Inhibition of Spinal 5-HT3 Receptor and Spinal Dorsal Horn Neuronal Excitability Alleviates Hyperalgesia in a Rat Model of Parkinson’s Disease

Pain in Parkinson’s disease (PD) is increasingly recognized as a major factor associated with poor life quality of PD patients. However, classic therapeutic drugs supplying dopamine have limited therapeutic effects on PD-related pain. This suggests that there is a mechanism outside the dopamine system that causes pain in PD. Our previous study demonstrated that 6-OHDA induced PD model manifested hyperalgesia to thermal and mechanical stimuli and decreased serotonin (5-hydroxytryptamine; 5-HT) in the spinal dorsal horn (SDH). Several 5-HT receptor subtypes have been confirmed to be associated with nociception in the spinal cord, such as 5-HT1A receptor, 5-HT1B receptor, 5-HT2 receptor, 5-HT3 receptor, and 5-HT7 receptor. Most research has shown that 5-HT1A receptor and 5-HT3 receptor play a key role in pain transmission in the spinal cord. We hypothesized that hyperalgesia of 6-OHDA rats may be related to increased excitability of SDH neurons, and functional change of 5-HT3 receptor may reverse the hyperalgesia of 6-OHDA lesioned rats and decrease cell excitability of SDH neurons. To test this hypothesis, we used whole-cell patch-clamp and pharmacological methods to evaluate the effect of 5-HT3 receptor and 5-HT1A receptor on the hyperalgesia of 6-OHDA rats. The results suggested that increased excitability in SDH neurons could be reversed by 5-HT3 receptor antagonist ondansetron (20 μmol/L) and palosetron (10 μmol/L), but not 5-HT3 receptor agonist m-CPBG (30 μmol/L) and SR 57,727 (10 μmol/L), 5-HT1A receptor agonist 8-OH DPAT (10 μmol/L) and eptapirone (10 μmol/L) and 5-HT1A receptor antagonist WAY-100635 (10 μmol/L) and p-MPPI (10 μmol/L). Intrathecal injection of ondansetron (0.1 mg/kg) but not m-CPBG (0.1 mg/kg), 8-OH DPAT (0.1 mg/kg), and WAY-100635 (0.1 mg/kg) significantly attenuated the mechanical hyperalgesia and thermal hyperalgesia in 6-OHDA lesioned rats. In conclusion, the present study suggests that inhibition of spinal 5-HT3 receptor and SDH neuronal excitability alleviates hyperalgesia in PD rats. Our study provides a novel mechanism or therapeutic strategy for pain in patients with PD.


Introduction
Parkinson's disease (PD) is the second most common neurodegenerative disorder in older people and is clinically characterized by motor symptoms of tremor, rigidity, akinesia, and dystonia. Pain as a nonmotor symptom in PD is increasingly recognized as a major factor associated with poor life quality and more than motor symptoms are [10]. The prevalence of pain in PD patients has been reported to range from 40 to 85% [7]. According to the Ford classification, there are five main subtypes of PD-related pain, musculoskeletal, dystonic, radicular and central pain, and akathisia [17]. Although pain is common in PD patients, there are no guidelines or standard remedies for the management of PD-associated pain [3]. Therefore, there is an urgency to investigate the pathogenesis of pain in PD to discover novel and effective treatments. Classic therapeutic drugs supplying dopamine have limited therapeutic effect on PD pain [16]. This suggests that there is a mechanism outside the dopamine system that causes pain in PD.
Clinical research has demonstrated that serotonergic dysfunction is associated with pain in PD [42]. Our previous study has also shown that a PD rat model induced by bilateral lesions of the substantia nigra pars compacta (SNpc) manifested hyperalgesia to thermal and mechanical stimuli. This hypersensitivity could be attributed at least partially to the decreased serotonin (5-hydroxytryptamine; 5-HT) content in the spinal dorsal horn (SDH) [43]. Whether the decreased 5-HT content has an effect on SDH neurons requires further investigation.
5-HT is one of the main neurotransmitters involved in the descending inhibitory system, and its projection from rostral ventrolateral medulla to the spinal cord is thought to be related to pain modulation. Several 5-HT receptor subtypes have been confirmed to be associated with nociception in the spinal cord, including 5-HT1A receptor, 5-HT1B receptor, 5-HT2 receptor, 5-HT3 receptor, and 5-HT7 receptor [34]. Previous studies have demonstrated that 5-HT1A receptor, 5-HT3 receptor, and 5-HT7 receptor are highly expressed in the SDH and associated with nociceptive regulation [13,39,40]. Most research has shown that 5-HT1A receptor and 5-HT3 receptor play a key role in pain transmission in the spinal cord [4]. 5-HT3 receptor, the only ionotropic 5-HT receptor, is a pentameric channel permeable to cations, causing the depolarization of neurons and increased excitability. 5-HT1A receptor is a G-protein-coupled receptor, negatively coupled with adenylyl-cyclase, causing the opening of potassium channels, the closing of calcium channels, and inducing neuronal hyperpolarization [27,31]. Therefore, the function of the 5-HT1A receptor and 5-HT3 receptor is related to the excitability of SDH neurons.
Many studies have shown that excitability of SDH neurons is increased in chronic neuropathic pain [14]. In consequence, we hypothesized that hyperalgesia of the 6-OHDA-induced PD model may be related to increased excitability of SDH neurons, and functional change of 5-HT3 receptor can reverse hyperalgesia of 6-OHDA lesioned rats and decrease excitability of SDH neurons. To test this hypothesis, we used wholecell patch-clamp and pharmacological methods to evaluate the effect of 5-HT3 receptor and 5-HT1A receptor in SDH on the hyperalgesia of the 6-OHDA PD rat model. Our study provides a novel mechanism or therapeutic strategy for pain in patients with PD.

Animals
Adult male Sprague-Dawley rats (150-180 g) were housed (five rats per cage) at a controlled temperature of 22-25 °C, 40-60% relative humidity, and a 12-h light/dark cycle with unlimited access to food and water. All experiments were approved by the Animal Use and Care Committee of Soochow University and followed the guidelines of the International Association for the Study of Pain.

6-OHDA-Induced PD Rat Model
Rats were anesthetized with 3% isoflurane-induced, as soon as the loss of righting reflex and anesthesia was maintained with 1.5% isoflurane and then placed in a stoelting stereotaxic apparatus (RWD Co., Shenzhen, China). Perforations were slowly drilled into the skull to allow for the insertion of a 10-μL Hamilton syringe, using the following stereotactic coordinates (from a rat brain atlas) − 5.3 mm anteroposterior, ± 1.8 mm mediolateral, and − 7.8 mm dorsoventral (from the dura) from the bregma. The animals were injected with 8 μg 6-OHDA (dissolved in 4 μL saline containing 0.02% ascorbic acid) or 4 μL saline (containing 0.02% ascorbic acid) on each side at a rate of 0.5 μL/min. The syringe remained in place for 8 min after completion of the injection and was then slowly retracted. The rats were transferred to a recovery cage with soft, non-particular bedding and were placed on the side for comfortable breathing. The experimenter monitored the animals until they were fully alert, ambulant, and started drinking and transferred them to the animal facility [43].

Rotarod Test
Three weeks after surgery, the rats were subjected to a rotarod test to evaluate motor coordination and balance.
Before the formal experiment, the rats were trained for 3 consecutive days on the rotarod system (SANS, Jiangsu Province, China) at an increasing speed (4 to 25 r/min at a rate of 0.5 r/min). The trained rats were tested three times at a speed of 25 r/min, and the mean duration was recorded to analyze.

Open Field Test
The open field test was used to evaluate animals' locomotor performance and anxiety behavior. Animals were placed in a square black plank (1 m × 1 m), surrounded by a 40-cm white wall, and divided into a 25 × 25-cm square by white lines. Each animal was allowed to explore the box freely for 10 min. Their behavior was recorded by an overhead camera. At the end of each trial, 75% alcohol was used to clean the box.

Mechanical Allodynia
The mechanical threshold was evaluated with E-von Frey (Mgo Basile, Italy) or von Frey filaments (Aesthesio, Dan-Mic Global, San Jose, CA, USA) using the up-down paradigm. The rats were placed in Plexiglas chambers on a wire mesh platform for 0.5-1 h. The calibrated Von Frey filaments were applied to the plantar surface of rats' right hind paw with sufficient force to bend the filaments for 10 s or until the rat withdrew. The threshold was calculated as the force of the smallest filament causing the withdrawal behavior [44].

Thermal Allodynia
The tail flick test was used to assess the thermal threshold of the animal. Radiant heat was applied to the tail using a tail flick apparatus (Mgo Basile, Italy). The radiant heat density was adjusted to yield the tail flick reaction on sham rats in 10-12 s. A cut-off of 15 s was set to prevent the risk of burns. The time from the laser beginning to the time the animal flicked its tail was recorded as the thermal threshold to analyze.

Electrophysiology
After incubation for 1 h, one of the spinal cord slices was transferred to the recording grooves of nylon mesh, and the slice was fixed with a U-shaped nylon mesh. The slices were recorded at room temperature, perfused with oxygenated recording fluid at a rate of 1.5 mL/min throughout. Neurons used for recording in lamina II of SDH were visualized with an Olympus BX51WI microscope with a 40 × water-immersion objective, infrared differential interference contrast (IR)-DIC. Recordings were performed in current-clamp or voltage-clamp mode at a holding potential of − 70 mV, unless otherwise indicated. The pipettes (4-8 MΩ tip resistance) were filled with internal solution (in mM):133 Kgluconate, 0.6 EGTA, 8 NaCl, 2 Mg-ATP, 0.3 Na-GTP, and 10 HEPES (pH = 7.2-7.3). Data were acquired using EPC10 amplifier and patchmaster software (HEKA, Germany), filtered and sampled at 5 kHz with a Bessel filter amplifier. Analysis was done with Clampfit software (pClamp10, Molecular Devices, USA). The resting potential was recorded immediately after the action potential (AP) configuration was executed. The firing patterns were evaluated using 1.5 s depolarizing current (step 20 pA) in the current clamp.

Western Blotting
Immunostaining was carried out using anti-TH antibody (1:5000, T1299, Sigma, USA) and mouse anti-β actin antibody (1:5000, A3854, Sigma, USA). Anti-mouse secondary antibody was used for mouse antibodies. Immunoreactive bands were obtained by clinx science instrument (Clinx, China). Densitometric analysis was performed using ImageJ software (National Institutes of Health, USA). The drug concentration used in the patch clamp test was based on previous studies [1,22,25,26,45]. For behavioral tests, ondansetron (0.1 mg/kg), m-CPBG (0.1 mg/kg), WAY-100635 (0.1 mg/kg), and 8-OH DPAT (0.1 mg/kg) were dissolved in 0.9% normal saline and injected intrathecally into the L4-L6 spinal cord via a microsyringe. The rats were given persistent anesthesia with isoflurane and placed on a board with the abdomen facing down and the L3-L5 spinal segments curved. The tail swing of the rats during intrathecal injection was considered successful. The drug (10 μL/rat) was slowly injected, and the needle was left for at least 30 s to ensure that the drug did not reflux [12,30]. The drug concentration used in behavioral test was based on previous studies [15,33].

Statistical Analysis
Data are presented as mean ± SEM. Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software, La Jolla, CA, USA). Normality was first checked for all data before analysis. Significance was determined using Student's t test, one-way ANOVA followed by Tukey's post hoc test and two-way ANOVA. P < 0.05 was considered significant.

Decreased Mechanical and Thermal Pain Threshold in 6-OHDA Rats
We observed the behavioral phenotypes of 6-OHDA rats at 4 weeks after 6-OHDA injection. The rotarod test showed shorter retention time in 6-OHDA rats than in sham rats ( Fig. 1a, P < 0.001, two-sample t test). The open field test had no difference in movement distance between 6-OHDA and sham rats (Fig. 1b, P > 0.05, two-sample t-test). Compared to sham rats, thermal pain threshold (tail-flick latency, TFL) and mechanical pain threshold (PWT) were significantly reduced in 6-OHDA rats (Fig. 1c, d, P < 0.01, P < 0.001, two-sample t test). These behavioral tests showed that the passive movement ability of 6-OHDA rats was impaired, the active locomotion ability was normal, and mechanical and thermal pain thresholds were reduced.
To confirm the 6-OHDA-induced PD model, western blotting was used to proceed with tyrosine hydroxylase (TH) quantification. TH protein level was decreased in the striatum of 6-OHDA rats (Fig. 1e, f, P < 0.01, two-sample t-test).

Classification of SDH Neuron Firing Pattern
Our previous study demonstrated that 5-HT content of 6-OHDA rats was decreased in the SDH, indicating that the 5-HT system is involved in hyperalgesia of 6-OHDA lesioned rats [43]. In addition, as two main functional 5-HT receptors, 5-HT3 receptor and 5-HT1A receptor were related to cell excitability in the spinal cord [27,31]. Therefore, to further explore the mechanisms of 5-HT3 receptor and 5-HT1A receptor in pain hypersensitivity of 6-OHDA rats, a whole-cell patch-clamp was used to detect the action potential of SDH neurons on the outside part of Lamine II of the fourth to sixth lumbar spinal and group data (f) for striatal TH protein levels (n = 3 per group). Two-sample t test was used for analysis. **P < 0.01; ***P < 0.001 versus sham group; ns, not significant cord (L4-L6) in sham group and 6-OHDA lesioned group (Fig. 2c). The action potential patterns of recorded neurons are divided into five categories: tonic pattern, delayed pattern, phasic pattern, gap pattern, and single pattern. The tonic pattern is characterized by an action potential discharge that persists during the whole current step. The delayed pattern is characterized by the action potential generated after a delay. The phasic pattern is typically restricted to one, a few, or a short burst of action potentials, whereafter the activity quickly returns to the resting state. The gap firing pattern is a response to injected depolarizing current in which an initial action potential is followed by a long interspike interval and then regular firing. The single pattern is characterized by an action potential discharge that initiates a single spike during the whole current step (Fig. 2a).
We recorded 313 SDH neurons in the spinal cord slice from 6-to 8-week-old rats. Among 161 neurons from spinal cord slices of sham rats, 112 exhibited a tonic pattern, 20 a delayed pattern, 12 a phasic pattern, seven a gap pattern, and 10 a single pattern when current steps were applied from a membrane potential of about − 70 mV. Similar to the discharge classification results of the sham group, among 152 neurons of 6-OHDA rats, 99 exhibited a tonic pattern, 20 a delayed pattern, 13 a phasic pattern, 11 a gap pattern, and nine a single pattern when current steps were applied from a membrane potential of about − 70 mV (Fig. 2b). Because most recorded neurons exhibited tonic firing patterns, we treated only tonic-type neurons, and neurons with other firing types were not included in the statistics.

Increased Excitability of SDH Neurons in 6-OHDA Rats
We compared the action potential parameters of neurons showing a tonic firing pattern in the 6-OHDA group and the sham group. The resting membrane potential of neurons in 6-OHDA rats was significantly increased (Fig. 3a, P < 0.001, two-sample t-test). However, the threshold and amplitude of action potential remained unchanged compared to those in sham rats (Fig. 3b, c, P > 0.05, two-sample t test). Rheobase, AP half width and Rin did not change, too (see in the supplementary material). The number of action potentials was increased under 40-80 pA current stimulation in the SDH of 6-OHDA rats compared to sham rats (Fig. 3d, e, P < 0.001, two-sample t test). These results suggested the increased excitability of SDH neurons in the 6-OHDA-induced PD rat model. Because the parameters of action potential of SDH neurons had the best uniformity at an injection current of 40 pA, we focused on the changes in action potential of SDH neurons at 40 pA before and after drug administration.

Effect of 5-HT3 Receptor Agonist and Antagonist on Excitability of SDH Neurons
To examine the effect of the 5-HT3 receptor involved in the excitability of SDH neurons, AP was recorded in the spinal cord slice in the presence of pre-and post-bath application The representative trace of typical neurons showed that bath application of ondansetron or palonosetron caused a significant decrease in AP frequency in SDH neurons under 40 pA current stimulation (Fig. 4a, c, d, f, P < 0.05, P < 0.01, P < 0.001, one-way ANOVA). Ondansetron or palonosetron did not significantly affect SDH neural membrane potentials in the sham group and 6-OHDA group (Fig. 4b, e, P < 0.05, P < 0.001, one-way ANOVA). However, the representative trace of typical neurons showed that bath application of m-CPBG or SR57727 had no significant effect on AP frequency in SDH neurons under 40 pA current stimulation (Fig. 4g, i, j, l, P < 0.05, oneway ANOVA). Similarly, m-CPBG or SR 57,727 did not significantly affect SDH neural membrane potentials in the sham group and 6-OHDA lesioned group (Fig. 4h, k, P < 0.05, P < 0.01, one-way ANOVA).

Effect of 5-HT1A Receptor Agonist and Antagonist on Excitability of SDH Neurons
We examined the role of the 5-HT1A receptor involved in the excitability of SDH neurons by application of 5-HT1A receptor selective antagonist WAY-100635 (10 μmol/L) or p-MPPI (10 μmol/L) and 5-HT1A receptor agonist 8-OH DPAT (10 μmol/L) or eptapirone (10 μmol/L) in the Krebs resolution. The representative trace of typical neurons showed that bath application of WAY-100635, p-MPPI, eptapirone, and 8-OH DPAT had no significant effect on AP frequency in SDH neurons under 40 pA current stimulation (Fig. 5a, c, d, f, g, i, j, l, P > 0.05, one-way ANOVA). WAY-100635, p-MPPI, eptapirone, and 8-OH DPAT did not Resting membrane potential (a), action potential threshold (b), and action potential amplitude (c) were analyzed. (n = 18 for sham group and n = 28 for 6-OHDA group). d Action potential frequency was counted under 40 pA, 80 pA, and 120 pA current stimulation. e Statistical plot of action potential frequency (n = 18 for sham group and n = 28 for 6-OHDA group). Two-sample t test was used for analysis. ***P < 0.001 versus sham group; ns, not significant significantly affect SDH neural membrane potentials in the sham group and 6-OHDA group (Fig. 5b, e, h, k, P > 0.05, one-way ANOVA).

Discussion
In the present study, our 6-OHDA PD rat model induced by lesions of dopaminergic neurons in the SNpc developed thermal and mechanical hypersensitivity at 4 weeks after surgery. Electrophysiological results suggested that SDH neurons had increased excitability, and 5-HT3 receptor antagonist ondansetron and palonosetron reversed this hyperexcitability. Intrathecal injection of ondansetron significantly attenuated the mechanical hyperalgesia and thermal hyperalgesia in the 6-OHDA lesioned rats. Our findings suggest that inhibition of spinal 5-HT3 receptor and SDH neuronal excitability alleviates hyperalgesia of 6-OHDA lesioned rats.
According to Braak PD staging, the spinal cord can be affected in an early phase of the disease [6]. Clinical research has suggested that early PD patients exhibit increased spinal nociceptive responses compared with healthy controls [5]. These studies have indicated that spinal cord lesions are closely related to pain in PD. In a transgenic mouse A53T PD model, α-synuclein is highly expressed in the spinal cord  (g, j). Resting membrane potential (h, k) and AP frequency (i, l) were analyzed. n = 6-9 for sham group and n = 6-8 for 6-OHDA group. One-way ANOVA followed by Tukey's post hoc analysis *P < 0.05; **P < 0.01; ***P < 0.001 versus as indicated; ns, not significant [38], and a humanoid study has shown that spinal cord stimulation alleviates motor deficits in a primate model of PD [37], which suggest that the spinal cord is a promising target for treatment of pain in PD. Our previous study has shown that 6-OHDA lesioned rats manifest hyperalgesia in response to thermal and mechanical stimuli, and this hypersensitivity could be attributed partially to the decreased 5-HT content in the SDH [43]. These results suggest that the spinal cord and its 5-HT receptors are involved in PD-related pain, but the underlying mechanisms are still elusive.
To explore what role the spinal cord plays in pain in PD, we investigated the electrophysiological properties of spinal Fig. 6 Effects of 5-HT3 receptor agonist and antagonist on thermal and mechanical hypersensitivity in 6-OHDA-lesioned rats. a, b Intrathecal injection of ondansetron (0.1 mg/kg) significantly attenuated mechanical and thermal hypersensitivity in 6-OHDA rats. c, d Intrathecal injection of m-CPBG (0.1 mg/ kg) did not inhibit mechanical and thermal hypersensitivity in 6-OHDA rats. n = 3-7 for each group, *P < 0.05, **P < 0.01 versus 6-OHDA + NS group, two-way ANOVA Fig. 7 Effects of 5-HT1A receptor agonist and antagonist on thermal and mechanical hypersensitivity in 6-OHDAlesioned rats. a, b Effect of intrathecal injection of 8-OH DPAT (0.1 mg/kg) on mechanical and thermal hypersensitivity of 6-OHDA rats. c, d Intrathecal injection of WAY-100635 (0.1 mg/kg) did not affect mechanical and thermal hypersensitivity in 6-OHDA rats. n = 5-8 for each group, two-way ANOVA cord slices from the PD rat model. We recorded 161 neurons from the sham group and 152 from the 6-OHDA group. Consistent with previous research [29], our results demonstrated that, among the recorded neurons, the number of neurons exhibiting a tonic pattern was the largest (74-76%). The tonic firing pattern is deemed to be a hallmark of spinal inhibitory interneurons, in contrast to spinal excitatory interneurons that show delayed or gap firing patterns [41]. Moreover, the spinal inhibitory interneurons play a key role in the pain transmission in SDH. We compared the action potential parameters of neurons showing a tonic firing pattern in the 6-OHDA group and the sham group. Neurons with tonic firing pattern had increased action potential frequency in the 6-OHDA lesioned group compared with the sham group, suggesting that these neurons transmit a higher level of nociceptive information to the central nervous system. Consistently, Keri-Ann Charles et al. [9] have reported that wide dynamic range neurons (spinal cord, lamina V) have a higher discharge frequency in 6-OHDA lesioned rats.
We explored the exact functional role of 5-HT3 receptor and 5-HT1A receptor in the hyperexcitability of SDH neurons in 6-OHDA lesioned rats. In terms of electrophysiology, bath application of 5-HT3 receptor antagonist ondansetron or palonosetron reversed the increased cell excitability of SDH neurons. Tomoyose et al. have reported that inhibition of 5-HT3 receptor inhibits excitatory synaptic transmission in the SDH, and Xu et al. demonstrated that inhibition of 5-HT3 receptor alone had no significant effect on inhibitory synaptic transmission in the SDH. Thus, the inhibitory effect of ondansetron and palonosetron on the excitability of SDH neurons may be attributed to attenuation of excitatory synaptic transmission. In terms of behavior tests, our results showed that intrathecal injection of ondansetron significantly attenuated the hyperalgesia of 6-OHDA rats. Consistent with our results, intrathecal administration of the 5-HT3 receptor antagonist ondansetron has been reported to inhibit spontaneous pain behavior following intraplantar formalin injection [11] and reverse mechanical allodynia in a model of spinal cord injury neuropathic pain [28,33]. However, Christopher M. Peters et al. have reported the lack of analgesic efficacy of spinal ondansetron on thermal and mechanical hypersensitivity following spinal nerve ligation in rats. The discrepancy may be due to dual effects of 5-HT3 receptor activation on distinct populations of neurons in the SDH, resulting in a lack of effect on behavioral outcomes.
Previous studies have shown that intrathecal administration of selective 5-HT3 receptor agonist (SR 57,227 and m-CPBG) alleviates hyperalgesia in rats with spinal nerve ligation [21]. Surprisingly, we found that m-CPBG or SR 57,727 had no obvious effect in electrophysiological and behavioral tests. We tried a high concentration of m-CPBG or SR 57,727, but still saw no obvious effect. Several recent functional studies have demonstrated that the dominant effect of 5-HT3 receptor activation in the spinal cord is antinociceptive by promoting GABAergic inhibitory synaptic transmission [19,20,45]. GABA interneurons in the SDH of 6-OHDA lesioned rats may have changes in cell structure and function, resulting in inability of m-CPBG to exert an analgesic effect. Future studies are warranted to examine the function of the GABAergic system in 6-OHDA rats.
Previous studies have shown that 8-OH DPAT produces antinociception in a model of carrageenan-induced inflammation and neuropathic pain [23,27]. Sagalajev et al. showed that intrathecal injection of 5-HT1A receptor antagonist WAY-100635 reversed hyperalgesia induced by high-dose glutamate in central amygdala [36]. It is unusual that our research showed that bath application or intrathecal injection of 5-HT1A receptor antagonist WAY-100635, p-MPPI, and 5-HT1A receptor agonist 8-OH DPAT or eptapirone had little effect on SDH neuronal excitability and hyperalgesia in 6-OHDA lesioned rats. The drug dose was selected according to previous studies [1,15,22,25,26]. However, the effects of 5-HT1A agonists and antagonists may be different in 6-OHDA lesioned rats, so more appropriate drug concentrations need to be investigated.
Huang et al. have indicated that the 5-HT3 receptor was expressed in the GABAergic neurons in the mouse SDH [24]. Some researchers have reported that GABAa antagonists can reverse the antinociception and electrophysiological effects of 5-HT3 receptor antagonists [2]. This suggests that 5-HT3 receptor in the spinal cord changes the function of GABA neurons, thereby affecting the excitability of projection neurons to further exert analgesic effects. This underlying analgesic mechanism of 5-HT3 receptor needs to be further explored.
Descending pain modulation includes the serotonin and norepinephrine systems. Intrathecal injection of clonidine, an alpha 2 adrenoceptor agonist, can attenuate pain hypersensitivity in 6-OHDA lesioned PD rat model [8]. Hayashida et al. demonstrated that alpha 2 adrenoceptor-mediated antihypersensitivity can be weakened by the blockade of spinal 5-HT3 receptor in rats with spinal nerve ligation, via reduction of basal GABA tone in the spinal cord, indicating a common mechanism to reduce neuropathic pain between spinal alpha 2 adrenoceptor and 5-HT3 receptor [21]. A potential linkage between spinal alpha 2 adrenoceptor and 5-HT3 receptor in pain regulation may exist in 6-OHDA lesioned PD rat model.
In summary, we found that inhibition of spinal 5-HT3 receptor and SDH neuronal excitability alleviates hyperalgesia of 6-OHDA lesioned PD rats.