Peggy Mason - US grants

APPLICANT'S ABSTRACT: The proposed experiments are designed to lead to a better understanding of the neural circuitry that underlies physical dependence on acute and chronic opioids. Opioid physical dependence is defined by the appearance of a variety of physiological and psychological symptoms that occur when an opioid agonist is terminated or an antagonist administered. This opioid abstinence syndrome includes spontaneous pain as well as increases in autonomic outflow that produce tachycardia, tachypnea and an elevated blood pressure. The proposed experiments will examine the neural basis for the nociceptive facilitation and the sympathoexcitation that occur during opioid withdrawal. The rostral ventromedial medulla (RVM), the ventrolateral medulla (VLM), the pontine locus coeruleus (LC) and the midbrain periaqueductal gray (PAG) all contain opioid sensitive neurons and have been implicated in one or more of the components of opioid physical dependence. The RVM is a nociceptive modulatory region that contains a population of cells that facilitate nociceptive transmission during opioid withdrawal. RVM neurons project to both the spinal dorsal horn and the VLM. The VLM, an autonomic and somatic modulatory region, projects to the spinal dorsal and intermediate horns. The VLM is a major source of afferents to the LC and is required for the activation of LC neurons during opioid withdrawal. The LC, a noradrenergic nucleus important in the control of overall arousal and vigilance, has been implicated in the production of most components of the opioid abstinence syndrome. Regional glucose utilisation is elevated in both the LC and the PAG during naloxone- precipitated withdrawal from chronic opioids. PAG stimulation modulates somatic and autonomic activity and contains cells that project to both the VLM and RVM. The brainstem pathways that produce the nociceptive facilitation and autonomic responses of the opioid abstinence syndrome will be traced by blocking synaptic transmission in selected brain regions during naloxone precipitated withdrawal from systemic opioids. The contribution of the RVM, VLM, and LC to the nociceptive and cardiopulmonary components of chronic and acute opioid physical dependence will be described and compared. To further define the site or sites of opioid action necessary for nociceptive facilitation and autonomic arousal during physical dependence, naloxone will be administred following the microinjection of a low, medium and high dose of morphine into the opioid-sensitive RVM, VLM and LC. Electrophysiological experiments will detail the physiological characteristics of single VLM and PAG neurons during acute and chronic opioid physical dependence. A high and a low dose of morphine will be tested in both the acute and chronic experiments. Furthermore, the afferents responsible for the activity of VLM neurons will be examined by inactivating RVM and PAG neurons during opioid withdrawal. This experiment will also be performed in animals treated both acutely and chronically with either a high or low dose of morphine.

DESCRIPTION (from the applicant's description): Despite pharmacological evidence that serotonin is important in pain modulation and opioid analgesia, electrophysiological studies have consistently demonstrated that the activity of presumed serotonergic neurons does not change during either pain behaviors or analgesia. The proposed experiments are designed to provide a better understanding of the physiology and anatomy of serotonergic neurons in the rat pontomedullary raphe magnus (RM) and adjacent nucleus reticularis paragigantocellularis pars alpha (NRPGa) of the anesthetized and unanesthetized rat. The specific experiments proposed are: 1) The first aim of this proposal is to determine an unequivocal physiological marker for pontomedullary serotonergic neurons in anesthetized rats. Using extracellular recording, RM/NRPGa cells will be classified according to criteria that may be useful in identifying serotonergic neurons, such as a long duration action potential, a slow and steady discharge rate, response to midbrain stimulation and sensitivity to high concentrations of a general anesthetic. The physiology of serotonergic cells will then be directly tested by electrophysiologically characterizing neurons, intracellularly labeling them and processing them for serotonin immunocytochemistry. In this way, the applicant hopes to identify the common physiological traits that are shared by serotonergic neurons but are not present in non-serotonergic RM/NRPGa cells. The somatodendritic morphology and axonal projections of physiologically characterized and intracellularly labeled serotonergic cells will be described and compared to that of non- serotonergic neurons in the same region. 2) The physiological characterization of serotonergic RM/NRPGa cells will be extended to the unanesthetized rat. Recordings will be made from pontomedullary serotonergic units in chronically implanted, freely behaving and unrestrained rats. By using criteria established in the first aim, units will be classified as serotonergic or non-serotonergic in the chronically instrumented but anesthetized rat. Anesthesia will then be discontinued and the cell will be recorded and recharacterized in the wake, unanesthetized rat. The influence of behavioral state on unit activity will be studied by recording cell discharge patterns during spontaneous changes in behavioral state. These experiments will determine whether serotonergic neurons and/or non-serotonergic cells in the rat RM/NRPGa alter their discharge rate in accordance with behavioral state. In the final set of proposed experiments, cell activity and the behavioral response to noxious stimuli will be simultaneously recorded during spontaneous changes in behavioral state. A linear regression analysis will be performed to determine whether the discharge rate of serotonergic cells correlates with the withdrawal latency. By using behavioral state as an experimental variable, the influence of serotonergic cell activity upon nociceptive responsiveness can be examined.

DESCRIPTION: (adapted from applicant's abstract) Neurons in the medullary raphe magnus (RM) are important in nociceptive modulation. The responses of RM cells to noxious heat and to analgesic doses of opioids have led to the hypothesis that OFF cells inhibit and ON cells facilitate nociceptive transmission in the anesthetized animal. However, little is known of how RM contributes to nociceptive modulation in behaving animals. The proposed experiments focus on the physiology of non-serotonergic RM cells in unanesthetized, freely behaving rats. The experiments proposed in Aim 1 compare the neuronal responses to noxious heat and to systemic morphine in anesthetized and unanesthetized conditions. The remaining aims will further our understanding of how RM cells are activated in behaving rats. Recent experiments suggest that non-serotonergic RM cells have state-dependent discharge in a pattern that is correlated to their response to noxious heat. However, since only a small number of neurons were studied and since the protocol was not completed for many cells, our preliminary findings need to be confirmed and extended. Therefore, RM cells, characterized by their response to noxious heat, will be recorded in freely behaving, unrestrained rats as they cycle through waking, slow wave sleep and paradoxical sleep states. Unit discharge pattern and rate during sleep and wake states will be compared. Neuronal and behavioral responses, including motor and cardiovascular components, to thermal stimulation of either warm or noxious intensity, will be compared for stimuli applied during different behavioral states. Similarly, the responses of RM cells to innocuous mechanical and auditory stimulation will be tested and compared for stimuli applied during waking, slow wave sleep or paradoxical sleep states. Since sensory modulation commonly accompanies active movements, RM cell and motor responses to noxious thermal stimulation will be systematically studied during drinking, eating and grooming. The final aim will test whether RM cells contribute to the decrease in sleep time observed during chronic pain conditions. First, RM cell discharge will be continuously recorded during the development of arthritis and will be compared to the development of arthritic hyperalgesia and to the sleep/wake pattern exhibited by the animal. Second, microinjection of the GABAA receptor antagonist, bicuculline, which is known to activate RM OFF cells will be used to test whether OFF cell activation can increase sleep time in arthritic animals. To determine whether OFF cells act primarily on sleep/wake regulation or nociceptive transmission, the effects of bicuculline on sleep/wake pattern and spontaneous pain behaviors will be compared.

DESCRIPTION (provided by applicant): Visceral nociception, like cutaneous nociception, is subject to descending modulatory influences from the medullary raphe magnus (RM). However, little is known about if and how RM cells contribute to: 1) visceral stimulus evoked nocifensive reactions; and 2) visceral stimulus evoked suppression of cutaneous nociception. The proposed experiments use colorectal distension (CRD) as a model visceral stimulus to explore these issues. There are 6 aims: . Aim 1A: Identify the spinal trajectory of afferents that carry ascending CRD information to RM cells. . Aim 1B: Identify the contributions of descending modulatory input, arising from RM and elsewhere, to CRD-evoked cardiovascular and visceromotor reactions. . Aim 2: Determine the effect of RM cellular inactivation on behavioral reactions to CRD. . Aim 3: Identify the physiological characteristics of neurons that discharge in a pro-nociceptive manner, with increasing excitatory responses to increasing intensities of CRD stimulation. . Aim 4: Determine the spinal pathway taken by descending modulatory input, from RM and elsewhere, to the lumbosacral spinal cord. . Aim 5: Establish the role of RM cellular activation in heterotopic suppression of cutaneous nociception by a noxious visceral stimulus. . Aim 6: Aim 3: Identify the physiological characteristics of neurons that may subserve the antinociceptive effects of CRD stimulation. The proposed experiments will test the novel hypothesis that RM's effects on spinal nociception consists of a "pro-nociceptive" component that is necessary for the normal behavioral reaction to a noxious visceral stimulus in addition to the better-studied "inhibitory modulation" component.

DESCRIPTION (provided by applicant): The goal of this training program is to produce students with a broad multi-disciplinary background that enables them to use a variety of approaches to solve important problems in the field of neurobiology. To this end, students complete courses that span neurobiology from the neuroanatomical, neurophysiological, and developmental to the cellular, molecular and behavioral. Formal course work is augmented by weekly seminars, journal clubs, and ah annual retreat. A group of 48 trainers provide excellent opportunities for students to pursue their diverse research interests with experienced and skilled mentors. Since our last submission, the number of applicants to-Neurobiology has doubled an increase that has not been reflected in the number of training grant slots. We are therefore requesting that the number of positions on this training grant be increased to 12, to keep up with our recent and projected growth.

DESCRIPTION (provided by applicant): Long after their first use by humans, narcotics remain the most widely used and effective treatment for clinical pain despite their disadvantages: numerous side effects, the development of tolerance, and the opportunity for drug abuse. Genetic and molecular biological techniques are needed to make major advances in addressing the problems associated with narcotics but pain modulation has been almost exclusively studied in the rat. The goal of this proposal is to develop the mouse as a model for studying morphine analgesia and its side effects and thereby allow modern molecular and genetic methods to impact pain management. In the anesthetized rat, the discharge of cells in the medullary raphe magnus (RM) predicts the magnitude and speed of motor withdrawals from noxious stimulation both before and after systemic morphine. In contrast, our preliminary results in anesthetized mouse suggest that RM cell discharge predicts homeostatic adjustments but not withdrawals from noxious stimulation, either before or after morphine. The first aim of this proposal is to identify potential targets of RM cell modulation in the anesthetized mouse. The contribution of RM cells to opioid analgesia in awake animals, if any, has not been defined. The second aim of this proposal is to test whether murine RM cells mediate morphine analgesia in the awake mouse, even if, as our preliminary results suggest, they do not do so in the anesthetized mouse. In the rat, RM neurons protect important homeostatic functions, such as sleep and feeding, from interruption. To test whether this murine RM cells similarly defend homeostasis, the activity of RM neurons will be recorded while unanesthetized mice naturally cycle through sleep and wake states and drink water. A mechanistic model for opioid action in the genetically tractable mouse will allow development of therapeutic strategies that minimize side effects and maximize analgesia.

Project summary This small research grant will allow us to study the only known individual with a congenital lack of somatosensory afferents (KS) and to compare her motor performance, unconscious body schema, and conscious body image with those of IW, an individual with the rare condition of acquired somatosensory loss as well as to controls. KS lives in the United States and IW lives in Britain and we propose to study both individuals in the fully equipped laboratory of co-PI Chris Miall in Birmingham, UK. In both participants motor nerve function is intact, allowing us to investigate motor coordination through its effector body parts, working without peripherally originating haptic feedback. Funds are requested to support travel and the study of control participants for comparison. The importance of large-fiber sensory input for creating a body schema that in turn allows for the rapid and largely unconscious production of movement will be placed in stark relief by KS and IW. Two distinct solutions to a loss of such input, one based on a congenital lack and one on an acquired loss, will be identified. In the former case, a degree of automaticity based on enhanced visual proprioception is expected whereas movement in the face of acquired deafferentation is expected to depend on conscious and slow visual perception combined with cognitive oversight. Thus, our working hypothesis is that IW uses visual perception, cognitively demanding motor planning, and conscious body image to move in a consciously supervised and minimally automated manner. In contrast, we hypothesize that KS uses visual proprioception, an automated substitution of visual information for somatosensory proprioception, to support a body schema that automatically supports motor planning and execution. Aim 1 will test our hypotheses by asking KS and IW to make repeated iterations of previously learned movements, with and without cognitive load, and to learn new movements that range in complexity. In Aim 2, the unconscious body schema of IW and KS will be interrogated through reach estimation and testing for peri-personal perceptual facilitation. Finally, in Aim 3, we will directly compare KS?s and IW?s conscious body images. Results from the proposed experiments will uncover for the first time the relative roles of large-fiber somatosensory information in setting up and then maintaining for decades, sensorimotor function, body schema and body image.

The long-term goal of the University of Chicago Neuroscience Early Stage Scientist Training Program (NESSTP) is to diversify the Neuroscience research workforce. In order to increase underrepresented Scholars' readiness and success in the neuroscience research workforce, we propose to implement interventions at critical transition stages along the academic pathways. To accomplish this goal, we will take advantage of the unified campus and single faculty at the University of Chicago which hosts undergraduate, graduate and postdoc training in both basic science and medical areas. This will allow us to offer enhanced training for scholars at all three training levels, and also to emphasize cross-level mentoring in order to facilitate young scientists' development as leaders and trainers in Neuroscience. To enhance the undergraduate Neuroscience pipeline, we will provide research experiences, aid in articulating career goals, and facilitate graduate school preparation for our underrepresented (UR) students pursuing Neuroscience majors in the College. To support UR graduate students' and postdocs' academic and career pathways, we will provide professional skill development training, career exploration and networking opportunities to aid academic career success. Finally, we will foster a continuity of Mentorship across all career stages by introducing good mentoring practices for the trainees and their faculty mentors, pairing NESSTP trainees across stages to engage in near-peer mentoring, and establishing a pan-Neuroscience Mentoring Committee to establish good mentoring practices and promote a diverse and inclusive Neuroscience training community at the University of Chicago.