In 1982, Ruda was able to demonstrate that the endogenous, or internally produced, opioid enkephalin made direct synaptic contact with the spinal cord neurons projecting to the thalamus , implying that these projection pathways were a major site of this peptide's modulation of sensory input about pain. [A peptide is a specific type of amino acid.] Another neuropeptide of particular interest was dynorphin, a substance which normally "lies low" in the system, but becomes highly active in response to noxious stimuli. Although conspicuously absent from the nerves in the skin, this peptide is widely distributed in the brain and spinal cord. Ruda's studies showed that dynorphin synapsed directly onto the thalamic projection neurons, and was also active within the complex network of interneurons. Michael Iadarola had begun working on the molecular biology of neuropeptides in Erminio Costa's lab at NIMH in the early 1980s. He had measured dynorphin in the spinal cord by labelling and tracing its antibodies. He had also developed a method to clone predynorphin, the precursor protein which forms first in the genetic sequence that expresses dynorphin. But he needed a biological system that would allow him to look at real time changes in levels of the peptide. In the literature, he discovered an inflammation model used in the hind limbs of rats and realized that he could use the opposite, unaffected, limb as a control. Using this assay, he found that the painful synaptic input triggered a genetic regulatory sequence, called the enhancosome , that activated the transcription of the precursor protein and thus of dynorphin itself.

The enhancosome: twin phosphorylation and dephosphorylation reactions of promoter genes CREB and TATA regulate the transcription of dynorphin. Illustration courtesy of Michael Iadarola.

In 1986, Iadarola was invited to give a seminar at the NAB; Dubner and his colleagues decided that his genetic research would be an excellent complement to the existing research. Two years later, Iadarola and Ruda collaborated on an important paper in Proceedings of the National Academy of Sciences . They documented the increased biosynthesis of dynorphin - first the messenger RNA of predynorphin and then of dynorphin itself - in the spinal cord of the rat, in response to brief inflammation of the hind leg.

“The dynorphin connection weaves everything together in our work." -Mike Iadarola on dynorphin

Oral history interview with Michael Iadarola, 1999. Tapes and transcript to be deposited in the NIH History Office and the John C. Liebeskind History of Pain Collection, UCLA.

This impressive evidence of dynorphin activity in response to inflammation led the researchers to hypothesize that this peptide, like enkephalin, might be one of the body's endogenous analgesics. If dynorphin was administered to a normal animal, however, it proved to have an algesic effect; the animal became more sensitive to heat and touch stimuli. An extended series of studies by Robert Caudle while he was a research fellow at NIDR, have shown that dynorphin plays at least two roles in the nervous system's response to pain: inhibiting pain signals in acute injury, but having an excitatory, intensifying, effect on neural transmission in persistent, chronic pain.

Dynorphin synthesis in rat spinal cord cells receiving input from the inflamed hindlimb v. spinal cord cells receiving input from contralateral limb. From M. A. Ruda, M. J. Iadarola, L. V. Cohen, and W. S. Young III, In situ hybridization histochemistry and immunocytochemistry reveal an increase in spinal dynorphin biosynthesis in a rat model of peripheral inflammation and hyperalgesia. Proceedings of the National Academy of Sciences v. 85 (1988): 623.