Edita Navratilova, Ph.D. - US grants

Project Summary Multiple lines of evidence support wide-spread structural and functional maladaptations in brain circuits as a contributing factor promoting chronic pain. Dysregulation of descending pain modulatory circuits have been demonstrated in chronic pain patients. However, our understanding of the molecular mechanisms and brain circuits that underlie inhibition or facilitation of pain under normal and pathological conditions remains limited. Opioid analgesics engage these brain circuits, but non-addictive options to treat pain are urgently needed. In this proposal, we will investigate pain modulatory mechanisms in the amygdala, a structure involved in integration of pain-related sensory inputs and emotional processing. We will test the hypothesis that functionally opposing endogenous mu (MOR) and kappa (KOR) opioid circuits in the central nucleus of the amygdala (CeA) inhibit or promote, respectively, pain behavior. Bidirectional descending pain modulatory circuits have been characterized most extensively in the rostral ventromedial medulla (RVM). However, little is known about descending modulatory circuits above the brainstem. In the RVM, pro-nociceptive and anti-nociceptive functions are mediated by MOR-expressing ?pain ON? cells and KOR-expressing ?pain OFF? cells. We propose that similar functional organization with MOR- and KOR-expressing cells serving opponent roles in sensory and/or affective pain behavior also exists in the CeA. Importantly, the reciprocal function of the MOR and KOR circuits may represent a general organizational principle for pain modulation in other brain areas. We will use transgenic mouse models in combination with optogenetic and chemogenetic approaches and anterograde and retrograde tracing for the cell type-specific and projection-specific analysis of MOR and KOR circuits in the CeA and their contribution to pain behaviors in a neuropathic pain model (spinal nerve ligation). Specific Aim 1 will determine the anatomical characteristics of KOR and MOR cells in the CeA. Specific Aim 2 will explore the synaptic and cellular effects of KOR and MOR activation or inhibition and their interaction. Specific Aim 3 will assess the role of MOR and KOR circuits in pain behaviors using reflexive responses (mechanical and thermal thresholds) as well as complex behaviors related to affective and motivational qualities of pain (vocalization, conditioned place preference/avoidance). Eletrophysiological and behavioral outcomes will be assessed with cell specific manipulations under control conditions and in a neuropathic pain model in both male and female mice allowing the determination of possible sex differences in the amygdala circuit. The proposed studies will identify a previously unknown KOR-mediated pro-nociceptive amygdala circuit and its relationship to MOR-mediated anti-nociceptive mechanisms. Impaired MOR signaling and/or increased KOR signaling in the CeA may result in an inhibition-excitation imbalance of descending modulation to promote chronic pain. Importantly, the opposing MOR-KOR function in the CeA would provide the conceptual basis for the development of non-addictive therapies for chronic pain.

Post-traumatic headache (PTH) commonly occurs following mild traumatic brain injury (mTBI), also known as concussion. PTH is a secondary headache that often presents with a migraine-like phenotype and is subdivided as acute or persistent (PPTH) depending on whether it resolves within 3 months after injury. The pathophysiology of PTH and PPTH is not understood and no evidence-based treatments exist for these conditions. Critically, PPTH might differ from PTH, not only in the duration but also in underlying mechanisms and responsiveness to treatment. The reasons for emergence of PPTH in some patients remain unclear but may be related to risk factors including pre-existing migraine and the experience of a previous mTBI. We have developed an approach to investigate the mechanisms of PTH and PPTH as well as potential strategies for treatment. Using a weight drop method in male and female mice that recapitulates biomechanical properties and clinical features of mTBI, we have shown that a single mTBI is sufficient to induce clinically relevant PTH symptoms including an acute period of allodynia, elevated CGRP blood levels and lowered thresholds for induction of cortical spreading depression (CSD). Additionally, we have explored the concept that the transition from acute to chronic pain states may rely on a ?pain memory? that can be studied using the ?two-hit? model of hyperalgesic priming where a prior insult confers vulnerability to a subsequent provocative stimulus. Thus, following resolution of acute allodynia, mTBI mice transition into a long-lasting persistent phase (PPTH) where, remarkably, allodynia can be reinstated by physiologically relevant and common migraine triggers, including stress. CGRP is established in migraine pathogenesis and our data also suggest an important role in promoting PTH. Treatment with either a CGRP antibody or with onabotulinum toxin A (botox) prevents mTBI-related allodynia (PTH) as well as subsequent provoked allodynia representative of PPTH. However, blockade of CGRP after mTBI sensitization is established is ineffective in blocking provoked allodynia, while botox still maintains efficacy. We have hypothesized that mTBI results in CGRP release from meningeal afferents promoting PTH and central sensitization that underlies the development of PPTH, but that PPTH may be maintained in a CGRP-independent fashion. Additionally, we hypothesize that existing sensitization prior to a mTBI event will promote vulnerability to the development of CGRP-independent PPTH. We explore these hypotheses with two related but, independent, aims using behavioral, neurochemical, immunohistochemical and electrophysiolgical analyses. Aim 1 will determine whether, and when currently available therapies can block mTBI-related outcomes relevant to PTH and if these treatments can prevent the expression of PPTH. Aim 2 will determine if prior sensitization promotes more severe, long-lasting and CGRP-resistant PPTH. Our studies will fill in significant knowledge gaps about the role of CGRP in promoting PTH and the importance of pre-existing sensitization in establishing CGRP- independent PPTH. Such information will influence treatment as well as guide the discovery of new therapies.

Project Summary: Many patients suffer from chronic pain in the absence of identifiable injury. Such pains are termed ?functional? and include irritable bowel syndrome, temporomandibular joint disorder, fibromyalgia, migraine and others. For reasons that are not understood, almost all functional pain syndromes (FPS) are female prevalent. FPS patients experience pain-free interictal periods punctuated by attacks of pain. The frequency of attacks is predictive of risk of chronification. Pain episodes thus produce a priming effect, establishing a state of increased vulnerability to future attacks, likely reflecting peripheral and central sensitization. FPS patients commonly identify stress as a key trigger of pain. Repeated stress may thus promote vulnerability and pain in a sexually dimorphic fashion. We have developed an injury-free rodent model of FPS based on hyperalgesic priming with repeated stress. Hyperalgesic priming produces a pain-free state of increased vulnerability that has been termed ?latent sensitization? (LS). Following induction of LS, normally subthreshold triggers can produce pain attacks, modeling the interictal and ictal periods of FPS. We will use this model to test the novel hypothesis that repeated stress activates kappa opioid receptor (KOR) signaling in the hypothalamus resulting in release of prolactin (PRL) and dysregulation of prolactin receptor (PRLR) isoform expression selectively in female nociceptors. PRL signals through homodimers of PRLR long and short (i.e., PRLR-L and PRLR-S) isoforms that respectively regulate transcription and pain. Repeated stress down-regulates PRLR-L promoting female-selective pain through stress-induced PRL/PRLR-S signaling. The balance of PRLR isoforms may therefore ?tune? female nociceptors to promote LS and pain from normally subthreshold stimuli. We will use genetic and chemogenetic manipulations along with anatomical, neurochemical, electrophysiological, pharmacological and behavioral studies in male and female mice to evaluate the role of dorsal root ganglion (DRG) PRLR-L down-regulation and stress-related hypothalamic KOR activation as essential mechanisms of LS and stress-related pain in females. Aim 1 will establish the effects of repeated stress on hypothalamic KOR signaling and PRL release. Aim 2 will establish a potential causal relationship of repeated stress or hypothalamic KOR activation on DRG PRLR isoform expression, neural excitability, LS and stress-related pain. Aim 3 will determine if KOR antagonists, DA agonists or a PRL antibody will prevent LS and FPS-like pain selectively in females. The proposed studies will characterize a previously unknown stress-related neuroendocrine link between hypothalamic KOR and PRL/PRLR signaling to promote female selective functional pain. Importantly, these studies will advance knowledge about previously unknown biological mechanisms and may unravel mechanisms for therapeutic interventions allowing improved therapy of FPS in women.

Project Summary ? Genetic Targeting Core Substance use disorders (SUD) are characterized by dysregulation of brain circuitry that involves diminished activity of the brain reward circuits, increased responsiveness of the stress circuits and impaired functioning of the executive cortical circuits. Neural changes are observed in the basal ganglia, extended amygdala and prefrontal cortical regions and encompass a wide range of endogenous neurotransmitters including dopamine, opioid peptides, endocannabinoids, corticotropin releasing factor (CRF), dynorphin, glutamate and others. It implies that these maladaptations may be causally responsible for behavioral changes often observed in chronic drug users, for example habit formation and compulsive drug taking, that further worsen the neuronal and behavioral signs of SUD. Restoration of normal functioning of the brain circuits is therefore a desirable goal of SUD therapies. However, the exact influence of chronic drug use on the addiction brain circuits and how manipulation of these circuits could prevent or treat substance use disorders remains to be elucidated. As part of the Center of Excellence in Addiction Studies, the Genetic Targeting Core (GT Core) will assist users in obtaining preliminary data on neural mechanisms of addiction that will form basis for future NIDA grant applications. The GT Core will provide services to target and manipulate the addiction circuits in a cell specific and circuits specific manner using state-of-the-art genetic techniques, including optogenetics, chemogenetics and CRISPR/Cas9 gene editing. Optogenetic strategies use microbial-based light activated ion channels (opsins) that allow fast neuronal activation (channelrhodopsins) or inhibition (halorhodopsins, archaerhodopsins), while chemogenetic approaches use engineered ion channels (PSAMs) or G protein- coupled receptors (DREADDs) that can be activated pharmacologicaly. CRISPR/Cas9 gene editing allows direct in vivo manipulation of the genome in rodents. The introduction of functional transgenes, such as opsins and DREADDs or editing of native genes requires in vivo delivery and expression of genetic material in desired cells. This is accomplished with viral or nonviral vector systems. Cell and circuit specificity can be further enhanced by the use of a specific Cre-driver mouse line. The GT Core will provide three main services to its users that correspond with the three Specific Aims: 1) assist with the selection of gene targeting methods, including the choice of the transgenic mouse line, and the type of viral vector; 2) optimize and implement gene delivery using high precision stereotaxic instruments; and 3) verify the efficacy of gene targeting using fluorescent microscopy or protein and gene quantification techniques. The GT Core will coordinate work with the Administrative, Behavioral and Neuroanalytical Cores to develop effective experimental strategies that involve several Cores. The close collaboration between our Cores ensures high expertise in all areas of the addiction research and will permit establishment of standardized outcome measures emphasizing scientific rigor and reproducibility.