Mary M. Heinricher - US grants

The rostral ventromedial medulla (RVM) has a well-documented role in modulation of nociceptive transmission, and several independent lines of evidence support a major contribution of this brainstem region to opiate antinociception. One approach to the analysis of opiate action is at the level of single unit neurophysiology. Neurons of the RVM can be divided into three classes based on the relationship of cell discharge to the occurrence of nocifensive reflexes such as the tail flick (TF). Cells of one class, termed "off-cells," pause just prior to withdrawal reflexes induced by noxious stimulation, and there is good evidence that these neurons are the RVM output neurons that inhibit nociceptive transmission and contribute to opiate antinociception. Off-cells are activated by morphine in doses which are sufficient to produce antinociception. This off-cell excitation is probably due to suppression of the activity of an inhibitory interneuron, since the direct cellular effects of opiates are generally effects of opiates are generally inhibitory. One likely candidate for the substance released by this inhibitory interneuron is the ubiquitous inhibitory neurotransmitter GABA. GABA is found in cell bodies and terminals in the RVM, and may contribute to the nociceptive modulating functions of this region. The overall aim of the proposed research is to determine the significance of GABA in inhibitory control of activity of these putative nociceptive modulating neurons in the RVM, specifically its role in opiate antinociception. Three general approaches will be used. First, the effects of RVM microinjection of GABA agonists and antagonists on the TF reflex and on morphine-induced antinociception will be determined in lightly-anesthetized rats. Second, extracellular single unit recording and iontophoresis will be used to determine the role of GABA-ergic inputs to physiologically-characterized RVM neurons. Blockade of the TF-related pause or spontaneously-occurring periods of quiescence in the off-cell by GABA antagonists would favor a role for GABA in inhibitory control of off-cells. Reversal of opiate-induced activation of off-cells by GABA agonists would be consistent with the notion that opiates activate off-cells by reducing GABA-mediated inhibition. Finally, recordings from single off-cells before and after pressure ejection of GABA antagonists in doses sufficient to block the TF will reveal any changes in cell activity that are correlated with changes in nocifensive responses. These studies will further our knowledge of intrinsic brain mechanisms for controlling nociceptive transmission. In particular, they will expand our understanding of opioid analgesia and the physiological role of endogenous opioid peptides.

DESCRIPTION: (Applicant's Abstract) The potent analgesic effects of opioids derive, in large part, from an action upon a network of neurons within the brainstem and spinal cord. This network has important links in the midbrain periaqueductal gray (PAG), the rostral ventral medulla (RVM) and spinal cord dorsal horn. The overall goal of this project is to increase our understanding of this pain-modulating system within the brainstem, with a focus upon how opioids influence the circuitry within the RVM. Our strategy involves the use of pharmacological tools such as iontophoresis and nanoinfusion in conjunction with single unit recording to identify factors that influence activity of RVM neurons in a functional context. This approach is possible because two populations of physiologically identifiable pain-modulating neurons have been characterized in the RVM of lightly anesthetized rats. "Off-cells," which show an abrupt cessation of firing just prior to occurrence of nocifensive reflexes, are invariably activated, although indirectly, by opioids. There is strong evidence that these neurons exert a net inhibitory effect on nociceptive processing. "On-cells" display a sudden increase in activity beginning just before the occurrence of nocifensive reflexes, and likely exert a permissive or even facilitating effect on nociception. On-cells are directly sensitive to opioids. This cell class thus has a key role as an avenue through which opioids are able to gain access to the nociceptive modulatory circuitry of the RVM. The focus of this proposal is on inputs that affect the firing of on-cells, and on how these inputs are affected by, or modify the actions of, opioids within the RVM. First, the contribution of excitatory amino acid neurotransmitters to the nocifensor reflex-related activation of a physiologically identified class of RVM neurons will be chacterized. Second, it is now well established that the analgesic actions of opioids can be modified by "anti-opioid" peptides including cholecystokinin and neurotensin, and we will determine the mechanisms through which these peptides enhance or limit the effects of opioids within the RVM. The importance of the RVM in the mechanisms of opioid analgesia is well documented. Because our understanding of the opioid-sensitive circuitry within the RVM allows us to use complementary behavioral and electrophysiological approaches, examination of the inputs responsible for activating the opioid-sensitive RVM neurons, and of the effects of anti-opioid peptides in this circuit should be particularly fruitful. These studies should not only advance our understanding of pain modulation, but insofar as they elucidate some of the factors that modify neuronal responses to opioids, could lead to improvements in the clinical use of analgesic opioid drugs.

DESCRIPTION:(adapted from applicant's abstract) The attention of investigators interested in pain and analgesia has in recent years been increasingly directed toward understanding the mechanisms that underlie persistent pain states. The most intense effort has been focused upon elucidating the now well-documented plasticity of elements in nociceptive transmission pathways, including primary afferent nociceptors and ascending transmission circuits. By contrast, the possibility that changes in descending modulatory systems might contribute to persistent pain states has received comparatively little attention. Nevertheless, there is now mounting evidence pointing to an important role for a well-characterized brainstem pain modulating system in hyperalgesia and persistent pain. This system, with links in the midbrain periaqueductal gray (PAG) and rostral ventromedial medulla (RVM), was once viewed as an "analgesia system" activated by acute stress or pain or by opioid analgesic drugs to inhibit spinal nociceptive processing. It is now known to be more complex, with a potential for bi-directional control of nociception. The aim of the present proposal is to analyze recruitment of this brainstem pain modulating system by interleukin-1beta (IL-1beta), a pro-inflammatory cytokine that orchestrates immune and neural responses to injury and infection. The applicants propose to use a combination of behavioral pharmacology and single cell recording methods to characterize the effects of IL-1beta on nociceptive responding, and to identify the central circuitry mediating these effects. They have found that administration of IL-1beta evokes a biphasic alteration in nociceptive responding, with a period of hyperalgesia followed by a later phase of hypoalgesia. They will therefore determine the time course of changes in nociceptive responding following systemic and intracerebroventricular administration of IL-1beta in both awake and isoflurane-anesthetized rats, and identify potential roles of prostanoids, NMDA, endogenous opioids or corticotropin releasing factor. Using a microinjection mapping technique, the investigators will test the hypothesis that IL-1beta acts directly within the hypothalamus and within RVM. They will further test the role of specific cell populations within the RVM using lesion and electrophysiological approaches. The role of brainstem pain modulating systems in opioid analgesia is well documented, but when and how this system is recruited to enhance pain is almost completely unknown. By elucidating mechanisms through which pro-inflammatory cytokines bring descending control systems into play, the proposed work should advance our understanding of the processes involved in pain modulation. In providing additional tools to manipulate these systems, this work may ultimately lead to improved clinical treatment of pain.

DESCRIPTION (provided by applicant): The attention of investigators interested in pain has in recent years been increasingly directed towards understanding mechanisms that form the basis for hyperalgesia and persistent pain states. The greatest effort has been on elucidating the now well-documented plasticity of elements in nociceptive transmission pathways, with demonstrated sensitization of both the primary afferent nociceptors and dorsal horn processing circuits. By contrast, the possibility that changes in brainstem descending modulatory systems might contribute to persistent pain states has received comparatively little attention. Nevertheless, there is now mounting evidence pointing to an important role for these modulatory systems in hyperalgesia and persistent pain. The best characterized modulatory system, with important links in the midbrain periaqueductal gray (PAG) and rostral ventromedial medulla (RVM), was long viewed as an "analgesia system," activated by acute stress or pain or by opioid analgesic drugs to inhibit spinal nociceptive processing. It is now thought to be more complex, enhancing or inhibiting nociception under different conditions. The overall goal of the work proposed here is to identify neural mechanisms for bi-directional control by this brainstem pain modulating system, focusing on two populations of putative nociceptive modulatory neurons that have been identified in the RVM: "off-cells," proposed to inhibit nociceptive processing, and "on-cells," proposed to facilitate nociception. Our approach will involve a combination of behavioral pharmacology with single cell recording techniques and microiontophoresis to understand how these physiologically distinct classes of RVM neurons might be recruited, and to further define the contribution of each class to pain modulation. The role of brainstem pain modulating systems in opioid analgesia is well documented, but neural mechanisms through which these systems contribute to pain facilitation are so far almost completely unknown. The proposed work should advance our understanding of central processes involved in bi-directional pain modulation, and should ultimately lead to improved clinical treatment of pain.

[unreadable] DESCRIPTION (provided by applicant): Prostaglandins have long been known to play an important role in peripheral mechanisms of nociceptor sensitization and hyperalgesia. However, there has been increasing awareness that prostanoids also act centrally, and new evidence suggests that prostaglandins exert their effects in the brain in part by recruiting a brainstem pain-modulating system with links in the midbrain periaqueductal gray and rostral ventromedial medulla (RVM). Our goal is to identify mechanisms through which PGE2 acting supraspinally might gain access to this brainstem descending modulatory system to facilitate nociception. Our approach will involve behavioral pharmacology and single cell recording studies in lightly anesthetized rats. We will focus on the medial preoptic area (MPO) because PGE2 acting in the MPO plays a central role in the hyperalgesia associated with illness, and because we recently demonstrated that PGEa in the MPO recruits the pain modulatory circuitry of the RVM. We will use inactivation studies to determine whether the altered nociception and thermogenesis produced by PGE2 in the MPO are mediated by functionally distinct cell classes in the RVM. We will also use selective prostaglandin E-type receptor agonists to determine if different receptors mediate the hyperalgesic and thermogenic effects of PGEa in the MPO. We will determine whether different RVM cell populations respond to PGE2 applied within the RVM itself, and test whether the pathway(s) from the MPO to the RVM for hyperalgesia and thermogenesis relay in the periaqueductal gray and/or dorsomedial hypothalamus. [unreadable] These studies will provide important insights into the supraspinal actions of PGE2, and into how higher structures such as the MPO influence brainstem pain modulating systems. Knowledge of the mechanisms through which prostaglandins recruit descending control systems will advance our understanding of the neural basis of pain modulation, and should ultimately lead to improved clinical treatment of pain. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): There is an urgent need for effective, non-opioid treatments for pain. Although the brain possesses pain- relieving (analgesic) circuits that are independent of endogenous opioids, little is known about substances capable of activating these systems. Dr. Lindsay Hough and colleagues at Albany Medical College discovered a novel class of non-opioid analgesic drugs derived from histamine antagonists. The prototype compound was named improgan. Improgan is highly effective in attenuating thermal and mechanical pain behaviors in rats and mice, but does not impair motor coordination or locomotor activity, and at least in limited testing, does not produce tolerance with daily dosing. Improgan's analgesic effects are not mediated by known opioid or histamine receptors. Nevertheless, improgan's actions appear to intersect with those of opioids in that focal application of improgan in the midbrain periaqueductal gray or in the rostral ventromedial medulla (RVM) produces effective antinociception. Moreover, the analgesic actions of intracerebroventricularly administered improgan are attenuated or blocked by inactivation of the RVM. Thus, improgan, like opioids, appears to activate an output from the RVM to produce its antinociceptive effect. Previous work from my laboratory has provided a number of insights into the actions of opioids in the RVM, and how these are translated into behaviorally measurable antinociception. The goal of the present experiments is to use our knowledge of opioid actions in the RVM as a framework for understanding the actions of improgan at the level of neuronal circuitry. We will use a combination of single cell recording and behavioral pharmacology to characterize the neural substrate of improgan analgesia within the RVM at the level of individual neurons and of the RVM circuit as a whole. These data will therefore provide mechanistic understanding of the link between improgan pharmacology and behavior. By comparing the neural mechanisms of this novel, non-opioid analgesic with the well-documented actions of opioids, these studies should provide new knowledge that may lead to development of clinically useful, non-opioid analgesic drugs.

Despite the high prevalence of migraine in the general population, our understanding of the underlying mechanisms remains incomplete. A variety of theories focused on peripheral neural or neurovascular mechanisms have been put forward, but none of these has so far received conclusive experimental support. An alternative proposal is that migraine is triggered, or at least maintained, by a "central generator" in the brain itself. We recently showed that the rostral ventromedial medulla (RVM), a region with a well documented role in pain modulation, contributes to behavioral hypersensitivity following dural inflammation, an animal model of migraine headache. The proposed studies will test the role of specific population of RVM neurons, termed "ON-cells", in this model. To accomplish this, we will record activity of identified RVM painmodulating neurons before and after dural inflammation and determine whether ON-cell mediate observed behavioral changes by manipulating the activity of this and other RVM cell classes using pharmacological tools. The experiments described in this proposal will attempt to delineate a more complete theory of migraine headache pain by extending the idea of "central sensitization" in migraine headache to brainstem modulatory systems. We expect to find important support for the idea of a "central generator" in migraine headache pain. This idea is attractive because a central dysfunction could potentially explain the multiple triggers for migraine attached, as well as the range of associated symptoms, including nausea and aversion to light and sound.

DESCRIPTION (provided by applicant: The attention of investigators interested in pain and analgesia has been increasingly directed beyond acute pain mechanisms towards processes that give rise to persistent pain states. There is now clear functional evidence that brainstem modulatory systems contribute to persistent pain associated with nerve injury and inflammation. The best characterized modulatory system has important links in the midbrain periaqueductal gray and rostral ventromedial medulla (RVM), and is recruited to enhance or inhibit nociception under different conditions. The present proposal focuses on the RVM. Over the last ten years, my laboratory has demonstrated that pain-inhibiting and pain- facilitating influences from the RVM are mediated by two classes of neurons, ON-cells, which exert a net facilitating influence on nociception, and OFF-cells, which have a net inhibitory action. The overarching goal of this proposal is to understand how activity- dependent changes in the properties and relationships of these neurons contribute to abnormal pain following nerve injury, and during chronic inflammation. Using a combination of single-cell recording and behavioral pharmacology, the proposed experiments will test whether changes in the mechanical thresholds of ON- and OFF-cells in nerve-injured animals are important for behavioral hypersensitivity, contrast changes in RVM neurons during chronic inflammation with those seen following nerve injury, and identify drivers of ON-cell activation in both models. A better understanding of molecular, cellular, and circuit-level mechanisms underlying chronic pain is essential if we are to develop better treatments for patients. There is now increasing evidence that pathological pain states are at least in part driven by changes in the brain itself. Descending modulatory pathways are known to mediate top-down regulation of nociceptive processing, transmitting cortical and limbic influences to the dorsal horn. These pathways are also intimately intertwined with ascending transmission through positive and negative feedback loops. Models of persistent pain that fail to include descending modulatory pathways are thus necessarily incomplete. By examining how the properties of known nociceptive modulatory neurons are transformed during the transition from acute to chronic pain, the present studies fill an important gap in our knowledge.

DESCRIPTION (provided by applicant): Migraine is the most common neurological disorder, and affects over 10% of the population in any given year, with over half of these individuals reporting severe impairment. For many patients, a severe, even disabling, component of the migraine attack is photophobia, yet neuroscientists are just starting to investigate the underlying neurobiological mechanisms. The present application tests the overarching hypothesis that brainstem pain-modulating circuits, already implicated in migraine-related pain, also contribute to migraine-related photophobia. This hypothesis is based on the entirely unexpected observation that pain-modulating neurons in the rostral ventromedial medulla, the final output of an important brainstem pain-modulating system, develop photoresponsiveness in animal models of migraine headache, although they do not respond to light under normal conditions. In three Specific Aims using the nitroglycerin migraine model in the rat, we will document light-evoked activity in identified pain-modulating neurons and determine whether this is specific to migraine. We will also determine whether pain- modulating systems contribute to light aversion and light-induced pain enhancement. Finally, we will identify possible pathways through which light gains access to pain- modulating systems. The present proposal brings together electrophysiological and behavioral approaches to show how light engages pain-modulating systems to produce photophobia. These studies will provide insights into the neurobiological mechanisms of migraine-related photophobia, fundamental information critical for developing new migraine treatments. PUBLIC HEALTH RELEVANCE: Migraine remains the most common neurological cause of disability, and current treatment options are ineffective for many patients. Migraine patients often avoid light, because light increases their headache pain. The work proposed in this application seeks to understand the role of the brain's own pain-modulating systems in photosensitivity during migraine, which should ultimately help us develop better treatments for migraine pain.

? DESCRIPTION (provided by applicant): There is now increasing evidence that pathological pain states are at least in part driven by changes in the brain itself. Descending modulatory pathways are known to mediate top- down regulation of nociceptive processing, transmitting cortical and limbic influences to the dorsal horn. Ascending pain transmission pathways are also intimately intertwined with these modulatory systems, forming positive and negative feedback loops. The output node of the best-characterized pain-modulating system is the rostral ventromedial medulla (RVM). Building on my laboratory's experience defining the outputs of RVM neurons, the studies in the present application fill an important gap, identifying a pathway through which noxious input reaches the RVM. My laboratory has demonstrated that pain-inhibiting and pain-facilitating influences from the RVM are mediated by two classes of neurons, ON-cells, which exert a net facilitating influence on nociception, and OFF-cells, which have a net inhibitory action. The overarching goal of this proposal is to begin to define nociceptive inputs to the RVM. Our preliminary data suggest that one important relay is the parabrachial complex. This region is the main target of nociceptive pathways arising from the superficial dorsal horn. We propose to test the role of the parabrachial complex in regulating the activity of RVM pain- modulating neurons, and to determine how noxious inputs are altered in chronic pain states. These studies will use in vivo single-cell recording from identified RVM ON- and OFF- cells, optogenetics, and pharmacological manipulations to test the hypothesis that the parabrachial complex is a direct relay for noxious input to RVM in acute thermal and mechanical nociception (Aim 1). The same approaches will then be used to determine how these nociceptive inputs to the RVM are modified in a model of chronic inflammatory pain (Aim 2). Complementing this in vivo work, parallel studies under Aim 3 will use in vitro electrophysiology in an adult RVM slice with optogenetic manipulation of defined terminals in the RVM to define the membrane mechanisms of parabrachial input to the RVM and determine how that is altered in chronic inflammation. By defining pathways through which noxious information reaches pain-modulating neurons at the membrane, individual neuron, and circuit level, we can begin to define how pain-modulating circuits are recruited in acute and chronic pain. This information is critical if we are ever to develop treatments addressing chronic pain as maladaptive brain disease.

PROJECT SUMMARY There is now increasing evidence that pathological pain states are dependent on changes in the brain itself. Descending modulatory pathways are known to mediate top- down regulation of nociceptive processing, transmitting cortical and limbic influences to the dorsal horn of the spinal cord. Ascending pain transmission pathways are also intimately intertwined with these modulatory systems, forming positive and negative feedback loops. The output node of the best-characterized pain-modulating system is the rostral ventromedial medulla (RVM). Building on the Heinricher laboratory's experience defining the outputs of RVM neurons, the studies in the present application fill an important gap, identifying a pathway through which noxious input reaches the RVM. The RVM has two pain-modulating cell types: ?ON-cells,? which exert a net facilitating influence on nociception, and ?OFF-cells,? which have a net inhibitory action. The overarching goals of the present proposal are to understand plasticity of this circuitry in chronic pain states, and how it is modulated by endogenous opioids and cannabinoids. We recently showed that the parabrachial complex (PB) is a critical relay of acute noxious information to the RVM. We propose to test the role of the PB in regulating the activity of RVM pain-modulating neurons, elucidate how opioids and cannabinoids modulate the activity of PB-RVM synapses, and determine how this connection is altered in chronic pain states. These studies will use in vivo single-cell recording from identified RVM ON- and OFF-cells, optogenetics, and pharmacological manipulations to test the hypothesis that the PB projection to the RVM is modulated following persistent inflammation (Aim 1). Complementing this in vivo work, parallel studies under Aims 2 and 3 will use in vitro electrophysiology in an adult RVM slice with optogenetic manipulation of identified PB-RVM terminals to define the membrane mechanisms of PB-RVM synapses and understand how these synapses are modulated by cannabinoids and opioids. We will also determine how this connection is altered in chronic inflammation. By defining pathways through which noxious information reaches pain- modulating neurons at the membrane, individual neuron, and circuit level, we can begin to define how pain-modulating circuits are recruited in acute and chronic pain. This information is critical if we are ever to develop treatments addressing chronic pain as maladaptive brain disease.

Project Summary Over a hundred million of Americans suffer from chronic pain and over ten million of Americans suffer from alcohol abuse or dependence. There is a bidirectional relationship between chronic pain and alcohol dependence. Thus, alcohol dependence is a major predictor of severity of chronic pain, and people with chronic pain conditions are more likely to use alcohol for pain relief. Unfortunately, a mechanistic understanding of alcohol-related pain sensitivity is lacking. Our studies have identified increased pain sensitivity in mice during withdrawal from voluntary alcohol self-administration. Consumption of alcohol in these mice reversed mechanical hypersensitivity produced by alcohol withdrawal. The increased pain sensitivity in mice undergoing withdrawal is consistent with increased pain in alcohol-dependent patients. In addition, we found increased pain sensitivity in control ?bystander? mice housed in the same room as mice undergoing alcohol withdrawal. The social transfer of hyperalgesia from mice undergoing withdrawal to the bystander mice involved olfactory cues. Such social transfer of hyperalgesia could affect co-dependent family members of alcoholic patients. Immunohistochemical analysis revealed differential activation of dorsomedial hypothalamus, anterior cingulate and anterior insular cortex in these animals. We hypothesize that the identified brain regions are differentially involved in alcohol withdrawal-induced hyperalgesia and socially-transferred hyperalgesia. The goal of this proposal is to address this hypothesis and further characterize the phenomena of alcohol withdrawal- and social transfer-induced hyperalgesia. This goal will be achieved in three Specific Aims: Aim 1 will further characterize the phenomenon of hyperalgesia in alcohol withdrawing and bystander mice by examining whether the observed thermal hyperalgesia is exaggerated in female bystander mice, involves negative affective states, anxiety or stress responses, or coexists with depression-like behaviors. Aim 2 will test whether alcohol-induced activation of neuronal populations within dorsomedial hypothalamus is necessary and sufficient for regulation of pain sensitivity and alcohol drinking behavior in mice. Aim 3 will test whether alcohol withdrawal and social transfer-induced activation of neurons of anterior cingulate and/or insula are necessary and sufficient for regulation of pain sensitivity.

PROJECT SUMMARY Many chronic pain patients complain of abnormal sensitivity to multiple sensory modalities, including light, sound, and smell. The underlying neural basis remains a puzzle and these claims are often viewed with suspicion, since sensory acuity per se is not enhanced, nor is there amplified processing in primary sensory pathways. The central thesis of this proposal is that dysfunction of brainstem pain-modulating systems, already thought to contribute to pain in these conditions, is also likely to play a role in multisensory hypersensitivity. This hypothesis is based on our unexpected observation that a subset of pain-modulating neurons in the rostral ventromedial medulla, the final output of an important brainstem pain-modulating system, respond to light. In three Specific Aims using single-cell recording approaches in rat, we will fully characterize the RVM response to light, generating stimulus-response functions and establishing the relevant spectrum. We will also determine whether photoresponsiveness of these neurons is enhanced in an animal model of migraine headache (where photosensitivity is well documented) or in a persistent inflammatory state. Finally, we will investigate ?top-down? modulation of photoresponsiveness from a higher brain structure known to contribute to stress-induced hyperalgesia and implicated in endocrine, autonomic, and behavioral responses to mild stress. The present proposal brings together electrophysiological and behavioral approaches to determine how light engages pain-modulating systems, effectively converting a visual stimulus to a somatic sensation, producing discomfort and aversion. These studies will provide fundamental insights into the neurobiological mechanisms of multisensory hypersensitivity, information critical to developing more appropriate treatments for migraine and other chronic pain disorders.

PROJECT SUMMARY The central nervous system has an intrinsic pain modulatory system that regulates nociceptive processing through descending projections from the brainstem to the spinal cord dorsal horn. The ventrolateral periaqueductal gray (vlPAG) integrates sensory information with input from higher cortical and subcortical areas, and sends projections to the rostral ventromedial medulla (RVM) that are relayed to the dorsal horn of the spinal cord. Both the vlPAG and RVM are heterogenous with respect to participating in multiple behavioral circuits. The proposed studies build on extensive previous work from the Heinricher laboratory that has defined the output from the RVM, showing that bidirectional pain control from this region is mediated by two physiologically defined cell classes, ?ON-cells? and ?OFF-cells,? that respectively facilitate and inhibit dorsal horn nociceptive transmission under different conditions. The Ingram laboratory has expertise studying opioid actions within the PAG and RVM, as well as adaptations in both areas with persistent inflammation. Proposed viral optogenetic strategies will map and define the vlPAG circuit that regulates RVM ON-cells involved in the facilitation of pain and elucidate underlying cellular mechanisms that shift the balance of RVM output from inhibition of pain to facilitation of pain with persistent inflammation. The combined expertise of the two laboratories will focus on identified PAG-RVM synapses using optogenetic stimulation of RVM terminals originating from the PAG. In vitro brain-slice recordings (Ingram lab) will examine the heterogeneity of PAG output to the RVM and PAG-RVM synapses, as well as cellular mechanisms of synaptic plasticity induced in persistent inflammation. These studies will use a fluorescent label for the ?-opioid receptor to differentiate presumed ON-cells from other classes in the slice to determine whether PAG terminals directly synapse on ON-cells, OFF-cells, or both, as well as what neurotransmitters are released. In vivo single-cell recording studies (Heinricher lab) will determine how inflammation-induced changes in PAG-RVM synapses control excitability of specific populations of RVM neurons and establish the link between these changes and pain behaviors. A better understanding of molecular, cellular, and circuit-level mechanisms that underlie pain is essential if we are to develop better treatments. By carefully mapping the descending projections from PAG to RVM during the development of persistent inflammation, and by tying these to defined RVM outputs and behavior, we can begin to determine the interactions in this complex network, and gain new insights into how pain-modulating systems are recruited and modulated in acute and chronic pain.