The focus of this proposal is on mechanisms underlying pain, in particular, the sensitization of nociceptive afferents. Specifically, I proposed to fill a critical gap in our understanding of these mechanisms by characterizing the contribution of neural activity, in particular, neural activity-dependent increases in intracellular Ca2+ in both the rapid and the longer-term sensitization of these afferents. Data from previous studies indicate that activity- dependent changes in gene expression are not only Ca2+ dependent, but dependent on the pattern of activity. Similarly, a number of ion channels that contribute to the establishment of excitability in sensory neurons are modulated by a variety of enzymes, that are themselves regulated by changes in intracellular Ca2+. In this context, I have proposed to focus on voltage-gated sodium channels (VGSCs) for a number of reasons, not the least of which include a) the essential role these channels play in neural excitability, b) evidence that both rapid post-translational modifications of these channels, and longer-term changes in channel expression, influence sensory neuron excitability, and c) because both rapid and longer-term changes in VGSCs have been shown to be Ca2+ and/or activity dependent. Finally, that the influence of activity on both rapid and longer-term changes in VGSCs properties may change in the presence of injury, is suggested by the observation that persistent inflammation results in dramatic changes in the regulation of intracellular Ca2+ in nociceptive afferents, which includes the almost complete loss of Na+/Ca2+ exchanger (NCX) activity in the sensory neuron somata. Thus, I hypothesize that dysregulation of intracellular Ca2+, particularly the decrease in NCX activity, contributes to inflammatory pain via the activity-dependent sensitization of nociceptive afferents secondary to both the rapid modulation of VGSCs, as well as longer-term changes in the pattern of expression of these channels. To test this hypothesis, I will characterize the influence of NCX blockade on rapid activity-dependent changes in the excitability and VGSC properties of mouse and human nociceptive afferents. I will then characterize the influence of NCX blockade on activity-dependent changes in transcription/translation on the excitability and VGSCs in mouse and human nociceptive afferents. The experiments proposed will not only serve as an excellent training vehicle enabling the mastery of a variety of powerful techniques and approaches, but will be the first to establish the relationship between the regulation of intracellular Ca2+ and both rapid and longer-term activity-dependent changes in afferent excitability and VGSC properties.