Rostral Ventromedial Medulla Research Articles

Involvement of supralemniscal nucleus (B9) 5-HT neuronal system in nociceptive processing: a fiber photometry study

Nociception is important perception that has harmful influence on daily life of humans. As to main pain management system, some descending pathways are called descending antinociceptive systems (DAS). As main pathways of DAS, it is well known that dorsal raphe (B6/B7) - rostral ventromedial medulla (B3) - spinal dorsal horn includes serotonergic system. However, possible role of supralemniscal (B9) serotonin (5-HT) cell group in pain management is still open question. In this study, we measured activities of B9 5-HT neuronal cell bodies and B9 5-HT neuron-derived axons located in the locus coeruleus (LC) and ventral tegmental area (VTA), which are also main players of pain management, using fiber photometry system. We introduced the G-CaMP6 in B9 5-HT neurons using transgenic mice carrying a tetracycline-controlled transactivator transgene (tTA) under the control of a tryptophan hydroxylase-2 (TPH2) promoter and site-specific injection of adeno associated virus (AAV-TetO(3G)-G-CaMP6). After confirmation of specific expression of G-CaMP6 in the target population, G-CaMP6 fluorescence intensity in B9 group and LC/VTA groups was measured in awake mice exposed to acute tail pinch and heat stimuli. G-CaMP6 fluorescence intensity rapidly increased by both stimuli in all groups, but not significantly reacted by nonnociceptive control stimuli. The present results clearly indicate that acute nociceptive stimuli cause a rapid increase in the activities of B9-LC/B9-VTA 5-HTergic pathways, suggesting that B9 5-HT neurons play important roles in nociceptive processing.

Daily administration of Sake Lees (Sake Kasu) reduced psychophysical stress-induced hyperalgesia and Fos responses in the lumbar spinal dorsal horn evoked by noxious stimulation to the hindpaw in the rats.

We tested whether Sake Lees (SL) had inhibitory effects on hyperalgesia in the hindpaw under psychophysical stress conditions. Male rats were subjected to repeated forced swim stress treatments (FST) from Day -3 to Day -1. Intraperiotoneal administration of SL which contained low concentration of ethanol (SLX) was conducted after each FST. On Day 0, formalin-evoked licking behaviors and Fos responses in the lumbar spinal cord (DH) and several areas within the rostral ventromedial medulla (RVM) were quantified as nociceptive responses. FST-induced hyperalgesia in the hindpaw was prevented by repeated SL and SLX treatments. Fos expression was significantly increased in DH and some areas within the RVM under FST, which was prevented by repeated SL or SLX. These findings indicated that daily administration of SL had the potential to alleviate stress-induced hyperalgesia.

Acupuncture for Pain Management: Molecular Mechanisms of Action

Acupuncture reduces pain by activating specific areas called acupoints on the patient's body. When these acupoints are fully activated, sensations of soreness, numbness, fullness, or heaviness called De qi or Te qi are felt by clinicians and patients. There are two kinds of acupuncture, manual acupuncture and electroacupuncture (EA). Compared with non-acupoints, acupoints are easily activated on the basis of their special composition of blood vessels, mast cells, and nerve fibers that mediate the acupuncture signals. In the spinal cord, EA can inhibit glial cell activation by down-regulating the chemokine CX3CL1 and increasing the anti-inflammatory cytokine interleukin-10. This inhibits P38 mitogen-activated protein kinase and extracellular signal-regulated kinase pathways, which are associated with microglial activation of the C-Jun N-terminal kinase signaling pathway and subsequent astrocyte activation. The inactivation of spinal microglia and astrocytes mediates the immediate and long-term analgesic effects of EA, respectively. A variety of pain-related substances released by glial cells such as the proinflammatory cytokines tumor necrosis factor [Formula: see text], interleukin-1[Formula: see text], interleukin-6, and prostaglandins such as prostaglandins E2 can also be reduced. The descending pain modulation system in the brain, including the anterior cingulated cortex, the periaqueductal gray, and the rostral ventromedial medulla, plays an important role in EA analgesia. Multiple transmitters and modulators, including endogenous opioids, cholecystokinin octapeptide, 5-hydroxytryptamine, glutamate, noradrenalin, dopamine, [Formula: see text]-aminobutyric acid, acetylcholine, and orexin A, are involved in acupuncture analgesia. Finally, the "Acupuncture [Formula: see text]" strategy is introduced to help clinicians achieve better analgesic effects, and a newly reported acupuncture method called acupoint catgut embedding, which injects sutures made of absorbable materials at acupoints to achieve long-term effects, is discussed.

The Modulation of Pain by Metabotropic Glutamate Receptors 7 and 8 in the Dorsal Striatum.

The dorsal striatum, apart from controlling voluntary movement, displays a recently demonstrated pain inhibition. It is connected to the descending pain modulatory system and in particular to the rostral ventromedial medulla through the medullary dorsal reticular nucleus. Diseases of the basal ganglia, such as Parkinson's disease, in addition to being characterized by motor disorders, are associated with pain and hyperactivation of the excitatory transmission. A way to counteract glutamatergic hyperactivation is through the activation of group III metabotropic glutamate receptors (mGluRs), which are located on presynaptic terminals inhibiting neurotransmitter release. So far the mGluRs of group III have been the least investigated, owing to a lack of selective tools. More recently, selective ligands for each mGluR of group III, in particular positive and negative allosteric modulators, have been developed and the role of each subtype is starting to emerge. The neuroprotective potential of group III mGluRs in pathological conditions, such as those characterized by elevate glutamate, has been recently shown. In the dorsal striatum, mGluR7 and mGluR8 are located at glutamatergic corticostriatal terminals and their stimulation inhibits pain in pathological conditions such as neuropathic pain. The two receptors in the dorsal striatum have instead a different role in pain control in normal conditions. This review will discuss recent results focusing on the contribution of mGluR7 and mGluR8 in the dorsal striatal control of pain. The role of mGluR4, whose antiparkinsonian activity is widely reported, will also be addressed.

Synaptic control of spinal GRPR+ neurons by local and long-range inhibitory inputs

Spinal gastrin-releasing peptide receptor-expressing (GRPR+) neurons play an essential role in itch signal processing. However, the circuit mechanisms underlying the modulation of spinal GRPR+ neurons by direct local and long-range inhibitory inputs remain elusive. Using viral tracing and electrophysiological approaches, we dissected the neural circuits underlying the inhibitory control of spinal GRPR+ neurons. We found that spinal galanin+ GABAergic neurons form inhibitory synapses with GRPR+ neurons in the spinal cord and play an important role in gating the GRPR+ neuron-dependent itch signaling pathway. Spinal GRPR+ neurons also receive inhibitory inputs from local neurons expressing neuronal nitric oxide synthase (nNOS). Moreover, spinal GRPR+ neurons are gated by strong inhibitory inputs from the rostral ventromedial medulla. Thus, both local and long-range inhibitory inputs could play important roles in gating itch processing in the spinal cord by directly modulating the activity of spinal GRPR+ neurons.

Serotoninergic pain modulation from the rostral ventromedial medulla (RVM) in chemotherapy-induced neuropathy: The role of spinal 5-HT3 receptors.

Chemotherapy-induced neuropathy (CIN) is a common complication of cancer treatment. Although CIN is treated with antidepressants that act at serotonin (5-HT) reuptake, the mechanisms of serotoninergic modulation of nociceptive transmission during CIN remain unknown, namely as to the involvement of the rostroventromedial medulla (RVM) and the role of spinal 5-HT3 receptors (5-HT3R). Male Wistar rats were injected with the cytostatic paclitaxel or vehicle solution. One month after CIN induction, we first studied the activation of RVM neurons, and then the activation of the local serotoninergic neurons. Immunostaining of phosphorylated extracellular signal-regulated kinase (pERK) indicated increased activation in paclitaxel-injected animals. The double immunohistochemistry of pERK and tryptophan hydroxylase (TpH) showed higher expression of TpH and increased co-localization of TpH and pERK of paclitaxel-injected animals, indicating that CIN is associated with increased activation of serotoninergic RVM neurons. The 5-HT content at the spinal dorsal horn assessed by HPLC was higher in paclitaxel-injected animals. The immunohistochemical analysis of 5-HT also showed increased expression at the superficial dorsal horn (laminae I-II) of paclitaxel-injected animals. The levels of 5-HT3R detected by immunohistochemistry were higher in the superficial dorsal horn (laminae I-II) of paclitaxel-injected animals. The intrathecal administration of the 5-HT3R antagonist ondansetron reversed mechanical and cold hypersensitivity in the von Frey and cold plate tests, respectively. The results indicate that CIN is associated with increased recruitment of descending 5-HT-mediated modulation from the RVM which affects the spinal serotoninergic system and probably accounts for pain hypersensitivity due to the pronociceptive role of spinal 5-HT3R.

Intra-CA1 Administration of Minocycline Alters the Expression of Inflammation-Related Genes in Hippocampus of CCI Rats.

Neuropathic pain is a chronic pain condition that is usually induced by peripheral nerve injury. Recent reports suggest that the inflammation-related cytokines accumulation in dorsal root ganglion, dorsal spinal cord, hippocampus, thalamus, and somatosensoric cortex are paralleled by pain responses in different animal models of neuropathic pain (Al-Amin et al., 2011; Sun et al., 2016; Chang et al., 2018; Liu et al., 2018). In the chronic constriction injury (CCI) and the spared nerve injury models of neuropathic pain in rats, an increase in interleukin 1 beta (IL-1β), interleukin 6 (IL-6), nerve growth factor (NGF), and glial cell-derived neurotrophic factor (GDNF) was observed in most brain regions (Al-Amin et al., 2011). The overproduction of tumor necrosis factor-α (TNF-α) may regulate synaptic plasticity in the rat hippocampus through microglia-dependent mechanism after spared nerve injury of the sciatic nerve (Liu et al., 2017). However, it is not clear whether other inflammation-related neuroactive substances will be affected after microglia activation in the rat hippocampus after peripheral nerve injury. It is clear that many kinds of toll-like receptors (TLRs) are expressed in the hippocampus and act as a type of pattern-recognition receptor that participate in inflammatory responses. TLR1 expression in the hippocampus was increased in the neurons, microglia, and astrocytes in seizure mice (Wang et al., 2015). TLR (2, 3, 4, 7, and 9) expression was upregulated in the hippocampus of restraint stressed rats (Timberlake et al., 2018). TLR2 and TLR4 in the rat hippocampus are related to the lipopolysaccharide (LPS)-induced neuron cell death (He et al., 2013; Henry et al., 2014). That TLR3-induces the increased expression of IL-1β in the rat hippocampus was suggested by Henry et al. (2014). TLR8, expressed in most regions of the brain, is associated with injury and neurite outgrowth (Ma et al., 2006). It is well known that TLR-dependent signaling is often associated with the overproduction and release of inflammatory cytokines in many different types of cells. However, the relationship between the changes of TLRs expression and microglia activation in hippocampus of CCI rats is not known. Previous studies reveal that chemokine production is enhanced in some neuroimmunological diseases accompanied by pathological pain (Cartier et al., 2005). CXCL13 is obviously upregulated in the spinal cord after spinal nerve ligation and induces astrocyte activation via its receptor CXCR5 (Zhang et al., 2017). Chemokine CCL2 (C-C motif ligand 2) in the rostral ventromedial medulla is related to the descending pain facilitation in nerve-injured rats (Guo et al., 2012). Expression of chemokines CCL2 and CCL3 was increased in the thalamus and hippocampus after severe spinal cord injuries (Knerlich-Lukoschus et al., 2011). The overproduction of IL-1β and CCL2 was found in the hippocampus of CCI rats (Fiore and Austin, 2018). Moreover, Lanfranco et al. reported that CCL5 gene expression was found in neurons and glial cells in the rat hippocampus (Lanfranco et al., 2018). However, no evidence directly addresses the relationship between microglia activation and chemokine accumulation in neuropathic hypersensitivity. It is clear that minocycline is an important modulator of the immune response and easily permeates the blood-brain barrier (Stolp et al., 2007; Vonder Haar et al., 2014). Clinically, minocycline can be administered by the intravenous route in patients with traumatic brain injury (Rojewska et al., 2014). More recent evidence suggest that minocycline is effective at reducing the spontaneous pain behavior in animal models of neuropathic pain, and that means it appears to be a promising analgesic drug (LeBlanc et al., 2011; Rojewska et al., 2014). In the present study, minocycline is applied to identify what inflammation-related genes at the hippocampus are closely related to the increased microglia activity in CCI-induced neuropathic pain rats.

Current understanding of premonitory networks in migraine: A window to attack generation.

To describe neuronal networks underlying commonly reported migraine premonitory symptoms and to discuss how these might precipitate migraine pain. Migraine headache is frequently preceded by a distinct and well characterized premonitory phase including symptoms like yawning, sleep disturbances, alterations in appetite and food intake and hypersensitivity to certain external stimuli. Recent neuroimaging studies strongly suggest the hypothalamus as the key mediator of the premonitory phase and also suggested alterations in hypothalamic networks as a mechanism of migraine attack generation. When looking at the vast evidence from basic research within the last decades, hypothalamic and thalamic networks are most likely to integrate peripheral influences with central mechanisms, facilitating the precipitation of migraine headaches. These networks include sleep, feeding and stress modulating centers within the hypothalamus, thalamic pathways and brainstem centers closely involved in trigeminal pain processing such as the spinal trigeminal nucleus and the rostral ventromedial medulla, all of which are closely interconnected. Taken together, these networks represent the pathophysiological basis for migraine premonitory symptoms as well as a possible integration site of peripheral so-called "triggers" with central attack facilitating processes.

Endocannabinoid and N-acylethanolamide levels in rat brain and spinal cord following systemic dipyrone and paracetamol administration.

The cannabinoid system has been suspected to play a role in the mechanisms of action of dipyrone and paracetamol. Our purpose was to measure the local endocannabinoid and N-acylethanolamide levels in the brain and spinal cord of rats following dipyrone and paracetamol administration. Nociception was assessed 1, 5, and 12 h following drug injections in Wistar rats, using tail-flick and hot-plate tests. The antinociceptive effects of dipyrone (150, 300, and 600 mg/kg, i.p.) and paracetamol (30, 100, and 300 mg/kg, i.p.) were observed. After administration of the highest doses of dipyrone and paracetamol, endocannabinoid (N-arachidonoylethanolamide (AEA), 2-arachidonoylglycerol (2-AG)) and N-acylethanolamide (palmitoylethanolamide (PEA), oleoylethanolamide (OEA)) levels were measured in the periaqueductal gray (PAG), rostral ventromedial medulla (RVM), and spinal cords of rats using tandem mass spectrometry with liquid chromatography. Increased 2-AG levels were observed in the PAG and the RVM 12 h after paracetamol injection; dipyrone exerted no action on 2-AG levels. Analgesic administrations led to a reduction in AEA levels in the RVM and spinal cord; similar decreases in PEA and OEA levels were observed in the RVM and the spinal cord. Dipyrone and paracetamol administrations appear to exert complicated effects on endocannabinoid and N-acylethanolamide levels in rats.

Anxiety-induced hyperalgesia in female rats is mediated by cholecystokinin 2 receptor in rostral ventromedial medulla and spinal 5-hydroxytryptamine 2B receptor.

BackgroundPreoperative anxiety is associated with postoperative hyperalgesia; however, few studies have investigated the mechanism underlying this association in female surgical patients. Research has suggested that ON cells in the rostral ventromedial medulla (RVM) receive nerve impulses via cholecystokinin 2 (CCK2) receptors, facilitating hyperalgesia. Additionally, the downstream serotonergic projection system from the RVM to the spinal cord has a dual regulating effect on pain responses, and the 5-hydoxytryptophan 2B (5-HT2B) receptor in spinal dorsal horn neurons is critically involved in mechanical allodynia.MethodsOvariectomized rats were treated with estrogen replacement, single prolonged stress (SPS), and plantar incision. Various receptor agonists and antagonists were then administered into the RVM and spinal cord to study the mechanism underlying postoperative hyperalgesia caused by preoperative anxiety in female rats.ResultsBehavioral testing revealed that preoperative SPS induced postoperative hyperalgesia, as well as the expression of the CCK2 receptor in the RVM and the expression of the 5-HT2B receptor, protein kinase Cγ (PKCγ), and phosphorylation of the N-methyl-d-aspartate receptor1 (p-NR1) in the spinal cord increased confirmed by Western blot. RVM microinjection of the CCK2 receptor agonist CCK-8 and intrathecal injection of the 5-HT2B receptor agonist BW723C86 both produced hyperalgesia in female rats after plantar incision, whereas the CCK2 receptor antagonist YM022, the 5-HT2B receptor antagonist RS127445, and the PKCγ inhibitor C37H65N9O13 decreased the rats’ sensitivity to the same stimulus. Additionally, electrophysiological analysis suggested that activation of the 5-HT2B receptor increased the whole-cell current (IBa) in superficial dorsal horn neurons through the PKCγ pathway.ConclusionOur study demonstrated that preoperative anxiety-induced postoperative hyperalgesia in female rats is associated with descending pain pathways. The CCK2 receptor in the RVM and spinal 5-HT2B receptor may play a role in this hyperalgesic effect.

Solving the Opioid Crisis: Respiratory Depression by Opioids as Critical End Point

Solving the Opioid Crisis: Respiratory Depression by Opioids as Critical End Point

Acute nociceptive stimuli rapidly induce the activity of serotonin and noradrenalin neurons in the brain stem of awake mice

Nociception is an important type of perception that has major influence on daily human life. There are some descending pathways related to pain management and modulation, which are collectively known as the descending antinociceptive system (DAS). Noradrenalin (NA) in the locus coeruleus (LC) and serotonin (5-HT) in the rostral ventromedial medulla (RVM) are components of the DAS. Most 5-HT neurons in the dorsal raphe (DR) have ascending projections rather than descending projections, and they project to the thalamus that modulates nociception. Both the DAS and the DR are believed to be involved in pain-emotion symptoms. In this study, we utilized a fiber photometry system to specifically examine the activity of LC NA neurons and RVM/DR 5-HT neurons using mice carrying tetracycline-controlled transactivator transgene (tTA) under the control of either a dopamine β-hydroxylase promoter or a tryptophan hydroxylase-2 promoter and site-specific infection of an adeno-associated virus carrying a TetO G-CaMP6gene. After confirmation of specific expression of G-CaMP6 in the target populations, changes in green fluorescent signal intensity were recorded in awake mice upon exposure to acute nociceptive stimulation consisting of a pinch and application of heat (55°C) to the tail. Both stimuli resulted in rapid and transient (<15s) increases in the activity of LC NA neurons and RVM/DR 5-HT neurons while the control stimuli did not induce any changes. The present results clearly indicate that acute nociceptive stimuli increase the activity of LC NA neurons and RVM/DR 5HT neurons and suggest a possible therapeutic target for pain treatment.

Development and Characterization of An Injury-free Model of Functional Pain in Rats by Exposure to Red Light

We report the development and characterization of a novel, injury-free rat model in which nociceptive sensitization after red light is observed in multiple body areas reminiscent of widespread pain in functional pain syndromes. Rats were exposed to red light-emitting diodes (RLED) (LEDs, 660 nm) at an intensity of 50 Lux for 8 hours daily for 5 days resulting in time- and dose-dependent thermal hyperalgesia and mechanical allodynia in both male and female rats. Females showed an earlier onset of mechanical allodynia than males. The pronociceptive effects of RLED were mediated through the visual system. RLED-induced thermal hyperalgesia and mechanical allodynia were reversed with medications commonly used for widespread pain, including gabapentin, tricyclic antidepressants, serotonin/norepinephrine reuptake inhibitors, and nonsteroidal anti-inflammatory drugs. Acetaminophen failed to reverse the RLED induced hypersensitivity. The hyperalgesic effects of RLED were blocked when bicuculline, a gamma-aminobutyric acid-A receptor antagonist, was administered into the rostral ventromedial medulla, suggesting a role for increased descending facilitation in the pain pathway. Key experiments were subjected to a replication study with randomization, investigator blinding, inclusion of all data, and high levels of statistical rigor. RLED-induced thermal hyperalgesia and mechanical allodynia without injury offers a novel injury-free rodent model useful for the study of functional pain syndromes with widespread pain. RLED exposure also emphasizes the different biological effects of different colors of light exposure. PerspectiveThis study demonstrates the effect of light exposure on nociceptive thresholds. These biological effects of red LED add evidence to the emerging understanding of the biological effects of light of different colors in animals and humans. Understanding the underlying biology of red light-induced widespread pain may offer insights into functional pain states.

A Novel Sympathoexcitatory Pathway between The Deep Superior Colliculus and The Rostral Ventromedial Medulla

IntroductionThe superior colliculus plays key roles in the immediate processing of threatening sensory stimuli and generates rapid behavioural responses that are critical for survival. We have previously found that disinhibition of the deep superior colliculus (dSC) unmasks coordinated respiratory, sympathetic and somatomotor outputs that are independent of processing in higher centres. We postulate that these effects are a result of direct innervation of autonomic and respiratory medullary neurons based on the results of neuroanatomical tracing studies that identified dSC synaptic contacts on spinally projecting neurons within the rostral ventromedial medulla (RVMM). In the present study we investigate the physiological significance of this relay and compare effects of dSC stimulation to activation of dSC‐RVMM terminals.MethodsWe used an AAV vector to express Channelrhodopsin2 (ChR2) in the dSC and chronically implanted a fibre optic cannula into the dSC. After transgene expression electrophysiology experiments were conducted under urethane anesthesia with rats instrumented to record blood pressure, diaphragmatic EMG and splanchnic sympathetic nerve activity (SNA). One subset of experiments then positioned the optrode in the RVMM to deliver light to the terminal projections from the dSC. For the other subset of experiments, extracellular recordings were made of spinally projecting RVMM neurons.ResultsdSC photoactivation evoked an increase in respiratory frequency (35±8.7%, P=&lt;0.01, N=7) and heart rate (11±2.6 bpm, P=&lt;0.01, N=7), and a modest change in blood pressure when compared to controls. Stimulus‐triggered averaging of SNA revealed short‐latency excitatory potentials in most cases. Photoactivation of ChR2‐expressing dSC‐RVMM terminals evoked similar effects on cardiorespiratory outputs as dSC stimulation, with an increase in respiratory frequency (14 ±9.2%, P&lt;0.001, N=6) and blood pressure (13 ±2 mmHg P&lt;0.01, N=7), as well as qualitatively similar sympathoexcitation. In preliminary experiments that recorded extracellular action potentials in bulbospinal RVMM neurons we recorded excitatory effects of dSC stimulation in 6/8 spontaneously active neurons.ConclusionWe conclude that optogenetic activation of dSC neurons that project to the RVMM can drive cardiorespiratory effects that are consistent with our previous findings and that dSC stimulation evokes excitatory effects in some RVMM bulbospinal neurons. This confirms our hypothesis of the existence of a previously uncharacterized excitatory pathway between the dSC and RVMM. Ongoing experiments will functionally characterize RVMM neurons that receive dSC input.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sex Differences in the Amygdaloid Projections to the Periaqueductal Gray During Inflammatory Pain in the Rat

The central nervous system is involved in modulation of pain and is sexually dimorphic in neural organization and function. Sex hormones are implicated in pain modulation and studies suggest that estradiol increases pain and decreases analgesia. Previous studies have identified that sex differences in descending modulation contribute to dimorphic pain and analgesia. The midbrain periaqueductal gray (PAG) is a critical brain region for descending inhibition of pain. The PAG projects to the rostral ventromedial medulla (RVM), which projects to the spinal cord to inhibit pain signals. In addition to pain, this descending system can be activated by endogenous (e.g. endorphins) and/or exogenous (e.g. morphine) substances and sex differences have been reported in the activation of this system during pain and analgesia. Forebrain structures, such as the amygdala, project to and engage the PAG descending circuit, however studies examining forebrain projections to the PAG have been conducted exclusively in males. Further, the central nucleus of the amygdala (CeA) seems to be involved in pain facilitation, while the medial nucleus (MeA) is involved in pain inhibition. The objective of the present study was to delineate the neural projections from the amygdala to the PAG in the female rat and determine if they are (1) sexually dimorphic in anatomical organization, (2) estradiol‐sensitive, and (3) activated by inflammatory pain. The retrograde tracer fluorogold (FG) was injected into the ventrolateral PAG of adult male (n=4) and female (n=4) Sprague Dawley rats. After 10 days, complete Freund's adjuvant (CFA) was injected into the right hind paw to induce inflammatory pain. Twenty‐four hours later, rats were perfusion fixed, brains were sectioned at 25 μm, then processed by immunohistochemistry. Fluorogold was visualized using diaminobenzidine (DAB) oxidation and Fos or estrogen receptor (ERα)was visualized using DAB oxidation in the presence of nickel. Heavy projections from the CeA and MeA to the PAG were observed in females, with a greater number of MeA neurons projecting to the PAG in females than the CeA. Overall, inflammatory pain induced greater Fos expression in the CeA and MeA of males. Interestingly, females had greater Fos expression in CeA‐PAG projection neurons, while in males Fos expression was greater in MeA‐PAG neurons. ERα expression was comparable between the sexes in the CeA, and higher in MeA neurons in females. Together our data indicate that although the MeA‐PAG projection is denser in females, inflammatory pain activates this pathway to a greater degree in males. This suggests that descending inhibition is more ‘engaged’ in males during inflammatory pain. On the other hand, inflammatory pain results in a greater activation of the CeA‐PAG pathway in females, suggesting a pain faciliatory mechanism in females. We are currently using confocal microscopy to determine potential sex differences in the neurochemistry of this system that underlies dimorphic modulation of the PAG during pain.Support or Funding InformationThis research was supported by NIH grants DA16272 and AR49555 to AZM and the STC Program of NSF IBN‐9876754. This work was funded, in part, by a TWU Quality Enhancement Program grant awarded to DJC.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

(353) Cell-Type Specific Modulation of RBM Neurons in Nociceptive Behaviors

The rostral ventromedial medulla (RVM) serves a critically important role in the processing of pain. Through a process known as descending modulation, the RVM can directly shape how somatosensory information, particularly pain, is integrated at the level of the spinal cord. Although it has been known for decades that the RVM participates in descending modulation, the identities of the neurons responsible for this phenomenon remain unclear. Here, we use Cre-dependent viral tracers and Cre-dependent designer receptor exclusively activated by designer drugs (DREADDs) within the RVM to dissect the functional circuitry underlying descending modulation. Furthermore, we use RNAscope to molecularly characterize the different subpopulations of RVM neurons. Identification of RVM-spinal cord circuits can provide insight into the cellular basis of descending modulation and can guide the development of improved therapies for pain.

(254) Functional and Anatomical Characterization of Descending Periaqueductal Gray (PAG) Projections and their Role in Pain Modulation

Endogenous analgesic pathways embody an alternative target for the development for chronic pain therapies. Previous studies have demonstrated the role of the ventrolateral periaqueductal gray (vlPAG) in descending pain modulation. It has been proposed that tonic GABAergic neurotransmission at the level of the vlPAG serves to inhibit efferent excitatory projections that mediate descending analgesia. Disinhibition of these projection neurons allows subsequent activation of rostral ventromedial medulla (RVM) neurons that inhibit nociception at the level of the spinal cord. However, lack of cell-type specificity in these studies has prevented the determination of the role of specific subsets of vlPAG neurons in analgesia. We have utilized recently developed genetic tools to selectively manipulate the activity of subclasses of vlPAG neurons to identify the circuit components critical for analgesia. Our data suggest that increasing the activity of glutamatergic vlPAG neurons or decreasing the activity of GABAergic vlPAG neurons results in increases in sensory thresholds in uninjured animals, while ameliorating hyperalgesia associated with chronic pain states. To further characterize descending pain circuitry at the level of the vlPAG, we decided to focus on efferent projections to the RVM. We hypothesize that these projections are partially responsible for the observed analgesia in our previous study. Population-specific anatomical tracing revealed that most of these projections are glutamatergic and originate from the ventrolateral columns of the PAG. Optogenetic stimulation of these PAG-RVM neurons results in elevation of thermal thresholds in uninjured animals and seems to reverse thermal hyperalgesia associated with inflammatory states. However, this is not the case in a model of neuropathic pain. In brief, our results provide direct experimental evidence supporting the GABA disinhibition hypothesis, highlighting the role of descending glutamatergic neurotransmission at the level of the PAG-RVM as a key component of endogenous analgesic pathways. This work is supported by NINDS F31NS103472 to JGGR.

Central Sensitization in Knee Osteoarthritis: Relating Presurgical Brainstem Neuroimaging and PainDETECT-Based Patient Stratification to Arthroplasty Outcome.

ObjectiveThe neural mechanisms of pain in knee osteoarthritis (OA) are not fully understood, and some patients have neuropathic‐like pain associated with central sensitization. To address this, we undertook the present study in order to identify central sensitization using neuroimaging and PainDETECT and to relate it to postarthroplasty outcome.MethodsPatients awaiting arthroplasty underwent quantitative sensory testing, psychological assessment, and functional magnetic resonance imaging (fMRI). Neuroimaging (fMRI) was conducted during punctate stimulation (n = 24) and cold stimulation (n = 20) to the affected knee. The postoperative outcome was measured using the Oxford Knee Score, patient‐reported moderate‐to‐severe long‐term pain postarthroplasty, and a range of pain‐related questionnaires.ResultsPatients with neuropathic‐like pain presurgery (identified using PainDETECT; n = 14) reported significantly higher pain in response to punctate stimuli and cold stimuli near the affected joint (P < 0.05). Neural activity in these patients, compared to those without neuropathic‐like pain, was significantly lower in the rostral anterior cingulate cortex (P < 0.05) and higher in the rostral ventromedial medulla (RVM) during punctate stimulation (P < 0.05), with significant functional connectivity between these two areas (r = 0.49, P = 0.018). Preoperative neuropathic‐like pain and higher neural activity in the RVM were associated with moderate‐to‐severe long‐term pain after arthroplasty (P = 0.0356).ConclusionThe psychophysical and neuroimaging data suggest that a subset of OA patients have centrally mediated pain sensitization. This was likely due to supraspinally mediated reductions in inhibition and increases in facilitation of nociceptive signaling, and was associated with a worse outcome following arthroplasty. The neurobiologic confirmation of central sensitization in patients with features of neuropathic pain, identified using PainDETECT, provides further support for the investigation of such bedside measures for patient stratification, to better predict postsurgical outcomes.

Microinjection of 26RFa, an endogenous ligand for the glutamine RF-amide peptide receptor (QRFP receptor), into the rostral ventromedial medulla (RVM), locus coelureus (LC), and periaqueductal grey (PAG) produces an analgesic effect in rats

26RFa is an endogenous ligand for the QRFP receptor. We previously found that intracerebroventricular injection of 26RFa produces an analgesic effect in a rat formalin test. In the present study, we directly tested the hypothesis that the analgesic effects of 26RFa in the formalin test are mediated in well-recognized regions of the descending inhibitory pain pathways, such as the rostral ventromedial medulla (RVM), locus coeruleus (LC), and periaqueductal grey (PAG) in rats. Injection cannulae were stereotaxically placed in the RVM, LC, or PAG through a burr hole. 26RFa (15 μg) or saline was delivered in a total volume of 0.5 μL. In a formalin test, 50 μL of 5% formalin was injected subcutaneously into the hind paw. In an antagonist study, idazoxan, an α-2 antagonist, or naloxone, an opioid receptor antagonist, was administered. Microinjection of 26RFa into the RVM had no effect compared with that in saline-injected rats. Microinjection of 26RFa into the LC contralateral, but not ipsilateral, to the formalin injection site significantly decreased the number of flinching behaviors compared with that of saline-injected rats. This effect was antagonized by intrathecal injection of idazoxan. Microinjection of 26RFa into the contralateral, but not ipsilateral, PAG produced an analgesic effect, and this effect was partly antagonized by intraperitoneal naloxone. These data suggest that 26RFa microinjected into the contralateral LC induced noradrenaline release in the spinal cord and produced an analgesic effect. In the contralateral PAG, 26RFa activated the opioid system, and some analgesic effects were mediated by opioid system activation.

Noninvasive vagus nerve stimulation alters neural response and physiological autonomic tone to noxious thermal challenge.

The mechanisms by which noninvasive vagal nerve stimulation (nVNS) affect central and peripheral neural circuits that subserve pain and autonomic physiology are not clear, and thus remain an area of intense investigation. Effects of nVNS vs sham stimulation on subject responses to five noxious thermal stimuli (applied to left lower extremity), were measured in 30 healthy subjects (n = 15 sham and n = 15 nVNS), with fMRI and physiological galvanic skin response (GSR). With repeated noxious thermal stimuli a group × time analysis showed a significantly (p < .001) decreased response with nVNS in bilateral primary and secondary somatosensory cortices (SI and SII), left dorsoposterior insular cortex, bilateral paracentral lobule, bilateral medial dorsal thalamus, right anterior cingulate cortex, and right orbitofrontal cortex. A group × time × GSR analysis showed a significantly decreased response in the nVNS group (p < .0005) bilaterally in SI, lower and mid medullary brainstem, and inferior occipital cortex. Finally, nVNS treatment showed decreased activity in pronociceptive brainstem nuclei (e.g. the reticular nucleus and rostral ventromedial medulla) and key autonomic integration nuclei (e.g. the rostroventrolateral medulla, nucleus ambiguous, and dorsal motor nucleus of the vagus nerve). In aggregate, noninvasive vagal nerve stimulation reduced the physiological response to noxious thermal stimuli and impacted neural circuits important for pain processing and autonomic output.