Glutamate and substance P are released by primary afferent fibers of the dorsal root ganglion (DRG) nerve terminals to transmit nociceptive signals to the central nervous system and to modulate pain signaling. Activation of presynaptic receptors on the DRG nerve terminal has been postulated to account for one critical level of control of synaptic coupling between the afferent DRG neuron and its target in the dorsal horn. The best known of these presynaptic regulatory receptors is of the metabotropic type. Activation of opiate and GABAB receptors, for example, has been suggested to inhibit release of substance P and glutamate from the DRG nerve terminal. However, ionotropic receptors are just beginning to be appreciated as important and powerful regulators of transmitter release as well. Recent evidence shows that ATP P2X receptors potently influence glutamate release from the DRG nerve terminal. The glutamate release is detected as large, synchronous release of multiple quanta of glutamate driven by regenerative action potential activity or as an increase in the frequency of mEPSC activity or quantal glutamate release. These studies suggest that elevation of ATP near the DRG nerve terminals, by a mechanism including Ca2+ dependent ATP release or release following spinal cord jury, will enhance signal output by increasing glutamate release during synaptic transmission. It may also evoke spurious signaling causing pain signals that are actually initiated in the spinal cord itself. The investigator plan's to address two main hypotheses that follow from these initial observations using electro-physiological and Ca2 imagining techniques to study synaptic transmission between DRG and dorsal horn neurons in co-culture and in spinal cord slices. The first hypothesis to be tested is that the presynaptic ATP P2X receptors expressed on DRG nerve terminals represent an important mechanism for regulating the release of the transmitters glutamate and substance P and that P2X receptor desensitization is a major determinant of the activity dependence of the P2X receptor presynaptic regulation. Activation of presynaptic ATP P2X receptors on DRG nerve terminals evokes a large synchronous release of multiple quanta of glutamate when action potential activity is allowed. ATP induces release of single quanta of glutamate when all action potential activity is suppressed. The second hypothesis we will test is whether ATP is able to depolarize the DRG nerve terminals by activation of Ca2+ permeable P2X receptors, that the depolarization initiates action potential activity at the DRG nerve terminal, and that these action potentials require the presence of TTX-resistant Na+ channels. The result of the studies in this proposal will be to provide insight on the role of presynaptic P2X receptors in sensory information processing as well as elucidate important pharmacological targets for the development of new pain therapies.
