title: Gephyrin clusters are absent from small diameter primary afferent terminals despite the presence of GABA(A) receptors.
abstract: Whereas both GABA(A) receptors (GABA(A)Rs) and glycine receptors (GlyRs) play a role in control of dorsal horn neuron excitability, their relative contribution to inhibition of small diameter primary afferent terminals remains controversial. To address this, we designed an approach for quantitative analyses of the distribution of GABA(A)R-subunits, GlyR α1-subunit and their anchoring protein, gephyrin, on terminals of rat spinal sensory afferents identified by Calcitonin-Gene-Related-Peptide (CGRP) for peptidergic terminals, and by Isolectin-B4 (IB4) for nonpeptidergic terminals. The approach was designed for light microscopy, which is compatible with the mild fixation conditions necessary for immunodetection of several of these antigens. An algorithm was designed to recognize structures with dimensions similar to those of the microscope resolution. To avoid detecting false colocalization, the latter was considered significant only if the degree of pixel overlap exceeded that expected from randomly overlapping pixels given a hypergeometric distribution. We found that both CGRP(+) and IB4(+) terminals were devoid of GlyR α1-subunit and gephyrin. The α1 GABA(A)R was also absent from these terminals. In contrast, the GABA(A)R α2/α3/α5 and β3 subunits were significantly expressed in both terminal types, as were other GABA(A)R-associated-proteins (α-Dystroglycan/Neuroligin-2/Collybistin-2). Ultrastructural immunocytochemistry confirmed the presence of GABA(A)R β3 subunits in small afferent terminals. Real-time quantitative PCR (qRT-PCR) confirmed the results of light microscopy immunochemical analysis. These results indicate that dorsal horn inhibitory synapses follow different rules of organization at presynaptic versus postsynaptic sites (nociceptive afferent terminals vs inhibitory synapses on dorsal horn neurons). The absence of gephyrin clusters from primary afferent terminals suggests a more diffuse mode of GABA(A)-mediated transmission at presynaptic than at postsynaptic sites.
authors.affiliation: Centre de Recherche de l'Institut Universitaire en Santé Mentale de Québec, Québec, Québec G1J 2G3, Canada, Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec H3G 1Y6, Canada, Département de Psychiatrie et Neurosciences, Université Laval, Québec, Québec G1K 7P4, Canada, Alan Edwards Center for Research of Pain, McGill University, Montréal, Québec H3A 0G1, Canada.
authors.forename: Louis-Etienne
authors.identifier: http://orcid.org/0000-0002-5514-0049
authors.initials: LE
authors.lastname: Lorenzo
authors.affiliation: Centre de Recherche de l'Institut Universitaire en Santé Mentale de Québec, Québec, Québec G1J 2G3, Canada, Department of Physics, McGill University, Montréal, Québec H3A 2T8, Canada.
authors.forename: Antoine G
authors.initials: AG
authors.lastname: Godin
authors.affiliation: Centre de Recherche de l'Institut Universitaire en Santé Mentale de Québec, Québec, Québec G1J 2G3, Canada.
authors.forename: Feng
authors.initials: F
authors.lastname: Wang
authors.affiliation: Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec H3G 1Y6, Canada.
authors.forename: Manon
authors.initials: M
authors.lastname: St-Louis
authors.affiliation: McGill University Health Centre, Montréal, Québec H3G 1A4, Canada.
authors.forename: Salvatore
authors.identifier: http://orcid.org/0000-0003-2368-4196
authors.initials: S
authors.lastname: Carbonetto
authors.affiliation: Department of Physics, McGill University, Montréal, Québec H3A 2T8, Canada, Department of Chemistry, McGill University, Montréal, Québec H3A 2K6, Canada, and.
authors.forename: Paul W
authors.initials: PW
authors.lastname: Wiseman
authors.affiliation: Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec H3G 1Y6, Canada, Alan Edwards Center for Research of Pain, McGill University, Montréal, Québec H3A 0G1, Canada, Department of Anatomy and Cell Biology, McGill University, Montréal, Québec H3A 2B2, Canada.
authors.forename: Alfredo
authors.initials: A
authors.lastname: Ribeiro-da-Silva
authors.affiliation: Centre de Recherche de l'Institut Universitaire en Santé Mentale de Québec, Québec, Québec G1J 2G3, Canada, Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec H3G 1Y6, Canada, Département de Psychiatrie et Neurosciences, Université Laval, Québec, Québec G1K 7P4, Canada, Alan Edwards Center for Research of Pain, McGill University, Montréal, Québec H3A 0G1, Canada, yves.dekoninck@crulrg.ulaval.ca.
authors.forename: Yves
authors.initials: Y
authors.lastname: De Koninck
mesh_terms: D000818:Animals; D015740:Calcitonin Gene-Related Peptide / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D037102:Lectins / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D009475:Neurons, Afferent / Q000502:physiology*; D017729:Presynaptic Terminals / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011963:Receptors, GABA-A / Q000378:metabolism*; D018009:Receptors, Glycine / Q000378:metabolism; D013116:Spinal Cord / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D037102:Lectins; D008565:Membrane Proteins; D011963:Receptors, GABA-A; D018009:Receptors, Glycine; C073441:gephyrin; D015740:Calcitonin Gene-Related Peptide
keywords: calcitonin gene-related peptide; dorsal root ganglion; dystroglycan; isolectin B4; pain; synaptic inhibition