PT  - JOURNAL ARTICLE
AU  - Louis-Etienne Lorenzo
AU  - Antoine G. Godin
AU  - Feng Wang
AU  - Manon St-Louis
AU  - Salvatore Carbonetto
AU  - Paul W. Wiseman
AU  - Alfredo Ribeiro-da-Silva
AU  - Yves De Koninck
TI  - Gephyrin Clusters Are Absent from Small Diameter Primary Afferent Terminals Despite the Presence of GABA<sub>A</sub> Receptors
AID  - 10.1523/JNEUROSCI.0159-14.2014
DP  - 2014 Jun 11
TA  - The Journal of Neuroscience
PG  - 8300--8317
VI  - 34
IP  - 24
4099  - http://www.jneurosci.org/content/34/24/8300.short
4100  - http://www.jneurosci.org/content/34/24/8300.full
SO  - J. Neurosci.2014 Jun 11; 34
AB  - Whereas both GABAA receptors (GABAARs) 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 GABAAR-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 GABAAR was also absent from these terminals. In contrast, the GABAAR α2/α3/α5 and β3 subunits were significantly expressed in both terminal types, as were other GABAAR-associated-proteins (α-Dystroglycan/Neuroligin-2/Collybistin-2). Ultrastructural immunocytochemistry confirmed the presence of GABAAR 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 GABAA-mediated transmission at presynaptic than at postsynaptic sites.