borchen, Germany. The recently described synaptophysin knockout mice (Eshkind and Leube, 1995) containing inactivated synaptophysin gene(s) on the X-chromosome were propagated by continuous inbreeding for several generations. For experimental analysis of littermates, female knockouts were ¢rst mated with wild-type C57BL/6 males. Heterozygous female o¡spring were then crossed with knockout male o¡spring thereby producing heterozygous/knockout female siblings and wild-type/knockout male siblings. To study the e¡ect of heterozygosity, heterozygous female animals were mated with C57BL/ 6 males generating wild-type and heterozygous female o¡spring. Animals were kept under constant laboratory conditions (12 h light, 12 h dark; lights on at 6.00 h and o¡ at 18.00 h; 100 lux at the bottom of the cages; 21 þ 2³C; 60% relative humidity; food and water ad libitum). For removal of tissue, mice (usually 9 weeks old) were anesthetized with ether and killed by decapitation or cervical dislocation either at 8.00 h in the light phase or at midnight during the dark phase under dim red light. The genotype was checked in each animal by genomic Southern blotting (Eshkind and Leube, 1995) and/or immunoblot analysis of postnuclear supernatants prepared from brain tissue (Leimer et al., 1996). Both retinae were routinely removed; one was usually processed for electron microscopical evaluation leaving the other as a reserve and/or for further analyses (polymerase chain reaction, immunoblotting, immuno£uorescence). Immuno£uorescence microscopy Eyeballs were frozen in isopentane that had been precooled in liquid nitrogen. To improve adhesion of tissue sections, glass slides were ¢rst cleaned by treatment with 1 M HCl for 30 min, washed twice for 5 min in water, rinsed with ethanol, and air-dried. Subsequently, they were treated brie£y with 3-aminopropyltrimethoxysilane (Sigma, St. Louis, MO, USA) that had been diluted in acetone (2% v/v), washed twice with

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acetone and air-dried. Cryostat sections (5^10 Wm thick) were mounted on the pretreated glass slides and dried for at least 1 day at room temperature. Sections were then treated with methanol for 5 min and with acetone for 10 min (both precooled to 320³C), and dried. Antibody incubations were as described (Leube et al., 1994). In some instances, primary antibodies were preadsorbed, prior to addition to the tissue section, with peptides that had been used for immunization (1 Wg/ml) for 30 min at room temperature in order to test their speci¢city. Epi£uorescence microscopy and recording were as recently described (Windo¡er et al., 1999). To prepare speci¢c antibodies against murine synaptoporin, peptides SyPo1 CHSSGQRYLSDPMEKHS and SyPo2 CGSSGGYSQQANLG were synthesized (Dr. Hans-Richard Rackwitz, DKFZ, Heidelberg, Germany) corresponding to non-overlapping regions of the cytoplasmic carboxy-terminus as determined from cDNA clone pPO2 (Eshkind and Leube, 1995); the amino-terminal cysteines were added for coupling. Peptides were coupled to maleimide-activated keyhole limpet hemocyanin (Pierce, Rockford, IL, USA). Guinea-pigs were immunized subcutaneously with 800 Wg coupled peptide, together with complete Freund adjuvant for the ¢rst injection or incomplete Freund adjuvant in subsequent injections, on days 0, 28, and 56. Blood was collected on day 61, and serum was used either directly or after a¤nity puri¢cation. For puri¢cation, peptides were coupled to UltraLink Iodoacetyl (Pierce) via sulfhydryls provided by the amino-terminal cysteines, according to the manufacturer's protocol. Antibody binding, the washing of the column, and elution were performed as described (Haass et al., 1996). In addition, the following primary antibodies were used: monoclonal antibodies against synaptophysin reacting either with the amino-terminus (kindly provided by Dr. Bertram Wiedenmann, Charite©, Humboldt University, Berlin, Germany; Eshkind and Leube, 1995) or the carboxy-terminus (antibody SY38; Wiedenmann and Franke, 1985) and polyclonal antibodies from rabbit against the cytoplasmic carboxy-terminus of synaptophysin (Dako, Hamburg, Germany). Secondary antibodies were: Cy3-conjugated goat anti-guinea-pig IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), Texas Red-conjugated goat anti-rabbit (Jackson ImmunoResearch Laboratories), and Cy2-conjugated goat anti-mouse IgG (Rockland, Gilbertsville, PA, USA).

viously described procedures (Biel et al., 1999). In brief, mice were dark-adapted (DA; overnight or for more than 6 h). Pupils were dilated with tropicamide eye drops (Mydriaticum Stulln, Pharma Stulln, Nabburg, Germany) and phenylephrine eye drops (Neosynephrin-POS 5%, Ursapharm, Saarbru«cken, Germany). Anesthesia was induced by s.c. injection of ketamine (Ketanest, Parke-Davis, Berlin, Germany; 66.7 mg/kg body weight), xylazine (Rompun, Bayer Vital, Leverkusen, Germany; 11.7 mg/kg body weight), and atropine (Atropin, Braun, Melsungen, Germany; 1 mg/kg body weight). Silver needle electrodes served as the reference (forehead) and ground (tail), and gold wire ring electrodes were used as active electrodes. Methylcellulose (Methocel, Ciba Vision, Wessling, Germany) was applied to ensure optimal electrical contact and to keep eyes hydrated during the entire procedure. The ERG equipment consisted of a Ganzfeld bowl, a DC ampli¢er, and a PC-based control and recording unit (Toennies Multiliner Vision, Jaeger/ Toennies, Ho«chberg, Germany). ERGs were recorded from both eyes simultaneously after placement of mice in the Ganzfeld bowl. The bandpass ¢lter cut-o¡ frequencies were 1 and 300 Hz. Single-£ash and £icker recordings were obtained under both scotopic and photopic conditions. Single £ash stimuli were presented with increasing intensities, ranging from 1034 cd/m2 to 25 cd/m2 , divided into 10 steps of 0.5 and 1 log cd/ m2 . Five to 10 responses were averaged with an inter-stimulus interval of 5 s (for 1034 , 1033 , 1032 , 3U1032 , 1031 , 3U1031 cd/ m2 ) or 17 s (for 1, 3, 10, 25 cd/m2 ). Flicker stimuli had an intensity of 3 cd/m2 with frequencies of 0.5, 2, 5, 10, 15, and 30 Hz. Light adaptation was performed with a background illumination of 30 cd/m2 presented for 10 min to reach a stable level of the photopic responses (Peachey et al., 1993). Furthermore, the time course of recovery from the photopic condition within a period of 40 min darkness was measured by using a £ash intensity of 1 cd/m2 .

Electron microscopy

The most likely candidate protein to compensate for the loss of synaptophysin in knockout mice is synaptoporin given the largely overlapping expression pro¢le of both physin isoforms (see Marque©ze-Pouey et al., 1991; Fykse et al., 1993). We therefore wanted to identify neuronal cell populations that expressed signi¢cant amounts of synaptophysin but no synaptoporin, and those that may have consequently exhibited de¢ciencies in synaptophysin null mice. To this end, antisera were prepared against two non-overlapping peptides corresponding to parts of the speci¢c cytoplasmic carboxy-terminus of murine synaptoporin (Eshkind and Leube, 1995). Using four di¡erent antisera and a¤nity-puri¢ed fractions thereof, we found that the retinal outer plexiform layer (OPL) was virtually devoid of synaptoporin immunolabel both during the light (Fig. 1C, D) and during the dark period (Fig. 1K, L), whereas a strong signal was detectable in the inner plexiform layer (IPL) in the same sections. The signal could be competed out with the peptides used for immunization but not with unrelated peptides of similar length and composition (data not shown). In contrast, antibodies directed against either the aminoor the carboxy-terminus of synaptophysin reacted strongly with both the IPL and the OPL of light-adapted (LA) mice (Fig. 1A, B), and the same expression pattern

Retinae were dissected from rapidly removed eyeballs and ¢xed in freshly prepared ¢xative, viz., 2% paraformaldehyde, 2.5% glutaraldehyde in phosphate-bu¡ered saline (PBS) for 15 h. Subsequently, tissue was rinsed in PBS containing 6.8% (w/v) sucrose, post¢xed in osmium tetroxide (2% w/v in PBS) for 90 min, washed three times in PBS, and dehydrated in a graded series of acetone. Tissue was £at-embedded in Epon (Serva, Heidelberg, Germany). Transverse sections (50^60 nm thick) were mounted onto one-hole Formvar-coated copper grids (Serva), stained with 8% (w/v) uranyl acetate (10 min), and contrasted with lead citrate for 5 min (Reynolds, 1963). Sections were viewed in an EM 109 (Zeiss, Oberkochen, Germany), and morphometry was carried out by means of a Morphomat (IDMS, IMA, Dortmund, Germany) directly at the microscope or on prints at a ¢nal magni¢cation of 80 000. All morphological analyses were performed blind, i.e., without knowledge of the genotype, by using coded specimens. Statistical analysis The data obtained are expressed as means þ S.E.M. For statistical analysis, Student's t-test or the Wilcoxon Mann^Whitney U-test was used. A value of 0.05 was regarded as signi¢cant. Electroretinography Electroretinograms (ERGs) were obtained according to pre-

RESULTS

Synaptoporin is not detectable in the outer plexiform layer of the retina in either wild-type or synaptophysin knockout mice

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Fig. 1. Indirect immuno£uorescence microscopy of murine retina depicting the distribution of the synaptic vesicle proteins synaptophysin (Sy; A, B, E, F, I, J) and synaptoporin (SyPo; C, D, G, H, K, L) in wild-type (wt) and synaptophysin knockout (ko) mice during light (light) and dark adaptation (dark). Synaptophysin was detected with antibodies directed against either the cytoplasmic amino-terminus (anti-N-Sy) or the cytoplasmic carboxy-terminus (anti-C-Sy). Note the complete absence of synaptophysin immunoreactivity in the IPL and OPL of knockout mice (brackets in E and F). Synaptoporin was identi¢ed by reacting with non-overlapping peptides corresponding to the cytoplasmic end of murine synaptoporin, i.e., either peptide SyPo1 (anti-SyPo1) or peptide SyPo2 (anti-SyPo2). Note the absence of synaptoporin immunoreactivity in the IPL of wild-type and knockout animals (bracket in C, D, G, H, K and L). Scale bar = 30 Wm.

was seen in DA mice (Fig. 1I, J). As expected, no signal was obtained with synaptophysin antibodies in knockout mice (Fig. 1E, F). Furthermore, the synaptoporin antibodies elicited, in knockout mice, a £uorescence pattern that was indistinguishable from that of wild-type controls (for LA animals compare Fig. 1G, H with Fig. 1C, D) indicating that no compensatory up-regulation of synaptoporin expression had occurred in the OPL in the absence of synaptophysin.

Vesicle-depleted pale-appearing and dilated rod photoreceptor terminals with multiple clathrin-coated vesicles are detectable in synaptophysin null mice The identi¢cation of the OPL as a distinct synaptic layer without signi¢cant amounts of synaptoporin prompted us to examine this area in synaptophysin knockout mice in comparison with that of wild-type littermates in detail by electron microscopy. Of all photo-

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receptors in the mouse retina, 98% are rods. The rod terminals contain a large number of densely clustered SVs with a narrow size distribution. Prominent features of these nerve endings are their electron-dense bodies, the so-called synaptic ribbons (SRs) that form variously shaped three-dimensional plates and are subject to dynamic changes depending on light exposure (for mouse, see Adly et al., 1999; Spiwoks-Becker et al., 2000). SVs are attached to the entire surface of SRs and to each other by thin stalks of up to 35 nm in length. The proximal SR ends are anchored by short ¢laments to the electron-dense arciform density, which is located underneath the plasma membrane in a region at which neurotransmitter vesicle exocytosis probably occurs (e.g., Gray and Pease, 1971; Brandon and Lam, 1983; RaoMirotznik et al., 1995). The presynaptic photoreceptor regions are in close contact with the conspicuous postsynaptic triads consisting of the dendritic process of the bipolar neuron facing the arciform density and £anking axonal extensions of two horizontal interneurons with scattered vesicles. In the ¢rst survey of knockout mice, unusually paleappearing terminals were noted in the OPL; these often occurred in groups and were interspersed between darker terminals. A typical example is shown in Fig. 2a depicting the less densely stained cytoplasm and the overall reduction of electron-lucent vesicles. Closely spaced vesicles were still attached to SRs whose morphology appeared normal (Fig. 2c). The arrangement of the synaptic complex was indistinguishable from that of wildtype controls and neighboring darker terminals in knockout mice. Many mitochondria were swollen and dilated and contained disrupted tubules. Pale rod terminals were found in LA and in DA knockout mice (Fig. 2). When present, approximately 18% of all terminals could be classi¢ed as pale. The number of animals with pale terminals was higher under light conditions (in seven of nine animals) than under dark conditions (in four of 12 mice). No pale-appearing cone pedicles were seen in any of the animals examined. In a few instances, pale terminals were also detected in heterozygous female ani-

131

mals (not shown), but none was observed in any of the control wild-type mice. Area measurements of pale-appearing terminals in LA and DA knockout animals showed that they were considerably larger (3.5 þ 0.20 Wm2 ; n = 22 terminals; P 6 0.001) than in neighboring dark terminals (2.7 þ 0.06 Wm2 ; n = 75 terminals) or dark terminals in wild-type mice (2.8 þ 0.06 Wm2 ; n = 105 terminals). Multiple omega-shaped pro¢les indicative of ongoing endo-/ exocytotic membrane tra¤cking could be identi¢ed easily in pale terminals (Fig. 2c) and appeared to be slightly more frequent in LA mice (1.67 þ 0.55 pro¢les per ribbon complex; n = 9 terminals) than in DA animals (0.5 þ 0.27 pro¢les per ribbon complex; n = 8 terminals). Remarkably, a large number of clathrin-coated vesicles was noted in pale terminals of LA knockout mice (4.51 þ 0.54% of all SVs; n = 15 terminals; Fig. 2c). Furthermore, a signi¢cant increase (P 6 0.001) of clathrincoated vesicles was seen in DA animals (12.48 þ 1.51% of all SVs; n = 8 terminals; Fig. 2d). Clathrin-coated vesicles often occurred in groups and were mostly located away from SRs. Coated pits were only seen in pale terminals of LA knockouts (Fig. 2c) but not in any of the DA knockouts. The number of synaptic vesicles is reduced in all rod photoreceptor terminals of synaptophysin-de¢cient mice in an activity-dependent manner More detailed analyses were undertaken to determine whether the darker rod terminals of synaptophysin knockout mice also di¡ered from those of wild-type siblings during light and/or dark adaptation. The major results are summarized in Table 1. No di¡erences were found in the number of rod terminals per 35-Wm OPL or in the number of terminals without synaptic body pro¢les between the di¡erent groups. Analysis of the synaptic body pro¢les (approximately 100 per animal) showed that neither their number per 35-Wm OPL nor their shape (rod-like, spherical or club-shaped) or length was di¡erent between the wild-type and knockout groups. In addi-

Table 1. Morphometric analysis of the ultrastructures in photoreceptor rod terminalsa of light-adapted wild-type (wt; n = 6) and synaptophysin knockout mice (ko; n = 9), and of dark-adapted wild-type (n = 9) and synaptophysin knockout mice (n = 12)b Light-adapted

Rod terminals/35-Wm OPL Rod terminals without synaptic body pro¢les (%) Number of synaptic bodies/35-Wm OPL Fraction of spherical pro¢les (% of total synaptic bodies) Fraction of club-shaped pro¢les (% of total synaptic bodies) Length of transversely sectioned, rod-like SRs (Wm) Rod terminals with multiple vacuoles (%)

Dark-adapted

wt

ko

wt

ko

36.2 þ 1.4 19.9 þ 5.0 28.5 þ 1.1 2.0 þ 1.0 1.4 þ 0.8 0.261 þ 0.020 27.8 þ 6.9

37.4 þ 0.9 16.1 þ 2.1 30.7 þ 1.1 2.3 þ 1.0 2.8 þ 1.6 0.271 þ 0.012 26.9 þ 4.8

35.7 þ 1.2 16.8 þ 2.7 28.4 þ 2.0 n.d.c 0.2 þ 0.2 0.246 þ 0.005 6.9 þ 2.3d

35.3 þ 1.1 17.1 þ 2.2 28.2 þ 0.9 n.d. 0.4 þ 0.2 0.269 þ 0.010 19.0 þ 4.3e

a

Pale-appearing rod terminals are not included. In each case, data were ¢rst averaged for each animal, and then the mean of the values was calculated and is given in the table together with the S.E.M. c n.d., not detectable. d Signi¢cant reduction in comparison with LA wild-type animals (P 6 0.01). e Signi¢cantly higher percentage in DA knockout mice than in DA wild-type controls (P 6 0.05). b

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Fig. 2. Electron microscopy comparing rod photoreceptor terminals of synaptophysin-de¢cient mice (ko) under light adaptation (light) and dark adaptation (dark). The survey views in panels a and b each show a pale terminal (*) with very few SVs next to darker terminals. Note that many SVs are still attached to SRs in pale terminals (details in c and d). Omega pro¢les (arrows in c), clathrin-coated pits, and coated vesicles are also abundant (circles). Multiple unusually £attened (closed arrowheads) and bizarre (open arrowheads) vesicles are marked in panels c and d. *, vacuoles; H, horizontal cell process; M, mitochondrion. Scale bars = 1 Wm in a and b, 0.5 Wm in c and d.

tion, no other morphological SR alterations could be detected (e.g., lamellation, electron density, contours). Close inspection of practically all darker rod terminals of knockout mice revealed multiple vesicle-free areas and regions with a reduced vesicle density, i.e., a lower number of vesicles per unit area (see Figs. 3^5). We therefore determined vesicle density quantitatively in dark terminals of wild-type and knockout mice (see Table 2). Blind

analyses were carried out on prints (magni¢cation 80 000) of randomly selected rod terminals (on average 10 per animal; minimum of four animals per group). This showed that the number of SVs per Wm2 was signi¢cantly reduced in LA knockout mice (19% reduction versus wild-type; see also Fig. 5). An even more pronounced decrease in vesicle density was seen in DA knockout animals, i.e., during times of increased SV exo-

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Fig. 3. Survey electron micrograph depicting rod photoreceptor terminals in the retina of a synaptophysin-de¢cient mouse in the middle of the dark period (ko dark). Note that the overall architecture and the triad arrangement of synaptic contact sites (H, horizontal cell process; B, bipolar cell dendritic extension) is normal. Multiple omega-shaped pro¢les indicative of active vesicle exo- and/or endocytosis can also be seen (small arrows). The number of SVs is reduced in comparison to terminals of wild-type animals. Branching tubule-like structures (long arrows) and irregularly shaped vacuolar structures (arrowhead) are labelled. M, mitochondrion. Scale bar = 0.5 Wm.

Table 2. Number of synaptic vesicles per Wm2 of photoreceptor terminal (classi¢ed as dark and pale) and per Wm synaptic ribbon in wild-type (wt) control mice and synaptophysin-de¢cient (ko) mice during light and dark adaptationa Light-adapted wt

Dark-adapted ko

wt

ko

160.3 þ 10.5b (n = 33) 125.8 þ 7.4 (n = 22)

209.5 þ 13.8 (n = 49) not detectable

133.5 þ 11.5c (n = 39) 114.3 þ 10.8 (n = 9)

38.5 þ 3.6 (n = 33) 36.1 þ 2.5 (n = 22)

38.3 þ 2.4d (n = 49) not detectable

33.3 þ 2.4e (n = 39) 39.2 þ 3.5 (n = 9)

2

Number of SVs per Wm of photoreceptor terminal Dark terminals 197.0 þ 10.3 (n = 38) Pale terminals not detectable Number of SVs per Wm of SR Dark terminals 45.8 þ 1.1 (n = 38) Pale terminals not detectable a

In each case, the entire visible photoreceptor terminals were analyzed and an area comprising approximately 1000 SVs in four to 10 randomly selected rod terminals was evaluated in each of four animals (except for the DA wild-type group, where ¢ve animals were examined), and the results were calculated for each animal individually. The values shown correspond to the mean of these determinations together with the S.E.M. The total number of rod terminals examined is given as n in parentheses. b Signi¢cant decrease in comparison with LA wild-type terminals (P 6 0.05). c Pronounced decrease in comparison with DA wild-type terminals (P 6 0.01). d Signi¢cant reduction in comparison with LA wild-type terminals (P = 0.03). e No statistically signi¢cant di¡erence with LA knockout terminals.

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Fig. 4. Electron microscopic comparison of dark-adapted (dark) rod photoreceptor terminals in wild-type (wt; a) and synaptophysin-de¢cient mice (ko; b). The terminals shown are sectioned horizontally thereby depicting long SR pro¢les that are £anked on either side by a horizontal cell process (H). Note that the number of SVs is considerably decreased in panel b (vesicle-free areas demarcated by *). Furthermore, the vesicle size is more heterogeneous (some large vesicles are labeled by open arrowheads in b), and the vesicle interior appears lighter in the knockout animals. Scale bars = 0.5 Wm.

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Fig. 5. Comparison of the ultrastructural morphology of light-adapted (light) rod photoreceptor terminals of wild-type (wt) and synaptophysin null mice (ko) demonstrating the reduced number of SVs and pleomorphic vesicle appearance in knockout animals. (a) Clustering of homogeneously sized SVs can be seen in the presynaptic regions, whereas in panel b, vesicles are less densely packed (some vesicle-free regions are labelled by *) and di¡er considerably in size and shape (ellipsoid vesicles are indicated by long arrows). Note also that the vesicle interior appears brighter in the knockout terminal than in the wildtype terminal. Short arrows, presynaptic vacuoles; H, horizontal cell process ; B, bipolar cell dendrite; M, mitochondrion. Scale bars = 0.5 Wm.

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Fig. 6. Histogram showing the relative frequency (in %) of SV diameter distribution (in steps of 10 nm) in rod photoreceptor terminals of wild-type mice (wt; a, c) and synaptophysin-de¢cient mice (ko; b, d) determined during the light (light-adapted) or dark phase (dark-adapted). The number (n) of SVs measured and the number of animals examined are given in each plot. Note the higher proportion of large vesicles in DA knockout mice (d) in comparison to DA wild-type animals (c).

cytosis and reformation (36% reduction versus wild-type; see Figs. 3 and 4). In contrast, vesicle density did not di¡er signi¢cantly between LA and DA wild-type animals (compare Figs. 5a and 4a). Pale terminals of knockout mice were evaluated separately (see also Table 2) and showed, as expected, the highest reduction of vesicle density (36% for LA mice and 45% for DA mice). In contrast to the apparent depletion of free cytoplasmic vesicles in dark terminals of knockout mice, only a slight and statistically non-signi¢cant reduction was observed for the number of SR-associated SVs (less than 40 nm away; Table 2). The reduction of SV number per Wm SR between light and dark adaptation was smaller in knockout than in wild-type animals; this may have been attributable to di¡erences in vesicle size (see below). Next, the comparatively rare omega-shaped endo-/exocytotic pro¢les (a particularly rich region is indicated by small arrows in Fig. 3) were quanti¢ed for each group

(four animals per group). As expected, a signi¢cant increase (P 6 0.001) was noted between LA (0.36 þ 0.09 per terminal; n = 50 terminals) and DA wild-type mice (1.45 þ 0.20 per terminal; n = 66 terminals). Interestingly, a comparable signi¢cant di¡erence was also determined between light adaptation (0.53 þ 0.12 per terminal; n = 47 terminals) and dark adaptation (1.84 þ 0.26 per terminal; n = 49 terminals) in knockout mice. Because of the compact clustering of vesicles and the electron-dense cytoplasm, it was not possible reliably to quantify the number of clathrin-coated vesicles in dark terminals of either knockout or wild-type animals. The cytoplasm of some rod endings contained one or more vacuolar structures (e.g., see Fig. 5) that had diameters between 80 nm and 200 nm and that were more abundant in LA than in DA mice. Remarkably, DA knockout mice showed signi¢cantly more endings with vacuoles (19.0%) than wild-type mice (6.9%), whereas in the LA groups, there was no di¡erence (27.8% versus

Table 3. Synaptic vesicle diameter (in nm) in di¡erent retinal synapses of wild-type (wt) and synaptophysin-de¢cient (ko) micea Light-adapted wt Rods Cones Rod bipolar cells Amacrine cells Horizontal cells

32.4 þ 1.7 33.0 þ 1.4 34.4 þ 1.3 29.2 þ 1.3 38.6 þ 2.3

Dark-adapted ko

(n = 359) (n = 333) (n = 207) (n = 156) (n = 218)

35.2 þ 1.8 36.0 þ 1.4 34.7 þ 1.6 31.9 þ 1.0 41.3 þ 2.8

(n = 473) (n = 281) (n = 225) (n = 166) (n = 161)

a

wt

ko

27.9 þ 1.0b (n = 512) 31.1 þ 2.2 (n = 217) 29.4 þ 2.1 (n = 210) 31.3 þ 1.5 (n = 213) 40.7 þ 2.6 (n = 236)

38.3 þ 1.5c (n = 717) 38.6 þ 0.7d (n = 275) 35.2 þ 0.7e (n = 238) 31.4 þ 1.9 (n = 142) 42.5 þ 3.7 (n = 174)

The number of vesicles measured in each instance is given as n in parentheses. In each animal examined an average vesicle diameter was determined and the value shown in the table represents the mean of these results together with the S.E.M. For analysis of rods six LA wildtype, nine LA knockout, nine DA wild-type and 12 DA knockout animals were used. For the examination of the other cell types four animals were used for each group. b Signi¢cantly reduced vesicle diameter in comparison with LA wild-type animals (P 6 0.01). c Signi¢cantly larger vesicle diameter than in rod terminals of control group (P 6 0.001). d Signi¢cantly larger vesicle diameter than in cone terminals of control group (P 6 0.05). e Signi¢cantly larger vesicle diameter than in rod bipolar neurons of control group (P 6 0.05).

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Fig. 7. Electron microscopy showing details of ultrastructural abnormalities in rod photoreceptors of synaptophysin-de¢cient mice (all dark-adapted except in b). (a) Nucleus (N) of a pale terminal with irregularly shaped heterochromatin surrounded by large amounts of euchromatin (arrowheads; see also arrowhead in b) di¡ering from the densely stained nuclei of terminals in wild-type animals. Clustered ribosome-like particles are seen in the perinuclear region (arrows in b and d) and in the vicinity of the synaptic ribbon complex (b). SVs appear brighter in knockout synapses than in wild-type synapses (see b) and frequently present pronounced membrane irregularities (e.g., arrows in c). Also note the swelling and disruption of mitochondria (M) in panels a and d. H, horizontal cell process. Scale bars = 1 Wm in a and d, 0.5 Wm in b, and 0.25 Wm in c.

26.9%; see also Table 1). In some instances, tubulovesicular elements were noted such as those demarcated by long arrows in Fig. 3. Synaptic vesicle morphology is di¡erent in rod photoreceptors of synaptophysin knockout mice To be able to de¢ne discrete morphological changes,

high magni¢cation pictures of terminals were prepared for direct comparison. The examples shown suggest that vesicle size may di¡er between wild-type and knockout animals during dark adaptation (Fig. 4) but not light adaptation (Fig. 5). To de¢ne these di¡erences more precisely, SV diameter was measured at the electron microscope level at a magni¢cation of 80 000 in ribbon synapses (approximately 4000 SVs) and other synapses

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Fig. 8. Graphic display of ERG results. In each graph, the top dotted line denotes the 95% quantile, the bottom dotted line the 5% quantile determined from the control groups. Gray box plots show results obtained with synaptophysin knockout animals depicting the median (cross; connected by black line), the 75% and 25% quantile corresponding to top and bottom end of the box, and the 95% and 5% quantile corresponding to top and bottom end of the bar. (A) Relationship between bwave amplitude on the ordinate and logarithm of £ash intensity applied as single £ashes on the abscissa (combined data from either two heterozygote female wild-type animals, or one female and two male synaptophysin knockout animals) under scotopic (upper graph) and photopic (lower graph) conditions. (B) Time course of recovery of the b-wave amplitude of LA mice (data from four wild-type males and ¢ve knockout males) during 38.5 min of dark adaptation. Ph: photopic (lightadapted) ERG. (C, D) Relationship between amplitude measurements on the ordinate and £icker frequency on the abscissa determined under scotopic (C) and photopic (D) conditions (combined data from either two heterozygous female controls, or one female and two male knockout animals). Note that the measurements determined for synaptophysin null mice do not di¡er signi¢cantly from those obtained in control animals.

(approximately 1500 SVs of amacrine and horizontal cells). These analyses, summarized in Table 3, con¢rmed that, in LA mice, no signi¢cant di¡erences existed between vesicle diameter determined in rod terminals or the other presynaptic regions of wild-type and knockout animals. However, SV size was signi¢cantly reduced from 32.4 þ 1.7 nm to 27.9 þ 1.0 nm in rod photoreceptors of DA wild-type animals, whereas it did not di¡er signi¢cantly between LA and DA synaptophysin null mice (35.2 þ 1.8 nm versus 38.3 þ 1.5 nm). Consequently, vesicles were considerably larger in DA knockout than in control animals. An increased vesicle diameter was also noted in the DA state in the other major ribbon-containing neurons of murine retina, i.e., cone and rod bipolar cells, but not in amacrine and horizontal cell terminals. The histograms in Fig. 6 depicting the diameter distribution of SVs in rods demonstrate that DA knockout mice show higher numbers of large SVs than DA wild-type animals. In the latter, more than 45% of the SVs were smaller than 30 nm, whereas in knockout mice, only 11% had a diameter of less than 30 nm. Finally, the size dis-

tribution of SVs was independent of the distance to the SRs in rods (data not shown). The direct comparison of wild-type and knockout rod terminals also showed that the SV interior was brighter in DA and LA knockout animals in relation to the controls that had been processed in parallel (see comparisons in Figs. 4 and 5; but also the surveys in Figs. 3 and 7b and high magni¢cation in Fig. 7c). Similarly, SVs of cones and rod bipolar cells appeared brighter in knockout than in wild-type synapses (not shown). Furthermore, many £attened and unusually shaped or distorted SVs were noted in knockout animals (see especially Figs. 2c, d and 7c), and regular spotlike membrane densi¢cations were frequently seen (Fig. 7c). Rod photoreceptors of synaptophysin-de¢cient mice show additional synaptic vesicle-unrelated abnormalities Occasionally, chromatin was signi¢cantly altered in the nuclei of knockout photoreceptors with fragmented

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heterochromatin that was separated by electron-lucent euchromatin at the nuclear periphery (Fig. 7a). The nuclear membrane was still visible and apparently intact. In addition, several rod photoreceptor endings contained multiple polyribosome-like structures in the cytoplasm (Fig. 7b, d); these were never seen in terminals of wildtype mice. Flash and £icker amplitudes are normal in the electroretinogram under scotopic and photopic conditions ERG was performed to determine whether the morphological alterations in photoreceptor synapses could be picked up by electrophysiological measurements. The scotopic single-£ash ERG amplitudes of knockout males, representing rod function only at low stimulus intensities, and a mixed response of rods and cones at higher intensities (usually above about 1031 cd/m2 ), were within normal limits (Fig. 8A). Moreover, the £ash ERG amplitudes recorded under photopic, rod-suppressive conditions were not di¡erent between synaptophysin null and control animals (Fig. 8A). The time course of b-wave recovery from LA conditions, examined to assess potential dynamic changes during dark adaptation, was the same in wild-type and knockout mice within 38.5 min after termination of background illumination (Fig. 8B). Furthermore, responses to trains of £ashes (£icker), under both scotopic (Fig. 8C) and photopic (Fig. 8D) conditions, were normal in male knockout mice when compared with heterozygous female controls.

DISCUSSION

The aim of this study was to identify defects in the seemingly normal synaptophysin-de¢cient mouse (Eshkind and Leube, 1995; McMahon et al., 1996). Assuming that some of the functions of synaptophysin are taken over by the closely related and often coexpressed isoform synaptoporin (Knaus et al., 1990; Fykse et al., 1993), we looked for neurons that do not express synaptoporin in either wild-type or knockout mice. The OPL, which contains synaptic complexes between retinal photoreceptors, bipolar cells, and horizontal interneurons, completely lacks synaptoporin (for other species, see Brandsta«tter et al., 1996) but is rich in synaptophysin (see also Wiedenmann and Franke, 1985; Schmied and Holtzman, 1989; Mandell et al., 1990; Eshkind and Leube, 1995; Brandsta«tter et al., 1996; von Kriegstein et al., 1999), whereas the IPL, which contains synaptic contacts between bipolar, amacrine, and ganglion cells, is strongly positive for both polypeptides. Immunoelectron microscopy has shown that photoreceptors express synaptophysin mostly in the abundant SVs and to a lesser extent in biosynthetic compartments (Schmied and Holtzman, 1989; Mandell et al., 1990). Photoreceptor ribbon synapses (Gray and Pease, 1971; Rao-Mirotznik et al., 1995; Vollrath and SpiwoksBecker, 1996) are especially suited for the examination of SV morphogenesis and function, because (1) they are extremely rich in SVs (for quantitation in cat, see Rao-

139

Mirotznik et al., 1995), (2) the recycling rate of SVs can be increased to very high levels simply by dark adaptation (Schae¡er and Raviola, 1978; Cooper and McLaughlin, 1983; cf. Rao-Mirotznik et al., 1995), and (3) membrane potential alterations can be measured by non-invasive methods, i.e., by ERG. Moreover, despite the morphological di¡erences in synaptic complex arrangement, the basic mechanisms of SV secretion and reformation do not di¡er from other conventional synapses (reviewed in von Gersdor¡ and Matthews, 1999). Thus, the originally described di¡erences in the molecular machinery (e.g., Grabs et al., 1996) have been corrected in the recent past and it is now thought that most presynaptic components are expressed in photoreceptors (von Kriegstein et al., 1999). Finally, because of the comparatively simple retinal architecture, synaptoporin-positive and -negative cell populations can be directly compared in the same specimen thereby providing ideal controls. The discrepancy between morphologic changes in the retinae of synaptophysin knockout mice and the absence of signi¢cant ERG changes shows the limitation of the latter method for discerning alterations in photoreceptor function other than those directly involved in the generation of sum potentials. One of our major morphologic ¢ndings in the photoreceptors of synaptophysin knockout mice is the reduction of SVs, thereby showing that synaptophysin is of importance for e¤cient SV formation. This is in accordance with the recent report of Daly et al. (2000) who have demonstrated that injection of inhibitory synaptophysin peptides corresponding to the cytoplasmic carboxy-terminus of synaptophysin into the squid giant synapse induces vesicle depletion during high frequency stimulation. A morphogenetic function of synaptophysin is also supported by the observed sorting of synaptophysin into special vesicles in various non-neuroendocrine cells, demonstrating the capacity of synaptophysin to segregate from other membrane proteins and to accumulate in particular membrane domains (Leube et al., 1989, 1994; Leimer et al., 1996; for contrasting observations in other cell types, see Johnston et al., 1989; Cameron et al., 1991; Linstedt and Kelly, 1991). The cholesterol-binding properties of synaptophysin o¡er an explanation of the way in which synaptophysin could determine this process (Thiele et al., 2000). The speci¢c protein^lipid interaction has been proposed to create microdomains with a tendency to form highly curved membranes, i.e., microvesicles. The enhanced vesicle depletion during times of elevated exocytotic activity further indicates that synaptophysin is a limiting, although non-essential, factor for SV recycling in vivo. This could be attributed to its involvement in either SV exocytosis and/or reformation of SVs or SV precursors (see Introduction; compare also with Zhang et al., 1998; Gad et al., 2000; Harris et al., 2000). We have found no signs of disturbed exocytosis in synaptophysin knockout mice given the largely normalappearing attachment and packing of SVs around SRs, presumably corresponding to the readily releasable SV pool (von Gersdor¡ and Matthews, 1999), the lack of increased exocytotic pro¢les near SRs, and the normal

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ERG. Instead, our observations point more to the importance of synaptophysin for SV reformation. First, the increase of plasma membrane in pale terminals of knockout mice is best explained as a consequence of reduced endocytotic membrane retrieval. Second, the increase in clathrin-coated vesicles in DA pale terminals could be interpreted as a compensatory mechanism to correct for impaired synaptophysin-dependent endocytosis. Third, the persistence of vacuoles as potential SV donor compartments (Schmidt et al., 1997; Schmidt and Huttner, 1998; for retina, see Schae¡er and Raviola, 1978; Cooper and McLaughlin, 1983; Cooper et al., 1983; Townes-Anderson et al., 1988) in DA knockout mice could be the consequence of reduced vesicle reformation. In addition, the phenotypic similarities to those reported by Daly et al. (2000) are striking, most notably the activity-dependent vesicle depletion and the increased clathrin-coated vesicle formation. These authors refer to preliminary data indicating that synaptophysin may form a calcium-dependent complex with dynamin, thereby directly in£uencing the endocytotic machinery. Alternatively, the altered SV composition in synaptophysin knockout mice could result in the rapid £attening of exocytotic vesicles, which would complicate and slow down SV reformation (Hannah et al., 1999; Thiele et al., 2000). When trying to understand the `incomplete' phenotype of synaptophysin knockouts, one should keep in mind that more than one mechanism may be involved in vesicle recycling. To date, two major pathways have been distinguished (Koenig and Ikeda, 1996; for retina see Cooper and McLaughlin, 1983; reviewed in Hannah et al., 1999; von Gersdor¡ and Matthews, 1999). The ¢rst, comparatively slow and complicated, pathway was proposed by Heuser and Reese (1973). It involves clathrin-coated intermediates, occurs distant to the active zone, and is calcium- and magnesium-independent. The other pathway, which is calcium- and magnesium-dependent and fast, is based on the `kiss and run' concept put forward by Ceccarelli et al. (1973) whereby SVs fuse only partially with the presynaptic membrane and are retrieved locally or from a speci¢c vacuolar compartment that remains in contact with the plasma membrane through a narrow neck. If synaptophysin only a¡ects one type of recycling, as has been suggested (Daly et al., 2000), it is not surprising that synaptophysin de¢ciency results only in mild defects in knockout mice. This interpretation is also supported by our observations that multiple clathrin-coated vesicles are readily identi¢ed in knockout animals indicating that the clathrinmediated pathway is not abolished by the loss of synaptophysin. On the contrary, the increased number of coated vesicles in DA pale terminals of knockout mice suggests increased usage of this presumably synaptophysin-independent pathway at this time. As would be expected in case of the participation of synaptophysin in the recycling of SVs, we have observed more pronounced vesicle depletion during increased transmitter

exocytosis and vesicle reformation in DA mice. Taken together, these results indicate that synaptophysin is of importance for a special type of SV recycling that does not involve clathrin-coated intermediates. We have identi¢ed pale-appearing terminals expressing considerably fewer SVs with a bright interior and a less electron-dense cytoplasm in knockout mice. We do not know why only some terminals exhibit this phenotype. To exclude genetic heterogeneities that could be responsible for this e¡ect, we used matched sibling pairs and have not observed a single pale terminal in any of the wild-type control animals. It is also possible that pale terminals, which often contain swollen mitochondria and altered chromatin, have exhausted their capacity to form vesicles and are destined for cell death. However, these pale terminals still exhibit light-dependent changes in the number of omega pro¢les, clathrin vesicles, and terminal size indicating ongoing SV recycling. Furthermore, the reduced number of pale terminals in DA mice and the observation that neither an increase in pale terminals nor increased retinal degeneration is noticeable in older animals (data not shown) argue against this notion. Di¡erences in vesicle diameter were only seen in DA animals when a reduction in vesicle size was noted in wild-type but not knockout mice. This size reduction may be a consequence of increased recycling as was originally proposed for the electric organ of Torpedo californica by Zimmermann and Whittaker (1974). Moreover, the di¡erences in vesicle diameter, shape, and membrane morphology must be considered in view of the capacity of synaptophysin to form complexes with other vesicle proteins (Bennett et al., 1992; Calakos and Scheller, 1994; Edelmann et al., 1995; Washbourne et al., 1995; Galli et al., 1996; Hannah et al., 1998) and to bind to cholesterol (Thiele et al., 2000) thereby determining vesicle composition and dimensions. As has been pointed out (Hannah et al., 1999; Thiele et al., 2000), these speci¢c interactions could be responsible for the formation of an intrinsic sca¡olding that is needed to maintain the high curvature of SV membranes. It may very well be that such a function is not an exclusive property of synaptophysin and its relatives but is shared by other integral vesicle membrane proteins with the same transmembrane topology, as suggested by Stenius et al. (1995) for the synaptogyrins and SCAMPs. Members of each of these families are also expressed in SVs (Brand and Castle, 1993; Stenius et al., 1995) and have been detected in the retinal OPL (von Kriegstein et al., 1999); this may explain the comparatively small di¡erences in vesicle diameter, di¡erences that are only uncovered during times of elevated neurosecretion.

AcknowledgementsöWe thank Ilse von Graevenitz, Sabine Thomas, and Karin Mai for expert technical help, and Dr. Reinhard Windo¡er for helpful discussions. The work was supported by the German Research Council (SFB 430 C2; Se 837/1-1).

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