PACAP/PAC1-R activation contributes to hyperalgesia in 6-OHDA-induced Parkinson’s disease model rats via promoting excitatory synaptic transmission of spinal dorsal horn neurons

Abstract

Pain is a common annoying non-motor symptom in Parkinson’s disease (PD) that causes distress to patients. Treatment for PD pain remains a big challenge, as its underlying mechanisms are elusive. Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor PAC1-R play important roles in regulating a variety of pathophysiological processes. In this study, we investigated whether PACAP/PAC1-R signaling was involved in the mechanisms of PD pain. 6-hydroxydopamine (6-OHDA)-induced PD model was established in rats. Behavioral tests, electrophysiological and Western blotting analysis were conducted 3 weeks later. We found that 6-OHDA rats had significantly lower mechanical paw withdrawal 50% threshold in von Frey filament test and shorter tail flick latency, while mRNA levels of Pacap and Adcyap1r1 (gene encoding PAC1-R) in the spinal dorsal horn were significantly upregulated. Whole-cell recordings from coronal spinal cord slices at L4–L6 revealed that the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in dorsal horn neurons was significantly increased, which was reversed by application of a PAC1-R antagonist PACAP 6–38 (250 nM). Furthermore, we demonstrated that intrathecal microinjection of PACAP 6–38 (0.125, 0.5, 2 μg) dose-dependently ameliorated the mechanical and thermal hyperalgesia in 6-OHDA rats. Inhibition of PACAP/PAC1-R signaling significantly suppressed the activation of Ca²⁺/calmodulin-dependent protein kinase II and extracellular signal-regulated kinase (ERK) in spinal dorsal horn of 6-OHDA rats. Microinjection of pAAV-Adcyap1r1 into L4–L6 spinal dorsal horn alleviated hyperalgesia in 6-OHDA rats. Intrathecal microinjection of ERK antagonist PD98059 (10 μg) significantly alleviated hyperalgesia in 6-OHDA rats associated with the inhibition of sEPSCs in dorsal horn neurons. In addition, we found that serum PACAP-38 concentration was significantly increased in PD patients with pain, and positively correlated with numerical rating scale score. In conclusion, activation of PACAP/PAC1-R induces the development of PD pain and targeting PACAP/PAC1-R is an alternative strategy for treating PD pain.

Introduction

Parkinson’s disease (PD) is one of the most common neurodegenerative movement disorders [1]. However, the characteristics of PD are diverse and complex. Hallmark feature of PD involves bradykinesia, resting tremor, rigidity, and postural instability; all of which belong to motor symptoms [2]. Not only that, PD also has nonmotor symptoms such as pain, depression, constipation, and sleep disorders, which affect quality of life [2]. Notably, pain is experienced by 30%–85% of patients with PD, and up to 42% had moderate to severe pain [3]. However, PD pain has been largely neglected and effective treatments are limited, with a critical factor being that the molecular-level mechanisms of PD pain remain elusive [4]. Although previous studies have shown that neuropeptides can be involved in the modulation of PD pain [5–7], unfortunately, the mechanisms of neuropeptide modulation of PD pain at the spinal cord level remain largely unknown.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a widely expressed neuropeptide encoded by Adcyap1 gene. It is highly conserved in evolution, and belongs to the family of vasoactive intestinal polypeptide (VIP)/glucagon/secretin. PACAP exhibits two biologically active forms of PACAP-27 and PACAP-38, with PACAP-38 being the predominant [8]. The distribution of PACAP is widespread and is characterized by pleiotropic effects, indicating that it plays an essential role in the regulation of biological functions. PACAP binds selectively to PAC1 receptor (PAC1-R), which shows high affinity for PACAP (K_d~0.5 nM) but low affinity for VIP (K_d~500 nM) [9, 10]. PACAP^−/− mice showed that acute visceral nociception was significantly attenuated and they did not exhibit neuropathic or inflammatory pain [11–13]. The reaction of the PAC1^−/− mice to the stimulus of chronic inflammatory pain was about 75% lower than that of wild-type mice [14]. PACAP/PAC1-R signaling plays significant roles in many pathophysiological processes such as migraine, emotion responses, nociceptive transmission, fear responses, regeneration, and immunity [15–19]. However, whether PACAP/PAC1-R is involved in the molecular mechanisms of PD pain remains unknown.

Activation of PACAP/PAC1-R regulates neuronal hyperexcitability in the hippocampus [20, 21]. Moreover, PACAP/PAC1-R plays a key role in the promotion and maintenance of neuropathic pain in the spinal cord and dorsal root ganglion [11, 22]. However, the PACAP/PAC1-R mechanism underlying the regulation of central sensitization in the spinal dorsal horn that mediates PD pain is still unknown.

In this study, we investigated whether PACAP/PAC1-R was involved in hyperalgesia in a rat model of 6-hydroxydopamine (6-OHDA)-induced PD [23–25]. We examined the expression of PACAP and PAC1-R, as well as the subsequent signals. We further investigated the role of PACAP/PAC1-R in regulating hyperalgesia and excitatory synaptic transmission in the spinal cord. In addition, a cross-sectional study was performed to explore the relationship between serum PACAP-38 levels and pain in patients with PD. In conclusion, our results indicate that the PACAP/PAC1-R signaling pathway at the spinal cord level plays an important role in the development of PD pain and may serve as a potential target for treatment.

Materials and methods

Animals

Adult male Sprague–Dawley (SD) rats, age 6–8 weeks, body weight 180–250 g, were purchased from Slaccas Laboratory (Shanghai, China). They were housed (2–5 rats per cage) at a constant temperature of 20–26 °C, 40%–70% relative humidity, and a 12-h light–dark cycle. The rats had free access to water and food. All experimental procedures were conducted in strict followed the principles of Laboratory Animal Care and Use, and followed the guidelines of the International Association for the Study of Pain, and were approved by the Institutional Animal Care and Use Committee of Soochow University (Suzhou, China).

PD rat model induction

Bilateral lesions of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) were induced by 6-OHDA. In order to protect the noradrenergic nerve terminals, rats were intraperitoneally (i.p.) administered desipramine (20 mg/kg, Sigma–Aldrich, St. Louis, MO, USA) 30 min prior to surgery. Rats were deeply anesthetized with 3% isoflurane (R510–22; RWD, Shenzhen, China) induction and maintained with 1.5% isoflurane. Rats were placed in a stereotaxic apparatus (RWD) for the injection of 6-OHDA or vehicle. Two micro-craniotomies were performed above the bilateral SNpc. The coordinates for the SNpc were 5.3 mm posterior of the bregma, ±1.8 mm lateral to the midline, and 7.5 mm deep relative to the bregma. Four microliters of 6-OHDA (Sigma–Aldrich, St. Louis, MO, USA) (2 µg/µL, dissolved in 0.2 mg/mL ascorbic acid in saline) was injected at a rate of 0.5 µL/min in two deposits using a 30-gauge needle Hamilton syringe that was held for 4 min. Sham rats underwent the same surgical procedure and received only the vehicle (4 µL of 0.2 mg/mL ascorbic acid in saline) at the same coordinates. Postsurgical care (food and water placed inside the cage for 1 weeks) was carried out similarly in the 6-OHDA-lesioned rats and sham rats. The experiments were carried out 3 weeks after modeling.

Rotarod test

Motor coordination was evaluated on a Rotarod (ZH-300; Zhenghua, China). Rats underwent training for 3 days, under an accelerating protocol from 4 to 40 rpm in 5 min. On 4 days, during the formal experiment, the experimental rats underwent the same protocol three times (being allowed to rest for at least 30 min between adjacent tests), and the latency to fall was recorded.

Open field test

To assess locomotor activity, the rats were tested in an open field uncovered box (100 cm × 100 cm × 40 cm) for 300 s. The experimental rats were gently placed in the behavioral test room to adapt to the environment 30 min before the formal experiment. The rats were tested one by one, and the bottom and inner wall of the open field area were sprayed with 75% ethanol and wiped dry before the next test. Total distance traveled and locomotor tracks were continuously recorded using a tracking system (ANY-maze; Stoelting, IL, USA) and data were automatically transmitted for further analysis.

von Frey filament test

The mechanical pain threshold of acclimated rats was measured with the up-down method, using von Frey filaments (Aesthesio; DanMic Global, San Jose, CA, USA). The first von Frey filament (10 g) was applied to the plantar surface of the left hind paw. If a withdrawal response was marked with X, the filament with the adjacent lower force was used. On the contrary, if the filament failed to elicit a withdrawal response, it was marked with O, and the adjacent higher filament force was applied. This paradigm continued until XOXOXO was recorded. The 50% paw withdrawal threshold was determined as described previously [26]: 50% threshold, g = (10^[Xf+kδ])∕10,000. The last filament applied in the XO series was referred to as X_f in the equation and was inserted as either the handle number or the log (g, target force). When the log (g, target force) was inserted as X_f in the formula, the division by 10,000 was omitted.

Tail flick test

The thermal pain threshold of the acclimated rats was measured using a radiant heat tail flick apparatus (Tail-flick Analgesia Meter, ZH-YLS-12A; Zhenghua, China). The radiant heat source irradiated the tail of the experimental animal 1–3 cm from the end. The cutoff time of heating was set to 15 s to prevent tail damage. Tail flick latency was recorded. Each rat underwent three tests at 30-s intervals. All of the above behavioral tests were conducted in a blinded manner.

Western blotting

The spinal dorsal horns of L4–L5 segments were removed, conducted in ice-cold 0.01 M phosphate-buffered saline (PBS), from deeply anesthetized rats, frozen in liquid nitrogen, and stored at −80 °C. Spinal dorsal horn tissues were homogenized by sonication in RIPA buffer (Beyotime Biotechnology, Shanghai, China) containing protease inhibitor (MedChemExpress, Shanghai, China) and phosphatase inhibitor (MedChemExpress) cocktail tablets. The homogenates were placed on ice for 20 min and centrifuged at 12,000 r/min for 20 min at 4 °C, and the supernatant was retained for protein detection. The protein concentration of the samples was determined by BCA protein assay kit (Thermo Scientific, Waltham, MA, USA). Samples containing equal amounts of protein were heated at 95 °C for 10 min in 1 × loading buffer before electrophoretic separation on 10%–12% SDS-PAGE, and transferred to 0.2-μm polyvinylidene fluoride membranes in an ice-water bath. The membranes were blocked with Tris–HCl buffer (TBST; 0.05 M Tris–HCl, 0.15 M NaCl, 0.05% Tween-20, pH 7.4) containing 5% nonfat powered milk at room temperature for 2 h. Membranes were incubated with primary antibody overnight on a table concentrator at 4 °C. Primary antibodies were: mouse anti-PACAP (1:1000, sc-166180; Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-PAC1-R (1:1000, sc-100315; Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-tyrosine hydroxylase (TH) (1:2500, T1299; Sigma–Aldrich, MO, USA), rabbit anti-p-ERK (1:2000, 4370; CST, Danvers, MA, USA), rabbit anti-ERK (1:1000, 4695; CST, MA, USA), rabbit anti-p-CaMKII (Thr286) (1:1000, 12716; CST, MA, USA), mouse anti-CaMKII (6G9) (1:1000, 50046; CST, MA, USA), rabbit anti-p-P38 (Thr180/Tyr182) (1:1000, 4511; CST, MA, USA), rabbit anti-P38 (D13E1) (1:1000, 8690; CST, MA, USA), anti-β-actin (1:5000, A3854; Sigma–Aldrich, MO, USA), mouse anti-β-tubulin (1:2000, T0198; Sigma–Aldrich, MO, USA), and mouse anti-GAPDH (1:5000, AC002; ABclonal Technology, Woburn, MA, USA). Membranes were washed with TBST buffer (3 × 10 min), and incubated with appropriate horseradish-peroxidase-conjugated secondary anti-mouse/rabbit IgG (1:5000; Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature. After membranes were washed with TBST buffer (3 × 10 min), the bands were visualized using electrochemiluminescence, and the gray values of blots were analyzed using Image J software (National Institutes of Health, Bethesda, MD, USA).

Quantitative real-time polymerase chain reaction (qPCR)

Total RNA was extracted from the spinal dorsal horn of the L4–L6 segment using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized using a reverse transcription kit (Thermo Fisher Scientific, MA, USA). qPCR was performed with SYBR-Green master mix (Thermo Fisher Scientific, MA, USA) and a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). GAPDH served as a house-keeping gene. qPCR specific primers were synthesized by RIBOBIO (Guangzhou, China) and the sequences are listed in Table 1. Relative mRNA expression levels of target genes were calculated using the 2^−ΔΔCT method.

Table 1.

Primer sequences for real-time qPCR.

Gene	Sequence
Pacap	forward 5′-ATGACCATGTGTAGCGGAGC-3′
	reverse 5′-CCGTCCTGATCGTAAGCCTC-3′
Adcyap1r1	forward 5′-CTGGAGAAGGTTCTAGTTGGTATGA-3′
	reverse 5′-ACTGGTCCAAGCCACTAAATGTT-3′
Vip	forward 5′-CACGCCCTATTATGATGTGTCC-3′
	reverse 5′-GATTCGTTTGCCAATGAGTGAC-3′
Vipr1	forward 5′-CGCTCATCTACACACGCCA-3′
	reverse 5′-CGGGAAAGGATACCCACTCTC-3′
Vipr2	forward 5′-AGTACAAGAGGCTCGCCAAGT-3′
	reverse 5′-GGAAGGAACCAACACATAACTCA-3′
Gapdh	forward 5′-GAACGGGAAGCTCACTGG-3′
	reverse 5′-GCCTGCTTCACCACCTTCT-3′

Measurement of PACAP-38

Concentrations of PACAP-38 were measured by ELISA (human, orb564381; rat, orb567582; Biorbyt, Cambridge, UK). ELISA was performed with a standard curve in each experiment.

Drug administration

For behavioral experiments, we purchased PACAP 1–38 (HY-P0221A, MedChemExpress), PACAP 6–38 (3236, Tocris Bioscience, Bristol, UK), PD98059 (P215, Sigma–Aldrich, MO, USA). The rats were anesthetized with isoflurane delivered intrathecally (i.t.) into the L4–L5 cerebrospinal fluid (CSF) via a microsyringe. The drug was slowly injected and the needle was held for 30 s. The swing of the rat’s tail during i.t. injection was the sign of successful administration. The dose of the drugs used in our study was determined based on previous research [16, 27, 28].

Patch-clamp recordings of spinal cord slices

Slice preparation

Patch-clamp recordings of spinal cord slices were performed as described previously [24, 29]. Rats were anesthetized with isoflurane and perfused with ice-cold sucrose-based artificial CSF, containing: 95 mM NaCl, 1.8 mM KCl, 0.5 mM CaCl₂, 1.2 mM KH₂PO₄, 7 mM MgSO₄, 26 mM NaHCO₃, 50 mM sucrose, 15 mM glucose, pH 7.2–7.4 (osmolarity adjusted to 310–320 mOsm with sucrose). The spinal cord segment containing L4–L6 was removed quickly and fixed in a 3% agar block. The agar block was placed in 31 °C Krebs solution containing: 95 mM NaCl, 1.8 mM KCl, 0.5 mM CaCl₂, 1.2 mM KH₂PO₄, 7 mM MgSO₄, 26 mM NaHCO₃, 50 mM sucrose, 15 mM glucose, pH 7.2–7.4 (osmolarity adjusted to 310–320 mOsm with sucrose) preoxygenated (95% O₂, 5% CO₂). The agar block was fixed on the stage of a vibrating microtome (VT1200S; Leica, Wetzlar, Germany). Coronal slices (300 μm in thickness) of spinal cord at L4–L6 were processed while the spinal cord was immersed in cold Krebs solution. Slices were transferred to an oxygenated Krebs solution and incubated for 1 h at 31 °C.

Electrophysiological recordings

After incubation for 1 h, one of the slices was transferred to the recording groove, and fixed with a nylon mesh in U-shape. During recordings, the recording chamber liquid uniform continuous perfusion with oxygenated artificial CSF, and the flow rate was set at 1.5 mL/min at room temperature. Whole-cell patch-clamp recordings were performed on neurons in lamina II of the spinal dorsal horn. Neurons were visualized using infrared differential interference contrast video microscopy with a water-immersion objective (40 × magnification, BX51WI; Olympus, Tokyo, Japan). Patch-clamp electrodes (5–10 MΩ tip resistance) were pulled using a P-97 puller (Sutter Instruments, Novato, CA, USA). Images of neurons recorded in the spinal cord slices reinforced with a CCD camera and were distinguished clearly on a computer displayer. The tip of the patch electrode was lowered slowly to the surface of the slice by a micromanipulator. Recordings were performed at a holding potential of −70 mV. For recording spontaneous excitatory postsynaptic currents (sEPSCs), the composition of the perfusing solution was as follows: 127 mM NaCl, 2.4 mM CaCl₂, 1.8 mM KCl, 1.3 mM MgSO₄, 1.2 mM KH₂PO₄, 26 mM NaHCO₃ and 15 mM glucose, pH 7.2–7.3. The composition of the internal solution in the electrodes was as follows: 8 mM NaCl,133 mM K-gluconate, 0.6 mM EGTA, 2 mM Mg-ATP, 8 mM NaCl, 0.3 mM Na-GTP, 10 mM HEPES, pH 7.2–7.3. Differentiation of sEPSCs from noise levels is based on the principle of selecting waveforms as “fast down-slow up”. For corresponding experiments, PACAP 6–38 (250 nM) and PD98059 (10 μM) were dissolved in artificial CSF and administered via perfusion. The concentrations of the drugs used in our study are based on previous studies [30, 31]. Data were filtered and sampled at 5 kHz with a Bessel filter amplifier, collected by Digi-data 1440A interface, MultiClamp 700B amplifier, and pClamp10 software (Molecular Devices, CA, USA).

Immunofluorescence

For immunofluorescent labeling, rats were anesthetized and transcardially perfused with ice-cold saline followed by 4% paraformaldehyde. The segments containing L4–L6 spinal cord were removed and post-fixed in 4% paraformaldehyde for 4 h, and dehydration was performed in a series of 10%–30% sucrose solutions. A series of spinal cord slices (20 µm) were processed using a cryostat (CM3050S; Leica, Wetzlar, Germany). After blocking with PBS containing 2% bovine serum albumin and 0.3% Triton X-100 for 2 h at room temperature, the slices were incubated with primary antibodies, including rabbit anti-PAC1-R (1:500, ab28670; Abcam, Cambridge, UK), mouse anti-NeuN (1:500, MAB377, Chemicon, Germany) at 4 °C overnight. After washing, the slices were incubated with species-appropriate secondary antibodies tagged with Alexa 488 (green) and Alexa 555 (red) (1:1000, Vector Laboratories, Newark, CA, USA) for 2 h at room temperature. After washing and airing, the slices were covered with 4′, 6-diamidino-2-phenylindole dihydrochloride (Vector Laboratories), which stained the nuclei. Images were captured using fluorescence microscopy (LSM 780; Carl Zeiss, Jena, Germany).

Adeno-associated virus generation and injection

For PAC1-R knockdown experiments, short hairpin RNA (shRNA) targeting Adcyap1r1 (gene of PAC1-R) was constructed and inserted in an adeno-associated virus (AAV) vector carrying green fluorescent protein (Obio Technology, Shanghai, China), sequence as follows: pAAV2/9-hSyn-EGFP-3 × Flag-miR30shRNA-(Adcyap1r1)-WPRE for the knockdown group and pAAV2/9-hSyn-MCS-EGFP-3 × Flag-miR30 for the scramble group. This shRNA specifically transduced neurons of rat and downregulated expression of PAC1-R.

For virus microinjection, rats were anesthetized with isoflurane and placed on a stereotactic frame. The rats were microinjected with 600 nL of virus pAAV2/9-hSyn-EGFP-miR30shRNA (Adcyap1r1) (3.69 × 10¹³ vg/mL) or scramble (3.12 × 10¹³ vg/mL) for each point into the bilateral spinal dorsal horn of L4–L6 (two points per side) with a microsyringe fitted with a glass electrode that was made using a P-97 puller (Sutter Instruments) at a slow rate (100 nL/min). The microsyringe was held for 5 min and withdrawn slowly to ensure that the drug did not reflux.

Participants

Patients with PD were from the Center of Parkinson’s Disease of the Affiliated Hospital of Xuzhou Medical University, Xuzhou, China. All patients were diagnosed by two experienced neurologists according to the criteria of International Parkinson and Movement Disorder Society (MDS) [32]. Exclusion criteria were: (1) other neurological disease, e.g., severe head trauma, cerebral infarction, Alzheimer’s disease, or epilepsy; and (2) severe hearing or visual loss, or history of peripheral neuropathy, severe osteoarthritis, or psychiatric disease. Sixty healthy, age- and gender-matched volunteer controls without a family history of PD were enrolled. Written informed consent was obtained from all participants. The research protocol was approved by the Ethics Committee of the Affiliated Hospital of Xuzhou Medical University (XYFY2021-KL165).

Clinical assessment

A specialist in movement disorders carried out the complete assessment. Demographic data and characteristics of PD were obtained from all patients, including age, gender, height, weight, education, smoking, hypertension, diabetes, Unified Parkinson’s Disease Rating Scale (UPDRS) part III (off), PD duration, levodopa equivalent daily dose and Hoehn–Yahr (H–Y) stage.

Pain assessment

During the assessment, patients were asked to report pain they had experienced, and characteristics of pain were recorded; e.g., location, duration, frequency, and therapy. Pain severity was evaluated with an 11-point numerical rating scale (NRS) (ranging from 0 “no pain” to 10 “worst pain imaginable”). According to the international classification of diseases (ICD)-11 pain grading method [33], pain severity is categorized based on the NRS scores as follows: no pain (0), mild (1–3), moderate (4–6), and severe (7–10). PD-related pain was divided into five categories: musculoskeletal pain, dystonic pain, radicular/neuropathic pain, central pain and akathisia [34]. Professional evaluation of PD-related pain was performed with the King’s Parkinson’s Disease Pain Scale (KPPS) [35].

Blood sample collection

Peripheral blood samples (4 mL) were collected from each participant in vacuum tubes without anticoagulant in the morning of the day for clinical assessment. Samples were allowed to clot at 4 °C for 1 h before centrifugation at 3000 r/min for 15 min at 4 °C. The samples were stored at −80 °C immediately until assay.

Statistical analysis

GraphPad Prism 8.3 was used for statistical analysis of the animal experiments. Data were checked for normal distribution and shown as the mean ± standard error of mean. All statistical analyses were based on the results of ≥ 3 independent biological samples, which were repeated on different experimental days. Two-tailed unpaired t-test, paired t-test, one-way analysis of variance (ANOVA), two-way repeated-measures ANOVA, or two-way ANOVA was used as indicated in the figure legends. Bonferroni’s test was used for multiple-comparison post hoc tests. The analysis of cumulative probabilities was performed using the Kolmogorov–Smirnov test. SPSS version 23 (IBM, Armonk, NY, USA) was used for statistical analysis of clinical cross-sectional study. Data were checked for normality and homogeneity of variance and presented as mean ± standard deviation, percentage or median (interquartile range). Numerical data were analyzed using the independent-sample t-test, one-way ANOVA if the data were normally distributed or the nonparametric test if the data were non-normally distributed. Categorical data were analyzed using Chi-square tests. Spearman’s correlation analyses were performed to evaluate the correlation between NRS and clinical variables. P < 0.05 was considered statistically significant.

Results

Bilateral lesions of SNpc DA neurons induce hyperalgesia and increase PACAP

A parkinsonian rat model was established by bilaterally microinjecting 6-OHDA into the SNpc (Fig. 1a, b). To confirm PD-like characteristics in 6-OHDA-treated rats, behavioral tests and Western blotting were performed. Bilateral injection of 6-OHDA into the SNpc induced motor deficiency, compared with the sham group (Fig. 1c). There was no change in locomotor activity, which was shown in the distance traveled in the open field test, in rats at 3 weeks after 6-OHDA injection (Fig. 1d). TH (a DA neuron marker) protein in the corpus striatum (Fig. S1a, b), and TH+ neurons were significantly reduced in 6-OHDA-lesioned rats (Fig. S1c, d). These data confirmed that a parkinsonian rat model was established as previously reported [23, 24]. To obtain information about the development of hyperalgesia in 6-OHDA-lesioned rats, we measured the mechanical and thermal pain threshold through nociceptive behavioral tests. Paw withdrawal threshold and tail flick latencies in response to von Frey filaments and radiant thermal stimuli were measured, respectively, 3 weeks after surgery (Fig. 1b). The 6-OHDA group had a significantly lower mechanical paw withdrawal 50% threshold and shorter tail flick latency than the sham group had (Fig. 1e, f).

Fig. 1. Behavior tests and level of PACAP-38 in spinal dorsal horn of rats after bilateral lesions of SNpc DA neurons with 6-OHDA.

a Diagram showing stereotactic injection of 6-OHDA into the SNpc to establish a PD rat model. b Timeline of experiments. c Change in rotarod test after bilateral 6-OHDA lesions, sham (n = 10) vs 6-OHDA (n = 12), (t = 6.01, ***P < 0.001, unpaired t-test). d Total distance in open field test of sham (n = 10) and 6-OHDA (n = 12) rats 3 weeks after surgery, (t = 0.03, P = 0.98, unpaired t-test). e, f 6-OHDA-lesioned rats developed mechanical hyperalgesia (t = 4.11, ***P < 0.001, unpaired t-test) and thermal hyperalgesia (t = 5.14, P < 0.001, unpaired t-test), sham (n = 10) vs 6-OHDA (n = 12). Real-time PCR results show mRNA level of Pacap (g) and Vip (h) in the spinal dorsal horn of L4–L6 in 6-OHDA rats compared with sham rats, Pacap (t = 9.37, P < 0.001, n = 4 rats for each group, unpaired t-test), Vip (t = 0.428, P = 0.684, n = 4 rats for each group, unpaired t-test). mRNA level of Adcyap1r1 (i), Vipr1 (j) and Vipr2 (k) in the spinal dorsal horn of L4–L6 in 6-OHDA rats compared with sham rats; Adcyap1r1, t = 4.07, P = 0.007; Vipr1, t = 1.62, P = 0.157; Vipr2, t = 0.03, P = 0.974; n = 4 rats for each group, unpaired t-test. l Representative Western blotting images. Western blotting showing expression of PACAP (m) and PAC1-R (n) in the spinal dorsal horn of L4–L6 in 6-OHDA rats compared with sham rats; PACAP, t = 3.99, P = 0.007; PAC1-R, t = 4.10, P = 0.006; n = 4 rats for each group, unpaired t-test). ns, not significant; **P < 0.01. Data are presented as mean ± SEM.

To explore expression of PACAP and VIP (the structurally related family member) in the 6-OHDA-induced model, mRNA levels of Pacap and Vip in the spinal dorsal horn were measured. qPCR showed that expression of Pacap was higher in 6-OHDA rats than in sham rats (Fig. 1g). However, the mRNA level of Vip did not show significant alteration (Fig. 1h). In addition, the mRNA level of PAC1-R was determined. Expression of Adcyap1r1 (gene encoding PAC1-R) in the spinal dorsal horn was higher in 6-OHDA rats than in sham rats (Fig. 1i). However, the mRNA levels of the other two receptors VIPR1 and VIPR2 did not change significantly in the spinal dorsal horn (Fig. 1j, k). These results suggest that PACAP and its selective receptor PAC1 may contribute to the pathology of the spinal dorsal horn in 6-OHDA rats. However, VIP, including the receptors VIPR1 and VIPR2, did not seem to have a significant role.

Western blotting showed that protein levels of PACAP and PAC1-R in the spinal dorsal horn of 6-OHDA rats were significantly higher than in the sham rats (Fig. 1l–n). The concentration of PACAP-38 in the CSF exhibited a negative correlation with the 50% threshold of mechanical paw withdrawal in rats (Fig. S2a, b). These results indicate that the parkinsonian rats presented with mechanical and thermal hyperalgesia, and that hyperalgesia developed in parallel with the activation PACAP/PAC1-R signaling in the spinal dorsal horn.

Inhibiting PACAP/PAC1-R by PACAP 6–38 attenuates hyperalgesia of 6-OHDA-lesioned rats

To determine whether activated PACAP/PAC1-R was involved in the hyperalgesia of 6-OHDA rats, we measured the mechanical paw withdrawal threshold and tail flick latency after intrathecal injection of PACAP 6–38, a PAC1-R antagonist. In 6-OHDA rats, a single injection of saline or PACAP 6–38 (0.125, 0.5, and 2 μg), PACAP 6–38 significantly reversed, dose-dependently, the paw withdrawal threshold 15 min after injection (Fig. 2a). The time course response curve showed that the effects of PACAP 6–38 (2 μg) persisted up to 30 min and 45 min post-injection for mechanical and thermal hyperalgesia, respectively (Fig. 2b, c). PACAP 6–38 had no effect on the mechanical paw withdrawal threshold and tail flick latency in the sham rats (Fig. 2b, c). These results indicate that the PACAP/PAC1-R may be involved in the development of hyperalgesia of 6-OHDA-induced PD rats.

Fig. 2. Inhibiting PACAP/PAC1-R by PACAP 6–38 attenuated hyperalgesia in 6-OHDA-lesioned rats.

a Von Frey tests were conducted on the left hind paw before and 15, 30, 45, and 60 min after PACAP 6–38 (0.125, 0.5, and 2 μg i.t.) was administered (time and dose interaction: F_{(12, 160)} = 3.82, P < 0.001; time: F _{(4, 160)} = 9.44, P < 0.001); dose: F _{(3, 40)} = 3.87, P = 0.016; ***P < 0.001 vs 0 (pre-treatment), ^###P < 0.001 vs Saline; n = 10–12 rats, two-way repeated measures ANOVA followed by Bonferroni’s post hoc test). b Reversal of mechanical hyperalgesia 3 weeks after 6-OHDA lesions induced by intrathecal injection of PACAP 6–38 (2 μg), (treatment and time interaction: F _{(12, 144)} = 3.54, P < 0.001; treatment: F _{(3, 36)} = 9.34, P < 0.001; time: F_{(4, 144)} = 4.02, P = 0.004; ***P < 0.001 vs 0 (pre-treatment), ^##P < 0.01 vs 6-OHDA + saline; n = 9–12 rats, two-way repeated measures ANOVA followed by the Bonferroni’s post hoc test). c Attenuation of 6-OHDA-lesion-induced thermal hyperalgesia by intrathecal injection of PACAP 6–38 (treatment and time interaction: F _{(12, 108)} = 2.75, P = 0.003; treatment: F _{(3, 27)} = 27.03, P < 0.001; time: F _{(4, 108)} = 2.57, P = 0.042; **P < 0.01, ***P < 0.001 vs 0 (pre-treatment), ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs 6-OHDA + saline; n = 6–10 rats, two-way repeated measures ANOVA followed by Bonferroni’s post hoc test). Data are presented as mean ± SEM.

Inhibiting PACAP/PAC1-R reverses the enhanced excitatory synaptic transmission

As is known to all spinal dorsal horn is the secondary neurons of sensory processing. An increasing body of evidence suggests that synaptic transmission in spinal dorsal horn neurons plays an important role in the development and maintenance of pain [29]. To investigate whether synaptic plasticity in the spinal dorsal horn of 6-OHDA-induced PD rats changes 3 weeks post-injection, we employed patch-clamp recordings in L4–L6 spinal cord slices to assess synaptic transmission. As illustrated in the representative traces of sEPSCs (Fig. 3a), the spontaneous excitatory synaptic transmission was intensified in the spinal dorsal horn neurons of 6-OHDA-lesioned rats compared with sham rats. We demonstrated that the frequency of sEPSCs was higher in the spinal dorsal horn neurons of the 6-OHDA-lesioned rats than in the sham rats (Fig. 3b). However, there was no significant difference in the amplitude of sEPSCs between the 6-OHDA-lesioned and sham rats (Fig. 3c).

Fig. 3. Inhibiting PACAP/PAC1-R signaling relieved the enhanced sEPSCs of the spinal dorsal horn neurons of 6-OHDA rats.

a Representative traces of sEPSCs recorded in the spinal dorsal horn neurons of 6-OHDA and sham rats at L4–L6. b Representative cumulative probability of the interevent intervals and summarized data of frequency of sEPSCs recorded in spinal dorsal horn neurons of sham and 6-OHDA rats at L4–L6 (D = 0.47, P < 0.001, K–S test; t = 4.49, P < 0.001, n = 8 cells for each group, unpaired t-test). c Representative cumulative probability and summarized data for the amplitude of sEPSCs recorded in spinal dorsal horn neurons of sham and 6-OHDA rats at L4–L6 (D = 0.10, P = 0.960, K-S test; t = 1.32, P = 0.21, unpaired t-test, n = 8 cells per group). d Representative traces of sEPSCs recorded in the neurons in spinal cord slices of 6-OHDA rats at L4–L6 pre- and post-incubated with PACAP 6–38 (250 nM). e Representative cumulative probability of the interevent intervals and summarized data of frequency of sEPSCs recorded in the spinal dorsal horn neurons of 6-OHDA rats pre- and post-incubated with PACAP 6–38 (D = 0.33, P < 0.001, K–S test; t = 7.79, P < 0.001, n = 6 cells, paired t-test). f Representative cumulative probability and summarized data for the amplitude of sEPSCs recorded in spinal dorsal horn neurons of 6-OHDA rats pre- and post-incubated with PACAP 6–38 (D = 0.13, P = 0.912, K–S test; t = 1.18, P = 0.292; n = 6 cells, paired t-test). ns, not significant; ***P < 0.001. Data are presented as mean ± SEM.

To investigate the effect of PACAP 6–38 on synaptic transmission in L4–L6 spinal dorsal horn neurons of 6-OHDA-induced PD rats, we measured sEPSCs before and after incubation with PACAP 6–38 (250 nM), applied via perfusion in spinal cord slices. Figure 3d shows the representative traces of sEPSCs recorded in spinal dorsal horn neurons of 6-OHDA rats pre- and post-incubated with PACAP 6–38. The frequency of sEPSCs was significantly decreased post-PACAP 6–38 compared with pre-PACAP 6–38 (Fig. 3e), whereas the amplitude of sEPSCs post-PACAP 6–38 was not altered (Fig. 3f). These findings suggest that the activation of PACAP/PAC1-R enhances the excitatory synaptic transmission, which may play an essential role in the development of hyperalgesia of 6-OHDA rats.

Activation of PACAP/PAC1-R pathway contributes to activation of CaMKII and ERK in the spinal dorsal horn of 6-OHDA-lesioned rats

The MAPK family is effector of the PACAP/PAC1-R pathway that plays an essential role in the excitability of neurons [36, 37]. To explore downstream signaling of the PACAP/PAC1-R pathway, which was activated in the spinal dorsal horn of 6-OHDA-lesioned rats, we detected expression of Ca²⁺/calmodulin-dependent protein kinase (CaMK)II, P38 and extracellular signal-regulated kinase (ERK) in the spinal dorsal horn by Western blotting. The 6-OHDA-induced lesions significantly increased protein expression of phospho (p)-CaMKII and p-ERK in the spinal dorsal horn (Fig. 4a–c). However, the protein expression of p-P38 was not changed after 6-OHDA lesion (Fig. 4a, d). Inhibiting PACAP/PAC1-R using PAC1-R antagonist by repetitive administration of PACAP 6–38 (2 μg i.t., daily for 5 consecutive days) significantly suppressed 6-OHDA-induced activation of CaMKII and ERK in the spinal dorsal horn of L4–L6 (Fig. 4e–g). However, PACAP 6–38 did not significantly affect p-CaMKII and p-ERK in the sham rats. These results demonstrate that inhibition of the PACAP/PAC1-R pathway suppressed the 6-OHDA-induced activation of CaMKII and ERK in the spinal dorsal horn. These findings suggest that CaMKII and ERK may be downstream of the activated PACAP/PAC1-R pathway in the spinal dorsal horn of 6-OHDA-lesioned rats.

Fig. 4. PACAP/PAC1-R upregulated the phosphorylation of CaMKII and ERK, but not P38, in the spinal dorsal horn of 6-OHDA rats.

a, b Expression of p-CaMKII in the spinal dorsal horn of L4–L6 in 6-OHDA rats compared with sham rats, (t = 9.54, P < 0.001, n = 4 rats for each group, unpaired t-test). a, c Expression of p-ERK in the spinal dorsal horn of L4–L6 in 6-OHDA rats compared with sham rats, (t = 6.81, P < 0.001, n = 4 rats for each group, unpaired t-test). a, d Expression of p-P38 in the spinal dorsal horn of L4–L6 in 6-OHDA rats compared with sham rats, (t = 1.33, P = 0.232, n = 4 rats for each group, unpaired t-test). e–g Consecutive intrathecal treatment with PACAP 6–38 (2 μg, daily for 5 consecutive days) reduced 6-OHDA-lesion-induced p-CaMKII (f) and p-ERK (g) activation in the spinal dorsal horn (f, group and treatment interaction: F _{(1, 8)} = 8.01, P = 0.022; group: F _{(1, 8)} = 22.00, P = 0.002, treatment: F _{(1, 8)} = 8.96, P = 0.017; g group and treatment interaction: F _{(1, 8)} = 14.43, P = 0.005; group: F _{(1, 8)} = 39.57, P < 0.001, treatment: F _{(1, 8)} = 18.90, P = 0.003; n = 3 rats, two-way ANOVA followed by the Bonferroni’s post hoc test). ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001. Data are presented as mean ± SEM.

Downregulation of PAC1-R in the spinal dorsal horn of L4–L6 alleviates hyperalgesia induced by 6-OHDA lesions

PACAP is a secreted polypeptide and has the characteristics of long-distance transport. We used downregulation of its specific receptor PAC1-R, which is located on the cell membrane, to disrupt PACAP/PAC1-R pathway at the genetic level. A previous immunofluorescence assay showed that PAC1-R immunoreactivity was principally colocalized with NeuN, a neuron-specific nuclear protein, rather than the marker of glial cells in the spinal dorsal horn [38]. Our immunofluorescence assay showed that PAC1-R colocalization with NeuN in the spinal dorsal horn was consistent with previous studies (Fig. S3). We constructed pAAV2/9 expressing a shRNA targeted against Adcyap1r1 (gene encoding PAC1-R) and used local RNA interference to downregulate PAC1-R in the spinal dorsal horn of L4–L6 (Fig. S4a–d). The range of pAAV-Adcyap1r1 infection confined to the bilateral spinal dorsal horn (Fig. S4e). All three strands of shRNA-Adcyap1r1 showed significant knockdown efficiency (Fig. S4f, g). Microinjection of virus did not change the total distance traveled in the open field test and latency to fall in rotarod test (Fig. S4h, i). No significant alterations were observed in the nociceptive behavior of rats with bilateral spinal dorsal horn expressing sh-scrambled or expressing sh-Adcyap1r1 (Fig. S4j, k).

To confirm the effect of PACAP/PAC1-R signaling on hyperalgesia induced by 6-OHDA, pAAV scramble and pAAV-Adcyap1r1 were microinjected into the L4–L6 spinal dorsal horn 1 weeks before induction of 6-OHDA lesions (Fig. 5a, b). Following the induction of 6-OHDA lesions, 3 weeks post-surgery, the thresholds of mechanical pain and thermal pain were significantly higher in rats expressing sh-Adcyap1r1, compared to those expressing sh-scrambled (Fig. 5c, d). However, under sham conditions following AAV injection, the threshold of mechanical pain and thermal pain remained unaltered (Fig. 5c, d). Downregulation of Adcyap1r1 gene expression suppressed activation of PAC1-R induced by 6-OHDA (Fig. 5e, g) and the subsequent signals CaMKII and ERK in the spinal dorsal horn (Fig. 5h–j). In contrast, downregulation of Adcyap1r1 gene expression did not alter the expression of PACAP in the spinal dorsal horn in the 6-OHDA-lesioned rats (Fig. 5e, f). These results indicate that downregulation of PAC1-R in the spinal dorsal horn alleviates hyperalgesia induced by 6-OHDA lesions.

Fig. 5. Microinjection of pAAV-Adcyap1r1 into the L4–L6 spinal dorsal horn alleviated 6-OHDA-lesion-induced mechanical allodynia and thermal hyperalgesia in rats.

a, b Schematic diagram showing experimental procedure for microinjection of Adcyap1r1 shRNA-expressing pAAV2/9 into the L4–L6 spinal dorsal horn of rats, stereoscopic brain injection 6-OHDA surgery and subsequent experiments. c Microinjection of pAAV-Adcyap1r1 into L4–L6 spinal dorsal horn alleviated 6-OHDA-lesion-induced mechanical pain threshold reduction (gene and group interaction: F _{(1, 37)} = 4.91, P = 0.033; gene: F _{(1, 37)} = 4.12, P < 0.05; group: F _{(1, 37)} = 15.93, P < 0.001; n = 10 or 11 rats, two-way ANOVA followed by the Bonferroni’s post hoc test). d Tail flick latency changes after microinjection of pAAV-Adcyap1r1 into L4–L6 spinal dorsal horn before induction of 6-OHDA lesions (gene and group interaction: F _{(1, 37)} = 4.54, P = 0.040; gene: F _{(1, 37)} = 4.38, P < 0.05; group: F _{(1, 37)} = 23.21, P < 0.001; n = 10 or 11 rats, two-way ANOVA followed by the Bonferroni’s post hoc test). Representative Western blots (e) and relative quantitative summary of PACAP (f) and PAC1-R (g) in the spinal dorsal horn of rats expressing shRNA-Adcyap1r1 or scrambled shRNA 3 weeks after stereoscopic brain injection of 6-OHDA or vehicle; (f) gene and group interaction: F _{(1, 8)} = 0.33, P = 0.580; gene: F _{(1, 8)} = 0.3221, P = 0.586; group: F _{(1, 8)} = 27.08, P < 0.001; n = 3 rats, two-way ANOVA followed by the Bonferroni’s post hoc test. g gene and group interaction: F _{(1, 8)} = 5.91, P = 0.041; gene: F _{(1, 8)} = 81.26, P < 0.001; group: F _{(1, 8)} = 7.25, P = 0.027; n = 3 rats, two-way ANOVA followed by the Bonferroni’s post hoc test. Representative Western blots (h) and relative quantitative summary of p-CaMKII (i) and p-ERK (j) in the spinal dorsal horn of rats expressing shRNA-Adcyap1r1 or scrambled shRNA; (i) gene and group interaction: F _{(1, 8)} = 7.92, P = 0.023; gene: F _{(1, 8)} = 14.67, P = 0.005; group: F _{(1, 8)} = 43.82, P < 0.001; j gene and group interaction: F _{(1, 8)} = 55.20, P < 0.001; gene: F _{(1, 8)} = 61.55, P < 0.001; group: F _{(1, 8)} = 101.50, P < 0.001; n = 3 rats, two-way ANOVA followed by the Bonferroni’s post hoc test. ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001. Data are presented as mean ± SEM. pAAV plasmid adeno-associated virus, shRNA short hairpin RNA.

Inhibition of ERK with PD98059 alleviates hyperalgesia induced by 6-OHDA via suppression of excitatory synaptic transmission

To further determine the role of ERK as subsequent signal of PACAP/PAC1-R in hyperalgesia of 6-OHDA-lesioned rats, we administered ERK antagonist PD98059 (10 μg, i.t.). The thresholds of mechanical pain and thermal pain in the 6-OHDA + PD98059 group were significantly increased compared with that in the 6-OHDA + DMSO group (Fig. 6a, b). To better understand the role of PACAP/PAC1-R signaling in PD pain, and its association with EKR, we pharmacologically stimulated PACAP/PAC1-R using PACAP 1–38, an established agonist for this receptor. When administered alone or in combination to control rats, PACAP 1–38 (10 μg, i.t.) decreased the thresholds for both mechanical and thermal pain. This effect was mitigated by prior application of PD98059 (10 μg, i.t.) in combination with PACAP 1–38 (Fig. 6c, d).

Fig. 6. Inhibition of ERK alleviated hyperalgesia and decreased excitatory synaptic transmission of spinal dorsal horn neurons of 6-OHDA-lesioned rats.

a Von Frey tests changes in the left hind paw were measured after PD98059 (10 μg i.t.) was administered (group and treatment interaction: F _{(1, 38)} = 4.14, P = 0.048; group: F _{(1, 38)} = 8.93, P = 0.005; treatment: F _{(1, 38)} = 4.15, P = 0.048; n = 10 or 11 rats, two-way ANOVA followed by the Bonferroni’s post hoc test). b Tail flick latency changes were measured after PD98059 (10 μg i.t.) was administered (group and treatment interaction: F _{(1, 38)} = 4.16, P = 0.048; group: F _{(1, 38)} = 21.92, P < 0.001; treatment: F _{(1, 38)} = 5.04, P = 0.031; PD98059 significantly increased the tail flick latency of 6-OHDA rats: P = 0.022; n = 10 or 11 rats, two-way ANOVA followed by the Bonferroni’s post hoc test). c Pretreatment with PD98059 (10 μg) mitigated PACAP 1–38-induced (10 μg) mechanical pain threshold reduction (F _{(3, 21)} = 6.52, P = 0.003; n = 6–7 rats, one-way ANOVA followed by the Bonferroni’s post hoc test). d Tail flick latency changes after intrathecal pretreatment with PD98059 (10 μg) before PACAP 1–38 (10 μg) administration (F _{(3, 21)} = 14.10, P < 0.001; n = 6 or 7 rats, one-way ANOVA followed by the Bonferroni’s post hoc test). e Representative traces of sEPSCs recorded in the neurons of spinal dorsal horn in 6-OHDA rats at L4–L6 pre- and post-incubation with PD98059 (10 μM). f Representative cumulative probability of the interevent intervals and summarized data of frequency of sEPSCs recorded in the spinal dorsal horn neurons of 6-OHDA rats pre- and post-incubated with PD98059 (D = 0.27, P < 0.001, K-S test; t = 6.49, P = 0.001; n = 6 cells, paired t-test). g Representative cumulative probability and summarized data for the amplitude of sEPSCs recorded in spinal dorsal horn neurons of 6-OHDA rats pre- and post-incubated with PD98059 (D = 0.09, P = 0.844, K-S test; t = 1.33, P = 0.24; n = 6 cells, paired t-test). ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001. Data are presented as mean ± SEM.

To further investigate the mechanism by which PD98059 attenuated hyperalgesia of 6-OHDA rats, we measured the sEPSCs of neurons in L4–L6 spinal cord slices of 6-OHDA-induced PD rats pre- and post-incubated with PD98059 (10 μM). Representative traces of sEPSCs pre- and post-incubated with PD98059 were shown in Fig. 6e. The frequency of sEPSCs post-PD98059 was significantly decreased compared with pre-PD98059 (Fig. 6f); however, the amplitude of sEPSCs post-PD98059 was not altered (Fig. 6g). These data suggest that PACAP-induced activation of ERK enhanced the excitatory synaptic transmission, eventually contributing to hyperalgesia in 6-OHDA rats.

Associations of serum PACAP levels with pain in patients with PD

To investigate whether the effect of PACAP on hyperalgesia in rodents can be extrapolated to humans, a clinical cross-sectional study was performed. A total of 132 patients with PD and 60 healthy controls (HCs) who were matched for age and gender were studied. Demographic and clinical characteristics of all subjects are shown in Table S1. There was a significant difference in serum PACAP-38 concentration among the HCs, PD without pain and PD with pain groups (Fig. S5). Multiple comparisons indicated that serum PACAP-38 concentration in the PD with pain group was significantly higher than that in the HCs group, has a trend for being significantly higher than that in the PD without pain group. We subcategorized the severity of PD pain based on the NRS scores. Serum levels of PACAP-38 were significantly higher in PD patients experiencing moderate and severe pain compared with those without pain or with mild pain (Fig. 7a). Correlation analysis revealed that serum concentration of PACAP-38 was positively correlated with NRS and KPPS (Fig. 7b, c, Table S2). Logistic regression analysis indicates that serum level of PACAP-38 may be one of the predictors for the pain severity in PD (Table S3).

Fig. 7. Correlation analysis of serum PACAP-38 levels and pain severity.

a Serum levels of PACAP-38 exhibited significant differences among different subgroups based on the NRS in PD patients (F = 4.133, P = 0.008; *P < 0.05, **P < 0.01, vs NRS 1–3; ^#P < 0.05, vs NRS 0; one-way ANOVA followed by the LSD post hoc test). b Correlation between serum levels of PACAP-38 and NRS score in patients with PD (Spearman r = 0.273, P = 0.002, n = 132). c Correlation between serum levels of PACAP-38 and KPPS score in patients with PD (Spearman r = 0.452, P < 0.001, n = 71). Data are presented as mean ± SD.

Discussion

Although PACAP is involved in neuropathic pain and neuronal excitability [11, 21], no studies have addressed whether and how PACAP regulates pain in PD. In the present study, we validated hyperalgesia in a rat model of PD, which is consistent with previous work in our laboratory and other researchers [23, 24, 39]. We used pharmacological methods and whole-cell patch-clamp techniques to examine the antinociceptive effects of PACAP 6–38, a potent and competitive antagonist of PAC1-R, in the spinal dorsal horn following bilateral lesions of SNpc DA neurons with microinjection of 6-OHDA. We demonstrated that the PACAP/PAC1-R signaling pathway played a critical role in the induction and persistence of hyperalgesia after lesion of SNpc DA neurons. Bilateral lesions of SNpc DA neurons potentially triggered hyperalgesia through the activation of PACAP/PAC1-R signaling. This process could regulate the synaptic transmission of nociceptive spinal dorsal horn neurons via the modulation of intracellular signaling activity, including the activation of ERK and CaMKII signals.

Previous studies have shown that monoamines, such as dopamine, participate in PD-related pain [40–42]. The management of PD-related pain remains challenging, and therapeutic approach is limited [4]. These findings indicate that additional mechanisms are involved in PD-related pain. Some studies have reported the role of PACAP/PAC1-R in PD, using either in vitro or in vivo models of PD, mainly focused on the effect for DA neurons and in substantia nigra-striatum system of brain [8, 43]. However, previous studies have shown that PACAP and PAC1-R were involved in the mechanisms of migraine, inflammatory pain, and neuropathic pain [11, 15, 16, 44, 45]. This contradiction might be explained by the differences in various pathological stages, pleiotropic character of PACAP, and observation time used, or may be related to the compensatory mechanism of the body against pathological damage. The main type of PD pain is musculoskeletal [3], and spinal cord lesions have been observed in PD patients and animal models of PD [46–48]. Therefore, research into the role of PACAP in PD models at the spinal cord level is helpful to elucidate the mechanisms of PD pain. Our recent work has shown that the excitability of spinal dorsal horn neurons was increased in adult rats with bilateral lesions of SNpc DA neurons [24]. In the present study, we showed that expression of PACAP and its selective receptor PAC1-R was significantly upregulated in the spinal dorsal horn of 6-OHDA rats, but not VIP and the receptors VIPR1 and VIPR2. Increased PACAP levels have been observed in the spinal dorsal horn of animal models of neuropathic pain [49]. 6-OHDA-lesioned rats show changes in nociceptive processing and increased neuronal excitability in the spinal dorsal horn neurons. The increased expression of PACAP and PAC1-R in the spinal dorsal horn of 6-OHDA-lesioned rats may be related to mechanisms such as neuroplasticity, stress response, changes in neuronal activity. The precise mechanism remains unclear, and further researches are needed to elucidate potential underlying mechanisms.

In addition, i.t. injection of PACAP 6–38 dose dependently reversed the mechanical pain in 6-OHDA rats. The time-response effect observed for PACAP 6–38 in the present study is consistent with prior research [16]. PACAP 6–38 acts as a competitive antagonist binds to PAC1-R, initiating rapid effects that might be accomplished through the rapid blockade of PACAP/PAC1-R signaling and modulation of ion channels. PACAP 6–38 may be rapidly metabolized or cleared from the site of action, leading to the gradual decline of its effects. The transient effect of PACAP 6–38 in our study could be attributed to several potential mechanisms, including metabolic processes, receptor desensitization, and the presence of compensatory mechanisms. The alleviation of hyperalgesia by PACAP 6–38 has also been observed in neuropathic and inflammatory pain states and migraine [16, 45, 49, 50]. PACAP induces pain-like syndrome after i.t. injection [27], and 73% of patients complained of migraine-like attacks after intravenous infusion of PACAP [51], which indicates that PACAP may be a sensory neurotransmitter. In our study, the stimulation of PACAP/PAC1-R using an agonist induced hyperalgesia.

Furthermore, downregulation of Adcyap1r1 gene expression by microinjection of AAV expressing sh-Adcyap1r1 in bilateral spinal dorsal horn to interrupt PACAP/PAC1-R signaling significantly alleviated the development of mechanical hyperalgesia and thermal hyperalgesia in 6-OHDA rats. We conclude that PACAP/PAC1-R signaling in the spinal dorsal horn is a new mechanism underlying hyperalgesia of 6-OHDA-induced parkinsonian rats.

Similar to rats suffering from medial forebrain bundle lesioned with 6-OHDA [48], SNpc-lesioned rats displayed hyperalgesia in parallel with hyperexcitability and central sensitization in the spinal dorsal horn neurons [24, 48]. However, no research has shown changes in synaptic transmission in rats with 6-OHDA-induced lesions. We observed that in the 6-OHDA-lesioned rats, the sEPSCs of the spinal dorsal horn neurons were enhanced relative to those in the sham rats, as shown by increased frequency. Pretreatment with PACAP uncoupled metabotropic glutamate receptor 5 inhibition on the autaptic EPSC, which is generated at a synapse formed by a neuron onto itself and recorded from cultured neurons [52]. Additionally, application of PACAP increased both frequency and amplitude of sEPSCs in neurons of mice brain slices [53]. We described for the first time the effect of PACAP/PAC1-R signaling on excitatory synaptic transmission at the spinal dorsal horn level. Hyperalgesia of 6-OHDA-lesioned rats may be associated with the enhanced sEPSCs of the spinal dorsal horn neurons. This was supported by the evidence that PACAP/PAC1-R signaling antagonist PACAP 6–38 significantly decreased the frequency of sEPSCs of the spinal dorsal neurons or elevated the pain threshold. We deduced that PACAP/PAC1-R signaling in the spinal dorsal horn neurons may induce excitatory synaptic transmission mechanism that underlies hyperalgesia in 6-OHDA-lesioned rats.

It has been demonstrated that PACAP 6–38 blocked PACAP-induced ERK activation [49]. In addition, PACAP augmented ERK phosphorylation, initiated calcium influx and increased CaMKII phosphorylation [36, 49, 54, 55]. Our results showed that activation of PACAP/PAC1-R signaling pathway in parallel with increased intracellular signaling cascades p-ERK and p-CaMKII in the spinal dorsal horn of L4–L6, rather than p-P38. However, 6-OHDA-lesion-induced activation of CaMKII and ERK in the spinal dorsal horn was significantly suppressed by administration of PACAP 6–38, and by interrupting PACAP/PAC1-R signaling via downregulating Adcyap1r1 gene expression in the spinal dorsal horn. PACAP regulated neuronal excitability via MEK/ERK signaling, and PD98059 attenuated chronic pain, inflammatory pain and orofacial neuropathic pain [28, 37, 49, 56]. In the current study, behavioral data demonstrated that inhibition of ERK attenuated hyperalgesia induced by PACAP/PAC1-R signaling in the 6-OHDA-lesioned rats, and i.t. pretreatment with PD98059 prevented hyperalgesia induced by PACAP 1–38. Additionally, Incubation of PD98059 significantly reduced the frequency of sEPSCs. Previous research has demonstrated that phosphorylated ERK modulates ion channels, such as potassium and sodium channels [20, 57], suggesting that PAC1-R-CaMKII-ERK signaling may regulate synaptic activity through ion channels. These data suggest that activation of ERK induced by PACAP/PAC1-R signaling may be involved in the development of hyperalgesia in part via enhancement of excitatory synaptic transmission of spinal dorsal horn neurons in 6-OHDA-lesioned rats.

Activation of PAC1-R has been shown to increase the frequency of sEPSCs in the suprachiasmatic nucleus [53], as well as the frequency of fast excitatory postsynaptic potentials upon PACAP38 application in dorsal root ganglion [58]. Moreover, evidence suggests that presynaptic PAC1-mediated signaling contributes to long-term potentiation, a process associated with synaptic plasticity [59]. Previous research has elucidated the involvement of PAC1-R in the modulation of presynaptic activity through the retrograde messenger nitric oxide (NO) [60]. Additionally, the crosstalk between NO and ERK signaling has been implicated in mediating nociception [61]. Therefore, it is hypothesized that a retrograde signaling mechanism may play a role in the regulation of presynaptic activity. However, further investigation is required to elucidate the underlying mechanisms of PAC1-R-CaMKII-ERK signaling on excitatory synaptic transmission in spinal dorsal horn neurons of 6-OHDA-lesioned rats.

Previous studies have shown that estrogen has an important effect on pain [62]. Additionally, the menstrual cycle of female rats may affect behavior of animals. Therefore, male rats were used in the present study to avoid the effects of estrogen on pain. We will further evaluate the sex differences in mechanisms mediating pain in future studies.

It has been demonstrated that serum PACAP levels of PD patients were inversely correlated with the score of attention/memory that was assessed with the nonmotor symptom scale [63]. Another study showed that CSF PACAP levels in patients with PD with dementia were similar to those in control participants [64]. Recent study showed PACAP-38 level was elevated in patients with deep brain stimulation, and no significant correlation between PACAP-38 level and score of UPDRS [65]. These findings indicate that further studies are needed to clarify the role of PACAP in different pathological states. In the present cross-sectional study, we focused on PD-related pain, which is an annoying nonmotor symptom of PD. Our observations showed that serum level of PACAP-38 was elevated in the PD with pain group. Notably, serum PACAP-38 levels were significantly elevated in PD patients with moderate and severe pain subgroups. The role of PACAP in pathophysiological processes is related to the disease state, with its serum levels changing along with the variations in pain. The prevalence of chronic pain is higher in elderly populations [66]. In this study, the HCs group had no history of chronic pain. Pain assessments and blood samples were collected from PD patients experiencing pain during their symptomatic periods. We also showed that serum PACAP-38 level was positively correlated with score of NRS and KPPS in patients with PD. Previous studies reported that PD-related pain was associated with depression, disease duration, levodopa equivalent daily dose, H–Y stage, and sleep disturbance, although the results were not consistent [3, 67]. The regression analysis suggested PD duration, PACAP-38, and H–Y stage were predictors of pain severity. Factors such as small sample size, selective patient populations, and assessment scale for data collection were reasons for these discrepancies [67].

However, pain in PD is heterogeneous and can be multifactorial in origin [34]. In our study, PD pain was classified according to the Ford classification [34], which is the most commonly used classification of pain among patients with PD. The major pain subtype is musculoskeletal pain (68% of those with pain) and consistent with previous descriptions [3]. The results of the present cross-sectional study should be considered in the light of its limitations, e.g., peripheral assessment of PACAP-38 in serum samples, small sample, absence of other nonmotor symptoms, and unknown mechanisms. Also, the selection bias of a single-center study should be considered. Therefore, a multicenter study with a larger sample and control for bias is needed to obtain more powerful results to verify the current findings. However, the exact origin of increased PACAP level in the spinal dorsal horn of 6-OHDA-lesioned rats and the PD with pain group is yet unknown, and further examinations are needed to uncover the precise mechanism.

In conclusion, we demonstrated that activation of PACAP/PAC1-R induced subsequent signals, which enhanced the excitatory synaptic transmission of the spinal dorsal horn, eventually leading to hyperalgesia in 6-OHDA-lesioned rats. Our clinical cross-sectional study showed that serum concentration of PACAP-38 positively correlated with scores of pain severity scales and served as a predictor for pain severity. These findings broaden our understanding of PD pain and provide a new perspective for therapy of pain related to PD.

Author contributions

CFL designed the study, and revised the manuscript. FW performed experiments, supervised the experiments and edited the manuscript. CJM collected data and edited the manuscript. LGD performed experiments, collected data, analyzed data and prepared figures and the manuscript. MQA performed experiments, collected data, and prepared figures. HYG, LGZ, CJL, and JBZ performed experiments and analyzed data. All the authors have read and approved the paper. The authors have no conflicts of interest to declare.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41401-023-01141-3.