GABAA and Glycine Receptor-Mediated Inhibitory Synaptic Transmission onto Adult Rat Lamina IIi PKCγ-Interneurons: Pharmacological but Not Anatomical Specialization

Abstract

Mechanical allodynia (pain to normally innocuous tactile stimuli) is a widespread symptom of inflammatory and neuropathic pain. Spinal or medullary dorsal horn (SDH or MDH) circuits mediating tactile sensation and pain need to interact in order to evoke mechanical allodynia. PKCγ-expressing (PKCγ+) interneurons and inhibitory controls within SDH/MDH inner lamina II (IIi) are pivotal in connecting touch and pain circuits. However, the relative contribution of GABA and glycine to PKCγ+ interneuron inhibition remains unknown. We characterized inhibitory inputs onto PKCγ+ interneurons by combining electrophysiology to record spontaneous and miniature IPSCs (sIPSCs, mIPSCs) and immunohistochemical detection of GABAARα2 and GlyRα1 subunits in adult rat MDH. While GlyR-only- and GABAAR-only-mediated mIPSCs/sIPSCs are predominantly recorded from PKCγ+ interneurons, immunohistochemistry reveals that ~80% of their inhibitory synapses possess both GABAARα2 and GlyRα1. Moreover, nearly all inhibitory boutons at gephyrin-expressing synapses on these cells contain glutamate decarboxylase and are therefore GABAergic, with around half possessing the neuronal glycine transporter (GlyT2) and therefore being glycinergic. Thus, while GABA and glycine are presumably co-released and GABAARs and GlyRs are present at most inhibitory synapses on PKCγ+ interneurons, these interneurons exhibit almost exclusively GABAAR-only and GlyR-only quantal postsynaptic inhibitory currents, suggesting a pharmacological specialization of their inhibitory synapses.

Synaptic Targets of Glycinergic Neurons in Laminae I–III of the Spinal Dorsal Horn

“…In addition, we assessed the dendritic location of synapses made by axon terminals of primary afferents, the function of which can be modulated by presynaptic glycinergic inhibition. For this reason, we selected the PKCγ-containing neurons which are known to receive substantial excitatory inputs from primary afferents and are under robust glycinergic pre- and postsynaptic inhibition [ 12 , 16 , 19 , 22 , 36 , 61 , 62 , 63 , 64 , 65 ]. In addition, their dendritic trees arborize in lamina II and lamina III [ 35 , 49 , 62 , 66 , 67 ], where we identified axo-axonic contacts between GlyT2 immunoreactive and IB4-binding as well as VGLUT1 immunoreactive axon terminals.…”

“…The relative contribution of GABAergic and glycinergic inhibition to spinal pain processing is, however, continuously under debate. The major points of this debate were clearly explicated in a recent paper [ 16 ] in which the authors investigated GABA A and glycine receptor-mediated inhibition of PKCγ-containing interneurons in inner lamina II of the spinal and medullary dorsal horn with pharmacological and anatomical methods. They claimed that nearly all (91.7%) inhibitory axon terminals making contact with these neurons contained GAD and approximately half of them (42.2% of all boutons) also expressed GlyT2; thus, nearly all of them could be regarded as GABAergic axon terminals, and half of the GABAergic axon terminals could also be considered glycinergic.…”

“…Even more remarkably, however, by recording sIPSCs and mIPSCs from the same set of synapses, it was found that approximately 70% of the quantal events were mediated by either glycine-only or GABA-only synaptic transmission. The major disagreement between morphological and physiological results, which was clearly presented in the article of El Khoueiry et al [ 16 ], became obvious several years ago and made the interpretation of data regarding the role of GABAergic and glycinergic inhibition in spinal pain processing quite ambiguous [ 1 , 2 , 9 , 10 , 12 , 14 , 23 ].…”

“…However, in addition to mixed GABA-glycine and GABA-only mIPSCs, glycine-only mIPSCs have been frequently recorded in neurons in the superficial spinal dorsal horn [ 9 , 10 ]. Furthermore, almost all physiological and pharmacological experimental data from the last two decades have substantiated the importance of glycine-only inhibition in the spinal dorsal horn, and its fundamental role in pain-related information processing [ 1 , 11 , 12 , 13 , 14 , 15 , 16 ]. There is compelling evidence that reduced glycinergic synaptic transmission induces central sensitization, hyperalgesia, and allodynia, leading to the development of chronic pain conditions [ 1 , 12 , 17 , 18 , 19 , 20 , 21 , 22 ].…”

“…Strongly reinforcing the importance of glycinergic inhibition, by using a combination of electrophysiology and optogenetics to quantify changes in inhibitory synaptic transmission onto neurons in the superficial spinal dorsal horn caused by the ablation of glycinergic neurons, Foster et al [ 1 ] found that more than 70% of the total inhibitory postsynaptic current amplitude recorded in excitatory neurons was blocked by the application of the glycine receptor antagonist strychnine and only the rest could be blocked by the GABA A receptor antagonist bicuculline. In addition, a series of studies in which inhibitory quantal postsynaptic currents were recorded in adult neurons within the superficial spinal dorsal horn showed that postsynaptic currents mediated by either glycine or GABA A receptors were common, but the incidence of mixed postsynaptic currents was much lower [ 16 , 23 , 24 , 25 ].…”