GABAergic basket cells expressing cholecystokinin contain vesicular glutamate transporter type 3 (VGLUT3) in their synaptic terminals in hippocampus and isocortex of the rat Jozsef Somogyi, 1,2 Agne Ás Baude, 1,3 Yuko Omori, 4 Hidemi Shimizu, 4 Salah El Mestikawy 5 , Masahiro Fukaya, 4 Ryuichi Shigemoto, 6,7 Masahiko Watanabe 4 and Peter Somogyi 1 1 MRC Anatomical Neuropharmacology Unit, Department of Pharmacology, Oxford University, Oxford OX1 3TH, UK 2 Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary 3 CNRS UMR 6150, Faculte  de Me Âdecine, IFR Jean Roche, Bd. Pierre Dramard, Marseilles, France 4 Department of Anatomy, Hokkaido University School of Medicine, Japan 5 INSERM Unite  513, Faculte  de Me Âdecine, 94010 Cre Âteil Cedex, France 6 Division of Cerebral Structure, National Institute for Physiological Sciences, School of Life Science, The Graduate University for Advanced Studies, Okazaki, Japan 7 CREST Japan Science and Technology Corporation, Kawaguchi, Japan Keywords: amine, GABA, neuropeptide, neurotransmitter, synapse Abstract Vesicular glutamate transporter type3 (VGLUT3) containing neuronal elements were characterized using antibodies to VGLUT3 and molecular cell markers. All VGLUT3-positive somata were immunoreactive for CCK, and very rarely, also for calbindin; none was positive for parvalbumin, calretinin, VIP or somatostatin. In the CA1 area, 26.8  0.7% of CCK-positive interneuron somata were VGLUT3-positive, a nonoverlapping 22.8  1.9% were calbindin-positive, 10.7  2.5% VIP-positive and the rest were only CCK- positive. The patterns of coexpression were similar in the CA3 area, the dentate gyrus and the isocortex. Immunoreactivity for VGLUT3 was undetectable in pyramidal and dentate granule cells. Boutons colabelled for VGLUT3, CCK and GAD were most abundant in the cellular layers of the hippocampus and in layers II±III of the isocortex. Large VGLUT3-labelled boutons at the border of strata radiatum and lacunosum-moleculare in the CA1 area were negative for GAD, but were labelled for vesicular monoamine transporter type2, plasmalemmal serotonin transporter or serotonin. No colocalization was found in terminals between VGLUT3 and parvalbumin, vesicular acetylcholine transporter and group III (mGluR7a,b; mGluR8a,b) metabotropic glutamate receptors. In stratum radiatum and the isocortex, VGLUT3-positive but GAD-negative boutons heavily innervated the soma and proximal dendrites of some VGLUT3- or calbindin-positive interneurons. The results suggest that boutons coexpressing VGLUT3, CCK and GAD originate from CCK-positive basket cells, which are VIP-immunonegative. Other VGLUT3-positive boutons immunopositive for serotonergic markers but negative for GAD probably originate from the median raphe nucleus and innervate select interneurons. The presumed amino acid substrate of VGLUT3 may act on presynaptic kainate or group II metabotropic glutamate receptors. Introduction The cell bodies and proximal dendrites of cortical pyramidal cells are innervated exclusively by GABAergic terminals, as revealed by immunocytochemical labelling for glutamic acid decarboxylase (GAD; Ribak, 1978) and g-amino butyric acid (GABA; Storm-Mathi- sen et al., 1983; Somogyi & Hodgson, 1985). At least two distinct populations of basket cells provide this somatic and perisomatic inhibition. One contains parvalbumin (PV; Katsumaru et al., 1988; Sik et al., 1995); the other expresses peptides derived from pro- cholecystokinin (CCK; Nunzi etal., 1985; Freund etal., 1986). Some of these latter cells also express vasoactive intestinal polypeptide (VIP; Acsady etal., 1996). Although the relative weight of these two distinct GABAergic inputs may differ amongst various cortical principal cells, this dual GABAergic innervation is found throughout the whole cortical mantle. Recent cloning (Fremeau, et al., 2002; Gras et al., 2002; Schafer et al., 2002; Takamori et al., 2002) and immunohistochemical loca- lization of the subtype 3 vesicular glutamate transporter (VGLUT3) revealed that many terminals on cell bodies of hippocampal pyramidal cells and interneurons are immunoreactive for VGLUT3 (Fremeau et al., 2002; Gras et al., 2002), where VGLUT3-positive terminals establish symmetrical synapses (typeII; Gray, 1959) similar to those made by GABAergic cells. This implies that at least one type of basket cell contains VGLUT3. Indeed, some GAD immunoreactive cells were labelled for VGLUT3 in the hippocampus (Fremeau et al., 2002). Therefore, we tested basket cell molecular markers for possible colocalization with VGLUT3. Furthermore, the whole cortical mantle is innervated by cholinergic and serotonergic afferents and both cholinergic and serotonergic neurons were shown to express VGLUT3 (Fremeau et al., 2002; Gras etal., 2002; Schafer etal., 2002). Hence, we tested the contribution of European Journal of Neuroscience, Vol. 19, pp. 552±569, 2004 ß Federation of European Neuroscience Societies doi:10.1111/j.1460-9568.2003.03091.x Correspondence: Dr Jozsef Somogyi, as above. E-mail: jozsef.somogyi@pharm.ox.ac.uk Received 7 August 2003, revised 10 October 2003, accepted 14 October 2003