Rostral Ventromedial Medulla Neurons Research Articles

Dissecting the interaction of pain and respiration at the level of the rostral ventromedial medulla via cell-type specific manipulations

Opioid drugs are highly effective for pain management, but they also cause respiratory depression. This life-threatening side-effect of opioid use occurs due to the interacting and/or overlapping neuronal circuits that regulate pain and breathing. Understanding the brain regions and cell-types that link pain and breathing will be essential to develop safer pain management strategies. Opioid analgesia involves altered firing patterns of neurons within the rostral ventromedial medulla (RVM). Although the RVM is known for its role in descending pain modulation, local administration of opioids within the RVM induces potent respiratory depression in addition to analgesia. The RVM has two main classes of neurons identified based on their firing response to pain stimuli. As their name suggests, “ON-cells” are activated during pain and “OFF-cells” are inhibited. Pharmacological and electrophysiological studies have shown that ON-cells express the μ-opioid receptor (MOR) and respond directly to opioids. Moreover, it is thought that the activity of ON-cells facilitates pain and respiration, and that opioid suppression of ON-cells contributes to respiratory depression. In contrast, OFF-cells do not appear to express MOR; however, activation of these neurons is required for opioid-induced analgesia. Therefore, changes in OFF-cells activity may more selectively affect pain thresholds. Due to a lack of selective pharmacological agents, directly testing these predictions has not been possible. Moreover, ON-cells can be excitatory or inhibitory, whereas OFF-cells are mostly inhibitory, adding another layer of complexity to their proposed functions. Here, we dissect these RVM subpopulations using modern intersectional genetic approaches to selectively manipulate RVM neurons based on expression of Oprm1 (the gene encoding MOR), as well as genetic markers for excitatory (Vglut2) or inhibitory (Vgat) neurotransmitter phenotypes. To examine the functional effects of Oprm1+ RVM neurons, an AAV designed to express Cre-dependent ChR2 was injected into the RVM of Oprm1Cre mice, followed by the implantation of an optical fiber. Respiratory activity of awake mice was then recorded using plethysmography during optogenetic stimulation of Oprm1+ RVM neurons. Consistent with the expected effects of ON-cells, breathing rate was increased to 5-6 Hz for the duration of the stimulation, and mice exhibited flinching and freezing behavior at light onset. In a second set of experiments, double transgenic Oprm1Cre; Vglut2FlpO mice were injected with an AAV that expresses ChR2 only when FlpO is present but Cre is absent (i.e. FlpO NOT Cre). Using this strategy, we were able to selectively target excitatory RVM neurons that do not express MOR. Optogenetic stimulations of these neurons during plethysmography revealed no changes in breathing or notable behavioral responses. However, paw withdrawal thresholds tested using Hargreaves’ method suggest that optogenetic activation of these neurons increases pain sensitivity. Our ongoing work will expand on these preliminary findings by using this approach to specifically manipulate Oprm1+;Vglut2+, Oprm1+;Vgat+, and Oprm1-;Vgat+ neurons to assess the roles of these RVM subpopulations in the regulation of pain and breathing. (NAB) R00HL145004; R01HL166317 (RSP) K01DA058543. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Dissecting neural circuits from rostral ventromedial medulla to spinal trigeminal nucleus bidirectionally modulating craniofacial mechanical sensitivity

Chronic craniofacial pain is intractable and its mechanisms remain unclarified. The rostral ventromedial medulla (RVM) plays a crucial role in descending pain facilitation and inhibition. It is unclear how the descending circuits from the RVM to spinal trigeminal nucleus (Sp5) are organized to bidirectionally modulate craniofacial nociception. We used viral tracing, in vivo optogenetics, calcium signaling recording, and chemogenetic manipulations to investigate the structure and function of RVM-Sp5 circuits. We found that most RVM neurons projecting to Sp5 were GABAergic or glutamatergic and facilitated or inhibited craniofacial nociception, respectively. Both GABAergic interneurons and glutamatergic projection neurons in Sp5 received RVM inputs: the former were antinociceptive, whereas the latter were pronociceptive. Furthermore, we demonstrated activation of both GABAergic and glutamatergic Sp5 neurons receiving RVM inputs in inflammation- or dysfunction-induced masseter hyperalgesia. Activating GABAergic Sp5 neurons or inhibiting glutamatergic Sp5 neurons that receive RVM projections reversed masseter hyperalgesia. Our study identifies specific cell types and projections of RVM-Sp5 circuits involved in facilitating or inhibiting craniofacial nociception respectively. Selective manipulation of RVM-Sp5 circuits can be used as potential treatment strategy to relieve chronic craniofacial muscle pain.

Direct and Indirect Nociceptive Input from the Trigeminal Dorsal Horn to Pain-Modulating Neurons in the Rostral Ventromedial Medulla.

The brain is able to amplify or suppress nociceptive signals by means of descending projections to the spinal and trigeminal dorsal horns from the rostral ventromedial medulla (RVM). Two physiologically defined cell classes within RVM, "ON-cells" and "OFF-cells," respectively facilitate and inhibit nociceptive transmission. However, sensory pathways through which nociceptive input drives changes in RVM cell activity are only now being defined. We recently showed that indirect inputs from the dorsal horn via the parabrachial complex (PB) convey nociceptive information to RVM. The purpose of the present study was to determine whether there are also direct dorsal horn inputs to RVM pain-modulating neurons. We focused on the trigeminal dorsal horn, which conveys sensory input from the face and head, and used a combination of single-cell recording with optogenetic activation and inhibition of projections to RVM and PB from the trigeminal interpolaris-caudalis transition zone (Vi/Vc) in male and female rats. We determined that a direct projection from ventral Vi/Vc to RVM carries nociceptive information to RVM pain-modulating neurons. This projection included a GABAergic component, which could contribute to nociceptive inhibition of OFF-cells. This approach also revealed a parallel, indirect, relay of trigeminal information to RVM via PB. Activation of the indirect pathway through PB produced a more sustained response in RVM compared with activation of the direct projection from Vi/Vc. These data demonstrate that a direct trigeminal output conveys nociceptive information to RVM pain-modulating neurons with a parallel indirect pathway through the parabrachial complex.SIGNIFICANCE STATEMENT Rostral ventromedial medulla (RVM) pain-modulating neurons respond to noxious stimulation, which implies that they receive input from pain-transmission circuits. However, the traditional view has been that there is no direct input to RVM pain-modulating neurons from the dorsal horn, and that nociceptive information is carried by indirect pathways. Indeed, we recently showed that noxious information can reach RVM pain-modulating neurons via the parabrachial complex (PB). Using in vivo electrophysiology and optogenetics, the present study identified a direct relay of nociceptive information from the trigeminal dorsal horn to physiologically identified pain-modulating neurons in RVM. Combined tracing and electrophysiology data revealed that the direct projection includes GABAergic neurons. Direct and indirect pathways may play distinct functional roles in recruiting pain-modulating neurons.

Descending Facilitation of Nociceptive Transmission From the Rostral Ventromedial Medulla Contributes to Hyperalgesia in Mice with Sickle Cell Disease

Sickle cell disease (SCD) is an inherited blood disorder that is associated with acute episodic and chronic pain. Mice with SCD have robust hyperalgesia mediated, in part, by sensitization of spinal dorsal horn neurons. However, underlying mechanisms are not fully understood. Since the rostral ventromedial medulla (RVM) is a major component of descending circuitry that modulates nociceptive transmission in the spinal cord, we examined if the RVM contributes to hyperalgesia in mice with SCD. Injection of lidocaine, but not vehicle, into the RVM eliminated mechanical and heat hyperalgesia in sickle (HbSS-BERK) mice without altering mechanical and heat sensitivity in naïve C57B mice. These data indicate that the RVM contributes to the maintenance of hyperalgesia in mice with SCD. In electrophysiological studies, we determined the changes in response properties of RVM neurons that might contribute to hyperalgesia in sickle mice. Recordings were made from single ON, OFF, and Neutral cells in the RVM of sickle and control (HbAA-BERK) mice. Spontaneous activity and responses of ON, OFF and Neutral cells evoked by heat (50 °C) and mechanical (26 g) stimuli applied to the hind paw were compared between sickle and control mice. Although there were no differences in the proportions of functionally-identified neurons or spontaneous activity between sickle and control mice, evoked responses of ON cells to heat and mechanical stimuli were increased approximately 3-fold in sickle mice as compared to control mice. Thus, the RVM contributes to hyperalgesia in sickle mice via a specific ON cell-dependent descending facilitation of nociceptive transmission.

Inhibition of itch by neurokinin 1 receptor (Tacr1) -expressing ON cells in the rostral ventromedial medulla in mice.

The rostral ventromedial medulla (RVM) is important in descending modulation of spinal nociceptive transmission, but it is unclear if the RVM also modulates spinal pruriceptive transmission. RVM ON cells are activated by noxious algesic and pruritic stimuli and are pronociceptive. Many RVM-spinal projection neurons express the neurokinin-1 receptor (Tacr1), and ON-cells are excited by local administration of substance P (SP). We hypothesized that Tacr1-expressing RVM ON cells exert an inhibitory effect on itch opposite to their pronociceptive action. Intramedullary microinjection of SP significantly potentiated RVM ON cells and reduced pruritogen-evoked scratching while producing mild mechanical sensitization. Chemogenetic activation of RVM Tacr1-expressing RVM neurons also reduced acute pruritogen-evoked scratching. Optotagging experiments confirmed RVM Tacr1-expressing neurons to be ON cells. We conclude that Tacr1-expressing ON cells in RVM play a significant role in the modulation of pruriceptive transmission.

Medullary kappa-opioid receptor neurons inhibit pain and itch through a descending circuit.

In perilous and stressful situations, the ability to suppress pain can be critical for survival. The rostral ventromedial medulla contains neurons that robustly inhibit nocioception at the level of the spinal cord through a top-down modulatory pathway. Although much is known about the role of the rostral ventromedial medulla in the inhibition of pain, the precise ability to directly manipulate pain-inhibitory neurons in the rostral ventromedial medulla has never been achieved. We now expose a cellular circuit that inhibits nocioception and itch in mice. Through a combination of molecular, tracing and behavioural approaches, we found that rostral ventromedial medulla neurons containing the kappa-opioid receptor inhibit itch and nocioception. With chemogenetic inhibition, we uncovered that these neurons are required for stress-induced analgesia. Using intersectional chemogenetic and pharmacological approaches, we determined that rostral ventromedial medulla kappa-opioid receptor neurons inhibit nocioception and itch through a descending circuit. Lastly, we identified a dynorphinergic pathway arising from the periaqueductal grey that modulates nociception within the rostral ventromedial medulla. These discoveries highlight a distinct population of rostral ventromedial medulla neurons capable of broadly and robustly inhibiting itch and nocioception.

Identification and characterization of rostral ventromedial medulla neurons synaptically connected to the urinary bladder afferents in female rats with or without neonatal cystitis.

The neurons in the rostral ventromedial medulla (RVM) play a major role in pain modulation. We have previously shown that early-life noxious bladder stimuli in rats resulted in an overall spinal GABAergic disinhibition and a long-lasting bladder/colon sensitization when tested in adulthood. However, the neuromolecular alterations within RVM neurons in the pathophysiology of early life bladder inflammation have not been elucidated. In this study, we have identified and characterized RVM neurons that are synaptically linked to the bladder and colon and examined the effect of neonatal bladder inflammation on molecular expressions of these neurons. A transient bladder inflammation was induced by intravesicular instillation of protamine sulfate and zymosan during postnatal days 14 through 16 (P14-16) followed by pseudorabies virus PRV-152 and PRV-614 injections into the bladder and colon, respectively, on postnatal day P60. Tissues were examined 96h postinoculation for serotonergic, GABAergic, and enkephalinergic expressions using in situ hybridization and/or immunohistochemistry techniques. The results revealed that>50% of RVM neurons that are synaptically connected to the bladder (i.e., PRV-152+) were GABAergic, 40% enkephalinergic, and about 14% expressing serotonergic marker tryptophan hydroxylase 2 (TpH2). Neonatal cystitis resulted in a significant increase in converging neurons in RVM receiving dual synaptic inputs from the bladder and colon. In addition, neonatal cystitis significantly downregulated vesicular GABA transporter (VGAT) with a concomitant increase in TpH2 expression in bladder-linked RVM neurons, suggesting an alteration in supraspinal signaling. These alterations of synaptic connectivity and GABAergic/serotonergic expressions in RVM neurons may contribute to bladder pain modulation and cross-organ visceral sensitivity.

G-Protein-Coupled Estrogen Receptor (GPER) in the Rostral Ventromedial Medulla Is Essential for Mobilizing Descending Inhibition of Itch.

Chronic itch is a troublesome condition and often difficult to cure. Emerging evidence suggests that the periaqueductal gray (PAG)-rostral ventromedial medulla (RVM) pathway may play an important role in the regulation of itch, but the cellular organization and molecular mechanisms remain incompletely understood. Here, we report that a group of RVM neurons distinctively express the G-protein-coupled estrogen receptor (GPER), which mediates descending inhibition of itch. We found that GPER+ neurons in the RVM were activated in chronic itch conditions in rats and mice. Selective ablation or chemogenetic suppression of RVM GPER+ neurons resulted in mechanical alloknesis and increased scratching in response to pruritogens, whereas chemogenetic activation of GPER+ neurons abrogated itch responses, indicating that GPER+ neurons are antipruritic. Moreover, GPER-deficient mice and rats of either sex exhibited hypersensitivity to mechanical and chemical itch, a phenotype reversible by the µ type opioid receptor (MOR) antagonism. Additionally, significant MOR phosphorylation in the RVM was detected in chronic itch models in wild-type but not in GPER-/- rats. Therefore, GPER not only identifies a population of medullary antipruritic neurons but may also determine the descending antipruritic tone through regulating µ opioid signaling.SIGNIFICANCE STATEMENT Therapeutic options for itch are limited because of an as yet incomplete understanding of the mechanisms of itch processing. Our data have provided novel insights into the cellular organization and molecular mechanisms of descending regulation of itch in normal and pathologic conditions. GPER+ neurons (largely GABAergic) in the RVM are antipruritic neurons under tonic opioidergic inhibition, activation of GPER promotes phosphorylation of MOR and disinhibition of the antipruritic GPER+ neurons from inhibitory opioidergic inputs, and failure to mobilize GPER+ neurons may result in the exacerbation of itch. Our data also illuminate on some of the outstanding questions in the field, such as the mechanisms underlying sex bias in itch, pain, and opioid analgesia and the paradoxical effects of morphine on pain and itch.

Physiological properties of pain-modulating neurons in rostral ventromedial medulla in female rats, and responses to opioid administration

Functional pain disorders disproportionately impact females, but most pain research in animals has been conducted in males. While there are anatomical and pharmacological sexual dimorphisms in brainstem pain-modulation circuits, the physiology of pain-modulating neurons that comprise a major functional output, the rostral ventromedial medulla (RVM), has not been explored in female animals. The goal of this study was to identify and characterize the activity of RVM cells in female, compared to male, rats. ON- and OFF-cells were identified within the RVM in females, with firing properties comparable to those described in males. In addition, both ON- and OFF-cells exhibited a sensitized response to somatic stimuli in females subjected to persistent inflammation, and both ON- and OFF-cells responded to systemically administered morphine at a dose sufficient to produce behavioral antinociception. These data demonstrate that the ON-/OFF-cell framework originally defined in males is also present in females, and that as in males, these neurons are recruited in females in persistent inflammation and by systemically administered morphine. Importantly, this work establishes a foundation for the use of female animals in studies of RVM and descending control.

Responses of neurons in rostral ventromedial medulla to nociceptive stimulation of craniofacial region and tail in rats

The rostral ventromedial medulla (RVM) plays a key role in the endogenous modulation of nociceptive transmission in the central nervous system (CNS). The primary aim of this study was to examine whether the activities of RVM neurons were related to craniofacial nociceptive behaviour (jaw-motor response, JMR) as well as the tail-flick response (TF). The activities of RVM neurons and TF and JMR evoked by noxious heating of the tail or perioral skin were recorded simultaneously in lightly anaesthetized rats. Tail or perioral heating evoked the TF and JMR, and the latency of the JMR was significantly shorter (P < 0.001) than that of the TF. Of 89 neurons recorded in RVM, 40 were classified as ON-cells, 27 as OFF-cells, and 22 as NEUTRAL-cells based on their responsiveness to heating of the tail. Heating at either site caused an increase in ON-cell and decrease in OFF-cell activity before the occurrence of the TF and JMR, but did not alter the activity of NEUTRAL cells. Likewise, noxious stimulation of the temporomandibular joint had similar effects on RVM neurons. These findings reveal that the JMR is a measure of the excitability of trigeminal and spinal nociceptive circuits in the CNS, and that the JMR as well as TF can be used for studying processes related to descending modulation of pain. The findings also support the view that RVM ON- and OFF-cells play an important role in the elaboration of diverse nociceptive behaviours evoked by noxious stimulation of widely separated regions of the body.

Medullary Kappa-Opioid Receptor Neurons Inhibit Pain and Itch Through a Descending Circuit

In perilous and stressful situations, the ability to suppress pain can be critical for survival. The rostral ventromedial medulla (RVM) contains neurons that robustly inhibit pain processing in the spinal cord through a top-down modulatory pathway. Although much is known about the role of the RVM in the inhibition of pain, the precise identities and mechanisms of pain-inhibitory neurons in the RVM have never been identified. We now expose a cellular circuit that inhibits pain and itch in mice. Through a combination of molecular, tracing, and behavioral approaches, we found that RVM neurons containing the kappa-opioid receptor (KOR) inhibit itch and pain. With chemogenetic inhibition, we uncovered that these neurons are required for stress-induced analgesia. Using intersectional chemogenetic and pharmacological approaches, we determined that RVM KOR neurons inhibit pain and itch through a descending circuit. Lastly, we identified a dynorphinergic pathway arising from the PAG that modulates nociception within the RVM. These discoveries highlight a distinct population of RVM neurons capable of broadly and robustly inhibiting itch and pain.

The cellular mechanism by which the rostral ventromedial medulla acts on the spinal cord during chronic pain.

Clinical therapies for chronic pain are limited. While targeted drugs are promising therapies for chronic pain, they exhibit insufficient efficacy and poor targeting. The occurrence of chronic pain partly results from central changes caused by alterations in neurons in the rostral ventromedial medulla (RVM) in the brainstem regulatory pathway. The RVM, which plays a key role in the descending pain control pathway, greatly contributes to the development and maintenance of pain. However, the exact roles of the RVM in chronic pain remain unclear, making it difficult to develop new drugs targeting the RVM and related pathways. Here, we first discuss the roles of the RVM and related circuits in chronic pain. Then, we analyze synaptic transmission between RVM neurons and spinal cord neurons, specifically focusing on the release of neurotransmitters, to explore the cellular mechanisms by which the RVM regulates chronic pain. Finally, we propose some ideas for the development of drugs targeting the RVM.

&lt;p&gt;Selective Ablation of Descending Serotonin from the Rostral Ventromedial Medulla Unmasks Its Pro-Nociceptive Role in Chemotherapy-Induced Painful Neuropathy&lt;/p&gt;

PurposeChemotherapy-induced painful neuropathy (CIPN) is a severe adverse effect of many anti-neoplastic drugs that is difficult to manage. Serotonin (5-hydroxytryptamine, 5-HT) is an important neurotransmitter in the rostral ventromedial medulla (RVM), which modulates descending spinal nociceptive transmission. However, the influence of the descending 5-HT from the RVM on CIPN is poorly understood. We investigated the role of 5-HT released from descending RVM neurons in a paclitaxel-induced CIPN rat model.MethodsCIPN rat model was produced by intraperitoneally injecting of paclitaxel. Pain behavioral assessments included mechanical allodynia and heat hyperalgesia. 5-HT content was analyzed by high-performance liquid chromatography (HPLC). Western blot and immunohistochemistry were used to determine tryptophan hydroxylase (Tph) and c-Fos expression. The inhibitors p-chlorophenylalanine (PCPA) and SB203580 were administrated by stereotaxical RVM microinjection. Ondansetron was injected through intrathecal catheterization.ResultsThe results demonstrated that Tph, the rate-limiting enzyme in 5-HT synthesis, was significantly upregulated in the RVM, and that spinal 5-HT release was increased in CIPN rats. Intra-RVM microinjection of Tph inhibitor PCPA significantly attenuated mechanical and thermal pain behavior through Tph downregulation and decreased spinal 5-HT. Intra-RVM administration of p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580 alleviated paclitaxel-induced pain in a similar manner to PCPA. Intrathecal injection of ondansetron, a 5-HT3 receptor antagonist, partially reversed paclitaxel-induced pain, indicating that 5-HT3 receptors were involved in descending serotoninergic modulation of spinal pain processing.ConclusionThe results suggest that activation of the p38 MAPK pathway in the RVM leads to increased RVM Tph expression and descending serotoninergic projection to the spinal dorsal horn and contributes to the persistence of CIPN via spinal 5-HT3 receptors.

PD24-06 MOLECULAR ALTERATION OF GABAERGIC PAIN MODULATORY PATHWAY IN THE ROSTRAL VENTROMEDIAL MEDULLA FOLLOWING ZYMOSAN-INDUCED CYSTITIS IN RATS

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology II (PD24)1 Apr 2020PD24-06 MOLECULAR ALTERATION OF GABAERGIC PAIN MODULATORY PATHWAY IN THE ROSTRAL VENTROMEDIAL MEDULLA FOLLOWING ZYMOSAN-INDUCED CYSTITIS IN RATS Bidyut K. Medda*, Sankar Addya, Bhavana Talluri, Faith Hoelzel, Maia Terashvili, Patrick Sanvanson, Reza Shaker, Jyoti N. Sengupta, and Banani Banerjee Bidyut K. Medda*Bidyut K. Medda* More articles by this author , Sankar AddyaSankar Addya More articles by this author , Bhavana TalluriBhavana Talluri More articles by this author , Faith HoelzelFaith Hoelzel More articles by this author , Maia TerashviliMaia Terashvili More articles by this author , Patrick SanvansonPatrick Sanvanson More articles by this author , Reza ShakerReza Shaker More articles by this author , Jyoti N. SenguptaJyoti N. Sengupta More articles by this author , and Banani BanerjeeBanani Banerjee More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000881.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The rostral ventromedial medulla (RVM) comprises of several subsets of neurons, which send descending projections to the spinal cord to modulate pain. These neurons are the major targets of efferent projections from the periaqueductal gray (PAG), the site of action of opioid-mediated analgesia. The majority of RVM originating descending neurons are GABAergic. Recently, we have documented microRNA (miRNA)-mediated transcriptional deregulation of spinal GABAergic system and visceral hypersensitivity (colon and bladder) in rats with zymosan-induced cystitis. The objective was to study the effect of noxious bladder insult on molecular expression of GABAergic RVM neurons of rats with zymosan-induced cystitis. METHODS: Two groups of Sprague Dawley rats (n=8 /group) were used in this study. Experimental and control groups received transurethral zymosan (1% in saline, 300ul) and saline respectively for 3 days from postnatal (P) days P58 through P60. Rats in both groups were euthanized on P60 and RVM tissues were collected for analysis. Gene expression profiles of experimental and control groups were evaluated using Rat Clariton S transcriptome arrays. miRNA expression profiles were examine using Affymatrix Gene Chips miRNA arrays. Individual gene was further evaluated using immunohistochemistry (IHC) and in situ hybridization (ISH). RESULTS: Intravesical zymosan treatment resulted in 4-fold downregulation of GABAA receptor subunit alpha 6 (GABAAα6) and glycine transporter Gly 2 (p<0.05 vs controls). Fourteen miRNAs demonstrated differential expression in zymosan treated rats (p<0.05 vs controls). The target gene prediction identified two upregulated miRNAs; miR-129-5p and miR-300 with complementary binding sites for 3’UTRs of GABAAα6 gene indicating a possible post-transcriptional deregulation of the RVM GABAergic pathway in zymosan-treated cystitis rats. A significant downregulation of RVM vesicular GABA transporter VGAT expression was observed in cystitis group by ISH (p<0.05 vs controls). The expression profile of GABAAα6 in RVM was validated by IHC. CONCLUSIONS: Cystitis results in miRNA-mediated transcriptional deregulation of GABA receptor and transporter expression in RVM neurons. This finding strongly suggests that the suppression of the RVM inhibitory transmission may contribute towards the development of spinal neuron hyperexcitability and bladder pain. Source of Funding: NIH R01DK099201 © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e534-e534 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Bidyut K. Medda* More articles by this author Sankar Addya More articles by this author Bhavana Talluri More articles by this author Faith Hoelzel More articles by this author Maia Terashvili More articles by this author Patrick Sanvanson More articles by this author Reza Shaker More articles by this author Jyoti N. Sengupta More articles by this author Banani Banerjee More articles by this author Expand All Advertisement PDF downloadLoading ...

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.

(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.

(151) - Modulation of chronic post-thoracotomy pain by NK-1 neurons in the rostral ventromedial medulla is not paralleled by changes spinal MAPKinase activation

Chronic post-operative pain after thoracotomy (CPTP) occurs in about half of the cases. Earlier findings in rat models of persistent neuropathic and inflammatory pain suggest that activation of different spinal Mitogen Activated Protein Kinases (MAPK) is critical for the initial and some longer-lasting pain, but no one has examined the MAPK activation pattern during chronic post-surgical pain. Recently we showed that ablation of Neurokinin-1 receptor (NK-1R)- expressing neurons in the rat rostral ventromedial medulla (RVM), by micro-injection of the specific neurotoxin SSP-SAP, (Sar9, Met(O2)11-Substance P conjugated to the ribosomal toxin saporin, 3 weeks before thoracotomy and rib retraction (TRR) was able to completely prevent CPTP, assayed by tactile hypersensitivity. Blank SAP, which has no cytotoxic activity, caused no change in CPTP. However, the same ablation procedure conducted post-surgically was less than fully effective to reverse these signs of CPTP. In the current study we tested the hypothesis that these differences in pain between pre-operative SSP-SAP (prevention), pre-operative Blank-SAP (control) and post-operative SSP-SAP (reversal) were correlated with the degree of long-term spinal MAPK activation (phosphorylation). We did this by assessing activated MAPKs, pERK, p-p38 and pJNK in spinal cord by Western blot and immunocytochemistry. Both assays revealed no differences in activated spinal MAPK levels assayed 5 weeks after surgery among the three groups, regardless of the pain level, showing that persistent pain in this model is not due to a persistent activation of spinal MAPKs.

Spinal nociceptive circuit analysis with recombinant adeno-associated viruses: the impact of serotypes and promoters.

Recombinant adeno-associated virus (rAAV) vector-mediated gene transfer into genetically defined neuron subtypes has become a powerful tool to study the neuroanatomy of neuronal circuits in the brain and to unravel their functions. More recently, this methodology has also become popular for the analysis of spinal cord circuits. To date, a variety of naturally occurring AAV serotypes and genetically modified capsid variants are available but transduction efficiency in spinal neurons, target selectivity, and the ability for retrograde tracing are only incompletely characterized. Here, we have compared the transduction efficiency of seven commonly used AAV serotypes after intraspinal injection. We specifically analyzed local transduction of different types of dorsal horn neurons, and retrograde transduction of dorsal root ganglia (DRG) neurons and of neurons in the rostral ventromedial medulla (RVM) and the somatosensory cortex (S1). Our results show that most of the tested rAAV vectors have similar transduction efficiency in spinal neurons. All serotypes analyzed were also able to transduce DRG neurons and descending RVM and S1 neurons via their spinal axon terminals. When comparing the commonly used rAAV serotypes to the recently developed serotype 2 capsid variant rAAV2retro, a >20-fold increase in transduction efficiency of descending supraspinal neurons was observed. Conversely, transgene expression in retrogradely transduced neurons was strongly reduced when the human synapsin 1 (hSyn1) promoter was used instead of the strong ubiquitous hybrid cytomegalovirus enhancer/chicken β-actin promoter (CAG) or cytomegalovirus (CMV) promoter fragments. We conclude that the use of AAV2retro greatly increases transduction of neurons connected to the spinal cord via their axon terminals, while the hSyn1 promoter can be used to minimize transgene expression in retrogradely connected neurons of the DRG or brainstem. Cover Image for this issue: doi. 10.1111/jnc.13813.

Administrations of thalidomide into the rostral ventromedial medulla produce antinociceptive effects in a rat model of postoperative pain.

The rostral ventromedial medulla (RVM) is highly involved in pain signal transmissions. Previous studies have shown that thalidomide is anti-nociceptive. Thus, we evaluated the neurobiological mechanisms of thalidomide in the RVM in the regulation of postoperative pain. We used a rat model of postoperative pain to investigate the effects of intra-RVM thalidomide treatments on postoperative pain, and evaluate the role of cannabinoid receptors in the effects of intra-RVM thalidomide treatments on GABAergic neurotransmission in the RVM neurons. We found intra-RVM thalidomide treatments reduced incisional surgery induced mechanical allodynia. This phenomenon was associated with attenuation of the frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs) and spontaneous IPSCs (sIPSCs) in RVM neurons. Furthermore, applications of WIN 55,212-3 mesylate, a non-selective cannabinoid receptor antagonist reversed the effects of repeated thalidomide treatment on the frequency but not the amplitude of mIPSCs and sIPSCs. Finally, we found that repeated thalidomide treatment robustly enhanced CB2 receptor expression, but slightly reduced CB1 receptor expression, in the RVM. These results suggested that the antinociceptive effects of thalidomide in the RVM likely involve the attenuation of GABA release, which are critically regulated by cannabinoid receptors.

Optogenetic Evidence for a Direct Circuit Linking Nociceptive Transmission through the Parabrachial Complex with Pain-Modulating Neurons of the Rostral Ventromedial Medulla (RVM).

The parabrachial complex (PB) is a functionally and anatomically complex structure involved in a range of homeostatic and sensory functions, including nociceptive transmission. There is also evidence that PB can engage descending pain-modulating systems, the best characterized of which is the rostral ventromedial medulla (RVM). Two distinct classes of RVM neurons, “ON-cells” and “OFF-cells,” exert net pronociceptive and anti-nociceptive effects, respectively. PB was recently shown to be a relay of nociceptive information to RVM ON- and OFF-cells. The present experiments used optogenetic methods in a lightly anesthetized rat and an adult RVM slice to determine whether there are direct, functionally relevant inputs to RVM pain-modulating neurons from PB. Whole-cell patch-clamp recordings demonstrated that PB conveys direct glutamatergic and GABAergic inputs to RVM neurons. Consistent with this, in vivo recording showed that nociceptive-evoked responses of ON- and OFF-cells were suppressed by optogenetic inactivation of archaerhodopsin (ArchT)-expressing PB terminals in RVM, demonstrating that a net inhibitory input to OFF-cells and net excitatory input to ON-cells are engaged by acute noxious stimulation. Further, the majority of ON- and OFF-cells responded to optogenetic activation of channelrhodopsin (ChR2)-expressing terminals in the RVM, confirming a direct PB influence on RVM pain-modulating neurons. These data show that a direct connection from the PB to the RVM conveys nociceptive information to the pain-modulating neurons of RVM under basal conditions. They also reveal additional inputs from PB with the capacity to activate both classes of RVM pain-modulating neurons and the potential to be recruited under different physiological and pathophysiological conditions.