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The Korean Journal of Pain logoLink to The Korean Journal of Pain
. 2026 Oct 1;39(4):506–520. doi: 10.3344/kjp.26029

The cerebrospinal fluid-contacting nucleus CGRP-RAMP1-PKA signaling pathway mediates trigeminal neuralgia and its mechanism

Bin Gui 1,2, Yang Bai 1,2, Yijun Zhang 1,2, Weiwei Yan 1,2, Zhi Yang 1,2, Xinling Wang 1,2, Jin Qian 1,2, Yu Peng 1,2, Licai Zhang 1,2,✉
PMCID: PMC13628115  PMID: 42687474

Abstract

Background

Trigeminal neuralgia (TN) is prevalent form of neuropathic pain (NP). Its regulatory circuits and mechanisms remain unclear. The authors’ previous work demonstrated that the cerebrospinal fluid-contacting nucleus (CSF-contacting nucleus) participates in the regulation of NP induced by sciatic nerve injury via the brain-CSF circuit. However, whether it mediates NP caused by trigeminal nerve injury has not been reported.

Methods

This study used viral retrograde tracing to examine neural projections between the CSF-contacting nucleus and the trigeminal ganglion (TG). Neuronal activity in TN model was assessed using immunofluorescence, and chemogenetic tools were used to test its role in pain behavior. Local calcitonin gene-related peptide (CGRP)-RAMP1-PKA signaling changes and their modulation were analyzed for effects on TN.

Results

Retrograde tracing confirmed neural projections between the CSF-contacting nucleus and the TG. In TN model rats, elevated c-Fos expression indicated its activation. Chemogenetic activation induced hyperalgesia in normal rats, whereas its inhibition reduced pain in TN rats. Molecular analyses revealed upregulation of CGRP, RAMP1, and components of the PKA pathway within the nucleus. Pathway inhibition via RAMP1 knockdown or olcegepant administration attenuated pain, with knockdown effect being reversible by cAMP.

Conclusions

This study reveals that the CSF-contacting nucleus is activated during TN and participates in central pain regulation through its local CGRP-RAMP1-PKA signaling pathway. Intervening in this pathway effectively modulates pain behavior, suggesting that this nucleus and its signaling system may represent novel therapeutic targets for treating TN. These findings provide experimental evidence for understanding the central mechanisms of TN and developing analgesic strategies.

Keywords: Calcitonin Gene-Related Peptide, Cerebrospinal Fluid, Cyclic AMP-Dependent Protein Kinases, Olcegepant, Receptor Activity-Modifying Protein 1, Trigeminal Ganglion, Trigeminal Neuralgia

INTRODUCTION

Trigeminal neuralgia (TN) is a common neuropathic pain (NP) disorder. Patients typically experience paroxysmal, electric shock-like severe pain in the trigeminal nerve distribution area, significantly impairing quality of life [1,2]. Research indicates that calcitonin gene-related peptide (CGRP) is highly expressed in the trigeminal ganglion (TG) during TN [3] and may play a key role in the initiation and sensitization of the condition [4-8]. However, the specific circuitry involved and the mechanisms of sensitization remain unclear. Consequently, clinically effective treatments for TN remain elusive.

The cerebrospinal fluid-contacting nucleus (CSF-contacting nucleus) is a unique brain nucleus first discovered and named by the authors’ research group [9]. Its existence has been confirmed in rat, mouse, and non-human primate macaque brains [10,11]. Its cell bodies reside in the brain parenchyma at the midbrain-pons junction. The nucleus receives extensive neural projections from the cerebral cortex [12], limbic system [13], diencephalon [14], brainstem, and spinal cord [15]. Its terminals extend into the CSF. This establishes a crucial pathway for substance transport, information transmission, and functional regulation between the brain and CSF [10,11]. The entire nervous system is immersed in CSF. Life activities and diseases mediated through this pathway are exceptionally diverse. The authors’ previous studies indicate that the CSF-contacting nucleus participates in regulating NP in spinal nerves induced by sciatic nerve transection [16-18]. However, no research has yet reported whether this nucleus mediates the regulation of TN.

This study aims to investigate whether the CSF-contacting nucleus participates in the pathological process of TN. It also aims to elucidate whether it mediates pain regulation through the CGRP-RAMP1-PKA signaling pathway. Using Sprague–Dawley (SD) rats as subjects, the authors examined the neural connectivity between the CSF-contacting nucleus and the TG via retrograde tracing. Immunofluorescence double-labeling was used to observe c-Fos proto-oncogene expression in CSF-contacting nucleus neurons of TN rats. Chemogenetic targeting of the CSF-contacting nucleus was used to assess its impact on rat TN behavior. Multiple modern molecular biology techniques were combined with quantitative behavioral methods. Using these, the authors investigated the expression of the CGRP-RAMP1-PKA signaling pathway, which mediates pain in the CSF-contacting nucleus of TN rats and the impact of inhibiting these signaling molecules on TN behavior. Expected outcomes will provide novel evidence for the CSF-contacting nucleus CGRP-RAMP1-PKA signaling pathway mediating TN. The study will reveal a novel pathway for CSF-contacting nucleus-mediated TN via the brain-CSF circuit. It will open new perspectives on neurohumoral pathways mediating pain, and offer fresh insights for the clinical diagnosis and treatment of NP.

MATERIALS AND METHODS

1. Animals

Male specific pathogen-free SD rats weighing 200–250 g were obtained from the Laboratory Animal Center of Xuzhou Medical University. The 192 rats were used in this study. All animal procedures adhered to the protocol reviewed and approved by the Xuzhou Medical University Laboratory Animal Ethics Committee (no. 202403T024). Animals were housed in a controlled environment at 22°C–25°C under a 12-hour light-dark cycle. They had free access to food and water.

2. TN model construction

Male SD rats were anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and subjected to chronic constriction injury of the infraorbital nerve (ION-CCI) [19]. After disinfection, a longitudinal skin incision of approximately 1 cm in length was made approximately 0.6 cm lateral to the nose and directly below the zygomatic arch. The subcutaneous tissues were bluntly dissected to fully expose the infraorbital nerve. Two sterile 5-0 chromic gut sutures, spaced approximately 2 mm apart, were each loosely tied around the nerve to achieve constriction, ensuring the narrowing of the nerve while maintaining patent epineurial blood flow and partial preservation of nerve conduction function. The incision was subsequently closed in layers under aseptic conditions. No antibiotics were administered postoperatively to avoid potential interference with the pain model. In the sham-operated (SHAM) group, animals underwent nerve exposure alone without ligation; the control group received no surgical intervention.

3. Pain behavioral assessment

The mechanical withdrawal threshold (MWT) was assessed using seven graded Von Frey filaments (Stoelting) with bending forces of 0.4, 0.6, 1.0, 1.4, 2.0, 4.0, and 6.0 g. Rats were acclimated in test cages for 10 minutes in a quiet environment. Each filament was then applied perpendicularly to the ipsilateral whisker pad area 5 times (2–3 seconds each, 15 seconds apart) in ascending order. A response was considered positive if any of the following reactions occurred: (1) rapid head withdrawal or avoidance of the stimulus; (2) attempted biting of the filament; or (3) three or more consecutive grooming strokes of the stimulated area with the forepaw [19]. The minimum stimulus intensity that elicited a positive response was recorded as the mechanical pain threshold at that time point and used for statistical analysis.

Facial grooming frequency was assessed by placing rats individually in transparent observation cages (20 × 10 × 13 cm) and continuously videotaping them during a 10-minute period of free activity using the Smarter video system (RWD Life Science), with recordings analyzed blindly by an observer unaware of the experimental group assignments [20].

4. LV and CSF-contacting nucleus stereotaxic localization

Lateral ventricle (LV) localization: stereotaxic coordinates were obtained from the Paxinos rat brain atlas, with the bregma as the origin. The injection coordinates for the rat LV were 1.0 ± 0.1 mm anteroposterior (AP), 1.6 ± 0.1 mm mediolateral (ML), and 4.0 ± 0.2 mm dorsoventral (DV).

CSF-contacting nucleus localization: referencing literature [10], coordinates derived from the formula were 8.24 ± 0.18 mm AP, 0.09 ± 0.01 mm mL, and 6.45 ± 0.11 mm DV.

5. CSF-contacting nucleus and TG labeling

Two µL of cholera toxin B subunit (CB)-Alexa Fluor 594 (C34777; Thermo Fisher) was injected into the LV using a microinjector (Hamilton) to label CSF-contacting nucleus.

Five µL of PRV-CAG-EGFP (BC-PRV-531-PLUS; Braincase) was injected into the CSF-contacting nucleus using a microinjector to retrograde label the TG. Tissue samples were collected on day 10 post-injection to allow for optimal viral expression and tracing.

6. Chemogenetic activation and inhibition of CSF-contacting nucleus

Principle of specificity: due to the physical barrier formed by the LV wall, only the CSF-contacting nucleus with axon terminals extending into CSF can absorb the virus, while other non-CSF-contacting nuclei within the brain cannot take it up.

Activation group: 300 nL of rAAV2/9-hSyn-DIO-hM3Dq-EGFP (BC-0144; Braincase) was injected into CSF-contacting nucleus using a microinjector.

Inhibition group: 300 nL of rAAV2/9-hSyn-DIO-hM4Di-mCherry (BC-0153; Braincase) was injected into the CSF-contacting nucleus using a microinjector.

Control group: 300 nL of rAAV2/9-hSyn-DIO-EGFP (BC-0244; Braincase) was injected into the CSF-contacting nucleus using a microinjector.

For all groups, 700 nL of rAAV-hSyn-SV40NLS-Cre (BC-0159; Braincase) was injected into the LV using a microinjector. Clozapine-N-oxide (CNO) (GC10822; GlpBio) was first dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution at a concentration of 3.33 mg/mL. This stock was subsequently diluted with phosphate-buffered saline to obtain the working solution at a concentration of 0.333 mg/mL (final DMSO concentration: 5%). CNO was administered intraperitoneally at a dose of 3.33 mg/kg (injection volume: 10 mL/kg) 1.5 hours before behavioral testing on day 14 post-injection.

7. Immunofluorescence

Following the extraction of brain tissue and TG, specimens underwent fixation with 4% paraformaldehyde, sucrose gradient dehydration, and embedding. Coronal sections (40 µm) were prepared using a Leica CM1900 cryostat microtome (Leica Microsystems). Sections were washed, permeabilized, and blocked in 10% donkey serum (Jackson) before incubation with primary antibodies: rabbit anti-RAMP1 antibody (ER62486, 1:500; HUABIO) and rabbit anti-c-Fos antibody (2250S, 1:400; Cell Signaling Technology) to bind target antigens. Secondary antibody: donkey anti-rabbit Alexa Fluor 488 (A-21206, 1:500; Thermo Fisher) was used for staining. Finally, images were acquired using an FV1000 laser confocal fluorescence microscope (Olympus) and quantified with ImageJ software.

8. Western blotting

After anesthesia, the rat brain was rapidly removed. Based on the authors’ previously established stereotaxic coordinates for the CSF-contacting nucleus, the brain was placed in a rat brain mold, and coronal slices were sectioned. Under a microscope, the CSF-contacting nucleus region was isolated. All samples were immediately frozen in liquid nitrogen and stored at −80°C until further processing. After brain tissue collection, samples were lysed using RIPA (P0013; Beyotime) buffer supplemented with PMSF (ST506; Beyotime). The lysates were then centrifuged (15 minutes, 12,000 rpm) to collect proteins, and the concentration was measured using the BCA kit (P0012; Beyotime). Following 12.5% SDS-PAGE electrophoresis and polyvinylidene fluoride membrane transfer, membranes were blocked with 5% nonfat milk and incubated with the following primary antibodies: rabbit anti-CGRP (HA722991, 1:1000; HUABIO), rabbit anti-RAMP1 (ER62486, 1:1000; HUABIO), rabbit anti-p-PKA (HA721864, 1:1000; HUABIO), mouse anti-GAPDH (60004-1-Ig, 1:10000; Proteintech), or rabbit anti-β-Tubulin (80713-1-RR, 1:10000; Proteintech) to detect target proteins. Due to the close molecular weights of p-PKA (40 kDa) and GAPDH (36 kDa), which precluded clear separation on the same membrane, β-tubulin (55 kDa) was uniformly used as the loading control for all target proteins in subsequent experiments, with the exception of the RAMP1 detection (where GAPDH was initially used), as shown elsewhere in this paper. All target proteins were detected under identical experimental conditions using the same batch of tissue samples. Secondary antibodies: horseradish peroxidase-labeled goat anti-rabbit or goat anti-mouse (A0208, A0216, 1:5000; Beyotime) were used to detect the primary antibodies. Images were captured using the Uvitech ECL imaging system (Alliance Q9 Advanced), and gray values were analyzed with ImageJ software.

9. RT-qPCR

Quantitative polymerase chain reaction (qPCR) sample collection followed the same procedure as described in the western blotting section. Total RNA was extracted from brain tissue using a rapid extraction kit (R451-50; GeneBetter). Total RNA was reverse-transcribed into cDNA using HiScript II Q RT SuperMix for qPCR (R223-01; Vazyme). Subsequent qPCR reactions were performed on a QuantStudio 7 Flex Real-Time Fluorescent Quantitative PCR System (Thermo Fisher) using 2X Universal SYBR Green Fast qPCR Mix (RK21203; ABclonal), with GAPDH (B662204; Sangon Biotech) as the internal reference gene. Gene expression levels were calculated using the 2−ΔΔCt method. All qPCR primer sequences are listed in Table 1.

Table 1.

Primers sequence for qRT-PCR

Gene Sequences
CGRP F 5’-CAGTGAAGAAGAAGCTCGCCTAC-3’
R 5’-GCCCTCAGCCTCCTGTTCC-3’
RAMP1 F 5’-CATTGCTGTCCACCACCGCTAC-3’
R 5’-GGAGCACAATGAAAGGGCAGAGG-3’
PKA F 5’-GGGTTCGCCAAGAGAGTCAA-3’
R 5’-TCAGTACACCCAATGCCCAC-3’
GAPDH F 5’-TGATGGGTGTGAACCACGAG-3’
R 5’-AGTGATGGCATGGACTGTGG-3’
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qRT-PCR: quantitative reverse transcription polymerase chain reaction, CGRP: calcitonin gene-related peptide.

10. Intervention of the CGRP-RAMP1-PKA signaling pathway

1) CGRP inhibition

Olcegepant (HY-10095; MedChemExpress) at 0.5 mg/µL was injected into the CSF-contacting nucleus via a cannula (RWD Life Science) to inhibit CGRP signaling.

2) RAMP1 silencing

Silencing group: 400 nL of rAAV-EF1α-DIO-EGFP-5'miR-30a-shRNA(rRAMP1)-3'miR-30a (BC-5275; Braincase) was injected into the CSF-contacting nucleus using a microinjector.

Control group: 400 nL of rAAV-EF1α-DIO-EGFP-5'miR-30a-shRNA(Scramble)-3'miR-30a (BC-1088; Braincase) was injected into the CSF-contacting nucleus using a microinjector.

For all groups, 700 nL of rAAV-hSyn-SV40NLS-Cre (BC-0159; Braincase) was injected into the LV using a microinjector.

3) PKA signaling activation

To activate PKA signaling, 1.25 mg/µL 8-Bromo-cAMP (HY-12306A; MedChemExpress) was injected through the cannula into the CSF-contacting nucleus.

11. Statistical analysis

All data are expressed as mean ± standard error of the mean. Statistical analysis was performed using GraphPad Prism 9.5.1 software. Comparisons between groups were conducted using independent samples t-tests (for two groups) or one-way/two-way analysis of variance (ANOVA) (for multiple groups). For behavioral data collected over multiple time points, two-way repeated measures ANOVA was used, followed by Tukey’s post hoc test for multiple comparisons. For other comparisons involving more than two groups, one-way ANOVA followed by Tukey’s post hoc test was applied. In all statistical tests, P < 0.05 was considered statistically significant.

RESULTS

1. Fiber projections exist from the CSF-contacting nucleus to the TG

Peripheral nerve tracer pseudorabies virus (PRV) was injected into the CSF-contacting nucleus (Fig. 1A). Clear positive labeling of neurons was observed in both the CSF-contacting nucleus and TG of the experimental animals (Fig. 1B, C). This suggests the existence of neural pathways connecting the TG to the CSF-contacting nucleus.

Fig. 1.

Fig. 1

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CSF-contacting nucleus and TG nerve projections. (A) Schematic diagram and injection site of tracer into the CSF-contacting nucleus. (B) Expression of tracer virus PRV within CSF-contacting nucleus (n = 4). (C) Retrograde labeling of tracer virus PRV in TG (n = 4). Scale bar: 100 μm. CSF-contacting nucleus: cerebrospinal fluid-contacting nucleus, TG: trigeminal ganglion, PRV: pseudorabies virus, LV: lateral ventricle, Aq: aqueduct.

2. During TN, neurons in the CSF-contacting nucleus are significantly activated

To label the CSF-contacting nucleus, CB (red) was injected into the LV, and the TN model was established using the ION-CCI method (Fig. 2A, B). MWT was assessed using Von Frey filaments in control (CON), SHAM, and TN rat groups over 0–21 days (P < 0.001, Fig. 2C). An observer blinded to experimental groupings analyzed video recordings of facial grooming frequency indicating pain-related responses in animals from days 0–21 (P < 0.001, Fig. 2D). Immunofluorescence double labeling visualized c-Fos/CB expression in the CSF-contacting nucleus of rats in the CON, SHAM, and TN groups (Fig. 2E). Statistical analysis of CSF-contacting nucleus c-Fos/CB expression was performed via one-way ANOVA (P < 0.001, Fig. 2F). TN rats exhibited significantly activated CSF-contacting nucleus neurons with markedly increased c-Fos expression, showing statistically significant differences compared to CON and SHAM groups.

Fig. 2.

Fig. 2

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Increased c-Fos expression in the CSF-contacting nucleus of TN rats. (A) Schematic diagram and injection site of CB into the LV. (B) Schematic diagram of TN model establishment. (C) Significant differences in MWT between the TN group and CON/SHAM groups, manifesting as hyperalgesia (n = 8). (D) Significant differences in grooming behavior between the TN group and CON/SHAM groups, with markedly increased grooming frequency (n = 8). (E) c-Fos expression in the CSF-contacting nucleus across all three groups (n = 5). Scale bar: 100 μm (immunofluorescence). (F) Quantitative analysis of c-Fos expression in the CSF-contacting nucleus shows significantly higher expression in the TN group compared to the CON and SHAM groups (n = 5). Data are presented as mean ± standard error of the mean. CSF-contacting nucleus: cerebrospinal fluid-contacting nucleus, CB: cholera toxin B subunit, LV: lateral ventricle, Aq: aqueduct, ION-CCI: chronic constriction injury of the infraorbital nerve, TN: trigeminal neuralgia, MWT: mechanical withdrawal threshold, CON: control, SHAM: sham-operated. **P < 0.01, ***P < 0.001, ns indicates no significant difference.

3. Chemogenetic targeting of CSF-contacting nucleus alters behavioral changes in TN Rat

Adeno-associated viruses carrying Cre and DIO were injected into the LV and CSF-contacting nucleus of the test animals, respectively. The CSF-contacting nucleus was activated using hM3Dq-EGFP and inhibited using hM4Di-mCherry (Fig. 3A). One and a half hours prior to behavioral testing, CNO was administered via intraperitoneal injection to chemogenically modulate the CSF-contacting nucleus via the Cre-loxP system (Fig. 3B). Using this strategy, the authors successfully achieved viral transfection and labeling of the CSF-contacting nucleus (Fig. 3C, D). Changes in MWT and statistical analysis were performed in normal rats following CSF-contacting nucleus activation (P < 0.01, Fig. 3E). Inhibition of the CSF-contacting nucleus alters the MWT in TN rats (P < 0.01, Fig. 3F). Activation of the CSF-contacting nucleus in normal rats induces hyperalgesia, while inhibition in TN rats significantly alleviates pain.

Fig. 3.

Fig. 3

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Chemogenetic regulation of CSF-contacting nucleus on MWT in TN rats. (A) Schematic of adeno-associated virus injection strategy: DIO-dependent virus injected into the CSF-contacting nucleus, Cre retrograde virus injected into the LV. (B) Timeline of chemogenetic modulation of the CSF-contacting nucleus and TN behavioral testing. (C) Expression of agonist-carrying adenovirus (hM3Dq-EGFP) in the CSF-contacting nucleus. (D) Expression of inhibitor-carrying adenovirus (hM4Di-mCherry) in the CSF-contacting nucleus. Scale bar: 100 μm. (E) Chemogenetic-specific modulation of the CSF-contacting nucleus in normal rats. Compared to the other two groups, the SHAM + hM3Dq group shows significantly reduced MWT and induced hyperalgesia (n = 8). (F) Chemogenetic modulation of the CSF-contacting nucleus in TN rats. Compared to the other three groups, the TN + hM4Di group shows a significant increase in MWT and pain relief (n = 8). Data are presented as mean ± standard error of the mean. CSF-contacting nucleus: cerebrospinal fluid-contacting nucleus, LV: lateral ventricle, Aq: aqueduct, CNO: clozapin-N-oxide, MWT: mechanical withdrawal threshold, TN: trigeminal neuralgia, SHAM: sham-operated. *P < 0.05, **P < 0.01, ***P < 0.001, ns indicates no significant difference.

4. Upregulation of CSF-contacting nucleus CGRP-RAMP1-PKA signaling molecule expression in TN rats

Based on chemogenetic evidence implicating the CSF-contacting nucleus in TN-related pain regulation, the authors proceeded to examine its molecular mechanisms. Previous studies on CGRP signaling in TN have primarily focused on the TG. However, their prior work has clearly demonstrated the involvement of the CSF-contacting nucleus in the pathological process of TN. To determine whether this nucleus regulates TN via the CGRP-RAMP1-PKA pathway, the nucleus was first labeled with CB (red) via LV injection, followed by immunofluorescence detection of RAMP1 (green). Results confirmed RAMP1 expression within the CSF-contacting nucleus, with significantly higher levels in TN rats compared to controls (P < 0.001, Fig. 4A, B). Western blot analysis further revealed that protein levels of CGRP (P < 0.01), RAMP1 (P < 0.001), and p-PKA (P < 0.001) in the CSF-contacting nucleus were significantly upregulated in TN rats compared to SHAM rats (Fig. 4C–F), suggesting that the TN surgery activated a local CGRP-RAMP1-PKA signaling cascade in this region. Furthermore, qPCR analysis also demonstrated significantly increased mRNA expression of CGRP, RAMP1, and PKA in TN rats (P < 0.01, Fig. 4G–I). These findings further support the notion that the CSF-contacting nucleus participates in TN through the CGRP-RAMP1-PKA pathway.

Fig. 4.

Fig. 4

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Expression of CSF-contacting nucleus CGRP-RAMP1-PKA signaling molecules in TN rats. (A) Representative immunofluorescence image showing RAMP1 expression within the CSF-contacting nucleus region. Scale bar: 100 μm. (B) Compared to the SHAM group, RAMP1 immunofluorescence intensity is significantly increased in the CSF-contacting nucleus region of the TN group (n = 4). (C–F) Compared to the SHAM group, protein expression levels of CGRP, RAMP1, and p-PKA are upregulated in the CSF-contacting nucleus of TN rats (n = 6). (G–I) mRNA expression levels of CGRP, RAMP1, and PKA are upregulated in the CSF-contacting nucleus of TN rats (n = 4). Data are presented as mean ± standard error of the mean. CSF-contacting nucleus: cerebrospinal fluid-contacting nucleus, CB: cholera toxin B subunit, Aq: aqueduct, CGRP: calcitonin gene-related peptide, TN: trigeminal neuralgia, SHAM: sham-operated. **P < 0.01, ***P < 0.001.

5. Knocking down RAMP1 within CSF-contacting nucleus alleviates TN, and this effect can be reversed by the PKA activator cAMP

To investigate whether downregulating RAMP1 expression in the CSF-contacting nucleus alleviates TN, this study employed a virus-mediated gene knockdown strategy. A dual viral injection procedure was performed: first, rAAV-hSyn-SV40NLS-Cre virus was injected into the LV, while simultaneously injecting RAMP1 knockdown (shRNA) or Scramble vectors into the CSF-contacting nucleus (Fig. 5A). Three weeks later, successful viral transduction within the target nuclei was confirmed by EGFP expression assessment (Fig. 5B, C). Behavioral results demonstrated that RAMP1 knockdown significantly alleviated TN-induced mechanical hyperalgesia compared to the control group (P < 0.001, Fig. 5D). Concurrently, RAMP1 knockdown effectively suppressed the TN-induced elevation of RAMP1 at both mRNA and protein levels, as well as the increase in PKA mRNA and p-PKA protein levels (P < 0.01, P < 0.001, Fig. 5E–I). To further validate these findings, a cannula was implanted into the CSF-contacting nucleus on day 3 post-TN surgery. Following recovery and adaptation, the PKA activator cAMP was administered via the cannula starting on day 7. Results demonstrated that cAMP administration significantly reversed the analgesic effect induced by RAMP1 knockdown (P < 0.001, Fig. 5D). Subsequent qPCR and western blot analyses revealed that RAMP1 knockdown suppressed not only its own expression but also PKA transcription (mRNA) and PKA activity (p-PKA levels). Notably, cAMP treatment restored PKA mRNA expression and p-PKA levels even when RAMP1 remained downregulated (P = 0.020, P < 0.001, Fig. 5E–I). This molecular trend aligned with the behavioral reversal, further emphasizing the critical role of the CGRP-RAMP1-PKA pathway in pain regulation mediated by the CSF-contacting nucleus. Importantly, neither viral nor pharmacological manipulations affected motor function in rats (Table 2), supporting the specific role of reduced RAMP1 expression within the CSF-contacting nucleus in alleviating TN.

Fig. 5.

Fig. 5

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Downregulating RAMP1 in CSF-contacting nucleus alleviates TN, and this effect can be reversed by PKA activators. (A) Schematic diagram of experimental design and timeline. (B, C) Representative fluorescence images confirming viral expression within the CSF-contacting nucleus. Scale bar: 100 μm. (D) Suppression of RAMP1 upregulation in the CSF-contacting nucleus of TN rats effectively alleviates pain behavior; however, subsequent administration of cAMP reverses this analgesic effect and restores hyperalgesia (n = 6). (E, F) Knockdown of RAMP1, a key component of the CGRP pathway in the CSF-contacting nucleus of TN rats, significantly downregulates its mRNA expression, whereas subsequent administration of cAMP reverses the decrease in PKA mRNA levels (n = 4). (G–I) Knockdown of RAMP1, a key component of the CGRP pathway in the CSF-contacting nucleus of TN rats, results in significant downregulation of its protein expression, while subsequent administration of cAMP reverses the decrease in PKA protein levels (n = 4). Data are presented as mean ± standard error of the mean. CSF-contacting nucleus: cerebrospinal fluid-contacting nucleus, LV: lateral ventricle, Aq: aqueduct, MWT: mechanical withdrawal threshold, TN: trigeminal neuralgia, CGRP: calcitonin gene-related peptide, SHAM: sham-operated. *P < 0.05, **P < 0.01, ***P < 0.001, ns indicates no significant difference.

Table 2.

Test of locomotor function post-injection of manipulation tools

Treatment group Placing Grasping Righting
SHAM + rAAV-CRE + rAAV-EGFP 5 (0) 5 (0) 5 (0)
SHAM + rAAV-CRE + rAAV-hM3Dq 5 (0) 5 (0) 5 (0)
SHAM + rAAV-CRE + rAAV-hM4Di 5 (0) 5 (0) 5 (0)
TN + rAAV-CRE + rAAV-EGFP 5 (0) 5 (0) 5 (0)
TN + rAAV-CRE + rAAV-hM3Dq 5 (0) 5 (0) 5 (0)
TN + rAAV-CRE + rAAV-hM4Di 5 (0) 5 (0) 5 (0)
TN + Scramble 5 (0) 5 (0) 5 (0)
TN + shRNA 5 (0) 5 (0) 5 (0)
TN + shRNA + Veh 5 (0) 5 (0) 5 (0)
TN + shRNA + cAMP 5 (0) 5 (0) 5 (0)
TN + Veh 5 (0) 5 (0) 5 (0)
TN + Olc 5 (0) 5 (0) 5 (0)
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Scores for placing, grasping, and righting reflexes were based on counts of each normal reflex exhibited in five trials. Values are presented as mean (standard error of the mean); n = 6 rats/group.

SHAM: sham-operated, TN: trigeminal neuralgia.

6. Specific blockade of CGRP within the CSF-contacting nucleus alleviates TN

To investigate how CGRP in the CSF-contacting nucleus participates in the activation of the CGRP-RAMP1-PKA pathway under TN conditions, this study administered the CGRP-specific antagonist olcegepant via local injection into this nucleus. On day 10 after TN modeling, a cannula was implanted into the CSF-contacting nucleus. Following a 4-day recovery period, olcegepant was administered via the cannula on day 14 (Fig. 6A). Behaviorally, the olcegepant-treated group exhibited significantly reduced pain-related responses compared to controls (P < 0.01, Fig. 6B). Western blot analysis revealed that olcegepant markedly decreased RAMP1 and p-PKA protein levels in the CSF-contacting nucleus of TN rats (P < 0.001, Fig. 6C–E). In addition, mRNA analysis revealed downregulated transcriptional expression of RAMP1 and PKA (P < 0.01, P < 0.001, Fig. 6F, G). Collectively, these data demonstrate that local inhibition of CGRP within CSF-contacting nucleus blocks the CGRP-RAMP1-PKA signaling cascade, thereby attenuating TN-associated pain.

Fig. 6.

Fig. 6

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Blocking CGRP within the CSF-contacting nucleus alleviates pain. (A) Experimental schematic and timeline. (B) Rats exhibited significantly reduced pain following olcegepant injection into the CSF-contacting nucleus (n = 6). (C–E) Protein levels of RAMP1 and PKA in the CSF-contacting nucleus are significantly reduced after olcegepant administration (n = 6). (F, G) mRNA levels of RAMP1 and PKA are reduced in the CSF-contacting nucleus after olcegepant treatment (n = 4). Data are presented as mean ± standard error of the mean. CSF-contacting nucleus: cerebrospinal fluid-contacting nucleus, TN: trigeminal neuralgia, MWT: mechanical withdrawal threshold, CGRP: calcitonin gene-related peptide, SHAM: sham-operated. **P < 0.01, ***P < 0.001, ns indicates no significant difference.

DISCUSSION

TN is a neuropathic disorder that severely impairs patients’ quality of life, and current pharmacological and surgical treatments often fail to provide adequate long-term relief due to limited efficacy or adverse effects [21–23]. Central sensitization plays a key role in the pathogenesis and maintenance of TN [24,25]. As a central nucleus associated with the CSF, the CSF-contacting nucleus may be involved in central pain regulation. Its unique brain-CSF interface may therefore offer a novel perspective for investigating these mechanisms. The present study indicates that the CSF-contacting nucleus contributes to TN through activation of the local CGRP-RAMP1-PKA signaling pathway.

The ION-CCI model is a commonly used model to simulate the clinical symptoms of TN, as it reproduces central sensitization and associated alterations in nociceptive pathways. Previous studies have demonstrated that ION-CCI induces sustained mechanical hyperalgesia in the ipsilateral facial region [19]. The CSF-contacting nucleus has been shown to participate in inflammatory pain [26], visceral pain [27], and NP [28–30], suggesting its close association with central pain regulatory mechanisms. In this study, using PRV trans-synaptic tracing, the authors observed labeled neurons in the TG following tracer injection into the nucleus, indicating a neural pathway connection between the CSF-contacting nucleus and the TG. Combined with their previous finding of projections from the spinal trigeminal nucleus (Sp5) to the CSF-contacting nucleus [15], these observations suggest that this nucleus may serve as a node linking peripheral signal afferents to central sensitization in TN. As a key relay station for orofacial nociception, Sp5 receives inputs from the TG and sends fibers to the parabrachial nucleus (PB), constituting an important pathway for TN pain transmission. Recent studies have further clarified that nociceptive information originating from the TG can be transmitted via Sp5 to the PB, thereby influencing the activity of the central amygdala and participating in the emotional and autonomic responses to pain [31,32]. Notably, the authors’ previous research also suggested that the CSF-contacting nucleus has fiber connections with the PB and amygdala [13,15], indicating that this nucleus may participate in this neural network, receiving pain signals through interactions with these nuclei. In the present study, upregulation of the neuronal activation marker c-Fos and chemogenetic manipulation further revealed that the CSF-contacting nucleus not only receives nociceptive information but may also actively participate in the transmission and modulation of pain signals.

CGRP is an important neuropeptide involved in nociceptive signaling [33], playing a role in transmitting peripheral noxious signals to the spinal cord. A key component of the canonical CGRP receptor complex, RAMP1, is present in spinal cord tissue, where the CGRP-RAMP1 axis participates in the fine-tuning of pain sensitivity [34,35]. Recent evidence suggests that this pathway is also highly active in the TG, contributing to the processing of mechanical pain [36,37]. Baggio et al. [38] demonstrated that in the peripheral TG, the CGRP-RAMP1-PKA axis drives migraine-associated hyperalgesia by activating CaV3.2 calcium channels, revealing its mechanism in peripheral pain sensitization. Furthermore, Yin et al. [39] discovered in skin fibroblasts that RAMP1 overexpression alone activates downstream transcription factor YAP via the Gαi3-PKA-CREB axis, promoting cell proliferation. This suggests the pathway functions as a core signaling axis across multiple systems. However, the function of this signaling cascade within the brain during TN remains unclear. Targeting RAMP1 or PKA within the CSF-contacting nucleus may offer novel therapeutic strategies for TN. The authors’ prior studies have confirmed associations between the CSF-contacting nucleus and multiple pain-related mediators, including substance P and TRPV1 [40,41]. In this study, using gene knockdown and pharmacological blockade approaches, it was found that downregulation of RAMP1, PKA mRNA, and p-PKA expression significantly alleviated hyperalgesia in TN rats, indicating that the CGRP-RAMP1-PKA signaling pathway is involved in the pathological process of TN. Specifically, CGRP reduced pain thresholds in rats by enhancing the phosphorylation levels of key proteins in the RAMP1-PKA pathway. Both RAMP1 knockdown and local injection of the CGRP-specific antagonist olcegepant effectively blocked downstream signaling of this pathway and alleviated pain [42], suggesting that local CGRP signaling and its binding to the receptor complex may be a key initiating step in pathway activation. Mechanistically, peripheral nociceptive input activates neurons in the CSF-contacting nucleus, promoting local CGRP synthesis and release. Released CGRP acts in an autocrine or paracrine manner on the CGRP receptor complex (CALCRL/RAMP1), potentially activating adenylyl cyclase via Gs protein and elevating the intracellular second messenger cAMP [43]. cAMP-dependent PKA phosphorylation is significantly enhanced during NP [44], and activation of p-PKA may amplify and sustain pain signals by enhancing neuronal excitability or modulating phosphorylation of downstream targets. Inhibition of each component of this pathway attenuated pain, whereas administration of the PKA activator cAMP reversed the analgesic effect induced by RAMP1 knockdown, providing further support for the possible involvement of this pathway in the pathological process of TN.

CSF-contacting nucleus neurons are not only capable of sensing the chemical environment of the CSF, but also express various neurotransmitter receptors and possess the function of secreting neuropeptides into the CSF, indicating a bidirectional and dynamic interaction with the CSF [45–49]. This suggests that exogenous drugs, once they enter the CSF, could potentially be detected by these neurons. Therefore, the CSF may serve both as a conduit for delivering therapeutic agents to this deep brain structure and as a medium for its output signals. Based on this, it was hypothesized that administering drugs via the CSF pathway (e.g., intrathecal injection of olcegepant) could offer a novel strategy for pain treatment. Furthermore, if capable of crossing the blood-CSF barrier, systemic delivery of olcegepant might also reach this nucleus through the CSF route. These questions require further investigation to clarify the feasibility and mechanisms of such administration routes.

This study has several limitations that warrant consideration. First, although PRV trans-synaptic tracing revealed labeled neurons in the TG following tracer injection into the CSF-contacting nucleus, the authors did not comprehensively investigate whether other nuclei might be involved in signal transmission between these two structures. Identifying potential intermediate nuclei within the TG and CSF-contacting nucleus pathway would contribute to a more complete understanding of the anatomical basis of TN and warrants further investigation. Second, while the authors confirmed the activation of the CGRP-RAMP1-PKA pathway in the CSF-contacting nucleus under TN conditions, their molecular analysis focused primarily on PKA mRNA expression and p-PKA protein levels. Previous studies have shown that [50], in experimental pain models, phosphorylated PKA can activate downstream effector molecules. However, the specific downstream effectors of this pathway and their role in TN remain to be determined. Furthermore, to avoid potential confounding effects of the estrous cycle, only male rats were used in this study. Previous studies have demonstrated sex differences in the clinical manifestations and pathological mechanisms of TN [36], and the role of the CGRP-RAMP1-PKA signaling pathway within the CSF-contacting nucleus in pain modulation in females requires further investigation.

In summary, these findings indicate that the CSF-contacting nucleus regulates TN through the CGRP-RAMP1-PKA pathway, providing a basis for understanding central sensitization and guiding targeted therapeutic development.

ACKNOWLEDGMENTS

We are grateful to Xuzhou Medical University and to all those who contributed to this study.

Footnotes

DATA AVAILABILITY

All data are included in this article, with additional datasets available from the corresponding author upon reasonable request post-publication.

CONFLICT OF INTEREST

No potential conflict of interest relevant to this article was reported.

FUNDING

This research is supported by the National Natural Science Foundation of China and the Original Exploration Program recommended by experts (Grant No. 82550903).

AUTHOR CONTRIBUTIONS

Bin Gui: Formal analysis, Investigation, Data curation, Writing/manuscript preparation; Yang Bai: Formal analysis, Investigation; Yijun Zhang: Investigation; Weiwei Yan: Study conception, Data curation; Zhi Yang: Investigation; Xinling Wang: Investigation; Jin Qian: Investigation; Yu Peng: Investigation; Licai Zhang: Study conception, Writing/manuscript preparation, Critical review, commentary or revision, Supervision, Project administration, Funding acquisition.

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