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Neuronal diversity in the pedunculopontine nucleus: topographical distribution, neurochemical nature and projections of calcium-binding protein expressing neurons Cristina Martinez-Gonzalez1, Marisela Morales2, Hui-ling Wang2, J. Paul Bolam1 and Juan Mena-Segovia1 MRC Anatomical Neuropharmacology Unit, Department of Pharmacology, University of Oxford, Mansfield Road, OX1 3TH, Oxford, U. K.1. Intramural Research Program, Cellular Neurophysiology, Biomedical Research Center, National Institute on Drug Abuse, 251 Bayview Blvd, Baltimore, MD 21224, USA2. This work was funded by the Medical Research Council UK. C.M.-G. is in receipt of a CONACyT studentship. The pedunculopontine nucleus (PPN) is a brainstem structure involved in motor control and arousal and consists of cholinergic, GABAergic and glutamatergic neurons. The PPN also contains populations of calcium-binding protein-expressing neurons. In order to define the neurochemical phenotype of these neurons we examined the expression of calbindin (CB) and calretinin (CR) together with other markers in the PPN. Sagittal sections of the rat brainstem were doubleimmunolabelled to reveal choline acetyltransferase (ChAT) and CB or CR. CB- and CR-positive neurons accounted for a large proportion of PPN neurons, they had a lower density in the rostral PPN, they rarely expressed ChAT, but about a third expressed both calcium-binding proteins. A combination of imunolabelling for CB and CR with in situ hybridisation for GAD65/67 or VGluT2 mRNA, revealed that about one third of the CB- or CR-expressing neurons were GABAergic and two thirds were glutamatergic. Double retrograde tracer injections in the subthalamic nucleus (STN) and the gigantocellular nucleus (GiN) showed two subsets of caudal PPN neurons with a distinct pattern of connectivity. Only a small proportion of neurons (< 10%) were labelled for both tracers, most were located in the caudal PPN and were non-cholinergic. In confirmation of previous studies we observed cholinergic neurons that have ascending projections to the STN or descending projections to the GiN. We also observed CR-expressing neurons projecting to both structures but did not observe CB-expressing neurons with descending projections to the GiN. Our findings demonstrate that subpopulations of CB or CR-expressing neurons have a GABAergic or glutamatergic phenotype, have different connectivity patterns and are more concentrated in the caudal PPN. They are thus likely to have distinct physiological functions. Our data support the notion that the PPN is a highly heterogeneous structure with topographical differences defining functional domains.