Gephyrin Clusters Are Absent from Small Diameter Primary Afferent Terminals Despite the Presence of GABAA Receptors

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 alpha 1-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 alpha 1-subunit and gephyrin. The alpha 1 GABA(A)R was also absent from these terminals. In contrast, the GABA(A)R alpha 2/alpha 3/alpha 5 and beta 3 subunits were significantly expressed in both terminal types, as were other GABA(A)R-associated-proteins (alpha-Dystroglycan/Neuroligin-2/Collybistin-2). Ultrastructural immunocytochemistry confirmed the presence of GABA(A)R beta 3 subunits in small afferent terminals. Real-time quantitativePCR(qRT-PCR) confirmed the results of light microscopy immuno-chemical analysis. These results indicate that dorsal horn inhibitory synapses follow different rules of organization at presynaptic versus postsynapticsites(nociceptive afferentterminals vs inhibitory synapses on dorsalhorn 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.