Pfl~igers Arch - Eur J Physiol (1996) 431 [Suppl] :R283--R284 © Springer-Verlag 1996 Synaptotagmin II immunoreactivity in normal and botulinum type-A treated mouse motor nerve terminals Pascal Juzans • Jordi Molgo - Lucette Faille • Denise Angaut-Petit Laboratoire de Neurobiologie Cellulaire et Molrculaire, C.N.R.S., F-91198 Gif-sur-Yvette, France Abstract. The distribution of synaptotagmin II, a synaptic vesicle protein, was examined ~ inamunohistochen~istry at norrnat mouse motor nerve terminals and after botulinum type-A treatment. An immunoreactivity to synaptotagmin II was detected in both control and bottdinum type-A treated motor nerve terminals including newly formed sprouts. These data, together with other reports showing the absence of synaptotagmin I at the neuromuscular junction, suggest that synaptotagmin II is the isoform involved in transmitter release at motor nerve terminals. Key words: Synaptotagmin II - synaptic vesicles - mouse - neuromuscular junction - botulinum t?pe-A toxin - terminal sprouting Introduction Synaptotagmin is a well characterized integral membrane protein of synaptic vesicles which functions as a Ca 2+ sensor and interacts with presynaptic neuronal proteins involved in neurotransmitter release [5]. Four homologous isoforms exist which are differentially distributed in the brain [4] as well as in the peripheral nervous system [6]. In the present work we looked for the presence of synaptotagmin II (Syt II) immunoreactivity at mouse motor nerve terminals in normal conditions and also after botulinum type-A (BoTx-A) treatment. BoTx-A not only temporarily inhibits acetylcholine release from motor nerve terminals but also triggers an ilnportant overgrowth of motor nerve endings from the original terminal arborizations [3]. This sprouting process may contribute to the functional recovery of neuromuscular transmission [1]. Thus, it was of interest to look for the presence of molecular components of s3~naptic vesicles in the sprouts in BoTx-A treated endings. Furthermore, we examined the distribution of Syt II inmaunoreactix4ty at normal terminals after sustained quantal acetylcholine release in order to evaluate the ability of the antibody to label regions where exocytosis occurs. Materials and methods Experiments were perfomled on isolated levator auris long~us (LAL) nerve- muscle preparations of adult Swiss mice. BoTx-A was injected in some animals as previously described [I], mice were saerified at various times after inje~ion. Some muscles were stained with the fluorescent pyridin 4-(4- diahylaminostyryl)-N-m~hyl-p)qridinium iodide (4-Di-2-ASP) [7]. For classical immunolabelling, muscles we're dissected in saline and fixed for 1 hour at 4°C with 4% formaldehyde in 0.1 M sodium phosphate buffer. They were then incubated overnight in a Syt II antibody, directed against the lumenal NH2-terminus of the protein [2], which was used at a concentration of 10 g~'rnl. The secondary- antibody was fluorescein-conjugated (Diagnostics Pasteur, France). Triton X-IO0 (0.2 %) was present in all solutions. Preparations were mounted in Fluoprep medium (bioMrrieux, France) and observed using a Sarastro-2000 eonfocal laser scanning microscope (Molecular Dynamics, USA). Prior to fixation of some control muscles, sustained quantal acetytchotine release was induced during 2 hours by adding La 3+ (0.4 mM) in a Ca 2+ free medium [8]. In these cases the primary' antibody was diluted in the medium (25gg/ml). In the next steps, the permeabilizing agent (Triton X-100) was omitted from all solutions. Results Syt-II immunoreacticity in normal terminals In LAL preparations, fixed at rest and subsequently permeabilized, the distribution of $3¢ II immunoreactivity was specific for motor terminals and unevenly distributed over the oblong areas that they occupy (Fig.lA). The pattern thus obtained was similar to the classical picture of the mouse neuromuscular arborization. These restflts indicate that Syt II (or at least a Syt II like protein) is the isoform which is present in the motor nerve terminals