Grants and Contracts Details
Latent central sensitization (LS) is a long-lasting form of sensitization in the spinal cord that is suppressed by the activation of pain inhibitory mechanisms. One of these mechanisms, established by work performed during the previous funding period, involves neuropeptide Y (NPY) and its receptors. We discovered that intrathecal NPY receptor antagonists or conditional genetic NPY depletion reinstated hyperalgesia, even when applied months after nerve injury or peripheral inflammation. Our overall hypothesis is that a recurring failure of NPY signaling may reflect the transition to a chronic state of lasting pain vulnerability, and our long-term goal is to prevent or alleviate chronic pain by facilitating endogenous NPY receptor analgesia in the CNS. The objective of the present application is to determine whether injury induces a sustained spinal release of NPY and Y1 receptor activation (Aim 1) that interrupts both TRP signaling at central terminals of DRG neurons (Aim 2) and GluN-AC1-Epac signaling in dorsal horn neurons (Aim 3). Aim 1 will test the hypothesis that injury induces a sustained NPY release and Y1 activation. Our progress indicates that tissue injury tonically increases NPY expression and NPY Y1 receptor (Y1) signaling and spinal anti-hyperalgesia, but whether this is due to tonic ligand-dependent Y1 activation has been difficult to study in the absence of a reliable assay of NPY release. To address this gap, we developed a new in situ assay of functional NPY release (evoked Y1 internalization), and will use it to evaluate spontaneous and stimulus-evoked spinal release of NPY at days, weeks, and months after nerve injury or inflammation. Aim 2 will test the hypothesis that NPY acts at Y1 on the central terminals of DRG neurons to silence TRPA1 and TRPV1-mediated pain. TRPA1 and TRPV1 channels are densely expressed on small DRG neurons that project to superficial laminae of the dorsal horn and serve as molecular receptors of noxious somatosensory stimuli. Aim 2a will use high-magnification confocal microscopy to determine whether tissue or nerve injury produces long-lasting increases in the expression of Y1 on the central terminals of primary afferent nociceptors. Aim 2b will use an intrathecal pharmacology approach to determine whether TRPA1 or TRPV1 antagonists prevent the ability of Y1 antagonists to reinstate hyperalgesia. Aim 3 will test the hypothesis that NPY inhibits Y1-expressing excitatory interneurons in the dorsal horn to silence nociceptive GluN„³AC1„³PKA/Epac signaling. Aim 3A. Neurophysiological recordings from Y1+ dorsal horn neurons. Our progress with a selective saporin-conjugated neurotoxin indicates that Y1-expressing interneurons in the dorsal horn contribute to hyperalgesia after nerve injury. To determine whether tonic ligand-dependent Y1 activity at these neurons maintains LS in a state of remission, we will allow Y1 neurons in lumbar spinal cord slices to uptake fluorescent NPY, and record from these neurons using patch clamp and Ca2+ imaging. We have the expertise to patch adult lamina II neurons taken 28d after injury, and to measure paired-pulse ratio to distinguish pre vs postsynaptic mechanisms at the synapse between primary afferents and their spinal neuron targets. We predict that conditional NPY deletion will increase the amplitude of EPSCs and [Ca2+]i mobilization on Y1-positive* lamina II neurons evoked by either dorsal root stimulation or laser-directed glutamate uncaging. Aim 3B. GluN contribution to LS. Our progress indicates that LS requires NMDA receptor activation of AC1 and the subsequent production of cAMP. Aim 3B delves deeper into this mechanism with evaluation of the essential NR2 subunits (GluN2A, GluN2B). Aim 3C. PKA/Epac contribution to LS. Adenylyl cyclase type 1 (AC1) is an enzyme that catalyzes the formation of cAMP in neurons and contributes to spinal mechanisms of chronic pain. To determine which target(s) of cAMP are tonically inhibited by Y1, we will use a pharmacological and shRNA approach to evaluate pain reinstatement after selective block of either protein kinase A (PKA) or Exchange protein directly activated by cAMP (Epac
|Effective start/end date||9/10/02 → 3/31/18|
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