title: Origin of calretinin-containing, vesicular glutamate transporter 2-coexpressing fiber terminals in the entorhinal cortex of the rat.
abstract: The entorhinal cortex of the rat (EC) contains a dense fiber plexus that expresses the calcium-binding protein calretinin (CR). Some CR fibers contain vesicular glutamate transporter 2 (VGluT2, associated with glutamatergic neurotransmission). CR-VGluT2 coexpressing fibers may have an extrinsic origin, for instance, the midline thalamic nucleus reuniens. Alternatively, they may belong to cortical interneurons. We studied the first possibility with anterograde and retrograde neuroanatomical tracing methods combined with CR and VGluT2 immunofluorescence and confocal laser scanning. The alternative possibility was studied with in situ hybridization fluorescence histochemistry for VGluT2 mRNA combined with CR immunofluorescence. In the anterograde tracing experiments, we observed many labeled reuniens fibers in EC expressing CR. Some of these labeled fibers contained immunoreactivity for VGluT2 and CR. In the complementary retrograde tracing experiments, we found retrogradely labeled cell bodies in nucleus reuniens of the thalamus that coexpressed CR. We also examined the colocalization of VGluT2 and CR in the entorhinal cortex by using in situ hybridization and CR immunofluorescence. In these experiments, we observed CR-immunopositive cortical neurons that coexpressed VGluT2. For the same sections, with CR as the principal marker and parvalbumin as a control marker, we found that parvalbumin neurons were negative for VGluT2 mRNA. Thus, CR-VGluT2-expressing axon terminals in EC belong to two sources: projection fibers from the thalamus and axon collaterals of local interneurons. VGluT2 expression is linked to the synaptic transmission of the excitatory neurotransmitter glutamate, so these thalamic CR-VGluT2 projection neurons and entorhinal CR-VGluT2 interneurons should be regarded as excitatory.
authors.affiliation: Department of Anatomy and Neurosciences, Graduate School of Neurosciences, Research Institute of Neuroscience, Vrije Universiteit Medical Center, 1007 MB Amsterdam, The Netherlands. fg.wouterlood@vumc.nl
authors.forename: Floris G
authors.initials: FG
authors.lastname: Wouterlood
authors.forename: Verena
authors.initials: V
authors.lastname: Aliane
authors.forename: Amber J
authors.initials: AJ
authors.lastname: Boekel
authors.forename: Elizabeth E
authors.initials: EE
authors.lastname: Hur
authors.forename: Laszlo
authors.initials: L
authors.lastname: Zaborszky
authors.forename: Pedro
authors.initials: P
authors.lastname: Barroso-Chinea
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Härtig
authors.forename: José L
authors.initials: JL
authors.lastname: Lanciego
authors.forename: Menno P
authors.initials: MP
authors.lastname: Witter
mesh_terms: D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives / Q000378:metabolism; D001931:Brain Mapping; D064032:Calbindin 2; D003911:Dextrans / Q000378:metabolism; D018728:Entorhinal Cortex / Q000166:cytology*; D005260:Female; D021621:Imaging, Three-Dimensional / Q000379:methods; D017403:In Situ Hybridization / Q000379:methods; D018613:Microscopy, Confocal / Q000379:methods; D009434:Neural Pathways / Q000502:physiology; D009474:Neurons / Q000166:cytology*; D010320:Parvalbumins / Q000378:metabolism; D017729:Presynaptic Terminals / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D064030:S100 Calcium Binding Protein G / Q000378:metabolism*; D013266:Stilbamidines / Q000378:metabolism; D050599:Vesicular Glutamate Transport Protein 2 / Q000378:metabolism*
chemical_list: C049774:2-hydroxy-4,4'-diamidinostilbene, methanesulfonate salt; C576512:Calb2 protein, rat; D064032:Calbindin 2; D003911:Dextrans; D010320:Parvalbumins; D064030:S100 Calcium Binding Protein G; D013266:Stilbamidines; D050599:Vesicular Glutamate Transport Protein 2; C076397:biotinylated dextran amine; D001710:Biotin