Several studies have suggested that 5-HT7 receptors are involved in nociceptive processing but the exact contribution of peripheral versus central 5-HT7 receptors still needs to be elucidated. In the present study, the respective roles of peripheral and spinal 5-HT7 receptors in the modulation of mechanical hypersensitivity were investigated under two different experimental pain conditions. In a first set of experiments, the selective 5-HT7 receptor agonist, E-57431, was systemically, intrathecally or peripherally (intraplantarly) administered to rats sensitized by intraplantar injection of capsaicin. Oral administration of E-57431 (1.25–10 mg/kg) was found to exert a clear-cut dose-dependent reduction of capsaicin-induced mechanical hypersensitivity. Interestingly, intrathecal administration of E-57431 (100 μg) also inhibited mechanical hypersensitivity secondary to capsaicin injection. In contrast, a dose-dependent enhancement of capsaicin-induced mechanical hypersensitivity was observed after local intraplantar injection of E-57431 (0.01–1 μg). In a second set of experiments, E-57431 was systemically or intrathecally administered to rats submitted to neuropathic pain (spared nerve injury model). Significant inhibition of nerve injury-induced mechanical hypersensitivity was found after intraperitoneal (10 mg/kg) as well as intrathecal (100 μg) administration of E-57431 in this chronic pain model. These studies provide evidence that, under sensitizing neurogenic/neuropathic conditions, activation of 5-HT7 receptors exerts antinociceptive effects at the level of the spinal cord and pronociceptive effects at the periphery. The antinociceptive effect mediated by central 5-HT7 receptors seems to predominate over the pronociceptive effect at the periphery when a selective 5-HT7 receptor agonist is systemically administered.
Serotonin (5-hydroxytryptamine [5-HT]) is involved in pain transmission, processing and control (Eide and Hole, 1993; Millan, 2002; Kayser et al., 2010). Much of pain research has focused on 5-HT1A, 5-HT1B/1D, 5-HT2A/2C and 5-HT3 receptors (Eide and Hole, 1993; Oyama et al., 1996; Obata et al., 2000; Kayser et al., 2002, 2010; Ahn and Basbaum, 2006; Colpaert, 2006; Pichon et al., 2010), but the role played by other receptors, including 5-HT7 receptors, just begins to be elucidated. Interestingly, both pronociceptive and antinociceptive roles have been suggested for 5-HT7 receptors.
A first series of investigations showed that systemic administration of selective 5-HT7 receptor agonists inhibited capsaicin-induced and nerve injury-induced hypersensitivity in mice. As expected from 5-HT7 receptor-mediated effects, antinociceptive-like responses elicited by agonists were reversed by 5-HT7 receptor antagonists, and pronociceptive effects were observed when selective 5-HT7 receptor antagonists were systemically administered (Brenchat et al., 2009, 2010). Nevertheless, the effect of systemically administered 5-HT7 receptor ligands could represent an overall balance of discrepant, opposite effects at different locations because 5-HT7 receptors are expressed at different sites compatible with a role in pain control, including dorsal root ganglia (DRG) (Pierce et al., 1997; Meuser et al., 2002; Doly et al., 2005) and spinal (Meuser et al., 2002; Doly et al., 2005) and supraspinal (thalamus, midbrain, pons and medulla) (Stowe and Barnes, 1998; Neumaier et al., 2001; Martin-Cora and Pazos, 2004) regions in the CNS.
Peripheral tissue injury causes the release of 5-HT from platelets and mast cells, which acts in combination with other inflammatory mediators to excite afferent fibers (Sommer, 2004). Indeed, convergent data in the literature suggest a pronociceptive role of 5-HT7 receptor at the periphery (Meuser et al., 2002; Rocha-González et al., 2005) based on investigations with nonselective 5-HT7 receptor agonists, such as 5-HT itself, 5-carboxamidotryptamine (5-CT; binding affinity Ki values at human 5-HT receptors: 5-HT1A = 0.34 nM; 5-HT1D = 0.70 nM; 5-HT7 = 0.93 nM) and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; binding affinity Ki values at human 5-HT receptors: 5-HT1A = 0.06 nM; 5-HT1D = 47 nM; 5-HT7 = 467 nM) (Boess and Martin, 1994; Doménech et al., 1997), administered locally (intraplantarly or intra-articularly) in the context of tissue injury and inflammation.
On the other hand, contradictory results have been reported with regard to the spinal contribution of 5-HT7 receptors in nociception. A pronociceptive role of spinal 5-HT7 receptors was initially proposed by Rocha-González et al. (2005), who found that spinal administration of the 5-HT7 receptor antagonist SB-269970 inhibited the increased formalin-evoked nociceptive responses elicited by intrathecal administration of 5-CT. However, other studies showed that intrathecal administration of 5-HT7 receptor antagonists inhibited the antinociceptive effects of morphine, tramadol and its metabolite O-Desmethyltramadol, and WIN 55,212-2 and ACEA, which led to the suggestion that activation of spinal 5-HT7 receptors, via descending serotonergic pathways, is required for opioid and CB1 receptor-mediated antinociception (Dogrul and Seyrek, 2006; Dogrul et al., 2009; Seyrek et al., 2010; Yanarates et al., 2010). Differences in animal models, experimental approaches and ligands used as pharmacological tools may account for the apparent discrepancies regarding the role played by spinal 5-HT7 receptors in nociception.
In an attempt to solve these discrepancies and to determine the respective contributions of spinal versus peripheral activation of 5-HT7 receptors, we compared the effects of systemic, intrathecal or peripheral administration of a selective 5-HT7 receptor agonist, E-57431 (binding affinity Ki values at human 5-HT receptors: 5-HT7 = 0.47 nM; 5-HT1D = 53 nM; 5-HT2A = 560 nM; and >1000 nM for the rest) (Brenchat et al., 2010), on mechanical hypersensitivity secondary to either intraplantar injection of capsaicin or spared injury to the sciatic nerve in the rat.
Male Wistar rats weighing 230–300 g (Charles River, France) were used in these studies. Rats were housed in groups of three, provided with food and water ad libitum and kept in controlled laboratory conditions with temperature maintained at 21 ± 1 °C and 12-h light/dark cycles with light on at 07:00 h. Experiments were carried out in a sound-attenuated, air-regulated, experimental room. All experimental procedures and animal husbandry were conducted according to ethical principles for the evaluation of pain in conscious animals (Zimmermann, 1983) and to ethical guidelines of the European Communities Council Directive of 24 November 1986 (86/609/ECC). The experimental work received approval by the Local Ethical Committee.
Capsaicin (8-methyl-N-vanillyl 6-nonamide) was purchased from Sigma-Aldrich Co. (Spain) and dissolved in 1% dimethylsulfoxide (DMSO). E-57431 (2-(2-(dimethylamino)ethyl)-4-(1,3,5-trimethyl-1H-pyrazol-4-yl)phenol hydrochloride) (MW 309.83 g/mol) was originated and synthesized by Laboratories Esteve (Barcelona, Spain). E-57431 is a potent 5-HT7 receptor agonist with more than 100× selectivity over other 5-HT receptor subtypes and non-5-HT receptors, transporters and ion channels (Brenchat et al., 2010). E-57431 was dissolved in 0.5% (hydroxypropyl)methyl cellulose (Sigma–Aldrich) for systemic administration (2 ml/kg), in physiological saline for intraplantar administration (10 μl), and in artificial cerebrospinal fluid (NaCl 147 mmol/l, KCl 2.7 mmol/l, CaCl2 1.2 mmol/l and MgCl2 0.85 mmol/l; CMA Microdialysis, N. Chelmsford, MA, USA) for intrathecal administration (10 μl).
2.3 Intrathecal catheterization
Chronic catheterization of the spinal subarachnoid space was performed as described previously (Storkson et al., 1996; Pogatzki et al., 2000). After basal measurements, surgery to generate intrathecal catheterization was carried out. Rats were anesthetized with pentobarbital (60 mg/kg, i.p.). The lumbar region was shaved, prepared with povidone-oidine, made kyphotic and incised 2–3 cm longitudinally in the midline at the level of the iliac crests. The space between the lumbar vertebrae L5 and L6 was punctured with a 22G hypodermic needle. Tail-flick or hind paw retraction indicated an intrathecal location. A 28G PU catheter (10 cm length, 0.36 mm OD; 0.18 mm ID, Alzet), reinforced with a teflon-coated stainless steel stylet, was advanced through the needle cranially. The needle and the stylet were removed and the catheter withdrawn so that 5 cm extended outside of the lumbar musculature. Superglue-3 gel (Loctite®) was used to fix the catheter to the fascia. The distal end of the 28G PU catheter was connected to an 8-cm length tube (0.84 mm OD; 0.36 mm ID) ended with an ALZET connection (1.02 mm OD; 0.61 mm ID). The catheter was tunneled under the skin to the cervical region, flushed with CSF and sealed with a cautery pen. The dead volume of the catheter was 20 μl. The skin was then closed and animals allowed to recover. Pharmacological evaluation was performed 3 days (capsaicin model) and 14 days (spared nerve injury) after catheterization. Catheterized rats had no detectable motor deficits.
2.4 Capsaicin model
Sensitization by intraplantar capsaicin injection results in hypersensitivity to both thermal and mechanical stimuli (Gilchrist et al., 1996). In this study, sensitization by intraplantar capsaicin injection was used to assess the effect of treatments on the response to mechanical stimulation (von Frey test) in rats.
Capsaicin was administered (50 μl) by intraplantar (i.pl.) injection into the mid dorsal surface of the right hind paw. An equal volume of solvent (1% DMSO) was used in the control group. The intraplantar injection was performed by means of a 100 μl 1710 TLL Hamilton® microsyringe (Teknokroma, Spain) with a 301/2- gauge needle. In a pilot study, different doses of capsaicin (0.1, 1, 10, 20 and 30 μg) were tested for their ability to induce mechanical hypersensitivity (i.e., allodynia) using von Frey filaments (data not shown). The capsaicin dose of 30 μg was able to induce robust and persistent paw withdrawal responses to subthreshold (i.e., allodynic) mechanical stimuli and was then used to evaluate the antinociceptive (i.e., antiallodynic) effects of the selective 5-HT7 receptor agonist E-57431. The dose of 10 μg of capsaicin was unable to induce mechanical hypersensitivity and was then used to explore possible pronociceptive (i.e., proallodynic) effects of E-57431. Basal measurements were always done before capsaicin or vehicle injection and drug treatments.
2.4.1 Systemic treatment
The antinociceptive effects of oral administration of the selective 5-HT7 receptor agonist E-57431 were investigated after intraplantar injection of capsaicin (30 μg/50 μl). Prior to pharmacological treatments, the von Frey test was done 30 min after intraplantar injection of capsaicin to discard animals that did not develop capsaicin-induced mechanical hypersensitivity. E-57431 (1.25, 2.5, 5 and 10 mg/kg) or vehicle was orally administered 30 min after intraplantar injection of capsaicin. Mechanical hypersensitivity was evaluated before capsaicin injection (basal), before administration (pre-treatment) and 30 min after oral administration of E-57431 or vehicle, corresponding to 60 min after capsaicin injection.
2.4.2 Intrathecal treatment
Intrathecal treatments were performed 3 days after intrathecal catheterization. Prior to pharmacological treatments, the von Frey test was done 30 min after intraplantar injection of capsaicin (30 μg/50 μl) to discard animals that did not develop capsaicin-induced mechanical hypersensitivity. E-57431 (100 μg/10 μl) or vehicle was intrathecally administered 30 min after intraplantar injection of capsaicin. Mechanical hypersensitivity was evaluated before capsaicin injection (basal), before intrathecal administration (time 0) and 30, 60 and 90 min after intrathecal administration of E-57431 or vehicle, corresponding to 60, 90 and 120 min after capsaicin injection.
2.4.3 Local intraplantar treatment
To study peripheral, local effects, E-57431 (0.01, 0.1 and 1 μg) or vehicle was intraplantarly administered into the right hind paw 10 min before capsaicin (10 μg/50 μl) or vehicle (DMSO 1%) injection into the same paw, and mechanical sensitivity was evaluated after a further 20 min period. To assess whether the effect of intraplantar injection of E-57431 was local, the 5-HT7 receptor agonist was also administered into the left (contralateral) hind paw while capsaicin was injected into the right hind paw, and the corresponding effect on mechanical hypersensitivity was assessed in the right hind paw.
2.5 Spared nerve injury
This partial denervation model of neuropathic pain was induced according to the method previously described by Decosterd and Woolf (2000). After basal measurements, surgery to generate nerve injury was carried out. Rats were anesthetized with pentobarbital (60 mg/kg i.p.) and the skin on the lateral surface of the thigh was incised. A section was made directly through the biceps femoris muscle to expose the sciatic nerve and its three terminal branches: the sural, common peroneal and tibial nerves. The procedure comprised ligation and axotomy of the tibial and common peroneal nerves leaving the sural nerve intact. The common peroneal and the tibial nerves were tight-ligated with 5.0 silk and sectioned distal to the ligation. Great care was taken to avoid any contact with or stretching of the intact sural nerve. Muscle and skin were then closed in two layers. Control, sham-operated rats underwent the same surgical procedure and the sciatic nerve was exposed, but neither ligated nor sectioned. Behavioral testing using von Frey filament stimulation was assessed on days 2, 7, and 14 after the surgical procedure in independent groups of nerveinjured and sham-operated rats to monitor the development of mechanical hypersensitivity.
2.5.1 Systemic and intrathecal treatments
On days 15–18 post-surgery, when neuropathic pain-related behaviors (i.e., mechanical allodynia) had clearly developed, two independent experiments using different routes of administration (intraperitoneal or intrathecal) were performed to investigate the effect of the selective 5-HT7 receptor agonist E-57431: (1) E-57431 (10 mg/kg) or vehicle was administered i.p. on day 15 post-surgery and von Frey filament testing was performed before nerve-injury (basal), before intraperitoneal administration (time 0) and 15, 30, 60 and 90 min thereafter; (2) three different doses of E-57431 (10 μg, 30 μg and 100 μg) or vehicle were administered intrathecally on days 15–18 post-surgery following a Latin square design and allodynia-like responses were determined before nerve-injury (basal), before intrathecal administration (time 0) and 15, 30, 60, 90 and 120 min thereafter.
2.6 von Frey test
Behavioral testing was performed in blinded conditions for drugs and doses tested in independent groups of rats. Mechanical hypersensitivity was quantified as previously described (Chaplan et al., 1994) by determining the pressure thresholds eliciting withdrawal of the ipsilateral and contralateral hind paws in response to graded stimulation with von Frey filaments applied onto the plantar surface. The skin area stimulated with von Frey filaments was the mid plantar surface of the hind paw in the capsaicin model (area surrounding the site of capsaicin injection in the ipsilateral paw) and the lateral plantar surface of the hind paws in the case of nerve-injured rats. Stimulation of the toes and heel was avoided. Rats were placed under plastic boxes above a wire mesh floor, which allowed full access to the paws. Behavioral acclimation was allowed for at least 30 min. Mechanical paw withdrawal thresholds were measured using the up-down testing paradigm (Dixon, 1980; Chaplan et al., 1994) by applying von Frey filaments in log increments of force (0.4, 0.6, 1, 2, 4, 6, 10, and 15 g) to the test area for about 3 s with an interstimuli interval of approximately 1 min. The 2-g stimulus was applied first. Whenever a withdrawal response to a given probe occurred, the next smaller von Frey filament was applied. Whenever a negative response occurred, the next higher force was applied. The test continued until: (1) the responses to four identical stimuli after the first change in response had been obtained or (2) a negative response to the highest von Frey filament or a positive response to the lowest von Frey filament had occurred. Abrupt paw withdrawal, licking, and shaking were regarded as positive responses. The 50% paw withdrawal threshold values were derived according to a previously described method (Chaplan et al., 1994).
At least a 50% reduction of the baseline threshold was established as acceptance criterion to select animals developing tactile allodynia both 30 min after capsaicin (30 μg) injection and 14 days after nerve injury. That is, the withdrawal response must be evoked when the paw is stimulated using a von Frey hair with bending force ≤7.5 g to select animals developing tactile allodynia for pharmacological studies.
2.7 Data analyses
Data are presented as mean paw withdrawal threshold in grams ± SEM. Statistical analysis to test significant differences among groups was made using ANOVA followed by Bonferroni's post hoc comparison. The level of significance was set at p < 0.05. Data analysis and graphing were done using GraphPad Prism software (version 4.0; GraphPad Software, Inc., USA).
3.1 Dose-response antinociceptive effect of orally administered E-57431 in the capsaicin model
Rats intraplantarly injected with capsaicin (30 μg) into the midplantar surface of the right hind paw (ipsilateral paw) developed mechanical hypersensitivity, evidenced by a reduction of the mechanical threshold triggering withdrawal of the ipsilateral paw in the von Frey test 30 min after injection (Fig. 1). In particular, 83% of rats injected with capsaicin developed mechanical hypersensitivity (≥50% reduction of the baseline threshold) and were thus selected to investigate the role of 5-HT7 receptors. No significant changes in the response to mechanical stimuli were observed in the contralateral paw (data not shown).
Oral administration of the selective 5-HT7 receptor agonist E-57431 30 min after capsaicin injection dose-dependently reversed capsaicin-induced mechanical hypersensitivity in the ipsilateral paw (Fig. 1). Significantly increased paw withdrawal thresholds in rats treated with E-57431 were found 30 min after oral administration of 5 and 10 mg/kg compared to vehicle-treated rats. Treatments with lower doses (1.25 and 2.5 mg/kg) of E-57431 did not significantly modify this evoked, pain-related behavior compared to vehicle treatment (Fig. 1). No significant changes in the response to mechanical stimuli were observed in the contralateral paw (data not shown).
3.2 Antinociceptive effect of intrathecally administered E-57431 in the capsaicin model
As activation of 5-HT7 receptors after systemic administration of E-57431 inhibited capsaicin-induced mechanical hypersensitivity in rats, we investigated whether this antinociceptive effect could have involved spinal 5-HT7 receptors. For this purpose, E-57431 was administered intrathecally after intraplantar injection of capsaicin.
Three days after intrathecal catheterization, 74% of rats injected with capsaicin (30 μg) into the mid-plantar surface of the right hind paw developed mechanical hypersensitivity (≥50% reduction of the baseline threshold) 30 min after capsaicin sensitization and were thus selected for the study (Fig. 2). No significant changes in the response to mechanical stimuli were observed in the contralateral paw (data not shown).
Intrathecal administration of E-57431 at the dose of 100 μg inhibited capsaicin-induced mechanical hypersensitivity in the ipsilateral paw (Fig. 2). Significantly increased paw withdrawal thresholds in rats intrathecally administered with E-57431 were found from 30 to 90 min post-treatment compared to vehicletreated rats. No significant changes were observed in the contralateral paw (Fig. 2).
3.3 Dose-response pronociceptive effect of E-57431 intraplantarly injected in the capsaicin model
Here we investigated whether peripheral activation of 5-HT7 receptors could exert pronociceptive effects (i.e., promote mechanical hypersensitivity). To assess this possibility, E-57431 or vehicle was administered locally, through intraplantar injection, and the dose of capsaicin was reduced from 30 to 10 μg, i.e. a dose unable to induce significant withdrawal responses when the paw was mechanically stimulated at subthreshold pressures, but providing a minimal sensitizing challenge to study pronociceptive effects of drugs, as previously reported (Brenchat et al., 2009). In these conditions, intraplantar injection of E-57431 dose-dependently enhanced mechanical hypersensitivity prompted by the local injection of capsaicin (10 μg) 30 min before. Significant pronociceptive effects were observed after injection of 1 μg of E-57431 (Fig 3). Lower doses of E-57431 (0.01 μg and 0.1 μg) did not significantly modify the paw withdrawal threshold compared to vehicle treatment. Interestingly, the pronociceptive effect of intraplantar E-57431 was not observed in rats injected with vehicle (1% DMSO) only (no capsaicin), suggesting that a minimal sensitizing challenge with capsaicin was needed for the 5-HT7 receptor agonist to exert its pronociceptive effect (Fig. 3). In contrast, intraplantar injection of E-57431 (1 μg) into the left (contralateral) hind paw was ineffective as it did not induce mechanical hypersensitivity in the right, ipsilateral paw receiving the 10 μg dose of capsaicin (Fig. 3). Accordingly, the pronociceptive effect of intraplantar administration of 1 μg of E-57431 into the ipsilateral paw was very probably mediated locally and did not involve any systemic action. No significant changes were observed in the contralateral paw (not shown).
3.4 Antinociceptive effect of intraperitoneally administered E-57431 in the spared nerve injury model of neuropathic pain
In order to assess the effect of the selective 5-HT7 receptor agonist E-57431 in another model of pain involving sensitization, we investigated whether mechanical allodynia could be affected by this ligand in rats rendered neuropathic by spared nerve injury. Mechanical hypersensitivity in nerve-injured animals was evidenced in 82% of rats by a reduction (≥50%) of the pressure threshold evoking withdrawal of the ipsilateral hind paw 14 days postsurgery compared to basal pre-surgery values (Fig. 4). Mechanical sensitivity to von Frey filament stimulation was unchanged in the contralateral paw of nerve-injured rats (not shown). In the same way, sham operation did not induce mechanical hypersensitivity as no significant changes in the response to mechanical stimulation were found in sham-operated rats 14 days after surgery compared to basal pre-surgery values (not shown).
Intraperitoneal administration of E-57431 at the dose of 10 mg/kg on day 15 post-surgery significantly inhibited mechanical hypersensitivity from 15 to 90 min after treatment, restoring the pressure threshold value triggering withdrawal of the nerveinjured, ipsilateral hind paw to basal pre-surgery levels (Fig. 4). No significant changes were elicited by E-57431 treatment in the contralateral paw of nerve-injured rats (not shown).
3.5 Antinociceptive effect of intrathecally administered E-57431 in the spared nerve injury model of neuropathic pain
Based on above results, we also tested whether the inhibition of nerve injury-induced mechanical hypersensitivity after systemic administration of E-57431 could be reproduced by selectively activating spinal 5-HT7 receptors. For this purpose, E-57431 was intrathecally administered on days 15–18 after spared nerve injury. Mechanical hypersensitivity in these animals was evidenced in 69% of rats by a reduction (≥50%) of the pressure threshold evoking withdrawal of the ipsilateral hind paw 14 days post-surgery compared to basal pre-surgery values (Fig. 5). In contrast, mechanical sensitivity to von Frey filament stimulation was unchanged in the contralateral paw of nerve-injured rats (data not shown).
Intrathecal administration of E-57431 (100 μg) inhibited nerve injury-induced mechanical hypersensitivity as shown by significant increases in pressure threshold values to trigger ipsilateral paw withdrawal 15, 60 and 90 min after treatment (Fig. 5). Lower doses of E-57431 (10 and 30 μg i.t.) were ineffective. E-57431 did not significantly modify pressure threshold values determined in the contralateral paw (not shown).
The present work provides demonstration in animal pain models involving central sensitization that activation of spinal 5-HT7 receptors exerts antinociceptive effects whereas activation of peripheral 5-HT7 receptors exerts pronociceptive effects. The antinociceptive effect at the central level seems to overcome the pronociceptive effect at the peripheral level as an overall antinociception is observed when 5-HT7 receptor agonists are systemically administered (Brenchat et al., 2009, 2010). As a pharmacological tool in the experiments described herein we used the new 5-HT7 receptor agonist E-57431, whose affinity, selectivity and functionality have been reported in detail (Brenchat et al., 2010).
Intradermal injection of capsaicin (i.e., capsaicin sensitization) is a useful model for correlative studies in pain conditions involving sensitization and behavioral hyperalgesia and allodynia (Gilchrist et al., 1996), and is regarded as a predictive model to assess antinociceptive action of analgesics in neuropathic pain (Joshi et al., 2006). In previous studies in mice, we showed that systemic administration of different selective 5-HT7 receptor agonists inhibited capsaicin-induced mechanical hypersensitivity (Brenchat et al., 2009). Similarly, we found that systemic administration of selective 5-HT7 receptor agonists, including E-57431, decreased partial nerve injury-induced mechanical and thermal hypersensitivity down to basal values found in control intact mice (Brenchat et al., 2010). Interestingly, no tolerance to the antinociceptive effects was evidenced following repeated (11 days) systemic administration of E-57431 (Brenchat et al., 2010).
In agreement with previous findings in mice, we found here that mechanical hypersensitivity could be reversed by acute systemic (oral or intraperitoneal) treatment with the selective 5-HT7 receptor agonist E-57431 in both the capsaicin and spared nerve injury models of pain signaling sensitization in rats. Our results also showed that intrathecal administration of E-57431 was as effective as systemic administration of the drug to inhibit mechanical hypersensitivity in both models. Interestingly, agonists acting at spinal 5-HT7 receptors cannot directly inhibit primary afferents or second-order nociceptive dorsal horn neurons because stimulation of the 5-HT7 receptor has excitatory effects (Lovenberg et al., 1993; Chapin and Andrade, 2001). Therefore, an indirect action through activation of 5-HT7 receptors localized on inhibitory interneurons is presumably required to inhibit nociceptive transmission. 5-HT7 receptors in the spinal cord have been described on primary afferents and astrocytes but are also located postsynaptically on local interneurons within the superficial laminae of the dorsal horn (Meuser et al., 2002; Doly et al., 2005). Actually, a previous study at the light microscope level showed that 5-HT7 receptors co-localize with GABA in neurons of the dorsal horn of the spinal cord (Brenchat et al., 2010) and it has been reported that spinal GABAergic interneurons, but not enkephalinergic interneurons, are involved in 5-HT7 receptor-mediated antinociception (Bourgoin et al., 2008; Viguier et al., 2009). This is based on the finding that intrathecal pretreatment with the GABAA receptor antagonist bicuculline, but not the GABAB receptor antagonist phaclofen or the opioid receptor antagonist naloxone, prevented the antihyperalgesic effects exerted by 5-HT7 receptor agonists in rats with constriction injury to the sciatic nerve (Bourgoin et al., 2008; Viguier et al., 2009). In line with these results, recent data also suggest that, at the spinal level, 5-HT7 receptor stimulation modulates pain signaling through Cl− conductance-dependent mechanisms (Viguier et al., 2010). Accordingly, activation of spinal inhibitory GABAergic interneurons could underlie, or at least contribute to, the analgesic effects of 5-HT7 receptor agonists. Finally, we previously reported a significant increase of 5-HT7 receptor immunoreactivity in the dorsal horn of the ipsilateral side of the spinal cord after sciatic nerve injury in mice (Brenchat et al., 2010). The upregulation of 5-HT7 receptors in the spinal cord after nerve injury suggests a “pain”-triggered regulation of receptor expression that may be relevant for the effectiveness of 5-HT7 receptor agonists. Indeed, 5-HT7 receptor upregulation might represent a compensatory, protective, spinal mechanism to reduce excessive nociception in neuropathic pain conditions.
In contrast to the antinociceptive (antiallodynic) effect found after systemic or spinal administration, a clear-cut pronociceptive (proallodynic) effect was found when E-57431 was administered intraplantarly into the ipsilateral hind paw injected with a low subactive dose of capsaicin. No effects were found when E-57431 was intraplantarly administered into the contralateral paw, suggesting that peripheral, local mechanisms (i.e., activation of 5-HT7 receptors on nerve endings) were responsible for its pronociceptive action. Interestingly, promotion of mechanical hypersensitivity did not occur when E-57431 was intraplantarly administered to rats receiving only vehicle (without capsaicin), indicating that a minimal sensitizing challenge with capsaicin was needed for the 5-HT7 receptor agonist to exert its pronociceptive action. This suggests that recruitment of additional mechanisms other than activation of 5-HT7 receptors is required to induce mechanical hypersensitivity at the periphery. In fact, 5-HT released from platelets and mast cells at the periphery acts on different 5-HT receptor subtypes and in combination with other inflammatory mediators to excite afferent nerve fibers (Sommer, 2004). Activation of 5-HT2A and 5-HT3 receptor subtypes present on C-fibers has already been reported to contribute to 5-HT-mediated peripheral pronociceptive effects (Obata et al., 2000; Sommer, 2004). Our data allow the conclusion that 5-HT7 receptors, in particular those expressed by small and medium-sized DRG neurons (Meuser et al., 2002; Doly et al., 2005), also probably contribute to 5-HT-mediated pronociceptive effects at the periphery. Indeed, this conclusion is supported by several convergent data in the literature: (1) intraplantar injection of the 5-HT7 receptor antagonist SB-269970 reduced formalin-induced nociception whereas intraplantar administration of non-selective 5-HT7 receptor agonists such as 5-HT itself and 5-CT increased formalin-induced nociceptive behavior (Rocha-González et al., 2005); (2) intra-articular injection of the mixed 5-HT1A/5-HT7 receptor agonist 8-OH-DPAT induced c-Fos expression in the dorsal horn of the rat spinal cord and this effect was prevented by intra-articular administration of the non-selective 5-HT7 receptor antagonist methiothepin (Meuser et al., 2002).
It is plausible that the antinociception resulting from activation of spinal 5-HT7 receptors could overcome the pronociceptive effect mediated by peripheral 5-HT7 receptors and thus be responsible for the overall antinociceptive effect found after systemic administration of selective 5-HT7 receptor agonists in conditions involving central sensitization (this study; Brenchat et al., 2009, 2010). Indeed, it was recently demonstrated that the antinociceptive effects of morphine, tramadol and its metabolite O-Desmethyltramadol, and CB1 receptor agonists were blocked by intrathecal administration of the selective 5-HT7 receptor antagonist SB-269970, which suggests that spinal 5-HT7 receptors are ultimately critical for opiate- and CB1-mediated antinociception, and act as effector targets for descending inhibitory serotonergic pathways (Dogrul et al., 2009; Seyrek et al., 2010; Yanarates et al., 2010).
Taken together, the data reported herein support the idea that 5-HT7 receptors play an important role in physiological mechanisms controlling nociception and pain, particularly in conditions involving sensitization of nociceptive pathways. Activation of 5-HT7 receptors may result in pronociceptive or antinociceptive effects depending on their location. Mechanical hypersensitivity is promoted when activation occurs at peripheral receptors and inhibited when activation concerns 5-HT7 receptors in the spinal cord, but an overall antinociception results from systemic administration of 5-HT7 receptor agonists. Further studies in other experimental pain conditions, focusing on other CNS locations and/or exploring underlying mechanisms, would be particularly useful for a thorough assessment of the therapeutic potentialities of the 5-HT7 receptor as a pharmacological target to develop new drugs to alleviate pain (i.e., clinical manifestations of neuropathic pain). Unfortunately, drugs selectively acting through central 5-HT7 receptor activation have not yet been clinically assayed.
We thank Mercè Olivet for her administrative assistance.