Selective distribution of GABA(A) receptor subtypes in mouse spinal dorsal horn neurons and primary afferents

In the spinal cord dorsal horn, presynaptic GABA(A) receptors (GABA(A)Rs) in the terminals of nociceptors as well as postsynaptic receptors in spinal neurons regulate the transmission of nociceptive and somatosensory signals from the periphery. GABA(A)Rs are heterogeneous and distinguished functionally and pharmacologically by the type of α subunit variant they contain. This heterogeneity raises the possibility that GABA(A)R subtypes differentially regulate specific pain modalities. Here, we characterized the subcellular distribution of GABA(A)R subtypes in nociceptive circuits by using immunohistochemistry with subunit-specific antibodies combined with markers of primary afferents and dorsal horn neurons. Confocal laser scanning microscopy analysis revealed a distinct, partially overlapping laminar distribution of α1-3 and α5 subunit immunoreactivity in laminae I-V. Likewise, a layer-specific pattern was evident for their distribution among glutamatergic, γ-aminobutyric acid (GABA)ergic, and glycinergic neurons (detected in transgenic mice expressing vesicular glutamate transporter 2-enhanced green fluorescent protein [vGluT2-eGFP], glutamic acid decarboxylase [GAD]67-eGFP, and glycine transporter 2 (GlyT2)-eGFP, respectively). Finally, all four subunits could be detected within primary afferent terminals. C-fibers predominantly contained either α2 or α3 subunit immunoreactivity; terminals from myelinated (Aβ/Aδ) fibers were colabeled in roughly equal proportion with each subunit. The presence of axoaxonic GABAergic synapses was determined by costaining with gephyrin and vesicular inhibitory amino acid transporter to label GABAergic postsynaptic densities and terminals, respectively. Colocalization of the α2 or α3 subunit with these markers was observed in a subset of C-fiber synapses. Furthermore, gephyrin mRNA and protein expression was detected in dorsal root ganglia. Collectively, these results show that differential GABA(A)R distribution in primary afferent terminals and dorsal horn neurons allows for multiple, circuit-specific modes of regulation of nociceptive circuits.