Metabotropic Glutamate Receptor 4- Immunopositive Terminals of Medium- Sized Spiny Neurons Selectively Form Synapses With Cholinergic Interneurons in the Rat Neostriatum ERIKO KURAMOTO, 1 FUMINO FUJIYAMA, 1 TOMO UNZAI, 1 KOUICHI NAKAMURA, 1,3 HIROYUKI HIOKI, 1 TAKAHIRO FURUTA, 1 RYUICHI SHIGEMOTO, 2,3 FRANCESCO FERRAGUTI, 4 AND TAKESHI KANEKO 1,3 * 1 Department of Morphological Brain Science, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan 2 Division of Cerebral Structure National Institute for Physiological Sciences School of Life Science, The Graduate University for Advanced Studies, Okazaki 444-8787, Japan 3 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Kawaguchi 332-0012, Japan 4 Department of Pharmacology, University of Innsbruck, Innsbruck A-6020, Austria ABSTRACT Metabotropic glutamate receptor 4 (mGluR4) is localized mainly to presynaptic membranes in the brain. Rat neostriatum has been reported to contain two types of mGluR4-immunoreactive axon varicosities: small, weakly immunoreactive varicosities that were distributed randomly (type 1) and large, intensely immunoreactive ones that were often aligned linearly (type 2). In the present study, most type 1 terminals formed asymmetric synapses on dendritic spines, whereas type 2 terminals made symmetric synapses on dendritic shafts, showing immunoreactivity for GABAergic markers. After depletion of neostriatal neurons, type 2 but not type 1 varicosities were largely decreased in the damaged region. When medium-sized spiny neurons (MSNs) were labeled with Sindbis virus expressing membrane-targeted green fluorescent protein, mGluR4 immunoreactivity was observed on some varicosities of their axon collaterals in immunofluores- cence and immunoelectron microscopies. Furthermore, type 2 varicosities were often positive for substance P but mostly negative for striatal interneuron markers and preproenkephalin. Thus, striatonigral/striato-entopeduncular MSNs are likely to be the largest source of type 2 mGluR4- immunopositive axon terminals in the neostriatum. Next, in the double-immunofluorescence study, almost all choline acetyltransferase (ChAT)-immunopositive and 41% of NK1 receptor- positive dendrites were heavily associated with type 2 mGluR4-immunoreactive varicosities. Neuronal nitric oxide synthase (nNOS)-positive dendrites, in contrast, seemed associated with only a few type 2 varicosities. Conversely, almost all type 2 varicosities were closely apposed to NK1 receptor-positive dendrites that were known to be derived from cholinergic and nNOS- producing interneurons. These findings indicate that the mGluR4-positive terminals of MSN axon collaterals selectively form synapses with neostriatal cholinergic interneurons. J. Comp. Neurol. 500:908 –922, 2007. © 2006 Wiley-Liss, Inc. Indexing terms: choline acetyltransferase; projection neurons; target neuron-specific localization; basal ganglia; confocal laser scanning microscopy; immunoelectron microscopy Grant sponsor: Ministry of Education, Science, Sports and Culture of Japan; Grant number: 16200025; Grant number: 16500217; Grant num- ber: 17022020; Grant number: 17022024; Grant number: 17650100; Grant sponsor: Japan Society for the Promotion of Science for Young Scientists; Grant number: 15-5638 (to K.N.). *Correspondence to: Prof. Takeshi Kaneko, Department of Morphologi- cal Brain Science, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan. E-mail: kaneko@mbs.med.kyoto-u.ac.jp Received 8 November 2005; Revised 13 July 2006; Accepted 24 Septem- ber 2006 DOI 10.1002/cne.21216 Published online in Wiley InterScience (www.interscience.wiley.com). THE JOURNAL OF COMPARATIVE NEUROLOGY 500:908 –922 (2007) © 2006 WILEY-LISS, INC.