Migraine and peripheral pain models show differential alterations in neuronal complexity.

OBJECTIVE: Our laboratory has recently shown that there is a decrease in neuronal complexity in head pain processing regions in mouse models of chronic migraine-associated pain and aura. Importantly, restoration of this neuronal complexity corresponds with anti-migraine effects of known and experimental pharmacotherapies. The objective of the current study was to expand this work and examine other brain regions involved with pain or emotional processing. We also investigated the generalizability of our findings by analyzing neuronal cytoarchitectural changes in a model of complex regional pain syndrome (CRPS), a peripheral pain disorder. METHODS: We used the nitroglycerin (NTG) model of chronic migraine-associated pain in which mice receive 10 mg/kg NTG every other day for 9 days. Cortical spreading depression (CSD), a physiological corelate of migraine aura, was evoked in anesthetized mice using KCl. CRPS was induced by tibial fracture followed by casting. Neuronal cytoarchitecture was visualized with Golgi stain and analyzed with Simple Neurite Tracer. RESULTS: In the NTG model, we previously showed decreased neuronal complexity in the trigeminal nucleus caudalis (TNC) and periaqueductal gray (PAG). In contrast, we found increased neuronal complexity in the thalamus and no change in the amygdala or caudate putamen in this study. Following CSD, we observed decreased neuronal complexity in the PAG, in line with decreases in the somatosensory cortex and TNC reported with this model previously. In the CRPS model there was decreased neuronal complexity in the hippocampus, as reported by others; increased complexity in the PAG; and no change within the somatosensory cortex. CONCLUSIONS: Collectively these results demonstrate that alterations in neuronal complexity are a feature of both chronic migraine and chronic CRPS. However, each type of pain presents a unique cytoarchitectural signature, which may provide insight on how these pain states differentially transition from acute to chronic conditions.

A non-convulsant delta-opioid receptor agonist, KNT-127, reduces cortical spreading depression and nitroglycerin-induced allodynia.

OBJECTIVE: The aim of this study was to determine if the non-convulsant delta-opioid receptor (DOR) agonist, KNT-127, could inhibit migraine-associated endpoints. BACKGROUND: The DOR has been identified as a therapeutic target for migraine. However, the development of delta agonists is limited as some ligands have seizurogenic properties, which may be related to their ability to induce receptor internalization. While both pro- and non-convulsant delta agonists can reduce migraine-associated allodynia, only the proconvulsant agonist, SNC80, has been shown to decrease cortical spreading depression (CSD). It is unclear if the ability of delta agonists to modulate cortical activity is related to the same signaling mechanisms that produce proconvulsant effects. METHODS: The effects of the non-convulsant delta agonist, KNT-127, were examined. Repetitive CSD was induced in female C57BL6/J (n = 6/group) mice by continuous application of KCl and the effect of KNT-127/vehicle (Veh) on both local field potentials and optical intrinsic signals was determined. To assess the effect of KNT-127 on established chronic migraine-associated pain, male and female C57BL6/J mice were treated with nitroglycerin (NTG; 10 mg/kg, ip) every other day for 9 days and tested with KNT-127 (5 mg/kg, sc) or Veh on day 10 (n = 6/group). DOR-enhanced green fluorescent protein mice (n = 4/group) were used to confirm the internalization properties of KNT-127 in the trigeminal ganglia, trigeminal nucleus caudalis, and somatosensory cortex. RESULTS: KNT-127 inhibited CSD events (t(10)  = 3.570, p = 0.0051). In addition, this delta agonist also reversed established cephalic allodynia in the NTG model of chronic migraine (F(1, 20)  = 12.80, p < 0.01). Furthermore, KNT-127 caused limited internalization of DOR in key migraine processing regions. CONCLUSIONS: This study shows that the antimigraine effects of DOR agonists can be separated from their proconvulsant effects. This data provides valuable information for the continued development of delta agonists for the treatment of migraine.

Emerging Treatment Targets for Migraine and Other Headaches.

Migraine is a complex disorder that is characterized by an assortment of neurological and systemic effects. While headache is the most prominent feature of migraine, a host of symptoms affecting many physiological functions are also observed before, during, and after an attack. Furthermore, migraineurs are heterogeneous and have a wide range of responses to migraine therapies. The recent approval of calcitonin gene-related-peptide based therapies has opened up the treatment of migraine and generated a renewed interest in migraine research and discovery. Ongoing advances in migraine research have identified a number of other promising therapeutic targets for this disorder. In this review, we highlight emergent treatments within the following biological systems: pituitary adenylate cyclase activating peptdie, 2 non-mu opioid receptors that have low abuse liability - the delta and kappa opioid receptors, orexin, and nitric oxide-based therapies. Multiple mechanisms have been identified in the induction and maintenance of migraine symptoms; and this divergent set of targets have highly distinct biological effects. Increasing the mechanistic diversity of the migraine tool box will lead to more treatment options and better patient care.

Soluble guanylyl cyclase is a critical regulator of migraine-associated pain.

Background Nitric oxide (NO) has been heavily implicated in migraine. Nitroglycerin is a prototypic NO-donor, and triggers migraine in humans. However, nitroglycerin also induces oxidative/nitrosative stress and is a source of peroxynitrite - factors previously linked with migraine etiology. Soluble guanylyl cyclase (sGC) is the high affinity NO receptor in the body, and the aim of this study was to identify the precise role of sGC in acute and chronic migraine. Methods We developed a novel brain-bioavailable sGC stimulator (VL-102), and tested its hyperalgesic properties in mice. We also determined the effect of VL-102 on c-fos and calcitonin gene related peptide (CGRP) immunoreactivity within the trigeminovascular complex. In addition, we also tested the known sGC inhibitor, ODQ, within the chronic nitroglycerin migraine model. Results VL-102-evoked acute and chronic mechanical cephalic and hind-paw allodynia in a dose-dependent manner, which was blocked by the migraine medications sumatriptan, propranolol, and topiramate. In addition, VL-102 also increased c-fos and CGRP expressing cells within the trigeminovascular complex. Importantly, ODQ completely inhibited acute and chronic hyperalgesia induced by nitroglycerin. ODQ also blocked hyperalgesia already established by chronic nitroglycerin, implicating this pathway in migraine chronicity. Conclusions These results indicate that nitroglycerin causes migraine-related pain through stimulation of the sGC pathway, and that super-activation of this receptor may be an important component for the maintenance of chronic migraine. This work opens the possibility for negative sGC modulators as novel migraine therapies.

Bradykinin receptor expression and bradykinin-mediated sensitization of human sensory neurons.

ABSTRACT: Bradykinin is a peptide implicated in inflammatory pain in both humans and rodents. In rodent sensory neurons, activation of B1 and B2 bradykinin receptors induces neuronal hyperexcitability. Recent evidence suggests that human and rodent dorsal root ganglia (DRG), which contain the cell bodies of sensory neurons, differ in the expression and function of key GPCRs and ion channels; whether bradykinin receptor expression and function are conserved across species has not been studied in depth. In this study, we used human DRG tissue from organ donors to provide a detailed characterization of bradykinin receptor expression and bradykinin-induced changes in the excitability of human sensory neurons. We found that B2 and, to a lesser extent, B1 receptors are expressed by human DRG neurons and satellite glial cells. B2 receptors were enriched in the nociceptor subpopulation. Using patch-clamp electrophysiology, we found that acute bradykinin increases the excitability of human sensory neurons, whereas prolonged exposure to bradykinin decreases neuronal excitability in a subpopulation of human DRG neurons. Finally, our analyses suggest that donor's history of chronic pain and age may be predictors of higher B1 receptor expression in human DRG neurons. Together, these results indicate that acute bradykinin-induced hyperexcitability, first identified in rodents, is conserved in humans and provide further evidence supporting bradykinin signaling as a potential therapeutic target for treating pain in humans.

Harmonized cross-species cell atlases of trigeminal and dorsal root ganglia.

Sensory neurons in the dorsal root ganglion (DRG) and trigeminal ganglion (TG) are specialized to detect and transduce diverse environmental stimuli to the central nervous system. Single-cell RNA sequencing has provided insights into the diversity of sensory ganglia cell types in rodents, nonhuman primates, and humans, but it remains difficult to compare cell types across studies and species. We thus constructed harmonized atlases of the DRG and TG that describe and facilitate comparison of 18 neuronal and 11 non-neuronal cell types across six species and 31 datasets. We then performed single-cell/nucleus RNA sequencing of DRG from both human and the highly regenerative axolotl and found that the harmonized atlas also improves cell type annotation, particularly of sparse neuronal subtypes. We observed that the transcriptomes of sensory neuron subtypes are broadly similar across vertebrates, but the expression of functionally important neuropeptides and channels can vary notably. The resources presented here can guide future studies in comparative transcriptomics, simplify cell-type nomenclature differences across studies, and help prioritize targets for future analgesic development.

PACAP-PAC1 receptor inhibition is effective in opioid induced hyperalgesia and medication overuse headache models.

Opioids prescribed for pain and migraine can produce opioid-induced hyperalgesia (OIH) or medication overuse headache (MOH). We previously demonstrated that pituitary adenylate cyclase activating polypeptide (PACAP) is upregulated in OIH and chronic migraine models. Here we determined if PACAP acts as a bridge between opioids and pain chronification. We tested PACAP-PAC1 receptor inhibition in novel models of opioid-exacerbated trigeminovascular pain. The PAC1 antagonist, M65, reversed chronic allodynia in a model which combines morphine with the migraine trigger, nitroglycerin. Chronic opioids also exacerbated cortical spreading depression, a correlate of migraine aura; and M65 inhibited this augmentation. In situ hybridization showed MOR and PACAP co-expression in trigeminal ganglia, and near complete overlap between MOR and PAC1 in the trigeminal nucleus caudalis and periaqueductal gray. PACAPergic mechanisms appear to facilitate the transition to chronic headache following opioid use, and strategies targeting this system may be particularly beneficial for OIH and MOH.

Bradykinin receptor expression and bradykinin-mediated sensitization of human sensory neurons.

Bradykinin is a peptide implicated in inflammatory pain in both humans and rodents. In rodent sensory neurons, activation of B1 and B2 bradykinin receptors induces neuronal hyperexcitability. Recent evidence suggests that human and rodent dorsal root ganglia (DRG), which contain the cell bodies of sensory neurons, differ in the expression and function of key GPCRs and ion channels; whether BK receptor expression and function are conserved across species has not been studied in depth. In this study, we used human DRG tissue from organ donors to provide a detailed characterization of bradykinin receptor expression and bradykinin-induced changes in the excitability of human sensory neurons. We found that B2 and, to a lesser extent, B1 receptors are expressed by human DRG neurons and satellite glial cells. B2 receptors were enriched in the nociceptor subpopulation. Using patch-clamp electrophysiology, we found that acute bradykinin increases the excitability of human sensory neurons, while prolonged exposure to bradykinin decreases neuronal excitability in a subpopulation of human DRG neurons. Finally, our analyses suggest that donor’s history of chronic pain and age may be predictors of higher B1 receptor expression in human DRG neurons. Together, these results indicate that acute BK-induced hyperexcitability, first identified in rodents, is conserved in humans and provide further evidence supporting BK signaling as a potential therapeutic target for treating pain in humans.

Harmonized cross-species cell atlases of trigeminal and dorsal root ganglia.

Peripheral sensory neurons in the dorsal root ganglion (DRG) and trigeminal ganglion (TG) are specialized to detect and transduce diverse environmental stimuli including touch, temperature, and pain to the central nervous system. Recent advances in single-cell RNA-sequencing (scRNA-seq) have provided new insights into the diversity of sensory ganglia cell types in rodents, non-human primates, and humans, but it remains difficult to compare transcriptomically defined cell types across studies and species. Here, we built cross-species harmonized atlases of DRG and TG cell types that describe 18 neuronal and 11 non-neuronal cell types across 6 species and 19 studies. We then demonstrate the utility of this harmonized reference atlas by using it to annotate newly profiled DRG nuclei/cells from both human and the highly regenerative axolotl. We observe that the transcriptomic profiles of sensory neuron subtypes are broadly similar across vertebrates, but the expression of functionally important neuropeptides and channels can vary notably. The new resources and data presented here can guide future studies in comparative transcriptomics, simplify cell type nomenclature differences across studies, and help prioritize targets for future pain therapy development.

Delta opioid receptors in Nav1.8 expressing peripheral neurons partially regulate the effect of delta agonist in models of migraine and opioid-induced hyperalgesia.

Migraine is one of the most common pain disorders and causes disability in millions of people every year. Delta opioid receptors (DOR) have been identified as a novel therapeutic target for migraine and other headache disorders. DORs are present in both peripheral and central regions and it is unclear which receptor populations regulate migraine-associated effects. The aim of this study was to determine if DOR expressed in peripheral nociceptors regulates headache associated endpoints and the effect of delta agonists within these mouse models. We used a conditional knockout, in which DOR was selectively deleted from Nav1.8 expressing cells. Nav1.8-DOR mice and loxP control littermates were tested in models of chronic migraine-associated allodynia, opioid-induced hyperalgesia, migraine-associated negative affect, and aura. Nav1.8-DOR and loxP mice had comparable effect sizes in all of these models. The anti-allodynic effect of the DOR agonist, SNC80, was slightly diminished in the nitroglycerin model of migraine. Intriguingly, in the OIH model the peripheral effects of SNC80 were completely lost in Nav1.8-DOR mice while the cephalic effects remained intact. Regardless of genotype, SNC80 continued to inhibit conditioned place aversion associated with nitroglycerin and decreased cortical spreading depression events associated with migraine aura. These results suggest that DOR in Nav1.8-expressing nociceptors do not critically regulate the anti-migraine effects of delta agonist; and that brain-penetrant delta agonists would be a more effective drug development strategy.

Neuronal complexity is attenuated in preclinical models of migraine and restored by HDAC6 inhibition.

Migraine is the sixth most prevalent disease worldwide but the mechanisms that underlie migraine chronicity are poorly understood. Cytoskeletal flexibility is fundamental to neuronal-plasticity and is dependent on dynamic microtubules. Histone-deacetylase-6 (HDAC6) decreases microtubule dynamics by deacetylating its primary substrate, α-tubulin. We use validated mouse models of migraine to show that HDAC6-inhibition is a promising migraine treatment and reveal an undiscovered cytoarchitectural basis for migraine chronicity. The human migraine trigger, nitroglycerin, produced chronic migraine-associated pain and decreased neurite growth in headache-processing regions, which were reversed by HDAC6 inhibition. Cortical spreading depression (CSD), a physiological correlate of migraine aura, also decreased cortical neurite growth, while HDAC6-inhibitor restored neuronal complexity and decreased CSD. Importantly, a calcitonin gene-related peptide receptor antagonist also restored blunted neuronal complexity induced by nitroglycerin. Our results demonstrate that disruptions in neuronal cytoarchitecture are a feature of chronic migraine, and effective migraine therapies might include agents that restore microtubule/neuronal plasticity.

Migraines are a common brain disorder that affects 14% of the world's population. For many people the main symptom of a migraine is a painful headache, often on one side of the head. Other symptoms include increased sensitivity to light or sound, disturbed vision, and feeling sick. These sensory disturbances are called aura and they often occur before the headache begins. One particularly debilitating subset of migraines are chronic migraines, in which patients experience more than 15 headache days per month. Migraine therapies are often only partially effective or poorly tolerated, making it important to develop new drugs for this condition, but unfortunately, little is known about the molecular causes of migraines. To bridge this gap, Bertels et al. used two different approaches to cause migraine-like symptoms in mice. One approach consisted on giving mice nitroglycerin, which dilates blood vessels, produces hypersensitivity to touch, and causes photophobia in both humans and mice. In the second approach, mice underwent surgery and potassium chloride was applied onto the dura, a thick membrane that surrounds the brain. This produces cortical spreading depression, an event that is linked to migraine auras and involves a wave of electric changes in brain cells that slowly propagates across the brain, silencing brain electrical activity for several minutes. Using these approaches, Bertels et al. studied whether causing chronic migraine-like symptoms in mice is associated with changes in the structures of neurons, focusing on the effects of migraines on microtubules. Microtubules are cylindrical protein structures formed by the assembly of smaller protein units. In most cells, microtubules assemble and disassemble depending on what the cell needs. Neurons need stable microtubules to establish connections with other neurons. The experiments showed that provoking chronic migraines in mice led to a reduction in the numbers of connections between different neurons. Additionally, Bertels et al. found that inhibiting HDAC6 (a protein that destabilizes microtubules) reverses the structural changes in neurons caused by migraines and decreases migraine symptoms. The same effects are seen when a known migraine treatment strategy, known as CGRP receptor blockade, is applied. These results suggest that chronic migraines may involve decreased neural complexity, and that the restoration of this complexity by HDAC6 inhibitors could be a potential therapeutic strategy for migraine.

Smaller amygdala volume and increased neuroticism predict anxiety symptoms in healthy subjects: A volumetric approach using manual tracing.

Volume reductions in the amygdala (AMY) have been found in patients with anxiety disorders, but findings are mixed in subclinical participants with high trait anxiety scores, in whom both reductions and increases in AMY volume have been identified. One potential reason for such discrepancies could be the employment of different methods to determine the AMY volume (i.e., manual tracing in psychiatric research vs. automated methods), in non-patient research. In addition to trait anxiety, smaller AMY volume has also been linked to neuroticism, a personality trait consistently linked to increased vulnerability to anxiety. However, it is not clear how AMY volume and neuroticism together may contribute to anxiety symptoms in healthy functioning. These issues were investigated in a sample of 46 healthy participants who underwent anatomical MRI scanning and completed questionnaires measuring trait anxiety and neuroticism. AMY volume was assessed using manual tracing, based on anatomical landmarks identified in each participant's anatomical image. First, smaller left AMY volume was linked to higher levels of neuroticism (p = .013) and trait anxiety (p = .024), which in turn were positively correlated with each other. Moreover, AMY volume had a significant indirect effect on trait anxiety through neuroticism (ab = - .009, 95% CI [- .019, - .002]). This effect was not bidirectional, as trait anxiety did not predict AMY volume through neuroticism. Collectively, these findings provide support for a brain-personality-symptom framework of understanding affective dysregulation, which may help inform the development of prevention and intervention paradigms targeting preservation of AMY volume and reduction of neuroticism, to protect against anxiety symptoms.

Forebrain delta opioid receptors regulate the response of delta agonist in models of migraine and opioid-induced hyperalgesia.

Delta opioid receptor (DOR) agonists have been identified as a promising novel therapy for headache disorders. DORs are broadly expressed in several peripheral and central regions important for pain processing and mood regulation; and it is unclear which receptors regulate headache associated symptoms. In a model of chronic migraine-associated pain using the human migraine trigger, nitroglycerin, we observed increased expression of DOR in cortex, hippocampus, and striatum; suggesting a role for these forebrain regions in the regulation of migraine. To test this hypothesis, we used conditional knockout mice with DORs deleted from forebrain GABAergic neurons (Dlx-DOR), and investigated the outcome of this knockout on the effectiveness of the DOR agonist SNC80 in multiple headache models. In DOR loxP controls SNC80 blocked the development of acute and chronic cephalic allodynia in the chronic nitroglycerin model, an effect that was lost in Dlx-DOR mice. In addition, the anti-allodynic effects of SNC80 were lost in a model of opioid induced hyperalgesia/medication overuse headache in Dlx-DOR conditional knockouts. In a model reflecting negative affect associated with migraine, SNC80 was only effective in loxP controls and not Dlx-DOR mice. Similarly, SNC80 was ineffective in the cortical spreading depression model of migraine aura in conditional knockout mice. Taken together, these data indicate that forebrain DORs are necessary for the action of DOR agonists in relieving headache-related symptoms and suggest that forebrain regions may play an important role in migraine modulation.

NOP receptor agonist attenuates nitroglycerin-induced migraine-like symptoms in mice.

Migraine is an extraordinarily prevalent and disabling headache disorder that affects one billion people worldwide. Throbbing pain is one of several migraine symptoms including sensitivity to light (photophobia), sometimes to sounds, smell and touch. The basic mechanisms underlying migraine remain inadequately understood, and current treatments (with triptans being the primary standard of care) are not well tolerated by some patients. NOP (Nociceptin OPioid) receptors, the fourth member of the opioid receptor family, are expressed in the brain and periphery with particularly high expression known to be in trigeminal ganglia (TG). The aim of our study was to further explore the involvement of the NOP receptor system in migraine. To this end, we used immunohistochemistry to examine NOP receptor distribution in TG and trigeminal nucleus caudalus (TNC) in mice, including colocalization with specific cellular markers, and used nitroglycerin (NTG) models of migraine to assess the influence of the selective NOP receptor agonist, Ro 64-6198, on NTG-induced pain (sensitivity of paw and head using von Frey filaments) and photophobia in mice. Our immunohistochemical studies with NOP-eGFP knock-in mice indicate that NOP receptors are on the majority of neurons in the TG and are also very highly expressed in the TNC. In addition, Ro 64-6198 can dose dependently block NTG-induced paw and head allodynia, an effect that is blocked by the NOP antagonist, SB-612111. Moreover, Ro 64-6198, can decrease NTG-induced light sensitivity in mice. These results suggest that NOP receptor agonists should be futher explored as treatment for migraine symptoms. This article is part of the special issue on Neuropeptides.

Targeted Nitric Oxide Synthase Inhibitors for Migraine.

Nitric oxide (NO) is a small gaseous signaling molecule that has important biological effects. It has been heavily implicated in migraine; and the NO donor, nitroglycerin, has been used extensively as a human migraine trigger. Correspondingly, a number of components of the NO signaling cascade have been shown to be upregulated in migraine patients. NO is endogenously produced in the body by NO synthase (NOS), of which there are three isoforms: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). Based on the accumulating evidence that endogenous NO regulation is altered in migraine pathogenesis, global and isoform-selective inhibitors of NOS have been targeted for migraine drug development. This review highlights the evidence for the role of NO in migraine and focuses on the use of NOS inhibitors for the treatment of this disorder. In addition, we discuss other molecules within the NO signaling pathway that may be promising therapeutic targets for migraine.