NEUROSYSTEMS Cellular expression and subcellular localization of secretogranin II in the mouse hippocampus and cerebellum Taisuke Miyazaki, 1 Miwako Yamasaki, 1 Motokazu Uchigashima, 1 Ayano Matsushima 2 and Masahiko Watanabe 1 1 Department of Anatomy, Hokkaido University School of Medicine, Sapporo 060-8638, Japan 2 Department of Physiology, Hokkaido University School of Medicine, Sapporo, Japan Keywords: fluorescent in situ hybridization, immunohistochemistry, large dense-core vesicles, neuropeptide, secretogranin Abstract Secretogranin II (SgII), or chromogranin C, is thought to participate in the sorting and packaging of peptide hormones and neuropeptides into secretory granules and large dense-core vesicle (LDCVs), and also functions as a precursor of neuropeptide secretoneurin. Although SgII is widely distributed in the brain and is predominantly localized at terminals of mossy fibers in the hippocampus and cerebellum and climbing fibers in the cerebellum, its cellular expression and ultrastructural localization remain largely unknown. In the present study, we addressed this issue in the adult mouse brain by multiple-labeling fluorescence in situ hybridization and immunofluorescence and by preembedding and postembedding immunoelectron microscopies. SgII was expressed in various neurons, distributed as either tiny puncta or coarse aggregates in the neuropil, and intensely accumulated in perikarya of particular neurons, such as parvalbumin-positive interneurons and mossy cells in the hippocampus and Purkinje cells in the cerebellum. Coarse aggregates were typical of terminals of mossy fibers and climbing fibers. In these terminals, numerous immunogold particles were clustered on individual LDCVs, and one or two particles also fell within small synaptic vesicle- accumulating portions. SgII was further detected as tiny puncta in neural elements lacking LDCVs, such as parallel fibers of cerebellar granule cells, somatodendritic elements of various neurons and Bergmann glia. Thus, SgII is present in LDCV and non- LDCV compartments of various neural cells. The wide subcellular localization of SgII may reflect diverse release sites of neuropeptides and secretorneurin, or suggests its role in the sorting and packaging of molecules other than neuropeptides in non- LDCV compartments. Introduction Glutamate, GABA and glycine are packaged into vesicles at nerve terminals, and are released to the synaptic cleft (De Camilli & Jahn, 1990). By constituting synaptic specialization as well as specific uptake and recycling systems, these classical neurotransmitters typically act with a short half-life of 5 ms, giving temporal and spatial specificity to signal transduction. On the other hand, more than 100 different neuropeptides are also used for interneuronal commu- nication, and exert profound and diverse effects on behaviors (Hokfelt et al., 2000). Neuropeptides are packaged into vesicles in neuronal perikarya, and are released from various neuronal elements, including nerve terminals, somata and dendrites (Morris & Pow, 1991; Ludwig & Leng, 2006). In most instances, classical neurotransmitters and neuropeptides coexist in the same neurons and in the same terminals, but are segregated into small synaptic vesicles (SSVs) and large dense- core vesicles (LDCVs), respectively (Torrealba & Carrasco, 2004; Salio et al., 2006). Chromogranins / secretogranins or granins are a class of acidic proteins stored in secretory granules of endocrine cells (Rosa et al., 1985a) and in LDCVs of neurons and neuroendocrine cells (Neuman et al., 1984; Somogyi et al., 1984; De Camilli & Navone, 1987; Navone & Di Gioia, 1988). Secretogranin II (SgII), or chromogranin C, is one of the granin family, and was initially found in the bovine anterior pituitary (Rosa & Zanini, 1981, 1983; Rosa et al., 1985a). SgII is thought to participate in the sorting and packaging of peptide hormones and neuropeptides into secretory granules and LDCVs (Rosa et al., 1985b; Gerdes et al., 1989; Courel et al., 2008). Furthermore, secretoneurin, a 33-amino-acid polypeptide cleaved from SgII by proteolytic processing (Kirchmair et al., 1993), and SgII itself have been shown to stimulate dopamine release from nigrostri- atal neurons (Rosa et al., 1985a,b; Eiden, 1987; Fischer-Colbrie et al., 1987; Huttner & Benedum, 1987; Wiedermann, 2000). Immunohistochemical studies on SgII (Rosa et al., 1985a; Cozzi et al., 1989) and secretoneurin (Marksteiner et al., 1993) have shown their wide regional distribution with predominant labeling in nerve terminals. Moreover, Rosa et al. (1985a) have revealed the locali- zation of SgII in secretory granules of the anterior pituitary. However, the subcellular localization of SgII in neurons of the central nervous system remains largely unknown. In the present study, we investigated the cellular expression and ultrastructural localization of SgII in adult mouse brains, particularly focusing on whether SgII is selective to LDCVs by analysing the hippocampus Correspondence: Dr M. Watanabe, as above. E-mail: watamasa@med.hokudai.ac.jp Received 20 July 2010, revised 22 August 2010, accepted 10 September 2010 European Journal of Neuroscience, Vol. 33, pp. 82–94, 2011 doi:10.1111/j.1460-9568.2010.07472.x ª 2010 The Authors. European Journal of Neuroscience ª 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience