For several years my laboratory has studied the neurochemical changes that take place in the neonatal rat spinal cord following repeated opiate administration as a cellular model of opiate analgesic tolerance. We have reported NMDA receptor-mediated increases in nociceptive primary afferent synaptic transmission associated with opiate tolerance. We have also discovered that the endocannabinoid 2-arachidonoylglycerol (2-AG), released in response to increased nociceptor activity, feeds back to inhibit such activity by acting at presynaptic CB1 receptors. Recently, we have found that, like nociception, itch sensation, also appears to be increased in animals previously exposed to repeated morphine injections - although this effect is largely masked by concurrent spinal endocannabinoid inhibition. After intrathecal administration of the CB1 receptor antagonist SR141716 (SR) significant increases in scratching behaviors are observed - particularly in rats previously treated with repeated morphine injections. We hypothesize that repeated opiate administration induces central sensitization of primary afferent neurotransmission of pruritus, thereby increasing the synthesis and release of 2-AG, which in turn feeds back to inhibit itch by binding to presynaptic CB1 receptors. The current application proposes to begin testing this hypothesis with three specific aims: 1) To verify that spinal endocannabinoids act at spinal CB1 receptors to inhibit itch. To test this hypothesis, we will first use immunocytochemistry to ascertain that, as in adults, both CB1 receptors and DAG lipase are present in the dorsal horn of neonatal rats. We will also intrathecally inject a different CB1 antagonist (AM251) or a 2-AG synthesizing enzyme (DAG lipase) inhibitor to study whether these means of inhibiting spinal 2-AG effects can also increase scratching behaviors in neonatal rats. Finally, we will attempt to replicate and extend our intrathecal SR studies to adult mice and compare these effects with those in littermate CB1 receptor knockout mice. 2) To study the mechanism by which anti-pruritic endocannabinoids are released in the spinal cord. Our data suggest that following repeated morphine injections not only are nociceptive primary afferents more active but primary afferents related to itch are also more excitable. We have previously found that morphine- induced primary afferent nociceptor sensitization is mediated predominately via NMDA receptors. In these studies we will test whether opiate-induced sensitization to itch is also mediated by NMDA receptors and whether it can be inhibited by the NMDA receptor antagonist MK801. Further, we will use Gastrin Releasing Peptide (GRP) to study whether application of this peptide, recently observed to produce spinally-mediated pruritus, can cause endocannabinoid release as evidenced by SR-induced increases in scratching. 3) To begin investigations of exogenous cannabinoid modulation of scratching behavior. Finally, we will use intrathecal injections of the cannabinoid WIN 55,212-2 in animals with GRP-induced scratching in hopes of demonstrating possible applications of cannabinoids for the treatment of itch. Thus, these studies may suggest novel anti-pruritic therapies for cannabinoids as well as providing new models and concepts to investigate the neural underpinnings of spinal interactions between pain and itch.
Like pain, the sensation of itch has important clinical significance in that chronic itch leads to considerable suffering. We have recently found evidence that endogenous substrates exist within the spinal cord which can inhibit itch and that these systems rely on endogenous cannabinoids. In these proposed preliminary studies, we seek to demonstrate what stimuli activate these endogenous itch-inhibiting systems, which endocannabinoid is primarily involved in inhibiting itch in vivo and what cannabinoid receptor these bioactive lipids bind to in producing their effect. Such studies may suggest novel anti-pruritic therapies for exogenous cannabinoids as well as providing new models and concepts to better understand the neurobiology of itch and, in particular, the spinal interactions between pain and itch.
