Differential mRNA Expression Patterns of the Synaptotagmin Gene Family in the Rodent Brain TOBIAS MITTELSTEADT, 1,2 GERALD SEIFERT, 3 ELENA ALVA ´ REZ-BARO ´ N, 1,2 CHRISTIAN STEINHA ¨ USER, 3 ALBERT J. BECKER, 1 AND SUSANNE SCHOCH 1,2 * 1 Institute of Neuropathology, University of Bonn, 53105 Bonn, Germany 2 Department of Epileptology, University of Bonn, 53105 Bonn, Germany 3 Institute of Cellular Neurosciences, University of Bonn, 53105 Bonn, Germany ABSTRACT Synaptotagmins are a large family of membrane-trafficking proteins. They are evolutionarily conserved and have 15 members in rodents and humans. Synaptotagmins-1, -2, and -9, are known to have an essential role as calcium sensors for fast synaptic release. Synaptotagmin-7 is a ma- jor calcium sensor for the exocytosis of large secretory vesicles in endocrine cells. The functional roles of most synaptotagmin isoforms remain unknown. Here we exam- ined whether synaptotagmins are expressed in the rodent brain in distinct patterns and whether individual neurons and astrocytes coexpress multiple synaptotagmin isoforms. We performed a systematic analysis of expression using radioactive in situ hybridization and quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR) as well as multiplex RT-PCR on a single-cell level. Our re- sults demonstrate that most synaptotagmins are expressed in the rodent brain in highly distinctive expression patterns, and that individual neurons express variable subsets of dif- ferent synaptotagmins. We also show that Syt-11 is the major isoform expressed in astrocytes. This study therefore supports the hypothesis that the functional properties of individual neurons and astrocytes are conferred by the spe- cific subset of synaptotagmins expressed in a cell. J. Comp. Neurol. 512:514 –528, 2009. © 2008 Wiley-Liss, Inc. Indexing terms: in situ hybridization; astrocytes; synapse; synaptic vesicle; calcium sensor; single cell multiplex PCR Synaptotagmins (Syts) are an evolutionarily conserved fam- ily of membrane-trafficking proteins. Seven isoforms have been identified in Drosophila (Adolfsen and Littleton, 2001) and 15 in humans and rodents, which share a common struc- tural organization with an N-terminal transmembrane domain, a central variable linker region, and two C-terminal C 2 - domains that potentially bind Ca 2 and/or phospholipids (Craxton, 2001, 2004; Sudhof, 2002) (http://smart.embl.de). Syt-1, the most extensively studied synaptotagmin isoform, is abundantly localized on synaptic vesicles and is known to be the major Ca 2 sensor for fast synchronous neurotransmitter release (Geppert et al., 1994; Fernandez-Chacon et al., 2001; Sun et al., 2007; Chapman, 2008). This function of Syt-1 is thought to be evolutionarily conserved (Jorgensen et al., 1995; Mackler et al., 2002; Yoshihara and Littleton, 2002). Several recent studies showed that in vertebrates Syt-2 and -9 also mediate fast Ca 2 -triggering of release in neurons. However, release induced by each isoform differs in its kinetics and apparent Ca 2 sensitivity (Nagy et al., 2006; Pang et al., 2006a,b; Xu et al., 2007). In contrast, Syt-12 was found to be a modulator of spontaneous synaptic vesicle fusion (Maximov et al., 2007). In chromaffin cells Syt-7 is the major calcium sensor for exocytosis and accounts, together with Syt-1, for most of the Ca 2 -triggered exocytosis in these cells (Schonn et al., 2008). The release properties of a neuron seem there- fore to be defined by the subset of synaptotagmins, which it expresses. In the rat brain 12 other synaptotagmin isoforms can be detected in addition to Syt-1, -2, and -9. They can be classi- fied according to their biochemical properties and by se- quence homology (Sudhof, 2002; Craxton, 2004). The individ- ual synaptotagmins exhibit diverging affinities for Ca 2 and phospholipids. Besides Syt-1, -2, and -9, Syt-3, -5, -6, -7, and -10 also bind to Ca 2 (Li et al., 1995a,b; Sugita et al., 2002; Additional Supporting Information may be found in the online version of this article. Grant sponsor: Deutsche Forschungsgemeinschaft; Grant numbers: Emmy Noether Program, SFB 645-A4, SFB/TR3-B8 (to S.S.), SFB/TR3-C6 (to A.J.B.), SFB/TR3-C1 (to C.S.), SPP 1172-SE774/3 (to G.S.); Grant spon- sor: European Community; Grant number: FP7-202167 (to C.S.); NGFNplus EMINET TP-5, -7 (to S.S., A.J.B.); Grant sponsor: Bonfor (to S.S., C.S., A.J.B.). *Correspondence to: Dr. Susanne Schoch, Sigmund Freud Str. 25, 53105 Bonn, Germany. E-mail: Susanne.schoch@uni-bonn.de Received 14 April 2008; Revised 20 August 2008; Accepted 15 October 2008 DOI 10.1002/cne.21908 Published online in Wiley InterScience (www.interscience.wiley.com). The Journal of Comparative Neurology 512:514 –528 (2009) © 2008 Wiley-Liss, Inc.