Chemogenetic Manipulation of Amygdala Kappa Opioid Receptor Neurons Modulates Amygdala Neuronal Activity and Neuropathic Pain Behaviors.

Neuroplasticity in the central nucleus of the amygdala (CeA) plays a key role in the modulation of pain and its aversive component. The dynorphin/kappa opioid receptor (KOR) system in the amygdala is critical for averse-affective behaviors in pain conditions, but its mechanisms are not well understood. Here, we used chemogenetic manipulations of amygdala KOR-expressing neurons to analyze the behavioral consequences in a chronic neuropathic pain model. For the chemogenetic inhibition or activation of KOR neurons in the CeA, a Cre-inducible viral vector encoding Gi-DREADD (hM4Di) or Gq-DREADD (hM3Dq) was injected stereotaxically into the right CeA of transgenic KOR-Cre mice. The chemogenetic inhibition of KOR neurons expressing hM4Di with a selective DREADD actuator (deschloroclozapine, DCZ) in sham control mice significantly decreased inhibitory transmission, resulting in a shift of inhibition/excitation balance to promote excitation and induced pain behaviors. The chemogenetic activation of KOR neurons expressing hM3Dq with DCZ in neuropathic mice significantly increased inhibitory transmission, decreased excitability, and decreased neuropathic pain behaviors. These data suggest that amygdala KOR neurons modulate pain behaviors by exerting an inhibitory tone on downstream CeA neurons. Therefore, activation of these interneurons or blockade of inhibitory KOR signaling in these neurons could restore control of amygdala output and mitigate pain.

Impaired amygdala astrocytic signaling worsens neuropathic pain-associated neuronal functions and behaviors.

Introduction: Pain is a clinically relevant health care issue with limited therapeutic options, creating the need for new and improved analgesic strategies. The amygdala is a limbic brain region critically involved in the regulation of emotional-affective components of pain and in pain modulation. The central nucleus of amygdala (CeA) serves major output functions and receives nociceptive information via the external lateral parabrachial nucleus (PB). While amygdala neuroplasticity has been linked causally to pain behaviors, non-neuronal pain mechanisms in this region remain to be explored. As an essential part of the neuroimmune system, astrocytes that represent about 40-50% of glia cells within the central nervous system, are required for physiological neuronal functions, but their role in the amygdala remains to be determined for pain conditions. In this study, we measured time-specific astrocyte activation in the CeA in a neuropathic pain model (spinal nerve ligation, SNL) and assessed the effects of astrocyte inhibition on amygdala neuroplasticity and pain-like behaviors in the pain condition. Methods and Results: Glial fibrillary acidic protein (GFAP, astrocytic marker) immunoreactivity and mRNA expression were increased at the chronic (4 weeks post-SNL), but not acute (1 week post-SNL), stage of neuropathic pain. In order to determine the contribution of astrocytes to amygdala pain-mechanisms, we used fluorocitric acid (FCA), a selective inhibitor of astrocyte metabolism. Whole-cell patch-clamp recordings were performed from neurons in the laterocapsular division of the CeA (CeLC) obtained from chronic neuropathic rats. Pre-incubation of brain slices with FCA (100 µM, 1 h), increased excitability through altered hyperpolarization-activated current (Ih) functions, without significantly affecting synaptic responses at the PB-CeLC synapse. Intra-CeA injection of FCA (100 µM) had facilitatory effects on mechanical withdrawal thresholds (von Frey and paw pressure tests) and emotional-affective behaviors (evoked vocalizations), but not on facial grimace score and anxiety-like behaviors (open field test), in chronic neuropathic rats. Selective inhibition of astrocytes by FCA was confirmed with immunohistochemical analyses showing decreased astrocytic GFAP, but not NeuN, signal in the CeA. Discussion: Overall, these results suggest a complex modulation of amygdala pain functions by astrocytes and provide evidence for beneficial functions of astrocytes in CeA in chronic neuropathic pain.

Mechanisms of pain in aging and age-related conditions: Focus on caregivers.

Pain is a complex, subjective experience that can significantly impact quality of life, particularly in aging individuals, by adversely affecting physical and emotional well-being. Whereas acute pain usually serves a protective function, chronic pain is a persistent pathological condition that contributes to functional deficits, cognitive decline, and emotional disturbances in the elderly. Despite substantial progress that has been made in characterizing age-related changes in pain, complete mechanistic details of pain processing mechanisms in the aging patient remain unknown. Pain is particularly under-recognized and under-managed in the elderly, especially among patients with Alzheimer's disease (AD), Alzheimer's disease-related dementias (ADRD), and other age-related conditions. Furthermore, difficulties in assessing pain in patients with AD/ADRD and other age-related conditions may contribute to the familial caregiver burden. The purpose of this article is to discuss the mechanisms and risk factors for chronic pain development and persistence, with a particular focus on age-related changes. Our article also highlights the importance of caregivers working with aging chronic pain patients, and emphasizes the urgent need for increased legislative awareness and improved pain management in these populations to substantially alleviate caregiver burden.

Hmgb1 Silencing in the Amygdala Inhibits Pain-Related Behaviors in a Rat Model of Neuropathic Pain.

Chronic pain presents a therapeutic challenge due to the highly complex interplay of sensory, emotional-affective and cognitive factors. The mechanisms of the transition from acute to chronic pain are not well understood. We hypothesized that neuroimmune mechanisms in the amygdala, a brain region involved in the emotional-affective component of pain and pain modulation, play an important role through high motility group box 1 (Hmgb1), a pro-inflammatory molecule that has been linked to neuroimmune signaling in spinal nociception. Transcriptomic analysis revealed an upregulation of Hmgb1 mRNA in the right but not left central nucleus of the amygdala (CeA) at the chronic stage of a spinal nerve ligation (SNL) rat model of neuropathic pain. Hmgb1 silencing with a stereotaxic injection of siRNA for Hmgb1 into the right CeA of adult male and female rats 1 week after (post-treatment), but not 2 weeks before (pre-treatment) SNL induction decreased mechanical hypersensitivity and emotional-affective responses, but not anxiety-like behaviors, measured 4 weeks after SNL. Immunohistochemical data suggest that neurons are a major source of Hmgb1 in the CeA. Therefore, Hmgb1 in the amygdala may contribute to the transition from acute to chronic neuropathic pain, and the inhibition of Hmgb1 at a subacute time point can mitigate neuropathic pain.

Pain-related cortico-limbic plasticity and opioid signaling.

Neuroplasticity in cortico-limbic circuits has been implicated in pain persistence and pain modulation in clinical and preclinical studies. The amygdala has emerged as a key player in the emotional-affective dimension of pain and pain modulation. Reciprocal interactions with medial prefrontal cortical regions undergo changes in pain conditions. Other limbic and paralimbic regions have been implicated in pain modulation as well. The cortico-limbic system is rich in opioids and opioid receptors. Preclinical evidence for their pain modulatory effects in different regions of this highly interactive system, potentially opposing functions of different opioid receptors, and knowledge gaps will be described here. There is little information about cell type- and circuit-specific functions of opioid receptor subtypes related to pain processing and pain-related plasticity in the cortico-limbic system. The important role of anterior cingulate cortex (ACC) and amygdala in MOR-dependent analgesia is most well-established, and MOR actions in the mesolimbic system appear to be similar but remain to be determined in mPFC regions other than ACC. Evidence also suggests that KOR signaling generally serves opposing functions whereas DOR signaling in the ACC has similar, if not synergistic effects, to MOR. A unifying picture of pain-related neuronal mechanisms of opioid signaling in different elements of the cortico-limbic circuitry has yet to emerge. This article is part of the Special Issue on "Opioid-induced changes in addiction and pain circuits".

Sex Differences in Comorbidities of Pediatric Craniosynostosis at Presentation.

INTRODUCTION: Craniosynostosis is a common pediatric presentation in which the premature fusion of one or more cranial sutures results in a misshapen skull. This birth defect is often associated with comorbidities due to structural impacts on nearby anatomical features. While there is some evidence for a male predominance among craniosynostosis patients, little has been investigated regarding sex differences in comorbidities of this condition. This study seeks to explore potential sexual dimorphisms in craniosynostosis patients at the time of presentation. METHODS: We conducted a retrospective, cross-sectional review of male and female non-syndromic craniosynostosis (NSC) patients between the ages of 1 month and 9 years that were evaluated at a 500-bed academic hospital or a 977-bed private hospital in Lubbock, TX, USA. Common comorbidities including ophthalmologic diagnoses, developmental delays, obstructive sleep apnea, chronic otitis media, hearing loss, chronic headaches, and seizure disorders were evaluated. The NSC cohort was compared to a similarly aged trauma group that represented the normal population. RESULTS: 175 NSC patients fit the inclusion criteria, of which 109 (62%) were male. A diagnosis of craniosynostosis was significantly associated with ophthalmological diagnoses (p < 0.0001), chronic otitis media (p < 0.0001), developmental delays (p < 0.0001), and hearing loss (p = 0.0047). Male NSC patients were less likely to present with ophthalmological diagnoses (p = 0.0010) or hearing loss (p = 0.0052) than females. CONCLUSIONS: Our findings expand on current literature evaluating possible comorbidities of NSC, particularly supporting the association with ophthalmological diagnoses, chronic otitis media, developmental delays, and hearing loss. We also report sex differences in ophthalmological diagnoses and hearing loss for NSC patients. These findings can serve to educate physicians of symptoms requiring prompt recognition and management in these patients.

Recent Advances in the Modulation of Pain by the Metabotropic Glutamate Receptors.

Metabotropic glutamate receptors (mGluR or mGlu) are G-protein coupled receptors activated by the binding of glutamate, the main classical neurotransmitter of the nervous system. Eight different mGluR subtypes (mGluR1-8) have been cloned and are classified in three groups based on their molecular, pharmacological and signaling properties. mGluRs mediate several physiological functions such as neuronal excitability and synaptic plasticity, but they have also been implicated in numerous pathological conditions including pain. The availability of new and more selective allosteric modulators together with the canonical orthosteric ligands and transgenic technologies has led to significant advances in our knowledge about the role of the specific mGluR subtypes in the pathophysiological mechanisms of various diseases. Although development of successful compounds acting on mGluRs for clinical use has been scarce, the subtype-specific-pharmacological manipulation might be a compelling approach for the treatment of several disorders in humans, including pain; this review aims to summarize and update on preclinical evidence for the roles of different mGluRs in the pain system and discusses knowledge gaps regarding mGluR-related sex differences and neuroimmune signaling in pain.

Kappa Opioid Receptor Blockade in the Amygdala Mitigates Pain Like-Behaviors by Inhibiting Corticotropin Releasing Factor Neurons in a Rat Model of Functional Pain.

Functional pain syndromes (FPS) occur in the absence of identifiable tissue injury or noxious events and include conditions such as migraine, fibromyalgia, and others. Stressors are very common triggers of pain attacks in various FPS conditions. It has been recently demonstrated that kappa opioid receptors (KOR) in the central nucleus of amygdala (CeA) contribute to FPS conditions, but underlying mechanisms remain unclear. The CeA is rich in KOR and encompasses major output pathways involving extra-amygdalar projections of corticotropin releasing factor (CRF) expressing neurons. Here we tested the hypothesis that KOR blockade in the CeA in a rat model of FPS reduces pain-like and nocifensive behaviors by restoring inhibition of CeA-CRF neurons. Intra-CeA administration of a KOR antagonist (nor-BNI) decreased mechanical hypersensitivity and affective and anxiety-like behaviors in a stress-induced FPS model. In systems electrophysiology experiments in anesthetized rats, intra-CeA application of nor-BNI reduced spontaneous firing and responsiveness of CeA neurons to peripheral stimulation. In brain slice whole-cell patch-clamp recordings, nor-BNI increased feedforward inhibitory transmission evoked by optogenetic and electrical stimulation of parabrachial afferents, but had no effect on monosynaptic excitatory transmission. Nor-BNI decreased frequency, but not amplitude, of spontaneous inhibitory synaptic currents, suggesting a presynaptic action. Blocking KOR receptors in stress-induced FPS conditions may therefore represent a novel therapeutic strategy.

Sex Differences in CGRP Regulation and Function in the Amygdala in a Rat Model of Neuropathic Pain.

The amygdala has emerged as a key player in the emotional response to pain and pain modulation. The lateral and capsular regions of the central nucleus of the amygdala (CeA) represent the "nociceptive amygdala" due to their high content of neurons that process pain-related information. These CeA divisions are the targets of the spino-parabrachio-amygdaloid pain pathway, which is the predominant source of calcitonin gene-related peptide (CGRP) within the amygdala. Changes in lateral and capsular CeA neurons have previously been observed in pain models, and synaptic plasticity in these areas has been linked to pain-related behavior. CGRP has been demonstrated to play an important role in peripheral and spinal mechanisms, and in pain-related amygdala plasticity in male rats in an acute arthritis pain model. However, the role of CGRP in chronic neuropathic pain-related amygdala function and behaviors remains to be determined for both male and female rats. Here we tested the hypothesis that the CGRP1 receptor is involved in neuropathic pain-related amygdala activity, and that blockade of this receptor can inhibit neuropathic pain behaviors in both sexes. CGRP mRNA expression levels in the CeA of male rats were upregulated at the acute stage of the spinal nerve ligation (SNL) model of neuropathic pain, whereas female rats had significantly higher CGRP and CGRP receptor component expression at the chronic stage. A CGRP1 receptor antagonist (CGRP 8-37) administered into the CeA in chronic neuropathic rats reduced mechanical hypersensitivity (von Frey and paw compression tests) in both sexes but showed female-predominant effects on emotional-affective responses (ultrasonic vocalizations) and anxiety-like behaviors (open field test). CGRP 8-37 inhibited the activity of CeA output neurons assessed with calcium imaging in brain slices from chronic neuropathic pain rats. Together, these findings may suggest that CGRP1 receptors in the CeA are involved in neuropathic pain-related amygdala activity and contribute to sensory aspects in both sexes but to emotional-affective pain responses predominantly in females. The sexually dimorphic function of CGRP in the amygdala would make CGRP1 receptors a potential therapeutic target for neuropathic pain relief, particularly in females in chronic pain conditions.

Sex differences in pain along the neuraxis.

Despite the overwhelming female-predominance in chronic pain disorders, preclinical pain studies have historically excluded females as research subjects. Male-biased explanations of pathological pain mechanisms may not fully translate to pain processes in females, necessitating the exploration of pain processing and modulation in both sexes at the preclinical and clinical levels. This review highlights historical trends in the study of sex differences within the pain field and examines the current literature regarding new techniques for the mechanistic analysis of pain modulation in males and females. A large body of evidence suggests that sex differences exist at the molecular, cellular, and systems levels of pain processing, likely influenced by a combination of genetic, hormonal, and neuroimmune factors that may differ at distinct levels of the neuraxis.

Case report: Medical student types journals during ketamine infusions for suicidal ideation, treatment-resistant depression, post-traumatic stress disorder, and generalized anxiety disorder.

Suicide is the most common cause of death in male resident physicians and the second most common cause of death in resident physicians overall. Physicians also experience high rates of major depressive disorder (MDD), post-traumatic stress disorder (PTSD), and burnout. These conditions frequently develop during medical school, and threaten not only physicians but the patients they care for. A 30-year-old medical student presented to our clinic with a history of treatment-resistant depression (TRD), generalized anxiety disorder (GAD), PTSD, and 5 years of daily suicidal ideation. Previous treatments included therapy, lifestyle modifications, and various combinations of six antidepressants. These interventions had little effect on the patient's mental health. The patient was treated at our clinic with an 8-month regimen of IV ketamine infusions and ketamine-assisted psychotherapy (KAP). The patient achieved remission from suicidality and PTSD within 1 month; and TRD and GAD within 7 months. The patient's Patient Health Questionnaire (PHQ-9) score decreased from 25 (severe depression) to 1 (not depressed). These findings suggest that ketamine and KAP may represent effective interventions for mental health applications in healthcare professionals. The patient made the unique decision to attempt to type narrative journals during four of his ketamine infusions (doses ranged from 1.8 to 2.1 mg/kg/h IV). The patient successfully typed detailed journals throughout each 1-h ketamine infusion. To our knowledge, these journals represent the first independently typed, first-person, real-time narratives of ketamine-induced non ordinary states of consciousness. The transcripts of these journals may provide useful insights for clinicians, particularly in the context of KAP.

Fear Extinction-Based Inter-Individual and Sex Differences in Pain-Related Vocalizations and Anxiety-like Behaviors but Not Nocifensive Reflexes.

Inter-individual and sex differences in pain responses are recognized but their mechanisms are not well understood. This study was intended to provide the behavioral framework for analyses of pain mechanisms using fear extinction learning as a predictor of phenotypic and sex differences in sensory (mechanical withdrawal thresholds) and emotional-affective aspects (open field tests for anxiety-like behaviors and audible and ultrasonic components of vocalizations) of acute and chronic pain. In acute arthritis and chronic neuropathic pain models, greater increases in vocalizations were found in females than males and in females with poor fear extinction abilities than females with strong fear extinction, particularly in the neuropathic pain model. Female rats showed higher anxiety-like behavior than males under baseline conditions but no inter-individual or sex differences were seen in the pain models. No inter-individual and sex differences in mechanosensitivity were observed. The data suggest that vocalizations are uniquely suited to detect inter-individual and sex differences in pain models, particularly in chronic neuropathic pain, whereas no such differences were found for mechanosensitivity, and baseline differences in anxiety-like behaviors disappeared in the pain models.

Association between Foramen Size and Febrile Seizure Status in the Pediatric Population: A Two-Center Retrospective Analysis.

Objective Febrile seizures have been shown to occur in 2 to 5% of children between the ages of 6 months and 5 years, making them the most common seizures of childhood. Multiple risk factors for febrile seizures have been identified; however, no investigation has been conducted to explore foramen size and associated venous drainage as a potential risk factor for experiencing febrile seizures. Of particular interest are the parietal foramen and the condylar canal, which conduct the parietal emissary vein and the occipital emissary vein, respectively. Emissary veins lack valves, allowing them to play a crucial role in selective brain cooling via a bidirectional flow of blood from the head's evaporating surface. Narrowed cranial apertures conducting these veins may lead to reduced cerebral venous outflow and delayed brain cooling, creating favorable conditions for a febrile event. This study seeks to explore the association between cranial aperture area and febrile seizure status. Methods A retrospective cross-sectional medical record review study from January 2011 to December 2017 was conducted at a 500-bed academic hospital and a 977-bed private hospital in Lubbock, Texas, United States. A total of 101 complex febrile seizure patients were compared with a similarly aged group of 75 trauma patients representing the normal population. Parietal foramen area and condylar canal area were electronically measured and defined as having "normal" or "below normal" area. Statistical Analysis Independent t -tests were used to compare foramen and canal areas by febrile seizure status. Logistic regression analyses were conducted to determine the association of small cranial aperture area with febrile seizure status. Results Below normal parietal foramen area had a strong association with febrile seizures in our patient population. Male sex, white race, and complete vaccination status were also found to have significant associations with febrile seizure status. Conclusion Our findings indicated that narrowed parietal foramen may be considered as a risk factor for febrile seizure development.

Child Abuse and Deformational Plagiocephaly in a West Texas Hospital System.

Background The aim of this study was to assess deformational plagiocephaly's (DP) predictive value in neglect and physical abuse (nonaccidental trauma [NAT]) within the pediatric population. In addition, we sought to characterize the prevalence of DP and NAT for our hospital's mostly rural catchment area. Methods Data on hospitalized patients diagnosed with NAT and/or neglect between 2012 and 2018 were collected via retrospective chart review. All enrolled children were younger than the age of 4 years at the time of diagnosis, and those without legible head computed tomographies or magnetic resonance images during their initial hospitalization were excluded. Utilizing neuroimaging, we calculated the cranial vault asymmetry index (CVAI) and cranial index for each patient to assess for DP. Differences between the two groups were assessed using Wilcoxon's rank-sum test for continuous variables and Fisher's exact test for categorical variables. A p -value of 0.05 or less was considered statistically significant. All analyses were conducted using SAS 9.4 (Cary, North Carolina, United States). Results The prevalence of DP within the combined cohort of NAT and neglect patients is 21%, similar to that reported in the literature for the general population (20-50%). There was no significance between the prevalence of DP and a history of NAT ( p > 0.1) or neglect ( p > 0.1). Furthermore, there was no correlation between CVAI and characteristics of initial presentation or history of trauma for either NAT ( p -values: 0.359 and 0.250, respectively) or neglect groups ( p -values: 0.116 and 0.770, respectively). Conclusion While there are many limitations to this study, our results suggest that abused children are no more likely to have history of DP than the general population, and the degree of DP is not associated with severity of trauma history or initial presentation. We hope the results of this study promote future investigations for unique and subtle predictive factors of child abuse/neglect.

Fear extinction learning ability predicts neuropathic pain behaviors and amygdala activity in male rats.

Background The amygdala plays a key role in fear learning and extinction and has emerged as an important node of emotional-affective aspects of pain and pain modulation. Impaired fear extinction learning, which involves prefrontal cortical control of amygdala processing, has been linked to neuropsychiatric disorders. Here, we tested the hypothesis that fear extinction learning ability can predict the magnitude of neuropathic pain. Results We correlated fear extinction learning in naive adult male rats with sensory and affective behavioral outcome measures (mechanical thresholds, vocalizations, and anxiety- and depression-like behaviors) before and after the induction of the spinal nerve ligation model of neuropathic pain compared to sham controls. Auditory fear conditioning, extinction learning, and extinction retention tests were conducted after baseline testing. All rats showed increased freezing responses after fear conditioning. During extinction training, the majority (75%) of rats showed a decline in freezing level to 50% in 5 min (fear extinction+), whereas 25% of the rats maintained a high freezing level (>50%, fear extinction-). Fear extinction- rats showed decreased open-arm preference in the elevated plus maze, reflecting anxiety-like behavior, but there were no significant differences in sensory thresholds, vocalizations, or depression-like behavior (forced swim test) between fear extinction+ and fear extinction- types. In the neuropathic pain model (four weeks after spinal nerve ligation), fear extinction- rats showed a greater increase in vocalizations and anxiety-like behavior than fear extinction+ rats. Fear extinction- rats, but not fear extinction+ rats, also developed depression-like behavior. Extracellular single unit recordings of amygdala (central nucleus) neurons in behaviorally tested rats (anesthetized with isoflurane) found greater increases in background activity, bursting, and evoked activity in fear extinction- rats than fear extinction+ rats in the spinal nerve ligation model compared to sham controls. Conclusion The data may suggest that fear extinction learning ability predicts the magnitude of neuropathic pain-related affective rather than sensory behaviors, which correlates with differences in amygdala activity changes.