Robert P. Yezierski - US grants

The anatomy and physiology of spinomesencephalic tract (SMT) cells will be studied using retrograde labeling and extracellular recording techniques, respectively. Spinal input to the mesencephalon has been documented in a variety of species including man. Projections to this region ascend in the ventral spinal cord along with the spinoreticular and spinothalamic tracts. While it is well established that the spinothalamic and spinoreticular projections are involved in the transmission of nociceptive information, by comparison little is known about the spinomesencephalic tract. Based on available clinical and experimental data this pathway is generally thought to be an important component in the neuronal apparatus responsible for the perception of pain. The present research is designed to examine the anatomical and functional organization of this pathway. To this end, retrograde tracing techniques will be used to determine the distribution of spinal neurons projecting to different midbrain regions in the rat, cat and primate. Particular attention will be given to the distribution of cells projecting to the deep layers of the superior colliculus and to areas known to be involved in the descending control of dorsal horn neurons or in the production of stimulation produced analgesia. Since the cells of origin of the SMT are thought to have an overlapping distribution with spinothalamic and spinoreticular tract cells, the possibility exists that the ascending axons of SMT cells may distribute collaterals to more than one postsynaptic target. To evaluate this question, experiments using two different retrograde labels delivered to non-overlapping injection sites will be carried out initially in the rat and if warranted in the cat and primate. The functional properties of the SMT will be studied using electrophysiological techniques to examine the afferent convergance and receptive field organization of identified SMT cells in the primate. The response of these cells to noxious/innocuous mechanical and thermal stimuli will be evaluated. These studies should provide further insight into the organization of ascending spinal pathways responsible for the transmission of sensory information. Moreover, it is felt that an important step towards understanding the interaction between ascending sensory pathways and descending control systems is to gain a better understanding of the afferent input to these systems.

Two major objectives of the present research plan are to study the: (a) neuronal circuitry and (b) synaptic mechanisms that contribute to the response and receptive field characteristics of cat spinomesencephalic tract (SMT) cells. These objectives will be addressed by three types of experiments. (1) The intracellular injection of horseradish peroxidase will be used to study the cell morphologies and dendritic arborizations of functionally characterized SMT cells. (2) Electrical stimulation with electrodes positioned at different brainstem levels will be used to map the distribution of sites producing excitatory and/or inhibitory effects on different classes of SMT cells. Spinal lesions will also be used to localize the spinal trajectory of descending pathways responsible for these effects and to determine the extent to which the complex receptive fields of SMT cells are due to the descending influence of supraspinal structures and/or local circuitry within the spinal cord. (3) Using intracellular recording techniques the inhibitory effects of peripheral stimulation on SMT cells will be studied to determine if these effects are due to pre- and/or postsynaptic mechanisms. The third objective of the research plan is to further evaluate the SMT projection to different midbrain regions. This will be studied by systematically mapping the termination site(s) of SMT axons using the technique of antidromic activation. Antidromic threshold maps will extend into the medial and lateral thalamus in an effort to study the collateralization of cat SMT axons. Previous studies related to the cat SMT have shown this pathway to be made up of a heterogenous population of neurons with varied receptive field and response properties, spinal origins, axonal trajectories and sites of termination. The potential involvement of this pathway in nociception is supported by the demonstration that the majority of SMT cells respond to noxious mechanical and thermal stimuli. Additional studies related to the anatomy and physiology of this pathway will contribute to our understanding of substrates potentially responsible for the multidimensional aspects of pain. In addition to providing a better understanding of the anatomy and physiology of SMT cells, the specific aims of the research outlined above are intended to extend our basic knowledge of sensory mechanisms in the spinal cord and particularly that related to nociception.

Two major objectives of the present research plan are to study the: (a) neuronal circuitry and (b) synaptic mechanisms that contribute to the response and receptive field characteristics of cat spinomesencephalic tract (SMT) cells. These objectives will be addressed by three types of experiments. (1) The intracellular injection of horseradish peroxidase will be used to study the cell morphologies and dendritic arborizations of functionally characterized SMT cells. (2) Electrical stimulation with electrodes positioned at different brainstem levels will be used to map the distribution of sites producing excitatory and/or inhibitory effects on different classes of SMT cells. Spinal lesions will also be used to localize the spinal trajectory of descending pathways responsible for these effects and to determine the extent to which the complex receptive fields of SMT cells are due to the descending influence of supraspinal structures and/or local circuitry within the spinal cord. (3) Using intracellular recording techniques the inhibitory effects of peripheral stimulation on SMT cells will be studied to determine if these effects are due to pre- and/or postsynaptic mechanisms. The third objective of the research plan is to further evaluate the SMT projection to different midbrain regions. This will be studied by systematically mapping the termination site(s) of SMT axons using the technique of antidromic activation. Antidromic threshold maps will extend into the medial and lateral thalamus in an effort to study the collateralization of cat SMT axons. Previous studies related to the cat SMT have shown this pathway to be made up of a heterogenous population of neurons with varied receptive field and response properties, spinal origins, axonal trajectories and sites of termination. The potential involvement of this pathway in nociception is supported by the demonstration that the majority of SMT cells respond to noxious mechanical and thermal stimuli. Additional studies related to the anatomy and physiology of this pathway will contribute to our understanding of substrates potentially responsible for the multidimensional aspects of pain. In addition to providing a better understanding of the anatomy and physiology of SMT cells, the specific aims of the research outlined above are intended to extend our basic knowledge of sensory mechanisms in the spinal cord and particularly that related to nociception.

The spinomesencephalic tract (SMT) is made up of several components each with varying sites of origin, physiological properties, spinal trajectories and midbrain termination sites. Midbrain regions associated with the termination of the SMT may be involved in sensory, motor and autonomic responses to different modalities of stimulation, including pain and temperature. Because of this it continues to be the long range objective of research related to the SMT to gain a better understanding of the different components of this projection system in order to better understand their role in the spinal and supraspinal processing of sensory information derived from cutaneous, muscle and visceral structures. The specific aims of the present research plan include evaluation of: (a) the origin and termination of SMT cells in the upper cervical cord; (b) trigeminal and spinal inputs to cervical SMT cells, including convergence from dural, skeletal muscle and cardiac afferent fibers; and (c) the contribution of propriospinal pathways to the functional properties of cervical SMT cells. Anterograde and retrograde tracing techniques will be used to study the termination and origin of SMT axons. These studies will include a quantitative evaluation of the terminal projection from different regions of the upper cervical cord to structures throughout the rostrocaudal extent of the midbrain. A quantitative evaluation of the laminar and segmental distributions of cells projecting to specific midbrain targets will also be carried out. The physiology of identified SMT cells will be studied with single-unit recording techniques similar to those used in previous studies. Trigeminal and spinal afferents will be activated by electrical, chemical and natural stimuli. Propriospinal pathways from the lumbosacral cord will be excited and/or inhibited using natural and electrical stimulation. The research plan addresses several important questions related to the organization of SMT components at different levels of the cord. The results of these studies should extend the basic understanding of spinal and supraspinal regions involved in the central processing of sensory, motor and visceral information in general, and specifically that related to craniofacial, muscle and cardiac pain. The results of the proposed studies should also contribute to the understanding of central pathways that may be involved in the multidimensional affective and motivational aspects of acute and chronic pain.

The spinomesencephalic tract (SMT) is made up of several components each with varying sites of origin, physiological properties, spinal trajectories and midbrain termination sites. Midbrain regions associated with the termination of the SMT may be involved in sensory, motor and autonomic responses to different modalities of stimulation, including pain and temperature. Because of this it continues to be the long range objective of research related to the SMT to gain a better understanding of the different components of this projection system in order to better understand their role in the spinal and supraspinal processing of sensory information derived from cutaneous, muscle and visceral structures. The specific aims of the present research plan include evaluation of: (a) the origin and termination of SMT cells in the upper cervical cord; (b) trigeminal and spinal inputs to cervical SMT cells, including convergence from dural, skeletal muscle and cardiac afferent fibers; and (c) the contribution of propriospinal pathways to the functional properties of cervical SMT cells. Anterograde and retrograde tracing techniques will be used to study the termination and origin of SMT axons. These studies will include a quantitative evaluation of the terminal projection from different regions of the upper cervical cord to structures throughout the rostrocaudal extent of the midbrain. A quantitative evaluation of the laminar and segmental distributions of cells projecting to specific midbrain targets will also be carried out. The physiology of identified SMT cells will be studied with single-unit recording techniques similar to those used in previous studies. Trigeminal and spinal afferents will be activated by electrical, chemical and natural stimuli. Propriospinal pathways from the lumbosacral cord will be excited and/or inhibited using natural and electrical stimulation. The research plan addresses several important questions related to the organization of SMT components at different levels of the cord. The results of these studies should extend the basic understanding of spinal and supraspinal regions involved in the central processing of sensory, motor and visceral information in general, and specifically that related to craniofacial, muscle and cardiac pain. The results of the proposed studies should also contribute to the understanding of central pathways that may be involved in the multidimensional affective and motivational aspects of acute and chronic pain.

The condition of pain following spinal cord injury (SCI) is one of many challenges facing patients coping with the physical and life threatening consequences of SCI. This condition continues to challenge health professionals with an incidence of 60-80 percent for all SCI patients. Nearly 40 percent of these patients report severe pain to the extent they would trade any chance of functional recovery for relief of pain. Without research directed towards understanding the mechanisms responsible for this condition it is unlikely that effective treatments will be developed. To this end, the focus of this proposal is directed towards evaluating the cellular events responsible for the development of spontaneous and evoked pain behaviors in a model developed to study the pathophysiological, biochemical, and molecular cascades responsible for different pain states associated with SCI. Over the past five years the excitotoxic model of SCI, which relies upon the intraspinal injection of the AMPA/metabotropic receptor agonist quisqualic acid (QUIS), has been used to simulate injury evoked elevations of glutamate and produce a pathological sequella similar to that following ischemic and traumatic SCI. The experiments of Specific Aim 1 will evaluate the relationship between the astrocytic, microglial, and neuronal responses to excitotoxic injury and the onset of spontaneous and evoked pain behaviors. The main focus of these experiments will be directed towards correlating the temporal profile of these responses along the longitudinal axis of the cord with the onset of pain behaviors following QUIS injections. Specific Aim 2 will focus on establishing a physiological correlate between cellular responses at different distances from the injury epicenter and changes in the functional state of sensory neurons following excitotoxic lesions. In Specific Aims 1-2 efforts will be made to evaluate the effects of the anti-inflammatory agent IL-10 and/or the iNOS inhibitor and NMDA antagonist agmatine on the relationship between the onset of pain behaviors and the cellular response and physiological changes following QUIS injury. Specific Aim 3 will focus on an evaluation of the biochemical changes following QUIS injections with a special emphasis on the initiating events responsible for the anatomical and functional changes in sensory circuits that underlie the onset of persistent pain following SCI. Of special interest will be the role of cytoskeletal proteins in the regulation of membrane fluidity and distribution of glutamate binding sites along with the role of inflammatory mediators in producing plastic changes in spinal sensory neurons. The long range goal of the proposed research is to understand the pathophysiological and biochemical mechanisms responsible for the progressive tissue damage in SCI, and to provide new directions for the development of therapeutic interventions for the prevention and treatment of chronic pain following injury.

DESCRIPTION (provided by applicant): Effective strategies in pain research and pain management include a growing emphasis on the collaborative efforts between clinicians and researchers working to identify novel therapeutic targets, predictive factors responsible for the onset of acute and chronic pain, and the evaluation of innovative strategies of pain management. For the continued development of novel pain management strategies a commitment is needed to multidisciplinary interventions utilizing state-of-the-art knowledge developed by individuals trained in the complexities of the biological and psychosocial components of pain. To prepare the next generation of pain scientists to meet these challenges we need to create training opportunities with programmatic structure that incorporate the diverse backgrounds and expertise of training faculty backed by a strong institutional commitment that exists in a collaborative environment. The training program developed at the University of Florida has taken strides to accomplish these objectives by offering training opportunities in eight diverse areas of research. At the foundation of the training program is an outstanding pain research community characterized by a spirit of collaboration responsible for a comprehensive program consisting of didactic and research components as well as required exposure to clinical and basic research environments. The program contains: (a) a well balanced core curriculum; (b) provisions to enhance diversity; (c) a component dealing with the ethical conduct of research; and (d) steps to ensure the program meets the needs of trainees in areas of research, education, and professional development. Due to the escalating prevalence of chronic pain conditions in our society, the University of Florida pain research community recognizes the need to produce a new generation of pain specialists that will be able to contribute to the improved understanding and effective management of acute and chronic pain. The goal of the program is to produce individuals equipped to develop independent clinical and/or basic science research programs and to instill in these individuals an appreciation for the benefits of collaborative, multidisciplinary, translational programs in meeting the present and future challenges in the field of pain research. RELEVANCE: The increasing challenge presented by the management of chronic pain requires new strategies of intervention. To meet these challenges the next generation of pain scientist will need to be trained in a way that emphasizes the application of research findings to the human condition. The proposed training program emphasizes integration and translation as well as a commitment to a new era of pain research.

DESCRIPTION (provided by applicant): How advancing age impacts biological systems responsible for the experience of pain represents a major challenge in the field of pain research. Acknowledging the fact that chronic pain in the elderly is a far more complex condition clinically, biologically, and therapeutically than pain in younger segments of the population underscores the need for integrative studies encompassing behavioral changes and biological mechanisms responsible for alterations in pain perception with advancing age. Over the past 20 years knowledge related to peripheral and central substrates of pain has seen significant advances. Unfortunately, there is little evidence that anything we have learned applies to the pain system in the elderly (animals or humans). An examination of cellular and molecular mechanisms responsible for the development of chronic pain reveals an undeniable overlap with mechanisms responsible for aging, thus providing the rationale for studies evaluating the interaction between the biological process of aging and the pathological condition of chronic pain. Three specific aims related to the impact of advancing age on thermal sensitivity will be addressed, including efforts to define the temporal characteristics of age-related changes in heat and cold sensitivity. Thermal sensitivity will be evaluated with a novel operant-based method of behavioral assessment. Animals will be evaluated longitudinally to study the cumulative effects of age on thermal sensitivity along with the impact of sensory changes on physical performance in male and female Fisher Hybrid (F344BN) rats ranging in age from 8-27 months. Since chronic inflammation is a common condition contributing to musculoskeletal pain, a primary cause of disability in the elderly, efforts will be made to examine the interrelationships among age, chronic inflammation, and changes in autonomic function following inflammatory injury. The predisposing influence of chronic injury early in life to the development of hypersensitivity later in life will also be evaluated. Potential central versus peripheral mechanism(s) for age-dependent changes in sensory function will be evaluated by examining age-dependent changes in spinal microglia and thermal receptor expression in dorsal root ganglia. The proposed research represents: (a) an essential step towards mechanistic studies related to age- dependent molecular and physiological mechanisms of pain;and (b) a pivotal link for translating basic pain research into clinical strategies related to pain disorders in the elderly. To accomplish these objectives a multidisciplinary team consisting of experts in pain and behavior, microglia, physical performance, and statistical analysis will be used to carry out the proposed Research Plan. The proposed studies represent an innovative approach that for the first time combines a longitudinal design with a clinically relevant strategy of operant behavioral assessment to study the effects of age on thermal sensitivity. PUBLIC HEALTH RELEVANCE: Chronic pain in the elderly is a far more complex condition clinically, biologically, and therapeutically than pain in younger segments of the population thus providing the rationale and motivation for studies directed towards understanding the impact of age on the development and maintenance of chronic pain. The proposed research represents: (a) an essential step towards mechanistic studies related to age-dependent behavioral, molecular and physiological mechanisms of pain;and (b) a pivotal link for translating basic pain research into clinical trial strategies related to pain disorders in the elderly.