Kenneth Eugene Miller - US grants

Excitatory amino acids (EAA's) are involved in the transmission or processing of sensory information in the spinal cord. Many of the neuronal elements, however, that use or are affected by EAA's presently are unknown. This project will examine excitatory amino acid circuitry in areas of the spinal dorsal horn related to the transmission and processing of sensory messages. The specific goal of this proposal is to determine the anatomical distribution and arrangement of spinal cord components involved in excitatory amino acid synaptic transmission. The distribution of EAA's or enzymes involved in the metabolism of EAA's will be examined using immunohistochemical techniques in the dorsal root ganglia and spinal cord of rats and cats at the light and electron microscopic level. Glutamate, aspartate, glutaminase, aspartate amino transferase, glutamine synthetase, glutamate dehydrogenase, and N-acetylaspartylglutamate will be studied. The EAA receptor subtype, NMDA, will be studied with a recently described antagonist, CPP. The binding characteristics of (3H)CPP will be determined on tissue sections and autoradiography (AR) will be used to study the anatomical distribution of (3H)CPP binding. Dorsal rhizotomy and neonatal capsaicin treatment will be used with (3H)CPP-AR to study changes in distribution or number of binding sites in response to a loss of primary afferents. Rhizotomy and capsaicin will used with immunostaining for EAA's or EAA enzymes to study putative EAA-containing primary afferents. The relationship of EAA's and EAA enzymes to primary afferent terminals will be examined with electron microscopy by combining immunohistochemistry with primary afferent labelling. Neurons with supraspinal projections will be labelled with retrograde tracers and immunohistochemistry will be used to determine the EAA content of these cells and the distribution of EAA fibers contacting these cells. The neurochemical content of fibers contacting EAA- containing spinal neurons will be studied with double labelling techniques. The results from experiments outlined in this proposal will reveal important information concerning the synaptic circuitry of excitatory amino acids in sensory pathways.

[unreadable] DESCRIPTION (provided by applicant): Pain is a debilitating complication during chronic inflammation, such as arthritis, and difficult to treat for long periods of time. We hypothesize that elevated glutamate release from sensory nerves is responsible, in part, for the enhanced pain sensitivities of chronic inflammation. We have discovered in a rat model of chronic inflammation that sensory nerves in skin/joints produce large amounts of glutamate and its synthetic enzyme, glutaminase. Chronic inflammation causes increased production of glutaminase in the neuronal cell bodies of the sensory nerves that is shipped to the skin and joints causing increased levels of glutamate. A chronic increase in the production and release of glutamate can stimulate glutamate receptors on sensory nerves to produce painful sensations. For the current proposal, we hypothesize that elevated levels of glutamate cause 1exaggerated painful responses during chronic inflammation. In addition, preliminary data indicate that nerve growth factor (NGF) as a retrograde signal and zeta-crystallin:quinone oxidoreductase (ZC) as a stabilizer of glutaminase mRNA are important for this chronic alteration in glutamate metabolism. We postulate, therefore, that NGF and ZC are responsible for altering glutaminase levels in primary sensory neurons during chronic inflammation. The following specific aims are presented: 1. Our hypothesis predicts that inhibition of glutaminase will reduce nociceptive responses and elevated glutamate levels during chronic inflammation. A glutaminase inhibitor will be administered to sensory neurons during chronic inflammation. 2. Our hypothesis predicts that glutaminase production in sensory neurons during chronic inflammation is regulated by ZC. A ZC inhibitor will be given to sensory neurons during the development of chronic inflammation. 3. Our hypothesis predicts that glutamate metabolism in sensory neurons can be modified by NGF. NGF administration to naive rats and NGF inhibition during chronic inflammation will be evaluated. Behavioral tests will determine responses to pressure and temperature. Biochemical & immunohistochemical analyses will evaluate alterations in glutamate and GT enzyme levels in the sensory cell bodies and sensory nerves. Results from these studies will give insight into the role of peripheral glutamate on nociceptive responses during inflammatory chronic pain. These results may be beneficial in the development of novel therapies for patients with intractable pain by addressing the regulation of glutamate in peripheral tissues. [unreadable] [unreadable]