Cell type-specific super-resolution imaging reveals an increase in calcium-permeable AMPA receptors at spinal peptidergic terminals as an anatomical correlate of inflammatory pain.

Spinal hyperexcitability is a key event in the development of persistent pain, and arises partly from alterations in the number and localization of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate receptors. However, determining precisely where these changes occur is challenging due to the requirement for multiplex labelling and nanoscale resolution. The recent development of super-resolution light microscopy provides new tools to address these challenges. Here, we apply combined confocal/direct STochastic Optical Reconstruction Microscopy (dSTORM) to reveal changes in calcium-permeable subunits of AMPA-type glutamate receptors (GluA1) at identified spinal cord dorsal horn (SCDH) peptidergic axon terminals in a model of inflammatory pain. L4/5 lumbar spinal cord was collected from adult male C57BL/6J mice 24 hours after unilateral hind paw injection of saline or 1% carrageenan (n = 6/group). Tissue was immunolabelled for markers of peptidergic axon terminals (substance P; SP), presynaptic active zones (Bassoon), and GluA1. Direct stochastic optical reconstruction microscopy revealed a 59% increase in total GluA1 immunolabelling in the SCDH in the carrageenan group, which was not detected by confocal microscopy. Cell type-specific analyses identified a 10-fold increase in GluA1 localized to SP structures, and identified GluA1 nanodomains that scaled with behavioural hypersensitivity, and were associated with synaptic release sites. These findings demonstrate that dSTORM has the sensitivity and power to detect nanoscale anatomical changes in the SCDH, and provides new evidence for synaptic insertion of GluA1-AMPA-Rs at spinal peptidergic nociceptive terminals in a model of inflammatory pain.

Clathrin-independent internalization of the human histamine H1-receptor in CHO-K1 cells.

The aim of the present study was to investigate the cellular pathway involved in histamine-stimulated internalization of the human H1-receptor in CHO-K1 cells expressing N-terminal myc-tagged H1-receptor (Myc-H1) or N-terminal myc-C-terminal green fluorescent protein (Myc-GFP H1) versions of the receptor. Studies of 3H-mepyramine binding and histamine-stimulated 3H-inositol phosphate accumulation in these cells showed that the Myc-H1 and Myc-GFP H1-receptors had identical pharmacology to the wild-type H1-receptor. The Myc-H1-receptor was rapidly internalized in CHO-K1 cells following stimulation with histamine (0.1 mM). This response occurred within 15 min, and could be prevented by the quaternary H1-receptor antagonist alpha-pirdonium. Similar data were obtained with the Myc-GFP H1-receptors. Internalization of the Myc-GFP H1-receptor was maintained in the absence of extracellular calcium and was not inhibited by the CAM kinase II inhibitor KN-62 (10 microM). Phorbol dibutyrate, an activator of protein kinase C, was also able to stimulate internalization of the H1-receptor. However, inhibition or downregulation of protein kinase C (which significantly modified histamine-stimulated inositol phosphate responses) was without effect on the internalization of the H1-receptor stimulated by histamine. Hypertonic sucrose did not prevent histamine-induced internalization of the Myc-GFP H1-receptor, but was able to attenuate internalization of transferrin via clathrin-mediated endocytosis in the same cells. In contrast, preincubation of cells with filipin or nystatin, which disrupts caveolae and lipid rafts, completely inhibited the histamine-induced internalization of the Myc-GFP H1-receptor, but was without effect on the sequestration of transferrin. The H1-receptor and cholera toxin subunit B were colocalized under resting conditions at the cell surface. Immunohistochemical studies with an antibody to caveolin-1 confirmed that this protein was also localized predominantly to the plasma membrane. However, following stimulation of CHO-Myc-GFP H1 cells with histamine, there was no evidence for internalization of caveolin-1 in parallel with the H1-receptor. These data provide strong evidence that the H1-receptor is internalized via a clathrin-independent mechanism and most likely involves lipid rafts.