title: Immunostaining for Homer reveals the majority of excitatory synapses in laminae I-III of the mouse spinal dorsal horn.
abstract: The spinal dorsal horn processes somatosensory information before conveying it to the brain. The neuronal organization of the dorsal horn is still poorly understood, although recent studies have defined several distinct populations among the interneurons, which account for most of its constituent neurons. All primary afferents, and the great majority of neurons in laminae I-III are glutamatergic, and a major factor limiting our understanding of the synaptic circuitry has been the difficulty in identifying glutamatergic synapses with light microscopy. Although there are numerous potential targets for antibodies, these are difficult to visualize with immunocytochemistry, because of protein cross-linking following tissue fixation. Although this can be overcome by antigen retrieval methods, these lead to difficulty in detecting other antigens. The aim of this study was to test whether the postsynaptic protein Homer can be used to reveal glutamatergic synapses in the dorsal horn. Immunostaining for Homer gave punctate labeling when viewed by confocal microscopy, and this was restricted to synapses at the ultrastructural level. We found that Homer puncta were colocalized with the AMPA receptor GluR2 subunit, but not with the inhibitory synapse-associated protein gephyrin. We also examined several populations of glutamatergic axons and found that most boutons were in contact with at least one Homer punctum. These results suggest that Homer antibodies can be used to reveal the great majority of glutamatergic synapses without antigen retrieval. This will be of considerable value in tracing synaptic circuits, and also in investigating plasticity of glutamatergic synapses in pain states.
authors.affiliation: Institute of Neuroscience and Psychology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK.
authors.forename: Maria
authors.initials: M
authors.lastname: Gutierrez-Mecinas
authors.affiliation: Department of Neurobiology, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Emily D
authors.initials: ED
authors.lastname: Kuehn
authors.affiliation: Department of Neurobiology, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Victoria E
authors.initials: VE
authors.lastname: Abraira
authors.affiliation: Institute of Neuroscience and Psychology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK.
authors.forename: Erika
authors.initials: E
authors.lastname: Polgár
authors.affiliation: Department of Anatomy, Hokkaido University School of Medicine, Sapporo 060-8638, Japan.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Watanabe
authors.affiliation: Institute of Neuroscience and Psychology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK. Electronic address: andrew.todd@glasgow.ac.uk.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Todd
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism*; D000072233:Homer Scaffolding Proteins / Q000378:metabolism*; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000378:metabolism; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D020671:Posterior Horn Cells / Q000378:metabolism* / Q000648:ultrastructure; D018091:Receptors, AMPA / Q000378:metabolism; D013569:Synapses / Q000378:metabolism* / Q000648:ultrastructure; D050599:Vesicular Glutamate Transport Protein 2 / Q000378:metabolism; D050495:Vesicular Inhibitory Amino Acid Transport Proteins / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D000072233:Homer Scaffolding Proteins; D008565:Membrane Proteins; D018091:Receptors, AMPA; C493477:Slc17a6 protein, mouse; D050599:Vesicular Glutamate Transport Protein 2; D050495:Vesicular Inhibitory Amino Acid Transport Proteins; C492244:Viaat protein, mouse; C073441:gephyrin; D018698:Glutamic Acid; C104722:glutamate receptor ionotropic, AMPA 2
keywords: confocal microscopy; excitatory interneuron; glutamatergic synapse; pain; primary afferent; spinal cord