Cerebrospinal Fluid-contacting Nucleus Research Articles

The recent research progress in neurobiological characteristics and pain regulation of the cerebrospinal fluid-contacting nucleus

AbstractThe ependymal epithelium forms the cerebrospinal fluid barrier, separating the brain and spinal cord from the cerebrospinal fluid. However, in specific regions of the central nervous system, there are neurons that directly interface with the cerebrospinal fluid, including neuronal bodies, dendrites, or axons, This constitutes what is referred to as the "cerebrospinal fluid contacting neurons system (CSF-CNS)". The research team led by Professor Zhang has successfully utilized cholera toxin subunit B coupled horseradish peroxidase complex (CB-HRP) to selectively label the specialized neuron system that interfaces with cerebrospinal fluid, pioneeringly designating it as the "cerebrospinal fluid-contacting nucleus", commonly referred to as the "CSF-contacting nucleus". For the first time, the discovery of the CSF-contacting nucleus provides compelling morphological evidence for the existence of a distinct neural structure within the brain parenchyma that establishes a connection with the cerebrospinal fluid, thereby suggesting its potential significance in facilitating material and information exchange between the brain parenchyma and cerebrospinal fluid. After conducting a comprehensive series of studies on the morphological structure, material expression, gene analysis and functional aspects of the CSF-contacting nucleus in rodents and non-human primates, it has been revealed that there are fibrous connections between the CSF-contacting nucleus and the cerebral cortex and subcortical nuclei being involved in the regulatory mechanisms of pain, cognition, learning and memory, emotion, addiction, stress and anxiety responses, visceral activity, olfaction, vision processing and perception, auditory processing, perception, motor control and coordination, homeostasis regulation including maintenance of body energy and fluid balance, as well as the control of sleep–wake cycles and synchronization of biological rhythms. Current experiments have confirmed that the CSF-contacting nucleus is related to pain, morphine dependence and withdrawal, learning and memory, as well as stress. This present article offers a comprehensive review of the neurobiological characteristics and recent advancements in pain regulation of the CSF-contacting nucleus. The aim is to provide novel insights into the investigation of pain regulation within bidirectional regulatory pathway between the brain and cerebrospinal fluid, with a specific focus on elucidating the role of the CSF-contacting nucleus as a bridge structure. Additionally, the objective of this research is to propose innovative strategies for pain management and associated disorders in the future.

Activation of P2X4 receptors in midbrain cerebrospinal fluid-contacting nucleus leads to mechanical hyperalgesia in chronic constriction injury rats.

Neuropathic pain is a refractory pain state, and its mechanism is still not clear. Previous studies have shown that the purine receptor P2X4R expressed on hyperactive microglia in the spinal cord is essential for the occurrence and development of neuropathic pain. The cerebrospinal fluid-contacting nucleus (CSF-contacting nucleus) in the midbrain has been found to play an important role in the descending inhibition system of modulation. However, there have been no studies on P2X4R in the CSF-contacting nucleus involved in neuropathic pain. To investigate whether P2X4R is expressed in the CSF-contacting nucleus and whether its expression in the CSF-contacting nucleus is involved in the regulation of neuropathic pain, we used a model of chronic sciatic nerve ligation injury (CCI) to simulate neuropathic pain conditions. Immunohistochemistry experiments were conducted to identify the expression of P2X4R in the CSF-contacting nuclei in CCI rats, and western blot analysis showed a significant increase in P2X4R levels 7days after modeling. Then, we packaged a P2rx4 gene-targeting shRNA in scAAV9 to knock down the P2X4R level in the CSF-contacting nucleus, and we found that CCI-induced mechanical hyperalgesia was reversed. In conclusion, P2X4R expressed in the CSF-contacting nucleus is involved in the process of neuropathic pain, and downregulating P2X4R protein in the CSF-contacting nucleus can reverse the occurrence and development of hyperalgesia, which could represent a potent therapeutic strategy for neuropathic pain.

TRPV1 and GABAB1 in the Cerebrospinal Fluid-Contacting Nucleus are Jointly Involved in Chronic Inflammatory Pain in Rats.

To assess the receptors of TRPV1 and GABAB1 receptors that were colocalized in cerebrospinal fluid contacting nucleus (CSF-contact nucleus) of chronic inflammatory pain (CIP) rats bringing inspiration for reducing chronic pain. A rat model of CIP was constructed by plantar injection of complete Freund's adjuvant (CFA), and the paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were measured 1, 3, 5, 7, 10, and 14 days after plantar injection. In the first part of the experiment, rats with CIP were divided into the immunofluorescence group and the coimmunoprecipitation (Co-IP) group (n = 6). Rats in the immunofluorescence group were injected with the retrograde tracer CB conjugated with Alexa Fluor 594 into the lateral ventricle two days before the injection of CFA into the plantar surface of the left paw. Three days later, rats that exhibited hyperalgesia were perfused, and their brains were extracted and used for double immunofluorescence staining of the CSF-contacting nucleus. Rats in the Co-IP group were anesthetized and dissected 3 days after CFA injection, and fresh brain segments containing the CSF-contacting nucleus were collected for Co-IP to assess the colocalization of TRPV1 and GABAB1 in the CSF-contacting nucleus (n = 6). In the second part of the experiment, SD rats were divided into the normal saline group (control group) and the CFA group. Fresh CSF-contacting nucleus-containing tissues were collected for Western blot analysis 3 days after plantar injection to observe the changes in TRPV1 and GABAB1 expression in the CSF-contacting nucleus. TRPV1 and GABAB1 were co-expressed in the CSF-contacting nucleus in rats with CIP, and their expression was upregulated. TRPV1 and GABAB1 in the CSF-contacting nucleus are jointly involved in CIP in rats, and there is a direct or indirect link between TRPV1 and GABAB1.

WITHDRAWN: The cerebrospinal fluid-contacting nucleus contributes to depression- like behaviors via MKP-1 in rats

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CSF-CN contributes to cancer-induced bone pain via the MKP-1-mediated MAPK pathway

Pain is a major complication of cancer and significantly affects the quality of life. Cerebrospinal fluid-contacting nucleus (CSF–CN) has been reported to be involved in the development of neuropathic pain and inflammatory pain. However, whether CSF-CN contributes to cancer-induced bone pain (CIBP) remains unknown. In this study, we aimed to illustrate the role of CSF-CN in the pathogenesis of CIBP and identify its potential mechanism via the MKP-1-mediated MAPK pathway. The Walker 256 cancer cells were injected into the tibia cavity of female Sprague-Dawley rats to induce CIBP models. Intracerebroventricular injection of cholera toxin subunit B– saporin (CB-SAP) was performed to “knockout” the CSF-CN. Morphine and LV-MKP-1 were applied. Mechanical and thermal hyperalgesia behaviors, double immunofluorescence staining and Western blot were conducted after CIBP induction. The results revealed that CIBP significantly reduced the mechanical withdrawal threshold and the thermal threshold. Double immunofluorescence staining revealed that c-Fos-positive neurons in CSF-CN were significantly higher in the CIBP group than that in the sham group. Targeted ablation of CSF-CN dramatically aggravated pain sensitivity. Moreover, MKP-1 was down-regulated in the CSF-CN after CIBP induction. Pharmacological intervention with morphine significantly ameliorated the mechanical and thermal hyperalgesia through reversing the down-expression of MKP-1 in the CSF-CN on day 14 after CIBP induction. Mechanically, overexpression of MKP-1 by LV-MKP-1 injection significantly relieved CIBP via inhibiting the expression of phosphorylated p38, which subsequently decreased the protein levels of Bax, cleaved caspase-3 and Iba-1, and reduced the mRNA levels of IL-1β, TNF-α and IL-6 in CSF-CN. In conclusion, CSF-CN contributed to CIBP via regulating the MKP-1-mediated p38-MAPK pathway. Future therapy targeting the expression of MKP-1 in the CSF-CN may be a promising new choice.

Novel Projections to the Cerebrospinal Fluid-Contacting Nucleus From the Subcortex and Limbic System in Rat.

Objective: To identify the novel projections received by the cerebrospinal fluid (CSF)-contacting nucleus from the subcortex and limbic system to understand the biological functions of the nucleus.Methods: The cholera toxin subunit B (CB), a retrograde tracer, was injected into the CSF-contacting nucleus in Sprague–Dawley rats. After 7–10 days, the surviving rats were perfused, and the whole brain and spinal cord were sliced for CB immunofluorescence detection. The CB-positive neurons in the subcortex and limbic system were observed under a fluorescence microscope, followed by 3D reconstructed with the imaris software.Results: CB-positive neurons were found in the basal forebrain, septum, periventricular organs, preoptic area, and amygdaloid structures. Five functional areas including 46 sub-regions sent projections to the CSF-contacting nucleus. However, the projections had different densities, ranging from sparse to moderate, to dense.Conclusions: According to the projections from the subcortex and limbic system, we hypothesize that the CSF-contacting nucleus participates in emotion, cognition, homeostasis regulation, visceral activity, pain, and addiction. In this study, we illustrate the novel projections from the subcortex and limbic system to the CSF-contacting nucleus, which underlies the diverse and complicated circuits of the nucleus in body regulations.

Monosynaptic Input Mapping of Diencephalic Projections to the Cerebrospinal Fluid-Contacting Nucleus in the Rat.

Objective: To investigate the projections the cerebrospinal fluid-contacting (CSF-contacting) nucleus receives from the diencephalon and to speculate on the functional significance of these connections.Methods: The retrograde tracer cholera toxin B subunit (CB) was injected into the CSF-contacting nucleus in SD rats according to the experimental formula of the stereotaxic coordinates. Animals were perfused 7–10 days after the injection, and the diencephalon was sliced at 40 μm with a freezing microtome. CB-immunofluorescence was performed on all diencephalic sections. The features of CB-positive neuron distribution in the diencephalon were observed with a fluorescence microscope.Results: The retrograde labeled CB-positive neurons were found in the epithalamus, subthalamus, and hypothalamus. Three functional diencephalic areas including 43 sub-regions revealed projections to the CSF-contacting nucleus. The CB-positive neurons were distributed in different density ranges: sparse, moderate, and dense.Conclusion: Based on the connectivity patterns of the CSF-contacting nucleus that receives anatomical inputs from the diencephalon, we preliminarily assume that the CSF-contacting nucleus participates in homeostasis regulation, visceral activity, stress, emotion, pain and addiction, and sleeping and arousal. The present study firstly illustrates the broad projections of the CSF-contacting nucleus from the diencephalon, which implies the complicated functions of the nucleus especially for the unique roles of coordination in neural and body fluids regulations.

Targeted Ablation of Distal Cerebrospinal Fluid-Contacting Nucleus Alleviates Renal Fibrosis in Chronic Kidney Disease.

The potential function of distal cerebrospinal fluid-contacting nucleus (dCSF-CNs) in chronic kidney disease (CKD) development is poorly understood. We hypothesized that dCSF-CNs might affect the renin-angiotensin system (RAS) in kidney injury progression, with dCSF-CNs ablation potentially alleviating local RAS and renal fibrosis in rats after five-sixths nephrectomy (5/6Nx). Part of rats were randomly administered artificial cerebrospinal fluid (aCSF) intracerebroventricularly (icv), followed by 5/6Nx or sham operation; and other part of rats were administered Cholera toxin B subunit conjugated with saporin (CB-SAP) for dCSF-CNs lesion before 5/6Nx. The effect of CB-SAP on dCSF-CNs ablation was confirmed by double immunofluorescence staining. RAS component, NOX2 and c-fos levels in the subfornical organ (SFO), hypothalamic paraventricular nucleus (PVN) and hippocampus, as well as tyrosine hydroxylase (TH) and c-fos positive cells in rostral ventrolateral medulla (RVLM) were assessed. Next, the levels of RAS components (angiotensinogen [AGT], angiotensin-converting enzyme [ACE], Ang II type 1 receptor [AT1R], angiotensin-converting enzyme 2 [ACE2], and Mas receptor), NADPH oxidases (NOX2 and catalase), inflammatory cytokines (monocyte chemotactic protein 1 [MCP-1] and IL-6), and fibrotic factors (fibronectin and collagen I) were assessed. Less CB-labeled neurons were found in dCSF-CNs of CB-SAP-treated rats compared with 5/6Nx animals. Meanwhile, CB-SAP downregulated AGT, Ang II, AT1R, NOX2, catalase, MCP-1, IL-6, fibronectin, and collagen I, and upregulated ACE2 and Mas receptor, compared with CKD rats. More TH and c-fos positive cells were found in RVLM of 5/6Nx rats but the number decreased after dCSF-CNs ablation. Targeted dCSF-CNs ablation could alleviate renal inflammation and fibrosis in chronic kidney injury by inhibiting cerebral and renal RAS/NADPH oxidase.

Activation of P2X3 receptors in the cerebrospinal fluid-contacting nucleus neurons reduces formalin-induced pain behavior via PAG in a rat model

The cerebrospinal fluid (CSF)-contacting nucleus is implicated in the descending inhibitory pathway in pain processing, whereas the cellular and molecular mechanisms underpinning CSF-contacting nucleus regulating pain signals remains largely elusive. ATP is evidenced to inhibit pain transmission at supraspinal level by the mediation of the receptor P2X, wherein its subtype P2X3 is identified as the most potent. Our present experiment investigated the functionality of P2X3 receptors in CSF-contacting nucleus in the formalin-evoked inflammatory pain. Immunofluorescence and western blot revealed the expression of P2X3 receptors in the CSF-contacting nucleus and their upregulated expression subsequent to administration of formalin in rat model. ATP (a P2X3 receptor agonist, 100nmol/5µl) by intracerebroventricular (i.c.v.) administration ameliorated pain behaviors and enhanced c-Fos immunoreactivity in the neurons of the periaqueductal gray (PAG), both of which were discounted by pre-administration of A-317491 (a selective P2X3 receptor antagonist, 25nmol/5µl). After the CSF-contacting nucleus was ablated by cholera toxin subunit B-saporin, ATP failed to induce analgesia, with the c-Fos immunoreactivity in the PAG neurons remaining intact. Our results validated that P2X3 receptors in the CSF-contacting nucleus are pivotal in inflammatory pain processing via the activation of PAG neurons.

Involvement of neurokinin 1 receptor within the cerebrospinal fluid‑contacting nucleus in visceral pain.

Studies have shown that the cerebrospinal fluid‑contacting nucleus (CSF‑CN) may be associated with the transduction and regulation of pain signals. However, the role of the CSF‑CN remains to be elucidated. Emerging evidence has suggested that neurokinin 1 receptor (NK1R) is important in the development of visceral pain and hyperalgesia, however, whether NK1R exists in the CSF‑CN and its exact role in visceral pain remain to be fully elucidated. In the present study, double‑labeled immunofluorescence staining and western blot analysis were performed to investigate this. It was revealed that NK1R was distributed in the CSF‑CN. Following the induction of visceral pain by formalin instillation, NK1R in the CSF‑CN was upregulated. In addition, by observing the behaviors of rats subjected to visceral pain, it was found that visceral pain was relieved by lateral intracerbroventricular injection of the NK1R antagonist, RP67580. These data provided a broader understanding of the role of NK1R in the CSF‑CN and demonstrated that the CSF‑CN was involved in acute visceral pain via the regulation of NK1R.

Cerebrospinal fluid-contacting nucleus mediates nociception via release of fractalkine.

Increasing evidence suggests that the cerebrospinal fluid-contacting nucleus (CSF-contacting nucleus) mediates the transduction and regulation of pain signals. However, the precise molecular mechanisms remain unclear. Studies show that release of fractalkine (FKN) from neurons plays a critical role in nerve injury-related pain. We tested the hypothesis that release of FKN from the CSF-contacting nucleus regulates neuropathic pain, in a chronic constriction injury rat model. The results show that FKN is expressed by neurons, via expression of its only receptor CX3CR1 in the microglia. The levels of soluble FKN (sFKN) were markedly upregulated along with the increase in FKN mRNA level in rats subjected to chronic constriction injury. In addition, injection of FKN-neutralizing antibody into the lateral ventricle alleviated neuropathic pain-related behavior followed by reduction in microglial activation in the CSF-contacting nucleus. The results indicate that inhibition of FKN release by the CSF-contacting nucleus may ameliorate neuropathic pain clinically.

Role of the RVM in Descending Pain Regulation Originating from the Cerebrospinal Fluid-Contacting Nucleus.

Evidence has suggested that cerebrospinal fluid-contacting nucleus (CSF-contacting nucleus) is correlated with the development and recurrence of pain. A recent research showed that the CSF-contacting nucleus acts as a component of the descending 5-hydroxytryptamine (5-HT) system and plays a role in descending pain inhibition. However, limited studies are conducted to investigate the relationship between the CSF-contacting nucleus and pain. In present study, we explored the effect of CSF-contacting nucleus on nociceptive behaviors in both normal and neuropathic rats via targeted ablation of the CSF-contacting nucleus in the brainstem, using cholera toxin subunit B-saporin (CB-SAP), a cytotoxin coupled to cholera toxin subunit B. The CB-SAP-treated rats showed aggravated thermal hyperalgesia and mechanical allodynia. Also, results from immunohistochemical experiments showed that rostral ventromedial medulla (RVM) received fiber projection from the CSF-contacting nucleus, which disappeared after ablation of the CSF-contacting nucleus, and the CB-SAP treated rats showed downregulation of c-Fos expression in the RVM as compared with the rats receiving i.c.v. injection of phosphate buffer saline (PBS). A significant downregulation of 5-HT-labeled neurons and tryptophan hydroxylase 2 (TPH2) as the marker of 5-HT cells in the RVM, and 5-HT expression in spinal dorsal horn in both normal and chronic constriction injury (CCI) rats after i.c.v. injection of CB-SAP was observed. These results suggested that RVM may be involved in descending pain modulation originating from the CSF-contacting nucleus.

Extracellular Signal-Regulated Kinase 5 in the Cerebrospinal Fluid-Contacting Nucleus Contributes to Neuropathic Pain in Rats

Background: The activation of mitogen-activated protein kinases (MAPKs) have been observed in synaptic plasticity processes of learning and memory in neuropathic pain. Cerebrospinal fluidcontacting nucleus (CSF-CN) has been identified with the onset and persistence of neuropathic pain. However, whether extracellular signal-regulated protein kinase 5 (ERK5), a member of MAPKs, in CSF-CN participates in neuropathic pain has not been studied yet. Objective: The aim of the present study was to identify the role of ERK5 in CSF-CN on the formation and development of neuropathic pain, and to investigate its possible mechanism. Study Design: Controlled animal study. Setting: University laboratory. Methods: After a chronic constriction injury (CCI) model was produced, BIX02188 was dissolved in 1% DMSO and injected into the lateral ventricles LV in a volume of 3 μl with different doses (0.1 μg, 1 μg, 10 μg). Mechanical allodynia and thermal hypersensitivity behavioral test, immunofluorescence, and western blot technique were used in this research. Result: Following CCI, mechanical allodynia and thermal hypersensitivity were developed within a day, peaked at 14 days, and persisted for 21 days. ERK5 was remarkably activated by CCI in CSFCN. Moreover, selective inhibiting of p-ERK5 expression in CSF-CN by BIX02188 could significantly relieve CCI-induced mechanical allodynia and thermal hypersensitivity, accompanying with the decreased phosphorylation of cAMP response-element binding protein (CREB) in CSF-CN. Limitations: More underlying mechanism(s) of the role of ERK5 in CSF-CN on the formation and development of neuropathic pain will be needed to explore in future research. Conclusion: These findings suggest activation of ERK5 in CSF-CN might contribute to the onset and development of neuropathic pain and its role might be partly accomplished by p-CREB. Key words: Neuropathic pain, extracellular signal-regulated kinase 5, distal cerebrospinal fluidcontacting neurons, cerebrospinal fluid-contacting nucleus, chronic constriction injury, cAMP response-element binding protein

Role of cerebrospinal fluid-contacting nucleus in sodium sensing and sodium appetite

The brainstem plays an important role in controlling sodium and water homeostasis. It is a major regulatory site for autonomic and motor functions. Moreover, it integrates cerebrospinal fluid (CSF) signals with neuronal and hormonal signals. Evidence suggests that the CSF-contacting nucleus (CSF-CN) transmits and integrates CSF signals, but, the definitive role of CSF-CN in sodium homeostasis is poorly understood. In this study, we used c-Fos as a marker of neuronal activity and causing colocalization of Nax channel and 5-HT. This proved that CSF-CN played a role in sensing the increase of CSF sodium level. Then, we determined the role of the CSF-contacting nucleus in increasing the sodium appetite of rats. So, we performed targeted lesion of the CSF-contacting nucleus in the brainstem using the cholera toxin subunit B-saporin (CB-SAP), a cytotoxin coupled to cholera toxin subunit B. The lesion of the CSF-CN showed decreased and degenerative neurons, while sodium appetite have increased and Fos immunocytochemistry detected neuronal activity in the lateral parabrachial nucleus (LPBN), but not in the subfornical organ (SFO) and organum vasculosum of the lamina terminalis (OVLT). These results indicate that the CSF-CN plays an important role in sensing CSF sodium level and satiating sodium appetite by influencing the LPBN but not SFO and OVLT. The Nax channel and 5-HT might be the molecular mechanisms through which contribute to sodium homeostasis.

Role of adrenomedullin in the cerebrospinal fluid-contacting nucleus in the modulation of immobilization stress

The contribution of the cerebrospinal fluid-contacting nucleus (CSF-contacting nucleus) and adrenomedullin (ADM) to the developmental modulation of stressful events remains controversial. This study explored the effects of endogenous ADM in the CSF-contacting nucleus on immobilization of stress-induced physiological parameter disorders and glucocorticoid hormone releasing hormone (CRH), rat plasma corticosterone expression, and verification of such effects by artificially lowering ADM expression in the CSF-contacting nucleus by targeted ablation of the nucleus. Immunohistochemical experiments showed that ADM-like immunoreactivity and the calcitonin receptor-like receptor (CRLR) marker were localized in the CSF-contacting nucleus. After 7 continuous days of chronic immobilization stress (CIS), animals exhibited anxiety-like behavior. Also, an increase in serum corticosterone, and enhanced expression of ADM in the CSF-contacting nucleus were observed, following activation by CIS. The intracerebroventricular (i.c.v.) administration of the ADM receptor antagonist AM22–52 significantly reduced ADM in the CSF-contacting nucleus, additionally, blocked the effects of ADM, meaning the expression of CRH in the hypothalamic paraventricular nucleus (Pa) and serum corticosterone level were increased, and the physiological parameters of the rats became correspondingly deteriorated. Additionally, the i.c.v. administration of cholera toxin subunit B-saporin (CB-SAP), a cytotoxin coupled to a cholera toxin subunit, completely eliminated the CSF-contacting nucleus, worsening the reaction of the body to CIS. The collective results demonstrated that ADM acted as a stress-related peptide in the CSF-contacting nucleus, and its lower expression and blocked effects in the nucleus contributed to the deterioration of stress-induced physiologic parameter disorders as well as the excessive expressions of stress-related hormones which were part of the hypothalamic–pituitary–adrenal (HPA) axis.

MTOR and Erk1/2 Signaling in the Cerebrospinal Fluid-Contacting Nucleus is Involved in Neuropathic Pain.

The cerebrospinal fluid-contacting nucleus (CSF-CN) has been demonstrated to be involved in neuropathic pain, but the underlying molecular mechanisms remain unclear. Previous work has shown that mTOR and ERK1/2 are important signaling pathways regulating neuropathic pain. However, studies on the interactions between these major pathways in neuropathic pain are very rare. Therefore, the purpose of this study is to determine whether mTOR and ERK1/2 exist in the CSF-CN and elucidate their alterations in neuropathic pain, especially, the crosstalk between them. Our results showed that mTOR and ERK1/2 were distributed in the CSF-CN, and their expression levels were increased in chronic constriction injury (CCI)-induced neuropathic pain. Furthermore, the injection of both the mTOR antagonist rapamycin and the ERK1/2 antagonist U0126 into the lateral ventricle of the brain attenuated CCI-induced neuropathic pain. Inhibition of the ERK1/2 pathway had little impact on mTOR signaling, but inhibition of the mTOR pathway significantly increased ERK/2 signaling. The coadministration of rapamycin and U0126 inhibited the rapamycin-induced upregulation of ERK, and had a greater effect on pain behaviors than did the single-drug administrations. These data extend our understanding of the relationship between mTOR and ERK in the supraspinal site and demonstrate that the CSF-CN participates in neuropathic pain via the regulation of mTOR and ERK1/2.

Role of the cerebrospinal fluid-contacting nucleus in the descending inhibition of spinal pain transmission

The brainstem is well recognized as a critical site for integrating descending modulatory systems that both inhibit and facilitate pain at the level of the spinal cord. The cerebrospinal fluid-contacting nucleus (CSF-contacting nucleus) distributes and localizes in the ventral periaqueductal central gray of the brainstem. Although emerging lines of evidence suggest that the CSF-contacting nucleus may be closely linked to transduction and regulation of pain signals, the definitive role of the CSF-contacting nucleus in pain modulation remains poorly understood. In the present study, we determined the role of the CSF-contacting nucleus in rat nocifensive behaviors after persistent pain by targeted ablation of the CSF-contacting nucleus in the brainstem using the cholera toxin subunit B–saporin (CB–SAP), a cytotoxin coupled to cholera toxin subunit B. Compared with CB/SAP, CB–SAP induced complete ablation of the CSF-contacting nucleus, and the CB–SAP-treated rats showed hypersensitivity in responses to acute nociceptive stimulation, and exacerbated spontaneous nocifensive responses induced by formalin, thermal hyperalgesia and mechanical allodynia induced by plantar incision. Furthermore, immunohistochemical experiments showed that the CSF-contacting nucleus was a cluster of 5-HT-containing neurons in the brainstem, and the spinal projection of serotonergic axons originating from the CSF-contacting nucleus constituted the descending 5-HT pathway to the spinal cord. CB–SAP induced significant downregulation of 5-HT in the spinal dorsal horn, and intrathecal injection of 5-HT significantly reversed hypersensitivity in responses to acute nociceptive stimulation in the CB–SAP-treated rats. These results indicate that the CSF-contacting nucleus 5-HT pathway is an important component of the endogenous descending inhibitory system in the control of spinal nociceptive transmission.

Involvement of Wnt5a within the cerebrospinal fluid-contacting nucleus in nerve injury-induced neuropathic pain

Studies have demonstrated that the cerebrospinal fluid-contacting nucleus (CSF-CN) is involved in neuropathic pain, but the underlying molecular mechanisms still largely remain obscure. Emerging evidence suggests that spinal Wnt5a plays a crucial role in regulation of chronic pain. However, little is known about the potential role of the supraspinal Wnt5a in the development of chronic pain. To investigate whether Wnt5a exists in the CSF-CN and its role in neuropathic pain, double-labeled immunofluorescence staining was used to identify the expression of Wnt5a in the CSF-CN and western blot analysis of the CSF-CN was employed to verify the alteration of Wnt5a protein in the process of neuropathic pain. In the present study, we demonstrated that Wnt5a is distributed in the CSF-CN and the Wnt5a protein was up-regulated by nerve injury-induced nociceptive stimuli. Furthermore, lateral intracerebroventricular injection of Wnt5a antagonist Box5 attenuated the chronic constriction injury (CCI)-induced neuropathic pain and down-regulated the expression of Wnt5a in the CSF-CN. These data extend our understanding of the role of Wnt5a in supraspinal site and demonstrate that the CSF-CN participates in nerve injury-induced neuropathic pain via the regulation of Wnt5a.

Role of cerebrospinal fluid-contacting nuclei in maintenance of chronic itch in rats

Objective To evaluate the role of cerebrospinal fluid-contacting nuclei (CSF-CN) in maintenance of chronic itch in rats.Methods Forty-two adult male Sprague-Dawley rats,aged 3 months,weighing 240-280 g,were used in the study.The experiment was performed in two parts.Part Ⅰ Forty-two Sprague-Dawley rats were randomly divided into 3 groups (n =14 each):control group (C group),acetone group (A group) and oxazolone group (O group).0.5％ oxazolone 15 μl was applied to the neck and back of rats in group O,while the equal volume of normal saline and acetone was applied in groups C and A,respectively.Application of the drug mentioned above was repeated on day 7,9,13,16,17,18,21 and 23 after the first stimulation in each rat in each group.Scratching behaviors were oberserved within 30 min after each stimulaiton.Six rats in each group were chosen and sacrificed after the last application of oxazolone,and the brains were obtained for determination of c-Fos expression in CSF-CN.Part Ⅱ Twenty-four rats were randomly divided into 3 groups (n =8 each):control group (C1 group),chronic itch group (group CI),and chronic itch + lesion group (CI + KA group).Chronic itch was induced by repeated application of oxazolone as previously described in CI and CI + KA groups.The chemical lesion of CSF-CN was performed at 6 h after 8th application of the drug.Then the scratching behaviors were observed within 30 min after 9th application of the drug.Results Part Ⅰ Compared with C group,the scratching behaviors were increased significantly at T4-8 in A group,and at T1-8 in O group (P ＜ 0.05),and the expression of c-Fos was up-regulated in O and A groups (P ＜ 0.05).Compared with A group,the scratching behaviors were increased significantly at T1-8 and the expression of c-Fos was up-regulated in O group (P ＜ 0.05).Part Ⅱ Compared with C1 group,the scratching behaviors were significantly increased in CI and CI + KA groups (P ＜ 0.05).The scratching behaviors were significantly reduced in CI + KA group compared with CI group (P ＜ 0.05).Conclusion CSF-CN is involved in the maintenance of chronic itch in rats. Key words: Neurons; Pruritus; Cerebrospinal fluid-contacting nucleus

Extracellular Signal-Regulated Kinase 5 in the Cerebrospinal Fluid-Contacting Nucleus Contributes to Morphine Physical Dependence in Rats

The cerebrospinal fluid-contacting nucleus (CSF-CN) may influence actual composition of the CSF for non-synaptic signal transmission via releasing or absorbing bioactive substances, which distributes and localizes in the ventral periaqueductal central gray of the brainstem. Previous studies demonstrated that CSF-CN was involved in neuropathic pain and morphine dependence. Thus, to identify whether extracellular signal-regulated kinase 5 (ERK5) distributed in the CSF-CN and its function on the formation and development of morphine physical dependence, morphine withdrawal-like behavioral test and immunofluorescent technique were used in this research. Morphine was subcutaneously injected by an intermittent and escalating procedure to induce physical dependence, which was measured by withdrawal symptoms. In this study, we found that horseradish peroxidase-conjugated toxin subunit B/p-ERK5 double-labeled neurons expressed in the CSF-CN of normal rats. ERK5 signaling pathway was remarkably activated by naloxone-precipitated withdrawal in the CSF-CN. Moreover, selective attenuation of p-ERK5 expression in the CSF-CN by lateral ventricle injection of BIX02188 could significantly relieve morphine withdrawal symptom. These findings confirmed that the activation of p-ERK5 in the CSF-CN might contribute to morphine physical dependence.