The aims of the present study were to

The aims of the present study were to further investigate the localisation of glycine and GABAA receptors on individual neurons and how closely these two receptor types were associated with each other by carrying out double immunofluorescent labelling and imaged using confocal laser scanning microscopy. The results of the present study show that in the human hypoglossal nucleus there are high levels of cellular immunoreactivity for both glycine receptors and the α1, α2, β2,3 subunits of the GABAA receptor. These are localised to cellular membranes on the soma and BAY 61-3606 of hypoglossal neurons, but that there is also a high degree of variability of the number of glycine receptor clusters and specific GABAA receptor subunits amongst individual neurons. This would indicate a high degree of variability of the GABAergic and glycinergic inputs onto individual hypoglossal neurons. The localisation of glycine receptors and GABAA receptor subunits investigated in this study generally agree with the results of glycine receptors and GABAA receptor subunits found in other studies. GABAA α1 and α2 subunits have been identified in rat hypoglossal nucleus (Fritschy and Mohler, 1995; Lorenzo et al., 2006) and GABAARα1 subunit was identified on ChAT staining motoneurons in the rat hypoglossal nucleus (O’Brien and Berger, 2001), however β2,3 subunits were only weakly stained in the rat hypoglossal nucleus (Fritschy and Mohler, 1995). Regional variations in GABAAR subunits were found throughout the rat hypoglossal nucleus and these could vary with age whereas glycine receptors were quite evenly distributed (O’Brien and Berger, 2001). Immunohistochemical staining of GABAergic terminals have been identified scattered throughout the nucleus in rat and primate hypoglossal nucleus. In addition, our study revealed small neurons with a bipolar appearance which were strongly labelled with the GABAAR α1 subunit which bear a strong resemblance to GABAergic interneurons identified in the macaque and rat hypoglossal nucleus (Aldes et al., 1988; Takasu et al., 1987). These small neurons appeared to have no detectable glycine receptor labelling associated with them.
Ethical statement
Acknowledgements We would like to thank the Neurological Foundation of New Zealand, The Neurological Foundation of New Zealand Human Brain Bank, The University of Auckland Doctoral Scholarships, The University of Auckland Faculty Research Development FundFRDF-3634745, The Health Research Council of New Zealand.
Introduction The inhibitory glycinergic interneurons within the dorsal horn of spinal cord receive the synaptic inputs from primary afferent fibers and innervate the excitatory nociceptive neurons (Lu et al., 2013). The glycine receptors (GlyRs)-mediated inhibitory synaptic transmission onto excitatory neurons plays a gating control over the flow of nociceptive information to higher brain regions (Foster et al., 2015). Following peripheral tissue injuries, the glycinergic inhibition is reduced (disinhibition), which is widely considered as a key contributor to inflammatory pain (Takazawa and MacDermott, 2010). Enhancement of GlyRs synaptic currents effectively alleviates chronic pain sensitization (Foster et al., 2015). Previous studies have demonstrated that the neuronal activities regulate GlyRs transportation to and from plasma membrane through exocytosis and endocytosis, which act in cooperation to determine the number of GlyRs at synapses (Dumoulin et al., 2009). In a couple of pathophysiological processes, the protein kinases-mediated phosphorylation alters the synaptic trafficking of GlyRs and fine-tunes the glycinergic efficacy (Acuna et al., 2016, Harvey et al., 2004, Manzke et al., 2010). In addition to phosphorylation, the ubiquitin conjugation has also been implicated in the dynamic regulation of GlyRs surface expression (Buttner et al., 2001). The ubiquitin modification requires the sequential action of E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes and E3 ubiquitin ligases (Rape, 2018). To date, hundreds of E3 ligase family members have been cloned and identified. A prevailing view is that E3 ubiquitin ligases are responsible for the recruitment of protein substrates and dictate the specificity of ubiquitination (O'Connor and Huibregtse, 2017). Based on the domain organization, the E3 ubiquitin ligases can be grouped into three subfamilies, which are characterized by the presence of Really Interesting New Gene (RING) domain, the Homologous to E6AP Carboxyl Terminus (HECT) domain and RING-Between-RING domain (RBR), respectively (Khoronenkova and Dianov, 2011, Lorenz, 2018).