Grant D. Nicol - US grants

Pro-inflammatory agents such as the prostaglandins (PG) are known to sensitize sensory neurons to noxious stimulation, resulting in a condition of heightened sensitivity known as hyperalgesia. At present, we know very little about the cellular mechanisms whereby PGs modulate the sensitivity and excitability of sensory neurons and the role PGs play in regulating the intensity or duration of the neurogenic inflammatory response. The studies outlined in this proposal are designed to elucidate those physiological mechanisms and PG-induced alterations in sensory transduction cascades that produce sensitization in sensory neurons of the dorsal root ganglion. The proposed studies utilize isolated sensory neurons of the rat dorsal root ganglion grown in cell culture. This model preparation permits investigation of the cellular mechanisms giving rise to PG-induced sensitization in the absence of potential modulating factors released by other cell types. Thus, the direct actions of pro-inflammatory agents to regulate neuronal excitability can be determined. The actions of PGs and excitatory chemical agents, such as bradykinin and capsaicin, are recorded electrophysiologically through utilization of whole-cell and perforated-vesicle patch-clamp recording techniques. Bradykinin and capsaicin are used to probe the levels of neuronal sensitization and thus elucidate whether PC modulation occurs at the level of second messenger mediated cascades or directly at the neuronal membrane. Initial studies will establish a fundamental understanding of the electrophysiological properties of the neuronal responses to bradykinin and capsaicin. This cellular response forms the basic probe to examine the state of neuronal sensitization. Subsequent studies, will further characterize the essential parameters required to produce PG-induced sensitization, such as PG concentration, type of PG, and the time course of PG action. Finally, we will establish the role of PG activation of second messenger- mediated transduction cascades in the physiological mechanisms regulating excitability or sensitivity of the response in sensory neurons. These studies should provide a basic understanding of the cellular mechanisms regulating sensory neuron excitability and the roles these processes play in the initiation and maintenance of neurogenic inflammation. This should also contribute to our understanding of chronic or long-term modifications of neuronal activity such as in long- term potentiation and facilitation. Ultimately, if we can isolate the ionic conductance)s) and establish the regulatory mechanisms producing this enhanced excitability, it will be possible to design drugs that selectively block this conductance or the altered pathway and thus, curb the pain and heightened sensitivity associated with chronic inflammatory conditions, such as rheumatoid arthritis.

Pro-inflammatory agents such as the prostaglandins (PG) are known to sensitize sensory neurons to noxious stimulation, resulting in a condition of heightened sensitivity known as hyperalgesia. At present, we know very little about the cellular mechanisms whereby PGs modulate the sensitivity and excitability of sensory neurons and the role PGs play in regulating the intensity or duration of the neurogenic inflammatory response. The studies outlined in this proposal are designed to elucidate those physiological mechanisms and PG-induced alterations in sensory transduction cascades that produce sensitization in sensory neurons of the dorsal root ganglion. The proposed studies utilize isolated sensory neurons of the rat dorsal root ganglion grown in cell culture. This model preparation permits investigation of the cellular mechanisms giving rise to PG-induced sensitization in the absence of potential modulating factors released by other cell types. Thus, the direct actions of pro-inflammatory agents to regulate neuronal excitability can be determined. The actions of PGs and excitatory chemical agents, such as bradykinin and capsaicin, are recorded electrophysiologically through utilization of whole-cell and perforated-vesicle patch-clamp recording techniques. Bradykinin and capsaicin are used to probe the levels of neuronal sensitization and thus elucidate whether PC modulation occurs at the level of second messenger mediated cascades or directly at the neuronal membrane. Initial studies will establish a fundamental understanding of the electrophysiological properties of the neuronal responses to bradykinin and capsaicin. This cellular response forms the basic probe to examine the state of neuronal sensitization. Subsequent studies, will further characterize the essential parameters required to produce PG-induced sensitization, such as PG concentration, type of PG, and the time course of PG action. Finally, we will establish the role of PG activation of second messenger- mediated transduction cascades in the physiological mechanisms regulating excitability or sensitivity of the response in sensory neurons. These studies should provide a basic understanding of the cellular mechanisms regulating sensory neuron excitability and the roles these processes play in the initiation and maintenance of neurogenic inflammation. This should also contribute to our understanding of chronic or long-term modifications of neuronal activity such as in long- term potentiation and facilitation. Ultimately, if we can isolate the ionic conductance)s) and establish the regulatory mechanisms producing this enhanced excitability, it will be possible to design drugs that selectively block this conductance or the altered pathway and thus, curb the pain and heightened sensitivity associated with chronic inflammatory conditions, such as rheumatoid arthritis.

DESCRIPTION (applicant's abstract): Increasing evidence suggests that activation of various components of the immune system contribute to chronic pain and inflammation. A number of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha), interleukin 1-beta (IL-1beta), and interleukin-6 (IL-6) are synthesized and released at sites of trauma and produce hyperalgesia in animal models of pain. One mechanism for cytokine-induced augmentation of pain perception could involve their direct actions on nociceptive sensory neurons to enhance excitability and/or sensitize them to physical or chemical stimuli. Despite abundant evidence that TNF-alpha, IL-1beta, and IL-6 produce hyperalgesia and inflammation, there is little information regarding their capacity to modulate intracellular signaling pathways that regulate sensory neuron function. The hypothesis of this proposal is that pro-inflammatory cytokines act directly on sensory neurons to enhance their excitability and sensitize these cells to noxious mechanical and chemical stimuli and, in turn, augment the release of neuroactive substances from these neurons. The proposed studies will utilize two approaches: patch-clamp electrophysiology to assess cytokine-induced alterations in membrane excitability in rat sensory neurons grown in culture and biochemical measurements of neuropeptide release in isolated sensory neurons grown in culture and an in vitro preparation of rat spinal cord slices. The aims of this proposal are: 1) to determine whether acute or chronic exposure to TNF-alpha, IL-1beta, or 1L-6 alters membrane excitability and/or sensitizes isolated sensory neurons to electrical or chemical stimuli, 2) to determine whether these pro-inflammatory cytokines stimulate and/or sensitize the release of SP and CGRP from rat sensory neurons grown in culture or from rat spinal cord slices; and 3) to determine the effects of pro-inflammatory cytokines on sphingolipid second messengers and diacylglycerol in sensory neurons and to establish causal relationships between changes in second messenger systems and cytokine-induced alterations in excitability and peptide release. Overall, the knowledge gained from these studies is critical for understanding the etiology of chronic pain and could eventually aid in designing interventions to alleviate the pain. The results of this work can increase the understanding of the cellular mechanisms mediating the interaction between the nervous system and the immune system and thus be applicable to other areas of neurobiology.

Inflammatory agents such as the prostaglandins (PGs) are known to sensitize sensory neurons to subsequent stimulation, resulting in a condition of heightened sensitivity known as hyperalgesia. At present we that inflammatory PGS enhance the sensitivity and excitability of sensory neurons through activation of the cAMP transduction cascade. However, very little is known regarding the regulatory mechanisms and transduction cascades controlling the intensity or duration of the enhanced sensitivity that results in neurogenic aspects of the inflammatory response and hyperalgesia. The proposed studies seek to establish the physiological mechanisms whereby the calcium,/nitric oxide/cGMP pathways lead to inactivation of the enhanced excitability or sensitization of sensory neurons. The hypothesis of this proposal is that cGMP, through activation of cGMP- dependent protein kinase (PKG) and its possible modulation of other intracellular mediators, alters the properties or state of various membrane currents to reverse the enhanced neuronal excitability produced by the cAMP/PKA pathway. The studies outlined in this proposal will use rat dorsal root ganglion cells grown in culture as a model system. The electrophysiological properties of these neurons will be examined with the patch-clamp technique. This allow measurement of membrane currents arising from the whole cell or single-ion channels. In conjunction with patch-clamp recordings, changes in the concentration of intracellular calcium, cAMP, and cGMP will be measured and correlated to the observed alterations in neuronal excitability. The specific aims of this proposal are: 1) To quantify the changes in intracellular calcium concentration using fluorescent calcium indicators and thus determine directly the contributions of calcium to the activation of signaling pathways involved in the inactivation of sensitization; 2) To establish a fundamental understanding of the different transduction cascades and to determine whether the inactivating pathways are specific to the nature of the stimulus or part of a more generalized cellular design; 3) To determine the specific intracellular mediators and their mechanisms of action that give rise to the inactivation of sensitization. This will provide an initial step in determining the specific target proteins that are modulated by the mediator(s) of inactivation. Ultimately, if we can establish the regulatory mechanism modulating the up and down regulation of excitability, it will be possible to design therapies that selectively modulate the altered pathway and thus curb the persistent pain and heightened sensitivity associated with chronic inflammatory conditions, such as rheumatoid arthritis.

[unreadable] DESCRIPTION (provided by applicant): Nerve growth factor (NGF) plays a critical role in the development and growth of sensory neurons. Seminal studies suggest that NGF may also play an important role in the regulating the sensitivity of sensory neurons to noxious stimulation. NGF levels are elevated in inflammatory exudates and is a potent causative agent in the production of both thermal and mechanical hyperalgesia. The behavioral findings suggest that the heightened sensitivity that occurs with inflammation may, in part, result from the actions of NGF on sensory neurons. There have been very few studies exploring the sensitizing actions of NGF on isolated neurons and their associated signaling pathways. Recent work in my laboratory demonstrates that NGF can rapidly augment the excitability of small diameter, capsaicin-sensitive sensory neurons through enhancement of the TTX-resistant sodium current and suppression of a voltage-dependent potassium current(s). Thus, NGF may have a significant impact on the state of neuronal excitability. The notion that NGF may be an important paracrine-type messenger in mediating the excitability of sensory neurons on a rapid time scale, perhaps less than one minute, is a completely unexplored idea. The Specific Aims outlined in this proposal are: Aim 1 will determine the effects of NGF on the excitability as well as the modulation of a variety of membrane currents that are critical in setting the firing level of the neuron. These studies will establish whether this sensitization results from activation of Trk A and/or p75 NTR. Aim 2 will follow an identical course of study to determine whether the other neurotrophins brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4), and neurotrophin-3 (NT-3) rapidly modulate the excitability and selected membrane currents. These studies will examine the role of Trk B, Trk C, and/or p75 receptors in modulating excitability. Aim 3 will establish whether glial cell-derived neurotrophic factor (GDNF) can rapidly modulate selected membrane currents in sensory neurons. GDNF plays a critical role in the growth and survival of a distinct population of non-peptidergic sensory neurons that lose their dependence on NGF. Aim 4 will explore the intracellular signaling pathways that mediate the rapid modulatory effects of NGF acting through Trk A and p75 NTRs, BDNF and NT-4 through Trk B, NT-3 through Trk C, and GDNF through the Ret kinase pathway. These studies will further our understanding of the cellular mechanisms and signaling pathways whereby neurotrophins acutely regulate the excitability of both nociceptive and non-nociceptive sensory neurons. A fundamental understanding of such events could lead to better designed compounds and therapies to facilitate the treatment of chronic inflammatory conditions. [unreadable] [unreadable]

Inflammation can augment dramatically the sensitivity of nociceptive sensory neurons. In some cases, the actions of inflammatory mediators are fairly well understood;however, there are signalling pathways for which we have very little knowledge. Such a pathway is the one in which the activating ligand is sphingosine 1- phosphate (S1P). Upon activation, S1P is released from a variety of immuno-competent cells and appears to play an important role in their chemotaxis and migration. S1 P is the endogenous ligand for a family of G protein-coupled receptors originally named EDG receptors (endothelial differentiation gene) and are now known as the S1 P receptor family (S1 PRJ. Our understanding of the role of S1 P in the onset and regulation of the inflammatory response is very limited, even in model systems. In our work on NGF, we discovered that externally applied S1 P significantly increased neuronal excitability and that these neurons expressed the mRNA for S1 PRs. Because of the emerging importance of S1 P in the onset of inflammation, this raises the question whether S1 P is an important primary messenger communicating between inflammatory cells and sensory neurons. To answer this question, three SAs are proposed: SA1 will establish which S1 PRs are expressed in sensory neurons and whether these receptors co-localize with specific defined populations of sensory neurons. SA2 will determine, using patch-clamp recording, which specific membrane currents are modulated by S1 P and how these changes lead to enhanced neuronal firing. S1 PRs involved in this sensitization will be determined by single-cell RT-PCR analysis and siRNA to knock-down the expression of specific receptors. SA3 in collaboration with Dr. Jun-Ming Zhang, will determine the capacity of S1 P to affect nociceptive behaviors in rats wherein S1 P is perfused directly onto the L5 DRG. Results from such studies will provide an important understanding of the potential communication between the immune and neuronal systems and lead to interventions that reduce the enhanced pain associated with inflammation.

DESCRIPTION (provided by applicant): Seminal studies suggested that the heightened sensitivity associated with inflammation may, in part, result from nerve growth factor (NGF) activation of nociceptive sensory neurons. My laboratory demonstrated that NGF/BDNF can rapidly increase the ability of sensory neurons to fire action potentials (APs) via p75 neurotrophin receptor (p75NTR) activation of the sphingolipid signaling cascade. The cellular mechanisms and downstream signaling pathways resulting from p75NTR activation are poorly understood. Our results demonstrate that p75NTR/ceramide activation of the novel atypical PKC, PKM¿ plays a key role in augmenting excitability and suggest that PKM¿ may have a critical role in the transition of acute to persistent sensitization as a result of PKM¿ becoming constitutively active. Proposed studies will use sensory neurons isolated from DRG of adult rats as a model system to explore the functional impact of p75NTR activation on membrane excitability. Knowledge gained from isolated neurons will be extended to studies exploring the role of these effector molecules in regulating nociceptive behavioral responses. These studies will be done in collaboration with Dr. Gary Strichartz at Harvard University. The specific aims are: Aim 1 will establish the role of p75NTR and its synthesis/activation of PKM¿. Also, p75NTR interaction with scaffolding proteins will determine their ability to regulate the activity of PKM¿ This work will focus on PKM¿-induced modulation of ion channels and their regulation of neuronal excitability. In collaboration with Dr. Gary Strichartz Aim 2 will establish the role of p75NTR-PKM¿ signaling cascades in the regulation of nociceptive behaviors in the intact animal. The strength of this multi-dimensional approach is that biochemical and molecular techniques will establish novel protein-protein associations and electrophysiology will establish the functional outcomes of those associations. These findings will be extended to studies in the intact animal to provide an understanding of the mechanisms whereby activation of p75NTR leads to heightened neuronal sensitivity. A fundamental understanding of these events is necessary so that better compounds and therapies can be designed to facilitate treatment of chronic inflammatory conditions. PUBLIC HEALTH RELEVANCE: Tissue injury and inflammation cause release of chemicals from a variety of cells near the injury site that results in increased sensitivity to painful stimulation. One of those key chemicals is nerve growth factor. Studies proposed in this application seek to understand how intracellular signaling pathways activated by nerve growth factor increase nerve sensitivity and thus lead to the increased pain responses.