Characterization of presynaptic proteins involved in synaptic vesicle exocytosis in the nervous system of Torpedo marmorata1

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Neuroscience Vol. 79, No. 1, pp. 285–294, 1997 Copyright ? 1997 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/97 $17.00+0.00 S0306-4522(96)00682-3

CHARACTERIZATION OF PRESYNAPTIC PROTEINS INVOLVED IN SYNAPTIC VESICLE EXOCYTOSIS IN THE NERVOUS SYSTEM OF TORPEDO MARMORATA J. HERREROS, F. AGUADO, J. M. CANALS, J. MARSAL and J. BLASI* Laboratori de Neurobiologia Cel.lular i Molecular, Departament de Biologia Cel.lular i Anatomia Patolo`gica, Facultat de Medicina, Hospital Prı´nceps d’Espanya, Universitat de Barcelona, Pavello´ de Govern. Feixa Llarga s/n. L’Hospitalet de Llobregat 08907, Spain Abstract––Synaptobrevin, SNAP-25 and syntaxin (SNAP receptor proteins) are molecular components that play a key role in the exocytotic machinery of synaptic vesicles. Their presence, distribution and interactions are reported in central and peripheral nervous systems of the electric fish Torpedo marmorata. These three proteins form a protein complex in all the nervous system regions tested, including the electric lobe and the electric organ which is innervated by pure cholinergic nerve terminals. Immunoblot analysis revealed a double protein pattern of SNAP-25 in the anterior brain and cerebellum, although a single protein band corresponding to SNAP-25 was observed in the electromotor system. Moreover, SNAP-25 showed a differential distribution in the electromotor system. It was present along nerve fibres and terminals that innervated the electric organ but it was not detected in nerve terminals at the electric lobe. Immunoisolation experiments using anti-synaptobrevin antibodies showed a tissue-specific co-existence of SNAP-25 and syntaxin with synaptobrevin in the immunoisolated organelles. In conclusion, the molecular components of the exocytotic machinery are shown to be conserved in Torpedo, although some differences mainly on SNAP-25, suggest a potential diversity in the regulation of neurosecretion. ? 1997 IBRO. Published by Elsevier Science Ltd. Key words: SNAP-25, synaptobrevin, syntaxin, SNARE proteins, SV2, electromotor system.

Recent advances in the membrane trafficking pathways have outlined molecular models for the synaptic vesicle life cycle. Synaptic vesicle and plasma membrane proteins as well as soluble factors have been involved in the fusion of synaptic vesicles with the plasma membrane.53 In particular, the plasma membrane proteins SNAP-25 (synaptosomal-associated protein of 25000 mol. wt) and syntaxin, together with the synaptic vesicle protein synaptobrevin, form a protein core involved in the docking and fusion of synaptic vesicles.51 This protein complex interacts with the soluble factors NSF (N-ethyl-maleimidesensitive factor) and SNAPs (soluble NSF attachment proteins) which are common elements in intracellular membrane fusion processes. Thus SNAP-25, syntaxin and synaptobrevin are considered SNARE (soluble NSF attachment protein receptors) proteins,51 directly involved in the regulated exocytotic pathway. The functional significance of these proteins was strengthened since they were shown to *To whom correspondence should be addressed. Abbreviations: BSA, bovine serum albumin, CHAPS, 3-[(3cholamidopropyl)dimethyl-ammonio]-1-propanesulphonate, EDTA, ethylenediaminetetra-acetate, NSF, N-ethylmaleimide-sensitive factor, PBS, phosphate-buffered saline, CNS, central nervous system, PNS, peripheral nervous system, SDS-PAGE, sodium dodecyl sulphatepolyacrylamide gel electrophoresis, SNARE, SNAPreceptor .

be the intracellular targets for clostridial neurotoxins.39 Recent studies have mainly focussed on the characterization of the assembly and disassembly of these protein complexes and their interaction with other synaptic proteins.8,53 Synaptobrevin (also known as VAMP, vesicle-associated membrane protein) is an integral protein of synaptic vesicles of 18–20000 mol. wt. It was originally cloned from Torpedo nervous system54 and later from several species,6,16,25,52,59 showing its high degree of conservation in evolution.52 SNAP-25 is a membraneassociated protein that was firstly characterized from mouse presynaptic terminals of the hippocampus.41 It has been involved in the neurite elongation.40 Syntaxin,26 an integral membrane protein mainly localized on the plasma membrane, was first identified from mouse retina as HPC-1 antigen.2 Syntaxin interacts with synaptotagmin,7 calcium channels31a,59a and Munc-1822a, suggesting a pivotal role in the events that lead to synaptic vesicle fusion with plasma membrane. SNAP-25, syntaxin and synaptobrevin are also found in non-neuronal secretory cells1,28,44,48,49 indicating a general role of these proteins in regulated exocytosis. Moreover, homologues of these proteins have been found in yeast,17 where they also form a protein complex10 involved in vesicular transport. The aim of this study is to explore the presence and the distribution of different molecular components of

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the synaptic machinery in the electromotor system of Torpedo. The electric organ of Torpedo marmorata is homologous to the neuromuscular junction. It is composed of electrocytes that are innervated in their ventral face and pile up into prisms.58 Indeed, synaptic preparations of high purity can be obtained from the electric organ.27,36,37 The cell bodies of the electromotorneurons, which send their axons to the electric organ, are located at the electric lobe in the brain stem. These neurons are innervated by the cells at the command nucleus, situated bilaterally in the mid-ventral medulla, which control the electric discharge.18,58 Using this electromotor system, it is possible to perform morphological and biochemical experiments on nerve terminals, axons and perikarya corresponding to the same type of neuron. Taking advantage of this model, we characterized the SNARE proteins both in the central nervous system (CNS) and peripheral nervous system (PNS). EXPERIMENTAL PROCEDURES

Materials Torpedo marmorata specimens were caught off the Mediterranean coast and mantained in artificial sea water. Antibodies á-Tubulin monoclonal antibody was from Sigma. HPC1/syntaxin,2 SV212 and SNAP-25 (Cl 71.1)20 monoclonal antibodies were kindly supplied by Drs C. Barnstable (New Haven, CT), K. Buckley (Boston, MA) and R. Jahn (New Haven, CT) respectively. Generation of the SNAP-25 rabbit antiserum was previously described.1 Same results were obtained with both anti-SNAP-25 polyclonal antiserum and Cl 71.1 monoclonal antibodies and they were used with no distinction or according to the requirements for double immunolabelling techniques. A specific antiserum against Torpedo synaptobrevin was generated in rabbits using as antigen a synthetic peptide that corresponds to the 14 amino acids of the cytosolic N-terminus of Torpedo synaptobrevin/VAMP-1.54 This peptide (MSAPPSGPAPDAQG) was produced in the Dept. Quı´mica Orga`nica, Chemical School, University of Barcelona. An additional cysteine residue was added at the C-terminus of the peptide to facilitate its coupling to keyhole limpet haemocyanine.21 Booster injections were given every fifteen days and rabbits were bled one week later. For immunoprecipitation or immunoisolation purposes, this antiserum was affinity purified using the synthetic peptide coupled to EAH Sepharose 4B (Pharmacia) according to manufacturer’s instructions. Immunocytochemistry Animals were anaesthetized with 3-aminobenzoic acid ethyl ester (tricaine; Sigma). Torpedo specimens were perfused with 0.2 M sodium cacodylate buffer, pH 7, followed by 4% paraformaldehyde-0.25% carbodiimide in the same buffer and afterwards postfixed for at least 2 h at 4)C in the same solution. Cryostat or Vibratome electric lobe sections of 40 µm were collected. Electric lobe sections were processed free-floating for immunofluorescence. Sections were incubated with 50 mM ammonium chloride in phosphatebuffered saline (PBS) for 30 min and then in a solution containing 5% bovine serum albumin (BSA), 0.25% glycine, 0.2% lysine, 0.25% gelatine, 0.3% Triton X-100 and 20% fetal calf serum in PBS for 1 h. After rinses with 0.2% Triton X-100 in PBS, sections were incubated overnight with

primary antibodies in 0.25% gelatine, 0.2% Triton X-100, 3% fetal calf serum in PBS, followed by incubation with Texas Red-linked antimouse or Fluorescein-linked antirabbit immunoglobulins (Amersham). Sections were mounted with ImmunoFluore mounting medium (ICN). For electric organ immunofluorescences, a similar procedure was used on dehydrated slide-mounted paraffin sections. Sections were examined in a Leica TCS 4D laser scanning confocal imaging system using an argon/krypton laser with excitation peaks at 488 nm (for Fluoresceinlabelled profiles) and 568 nm (for Texas Red-labelled profiles). Alternatively, small electric organ fragments were fixed and squashed directly between two slides. Squashed electric organ and some electric lobe sections were immunolabelled using the ABC kit (Vectastain; Vector) as described elsewhere1 and developed for peroxidase activity. Control sections were routinely processed by omitting the primary antibody, absorbing the antibody with the specific peptide or using the preimmune serum at an equivalent concentration. Biochemical methods Electric organ, electric lobe, cerebellum and the anterior brain of Torpedo and rat cerebral cortex were homogenized in PBS, pH 7.4, containing 1 mM EDTA and 1 mM phenylmethylsulphonyl fluoride (PMSF, Sigma) using a glass homogenizer. A postnuclear supernatant was obtained after a low speed centrifugation (3000 g, 2 min) and used for sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting analysis, immunoprecipitation and immunoisolation of synaptic vesicles. Electric organ synaptosomes were isolated according to Morel et al.36 Briefly, electric organ was finely chopped and equilibrated in a Torpedo physiological buffer. After different filtration steps, the homogenate (H) was centrifuged at 6000 g for 20 min. The supernatant (S) was discarded and the pellet (P) was resuspended and layered onto a discontinuous sucrose gradient: 0.3 M, 0.5 M and O.7 M sucrose. After centrifugation (22000 g for 40 min) aliquots of the soluble (C1) and the synaptosomal (C2, 0.3–0.5 M interface) fractions were collected. For all the fractions, 10 µg of protein was precipitated with 5% trichloracetic acid (30 min at 4)C) and centrifuged at 10000 g for 10 min. Pellets were resuspended in SDS-sample buffer containing 10% â-mercaptoethanol and the protein pattern was analysed by 12.5% SDS-PAGE31 and immunoblotting. Nitrocellulose membranes were blocked with 5% non-fat milk, 0.1% Tween-20 in Tris 10 mM, NaCl 140 mM, pH 7.4 buffer and processed as described elsewhere.23 Immunoreactive electrophoretic bands were revealed using Enhanced Chemiluminescence (ECL; Amersham) or [125I]Protein A (ICN). For immunoprecipitation, aliquots corresponding to 400–500 µg of protein from Torpedo electric organ or the different brain postnuclear supernatants were solubilized with 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulphonate] (CHAPS; Boehringer Mannheim) for 30 min at 4)C. After centrifugation (10000 g, 10 min), supernatants were incubated for 3 h at room temperature with affinitypurified anti-synaptobrevin antibodies (about 6 µg of IgG) previously coupled to Protein G Sepharose Fast Flow (Pharmacia). Beads were finally rinsed and resuspended in SDS-sample buffer without â-mercaptoethanol. Synaptic vesicles immunoisolation was performed using the postnuclear supernatants of the different tissue homogenates as starting material. These samples were incubated with anti-synaptobrevin antibodies previously coupled to protein G-Sepharose beads and processed as above. In these experiments no detergent was used. Pre-immune serum was used as control in the immunoprecipitation and immunoisolation experiments. Under this situation, no protein bands were immunodetected on the beads.

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Protein concentration was determined according to Bradford,9 using BSA as standard. In situ hybridizations Torpedo brains were removed and frozen in 2-methylbutane cooled in liquid nitrogen. Sections of 10 µm were obtained in a cryostat, dried on poly--lysine-coated slides and fixed in 4% paraformaldehyde in PBS. A pBluescript plasmid containing the entire open reading frame and the 3* untranslated region of the Torpedo SNAP-25 cDNA (TS11,47 generously supplied by Dr D. Larhammar, Uppsala, Sweden) was used as a template. TS11 was linearized with Xho I and transcribed in the presence of [35]S-UTP using T3 polymerasa (Promega). After alkaline hydrolysis, hybridization was performed essentially according to Me¨llstrom et al.35 The same hybridized sections, previously exposed to an X-ray film, were dipped in Kodak NTB2 emulsion and left at 4)C for six weeks. The emulsion was developed and sections were counterstained and observed under bright- and dark-field microscopy. In control experiments, a sense probe of similar size and specific activity showed no hybridization signal. RESULTS

Fig. 1. Immunoblotting of Torpedo CNS fractions. SNAP25, HPC-1/syntaxin, SV2 and synaptobrevin (SYB) were found in homogenized samples from anterior brain (AB), cerebellum (Ce) and electric lobe (EL) of Torpedo. R corresponds to a rat brain postnuclear supernatant. Note that the antibodies against Torpedo synaptobrevin do not recognize rat synaptobrevin and the double band pattern of SNAP-25 detected in AB and Ce. The amounts of SV2 are under the detection level in the EL homogenate. Lanes were loaded with equal amounts (10 µg/lane) of protein and developed using the ECL kit.

Biochemical characterization of SNAP-25, syntaxin and synaptobrevin in Torpedo brain and electric organ An antiserum against the amino acid sequence of the cytoplasmic N-terminus specific for Torpedo SYB was raised and characterized. Anti-synaptobrevin antibodies recognized a single band of 20000 mol. wt on immunoblotted homogenates from different parts of Torpedo brain (Fig. 1). Synaptobrevin from a rat brain homogenate was not recognized by this antiserum (Fig. 1), whereas it was immunodetected by rat specific anti-synaptobrevin II antibodies (not shown). Syntaxin, SNAP-25 and SV2, an integral glycoprotein of the synaptic vesicle membrane12 were analysed by immunoblot (Fig. 1). Rat and Torpedo SV2 showed different electrophoretic mobility (Fig. 1) as previously described.12 HPC-1/syntaxin was immunodetected as a single band in Torpedo tissues, whereas both syntaxin 1A and 1B7 were noticeable in the rat brain homogenate. SNAP-25 was consistently detected as a two electrophoretic band pattern in the anterior brain and cerebellum of Torpedo (Fig. 1), although in the cerebellum the band with higher mobility was fainter. This pattern was different in electric lobe (Fig. 1) and electric organ (see below and Fig. 2), where a single band corresponding to SNAP-25 was immunodetected. To rule out the possibility that this two band pattern was due to proteolysis, in vitro-translated SNAP-25 from the electric lobe was obtained47 and labelled with [35S]methionine. The translation product was mixed with the different tissues during the homogenization process. The sole band corresponding to the translation product from the electric lobe was visible by autoradiography in all the tissues, whereas the twoband pattern of SNAP-25 from the anterior brain and cerebellum was unaffected as analysed by immunoblot (data not shown). Moreover, both bands of SNAP-25 were not differently affected by treatment

Fig. 2. Immunoblot analysis of different samples (10 µg/ lane) from the isolation process of electric organ synaptosomes for synaptobrevin, SNAP-25, syntaxin and á-tubulin. SNAP-25, synaptobrevin and syntaxin concentrated in the electric organ synaptosomal protein fraction (C2). Bands were visualized using [125I]protein A. H, homogenate; S, first supernatant; P, first pellet; C1, soluble fraction; C2, synaptosomal-enriched fraction (corresponding to 0.3–0.5 M sucrose interface).

with hydroxylamine 1 M, an agent that selectively cleaves the thioester bonds with fatty acids (data not shown). Aliquots from the isolation process of electric organ synaptosomes were analysed by SDS-PAGE and immunoblotting (Fig. 2). Synaptobrevin, SNAP-25 and syntaxin showed a similar subcellular distribution pattern and concentrated in the synaptosomal enriched (C2) fraction. á-Tubulin displayed a different pattern compatible with a soluble protein. The anti-synaptobrevin antiserum was affinity purified and used for immunoprecipitation experiments. Synaptobrevin from CHAPS extracts was effectively immunoprecipitated from electric organ and different regions of the brain (Fig. 3A). Immunoblotting analysis also revealed that syntaxin and SNAP-25 co-immunoprecipitated with

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synaptobrevin in all tissues tested (Fig. 3A) in agreement with their association in a protein complex.51 For control immunoprecipitation experiments, anti-á-tubulin antibodies were also used. None of the components of the 20S complex was coimmunoprecipitated under these conditions (not shown). Immunoisolation of Torpedo synaptic vesicles with anti-synaptobrevin antiserum Affinity-purified anti-synaptobrevin antibodies were coupled to protein G-Sepharose beads and used to immunoisolate synaptic vesicles from different parts of Torpedo PNS and CNS. Synaptic vesicles from all samples contained SV2 and synaptobrevin, as expected for classical constituents of synaptic vesicles (Fig. 3B). Syntaxin was found in different amounts depending on the region of the brain. It was clearly detected in immunoisolated synaptic vesicles from the cerebellum, electric organ and electric lobe, but almost absent from the anterior brain synaptic vesicles (Fig. 3B). SNAP-25 was found to coimmunoprecipitate only in the electric lobe and the electric organ (Fig. 3B). In all cases, á1 Na+/K+ATPase and á-tubulin were not detected in the immunoisolated synaptic vesicles, indicating poor or null contamination by plasma membranes and soluble proteins (data not shown). Differential localization of SNAP-25 in the Torpedo electromotor system The cellular distribution of SNAP-25 and synaptobrevin was examined in the electromotor system by means of immunocytochemical techniques. AntiSNAP-25 antibodies immunostained nerve fibres in the electric organ, electric lobe and command nucleus (see below and Fig. 4). Immunolabelling was especially prominent in squashed electric organ (Fig. 4A), where the whole extensive branching of axons was immunostained. In order to study SNAP-25 distribution in more detail, double immunofluorescences were performed for SNAP-25 and synaptobrevin. As expected, antisynaptobrevin antiserum immunolabelled the nerve terminals that innervate the ventral face of electrocytes in the electric organ and the nerve terminals at the electric lobe (Fig. 5A and B). This immunostaining was identical to that of the synaptic vesicle protein SV2, widely used as a nerve terminal marker (Fig. 5D). Anti-SNAP-25 immunolabelled nerve terminals at the ventral face of electrocytes (Fig. 5B). This pattern was coincident with synaptobrevin immunostaining, as corroborated by co-localization analysis using confocal laser microscopy. The same approach was attempted in electric lobe and command nucleus sections. Double immunofluorescences for SNAP-25 and synaptobrevin in the command nucleus revealed

Fig. 3. A) Immunoprecipitation of synaptobrevincontaining protein complexes from CHAPS extracts of Torpedo electric organ and brain homogenates. Immunoblot analysis of the cerebellum (Ce), anterior brain (AB), electric lobe (EL) and electric organ (EO) immunoprecipitates for synaptobrevin (SYB), SNAP-25 and syntaxin. Note that the two bands of SNAP-25 immunodetected in the anterior brain and cerebellum are found in the immunoprecipitates. B) Immunoisolation of synaptic vesicles from Torpedo electric organ and brain homogenates. Immunoblot analysis of cerebellum (Ce), anterior brain (AB), electric lobe (EL) and electric organ (EO) immunoisolated fractions (p) and the remaining supernatants (s; aliquot of 25 µl) for synaptobrevin (SYB), SNAP-25, syntaxin and SV2. SV2 and synaptobrevin were consistently found in immunoisolated synaptic vesicles. Significant levels of syntaxin were also detected in all immunoisolated fractions except for the anterior brain, whereas SNAP-25 was detected in electric lobe and electric organ.

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Fig. 4. A) SNAP-25 immunostaining in squashed electric organ. SNAP-25-immunoreactive axons and their extensive arborization pattern are observed. Arrow indicates the electrocytes. Scale bar=58 µm. B) SNAP-25 immunostaining in an electric lobe section. Immunoreactive thick fibres (arrows) corresponding to neuronal processes and also thin nerve fibres (axons; arrowheads) are shown. Intense cytosolic immunostaining is also observed, especially in the perinuclear region (curved arrows); * indicates blood vessels. Scale bar=25 µm.

that both proteins co-localized in some nerve terminals (Fig. 5C), which form axodendritic and axosomatic synapses.18 SNAP-25 appeared to be restricted to neuronal processes (i.e. dendrites and/or nerve fibres) in the electric lobe (Fig. 5A). However, we failed to detect SNAP-25 at the electric lobe nerve terminals. Co-localization analysis confirmed poor co-localization of synaptobrevin and SNAP-25 in electric lobe nerve terminals. Furthermore, synaptobrevin-immunoreactive nerve terminals appeared aligned on SNAP-25-positive neuronal processes. These nerve terminals are reported to form en passant synapses on the dendrites of electromotorneurons.45 In addition, anti-SNAP-25 antibodies also immunolabelled the perikarya of electromotorneurons (Fig. 4 and Fig. 5). To examine the presence of SNAP-25 in electric lobe nerve terminals in more detail, in situ hybridizations for the SNAP-25 mRNA were performed on Torpedo sagittal brain stem sections containing the command nucleus, which innervate the electric lobe motorneurons (Fig. 6). The SNAP-25 mRNA was detected in neurons at the command nucleus, suggesting the synthesis of this protein by this nucleus. Hybridization signal was also found in dispersed neurons in the medulla. A high level of SNAP25 mRNA expression was detected in the perikarya of electromotorneurons (Fig. 6), suggesting a high degree of protein synthesis and the presence of apparent amounts of SNAP-25 in the electromotor system.

DISCUSSION

The electromotor system of Torpedo offers the advantage that both peripheral and central systems are easily accessible. In this report, we characterize different synaptic proteins involved in the synaptic mechanism of exocytosis,53 using this electromotor system. In particular, the localization and distribution of SNARE proteins, SNAP-25, synaptobrevin and syntaxin, have been investigated. Most of our knowledge on these proteins comes from studies performed using mammalian CNS preparations.8,53 However there is strong evidence that the molecular machinery for membrane fusion has been conserved during the phylogeny. Presence of neuronal SNARE proteins and their interactions From our results, it is clear that these proteins are present in Torpedo nervous systems as detected by immunoblotting and immunocytochemistry. They concentrate in the synaptosomal fraction isolated from Torpedo electric organ, as similarly shown for SNAP-25 in a mouse hippocampus synaptosomal fraction.41 Furthermore, it is demonstrated that the SNARE interaction in a protein complex51 is conserved in different regions of Torpedo CNS and PNS. Altogether, the results suggest that these proteins are highly conserved in evolution and support a common mechanism in exocytosis.17

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Fig. 5. Confocal image of double-immunofluorescence staining for SNAP-25 (monoclonal antibody Cl 71.1; red) and synaptobrevin (green) in electric lobe (A, D), electric organ (B) and command nucleus (C) sections. Immunostaining overlap is shown in yellow. Scale bars=10 µm. A) Anti-SNAP-25 stains nerve fibres and does not co-localize on synaptobrevin-immunoreactive nerve terminals (arrows), which appeared aligned on SNAP-25-positive nerve fibres in the electric lobe. B) SNAP-25 and synaptobrevin co-localize on nerve terminals that innervate the ventral face of electrocytes in paraffin-embedded electric organ. Arrow indicates SNAP-25-immunoreactive axons in transverse section. C) Anti-SNAP-25immunostained nerve fibres in the region of the command nucleus and also some nerve terminals. Many synaptobrevin-immunoreactive nerve terminals co-localize with SNAP-25, especially at those nerve terminals surrounding cell bodies of the command nucleus (arrows); * indicates a blood vessel. D) Anti-SV2 and anti-synaptobrevin-immunostained nerve terminals (arrows) in an electric lobe section.

However, some differences were observed in the electrophoretic pattern of SNAP-25 from some regions of the CNS. A double band pattern of SNAP-25 was constantly detected in Torpedo anterior brain and cerebellum, which is not a consequence of protein degradation or of different palmitoylation. Thus, this double pattern suggests there are two different forms of SNAP-25 in the Torpedo CNS that may differ in their sequence of amino acids. Both forms of SNAP-25 were involved in the SNARE interaction into a protein complex found in the anterior brain and cerebellum. Two isoforms of SNAP-25 (a and b) were described in mammalian4,5,42 and chicken3 nervous systems. Whereas SNAP-25b concentrates in presynaptic terminals at the onset of synaptogenesis,

SNAP-25a appears early in the development and is found in nerve fibres and perikarya,5,42 playing a role in axonal growth.40 Different isoforms of SNAP-25 were also previously described in goldfish46 but not in Torpedo, where SNAP-25 was cloned from a cDNA electric lobe library.47 Further experiments are needed to clarify the functional significance of these two SNAP-25 isoforms in the adult Torpedo CNS. Synaptic vesicles immunoisolation experiments Syntaxin can be found in synaptic vesicles as demonstrated by immunoisolation experiments. The absence of á1 Na+/K+-ATPase in the synaptic vesicle immunoisolated fractions almost exclude contamination of syntaxin from plasma membrane fragments.

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Fig. 6. In situ hybridization of SNAP-25 mRNA in Torpedo brain stem. A) Autoradiography of a sagittal section of the brain stem. Hybridization signal is found in the electric lobe (filled arrow), the command nucleus (empty arrow) and also some dispersed neurons of the medulla. Scale bar=0.7 mm. B) Dark-field photomicrograph of the same section after dipping in photographic emulsion. The command nucleus (empty arrow) and electromotorneurons of the electric lobe (filled arrows) are shown; * indicates blood vessels. Scale bar=96 µm. C) High levels of expression are found in the electromotorneurons cell bodies at the electric lobe. Scale bar=25 µm.

HPC- 1/syntaxin was previously localized on synaptic vesicles in lysed synaptosomes from rat cerebellum30, rat cortex and striatum sections.50 Using biochemical techniques it was also shown that syntaxin was present on immunoisolated synaptic vesicles.56 Therefore, although it has been described as a main component of the synaptic plasma membrane, it is concluded here that syntaxin may exist in these organelles. This indicates that this protein recycles through synaptic vesicles as previously suggested.56 The functional significance of the synaptic vesicle pool of syntaxin is strengthened since this pool is preferentially cleaved by botulinum neurotoxin type C1.56 The immunoisolated synaptic vesicles from the electric lobe also contained significant amounts of SNAP-25. Our interpretation of this finding is that anti-synaptobrevin antiserum immunoisolated synaptic vesicles but also some SNAP-25-containing vesicles from the cell bodies of electromotorneurons. Electromotorneuron cell bodies constitute up to 80% of the electric lobe tissue. Therefore, these immunoisolated SNAP-25-containing vesicles may be related to the abundant synthesis of this protein in these cells (note the anti-SNAP-25 antibodies immunostaining of the cytoplasm of the electromotorneurons in Fig. 4 as well as their levels of SNAP-25 mRNA in Fig. 6).15,32 Similarly, immunoisolation from the electric organ showed detectable amounts of SNAP-25,

which can be explained by immunoabsorbed vesicles involved in axonal transport of synaptic proteins such as SNAP-25,24 synaptobrevin and syntaxin. SNAP-25 immunocytochemistry In this study synaptobrevin54 and SV212 have been used as nerve terminal markers. Both proteins share a well stated specific localization on synaptic vesicles. Indeed, SV2 has been used as a nerve terminal marker in several species including the electric ray29,57,60 and is ubiquitous in all synapses.11,55 SNAP-25 was localized both in nerve terminals and axons of the electric organ, which also correlates with a SNAP-25 enrichment in a synaptosomal fraction isolated from the electric organ. The SNAP-25 distribution all along the axon agrees with previous results19,41 and suggests an additional function of SNAP-25 besides its involvement in neurotransmitter release. The inability to immunodetect SNAP-25 in the electric lobe nerve terminals could indicate that SNAP-25 may only be present in the dendritic component of these axodendritic synapses, although it was localized at electric organ and some command nucleus nerve terminals and fibres. This observation would be in agreement with a differential distribution and expression of SNAP-25,14,15,19a,38,41 and other synatic proteins such as synapsins,33,34 rab3A55 or

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syntaxin2,50 in distinct subsets of synapses. In this case, one would expect that another presynaptic protein could play the role of SNAP-25. However, the mRNA for SNAP-25 is present in the command nucleus where cell bodies of neurons that innervate the electric lobe are located. Therefore, these synapses may contain amounts of SNAP-25 which would be under the detection level of immunocytochemical techniques. Nevertheless, other possibilities should be considered: 1) the command nuclei contain the neurons that send their axons to the electric lobe. However, it is not clear that all the neurons located at these nuclei send their efferences to the electromotorneurons18 and the possibility that the detected mRNA may correspond to cells that do not project to the electric lobe remains open; 2) maybe the SNAP-25 epitope is not accesible to the antibodies56a (e.g., due to interaction with other proteins) in the axons and nerve terminals of the command nucleus neurons; 3) there may be a different form of the protein that is not recognized by the used antiSNAP-25 antibodies; 4) finally, factors such as mRNA stability43 or rapid degradation of the translated peptide22 cannot be ruled out. Special emphasis must be laid on immunostaining of electromotorneurons with anti-SNAP-25 antibodies. Each electromotorneuron displays an intense metabolic activity and innervate at least the area of three electroplaque cells in the electric organ.13,58 As shown in Fig. 4, an apparent immunolabelling of

SNAP-25 is achieved in the cytoplasm of these electromotorneurons. This immunolabelling may correspond to Golgi cisternae as suggested by other authors in dorsal root ganglia sensorial cells15 and correlates with an intense protein synthesis. Indeed, this is a common result in electric lobe sections obtained with most of the antibodies tested against different synaptic proteins. CONCLUSION

SNARE proteins are present in CNS and PNS of Torpedo marmorata where they form complexes in detergent solubilized samples. Since a double-band pattern of SNAP-25 is immunodetected on electrophoresed Torpedo anterior brain and cerebellum samples, the presence of different SNAP-25 isoforms in these tissues is suggested. Acknowledgements—We are indebted to Dr F. Gonza´lezAguilar for introducing us into the perfusion technique in Torpedo. We thank Dr D. Larhammar (Uppsala) for Torpedo SNAP-25 cDNA and Drs K. Buckley (Boston), R. Jahn (New Haven) and C. Barnstable (New Haven) for their gift of antibodies. We also thank S. Castel (Serveis Cientı´fico-Te`cnics, U.B.) for assistance in confocal microscopy, I. Go´mez de Aranda for synaptosomes isolation and Dr J. Garcia-Valero for helpful discussion. Supported by a grant from DGICYT (Ministerio de Educacio´n y Ciencia, Spain) to J.M.

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Note added in proof While this manuscript was in the reviewing stage, Shiff et al. (1996) Neurochem. Int. 29, 659–667, reported a double band pattern for syntaxin in Torpedo cerebellum using 0.5 M urea electrophoresis gels.