Gregory W. Terman - US grants

APPLICANT'S ABSTRACT: This application is a request for a Scientist Development Award. This award is sought to enable my full time research on the modulatory effects of opiates on spinal cord neurotransmission and synaptic plasticity. These new research experiences under the tutelage of my preceptor and additional consultants will equip me technically and practically to move forward quickly to become an independent researcher. Long term potentiation (LTP), the best studied model, to date, of synaptic plasticity within the CNS, involves a seemingly permanent increase in neuronal excitation following repeated activation of afferent input to that neuron and is thought to be the cellular basis of learning. Recent clinical and behavioral evidence has accumulated suggesting that much the same activity- dependent synaptic changes can take place in neural systems involved in pain perception. Intense painful stimuli can produce a central sensitization to further noxious stimulation in both laboratory animals and man. Behavioral pharmacologic studies of this sensitization suggest numerous similarities to the neurochemistry of LTP, including its reliance on activation of the NMDA class of glutamate receptors and its modulation by certain opiates. The studies proposed here involve the utilization of an in vitro spinal cord slice preparation to study the opioid modulation of primary afferent dorsal horn synapses in regions of the spinal cord known to be involved in nociceptive processing. These studies stem directly from our findings in the hippocampus that LTP induction can be blocked by kappa opioids. Initial experiments using the neonatal rat transverse spinal cord slice will focus on the actions of applied mu, kappa, and delta opioids in modulating both stimulation-evoked neural excitation in lamina I of the dorsal horn and activity-dependent plastic changes in this area. These studies will use single cell extracellular recording techniques first and will benefit from the in vitro preparation in enabling application of known concentrations of agonists and antagonists to the site of action and in allowing easy visualization of the recording electrode position. Positive extracellular findings will be further evaluated using whole cell voltage clamp techniques to study post-synaptic mechanisms of action. Further, retrogradely transported fluorescent markers from rostral spinothalamic tract lesions will be used to target specific presumably nociceptive spinothalamic cell responses. The effects of endogenous mu, delta and kappa opioids on spinal neurotransmission and synaptic plasticity will then also be evaluated. Finally, these results in neonatal spinal cord will be compared and contrasted to studies using an adult spinal cord slice preparation. Thus, using an in vitro model and a variety of electrophysiological techniques, we hope to provide insight into the neurochemical mechanisms of primary afferent neurotransmission and neuroplasticity and how mu, kappa, and delta opiates differentially act to modulate these mechanisms in the adult and neonate. Such information may ultimately lead to better pharmacological means of managing and/or preventing certain acute and chronic pain states.

[unreadable] DESCRIPTION (provided by applicant): Intense nociceptive stimuli can produce a central sensitization to later noxious stimulation in both laboratory animals and man. Long term potentiation (LTP), the best-studied cellular model of neuroplasticity within the CNS, involves a seemingly permanent increase in neuronal excitation following repeated activation of afferent input to that neuron. We have recently developed a model of LTP in the spinal cord slice preparation and begun to investigate its pharmacological and physiological characteristics using visualized whole cell voltage clamp recordings. Mu opiates inhibit induction of spinal LTP. Both exogenous and endogenous kappa opiates also inhibit LTP but act primarily by inhibiting maintenance mechanisms. [unreadable] [unreadable] In this proposal we plan to expand our studies of LTP in the spinal cord slice by: 1) using imaging techniques to specifically target dorsal horn nociceptors (i.e., selecting back-filled spinothalamic cells with three dimensional morphologies characteristic of nociceptors) for further experiments on kappa modulation of spinal LTP. [unreadable] [unreadable] 2) examining animals previously sensitized to noxious stimuli by inflammation (with resultant long-term anatomical and physiological changes in spinal pain circuitry) to correlate behavioral evidence of sensitization with electrophysiological evidence of spinal LTP including sensitivity to kappa opioids. [unreadable] [unreadable] 3) studying the dose related effects of dynorphin in modulating Lamina I neurotransmission and LTP, including differentiation of its kappa opiate and NMDA receptor activities. [unreadable] [unreadable] Such investigations of LTP in the spinal cord will lead to a better understanding of CNS neuroplasticity, in general, and nociceptive sensitization, in particular, and may ultimately lead to better pharmacological means of managing or preventing certain pain states.

Description (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.