Modulation of pain-associated hyperexcitability at central synapses of capsaicin-sensitive nociceptors

TRPV1-expressing (capsaicin-sensitive) afferents correspond largely to peptidergic nociceptors, whichplay an important role in both acute pain and chronic hyper-sensitive pain states. Investigation ofprocesses that can modulate function of their early central synapses in spinal dorsal horn could point theway to novel analgesics for chronic pain. We have developed a new method to quantify receptor-evokedCa2+ fluorescence responses of ex vivo synaptic preparations and use it here to measurecapsaicin-evoked responses in dorsal horn from control and pain state animals.Synaptoneurosomes (re-sealed presynaptic and closely apposed postsynaptic compartments) wereprepared from dorsal lumbar spinal cord of male Sprague-Dawley rats, under conditions designed tomaintain functional integrity, and loaded with a no-wash Ca2+ fluorophore (Calcium 5). Capsaicin orother agents (including ionomycin as a positive control) were added in vitro and responses measured byfluorometric plate reader.Responses to capsaicin showed concentration-dependent increases from 0.2-10 microM, were 5-6 foldgreater in dorsal than in ventral horn and were largely reversed by the TRPV1 antagonist AMG9810 orpresynaptically acting tetanus toxin. In addition the responses were inhibited by antagonists of AMPAorNMDA-type glutamate receptors, consistent with glutamatergic transmission from capsaicin-activatedpresynaptic terminals. Agents selective for several distinct subtypes of GluN2 subunit showeddifferential ability to inhibit capsaicin responses.We further explored the effects of endogenous analgesic mechanisms. In vitro addition of mu (and to alesser extent delta) opioids strongly attenuated capsaicin responses. In a model of chronic inflammatorypain (intraplantar Complete Freund’s Adjuvant), ex vivo responsiveness to capsaicin was increased in amanner completely reversed by NMDA receptor antagonists. This inflammation-inducedhypersensitivity at TRPV1 afferent central synapses was strongly attenuated by prior in vivoadministration of the TRPM8 agonist, icilin (200 microM topical to hindpaws, 15 min).These observations reveal quantifiable actions of established or novel analgesic targets impacting oncentral synapses of TRPV1-expressing nociceptors.