This project concerns the role of central nervous system (CNS) neurons in sensory processes especially as they relate to pain and the control of pain. The main broad questions are: what are the molecular mechanisms and what are the physiological mechanisms regulating spinal nociceptive neurochemistry? A model of peripheral inflammation has been developed to investigate the relationship between spinal cord neurons, in particular those containing the opioid peptides enkephalin and dynorphin (but also other neurotransmitters and neuropeptides) and abnormal primary afferent input. Alterations in gene expression are assessed by measurement of peptide, protein of mRNA levels and cellular localization techniques. Our present goal is to understand the molecular mechanisms controlling transcription of the dynorphin opioid gene. Transcriptional control involves specific DNA binding proteins that recognize nucleotide sequences in the promoter region of the dynorphin gene. These proteins are often first identified as oncogenes and we have shown that inflammation induces an increase in mRNA levels coding for the c-fos proto-oncogene. This nuclear protein is involved in transcriptional regulation and the increase in c-fos mRNA precedes the increase in dynorphin. Analysis of the Fos protein revealed a sequential, long-lasting increase in multiple Fos-immunoreactive proteins in nuclei isolated from dorsal spinal cord. The increase in Fos proteins far outlasts the elevation in c-fos mRNA and, essentially, occurs throughout the entire period of peripheral symptomology. These basic observations are being extended to other nuclear transcription factors such as NGFI-A and to an analysis of their binding sites on the dynorphin gene. Using our antibody to the c-fos protein we have also established a gel mobility shift assay to determine potential Fos binding sites on target sequences of DNA. We have shown that the Fos proteins synthesized during inflammation can form AP-1 complexes with a double stranded oligonucleotide containing the AP-1 consensus sequence. Current studies are using this technique to assess AP-1 like sites in the dynorphin and enkephalin promoters.
