S-(+)-fenfluramine-induced nociceptive behavior in mice: Involvement of interactions between spinal serotonin and substance P systems

1. Introduction 

Several lines of evidence suggest that the neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) plays an important role in the modulation of nociceptive transmission and that the major site of action of 5-HT is the spinal cord (for review, see Fields et al., 1991, Furst, 1999). Although multiple 5-HT receptor subtypes have been defined in the central nervous system (for review, see Barnes and Sharp, 1999), at least four types of 5-HT receptors (5-HT1, 5-HT2, 5-HT3 and 5-HT4) with several subtypes have been identified in the spinal cord (for a review, see Hoyer et al., 1994).

Substance P (SP) has been proposed to be an important neurotransmitter in primary sensory neurons involved in nociception (for a review, see Sakurada et al., 1997) and coexisted with 5-HT in bulbospinal neurons and SP-containing neurons which project to the raphe nuclei, the major site of 5-HT cell bodies (Johansson et al., 1981). Behavioral studies in mice have indicated that intrathecal (i.t.) administration of 5-HT (2μg) (Eide and Hole, 1991a) or a selective 5-HT2 receptor agonist (Eide and Hole, 1991b, Mjellem et al., 1993) produces the characteristic nociceptive behavior consisting of hindlimb scratching directed toward the flank, biting and/or licking of the hindpaw and the tail resembling the behaviors induced by neurokinin-1 (NK1) receptor agonists such as SP (Takahashi et al., 1987, Sakurada et al., 1991). The nociceptive behavior induced by 5-HT (2μg) is reduced by i.t. pretreatment with spantide, a non-selective NK1 receptor antagonist (Eide and Hole, 1991a). In contrast, i.t. administration of 5-HT (20μg) or the selective 5-HT1 receptor agonists produces the antinociceptive effect as measured by the mouse tail-flick test, which is reduced by pretreatment with SP (Eide and Hole, 1991a). These reports seem to indicate functional interactions between 5-HT and SP in the regulation of nociceptive transmission in the mouse spinal cord. However, the effects of using 5-HT receptor agonists do not necessarily reflect the physiological significance of the spinal interactions between 5-HT and SP systems in nociceptive transmission.

Thus, in the present study, we first examined the effect of i.t. administration into mice of S-(+)-fenfluramine, which releases 5-HT from axon terminals; as a result, S-(+)-fenfluramine produced the behavioral response consisting of scratching, biting and licking through the activation of 5-HT2 receptors subsequent to 5-HT release. We also examined the effects of a selective NK1 receptor antagonist and SP antiserum on the S-(+)-fenfluramine-induced behavioral response in order to clarify the physiological significance of the spinal interactions between 5-HT and SP systems in nociceptive transmission.

2. Materials and methods 

3. Results 

3.1. Behavioral response induced by i.t.-administered S-(+)-fenfluramine 

The i.t. administration of S-(+)-fenfluramine (0.1nmol) produced a characteristic behavioral response consisting of scratching, biting and licking, which peaked at 5–15min and almost disappeared at 20min after injection (Fig. 1b). As seen in Fig. 1a, a dose-dependent increase in the total time of scratching, biting and licking was observed following i.t. administration of S-(+)-fenfluramine in doses ranging from 0.01–0.1nmol. However, no further increase in the behavioral response was produced by injection of S-(+)-fenfluramine at a higher dose of 0.3nmol. In further experiments, 0.1nmol of S-(+)-fenfluramine and a 20min observation time were therefore used in combination with various drugs and antisera to test their inhibitory actions. It was then examined whether S-(+)-fenfluramine-induced behavior is reversed by morphine. As shown in Fig. 2, pretreatment with morphine (0.02–0.5mg/kg, i.p.) inhibited S-(+)-fenfluramine-induced behavior in a dose-dependent manner with an ID50 value of 0.19 (0.19–0.20)mg/kg, suggesting that the behavioral response is related to nociception.

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Fig. 1. (a) Effects of varying doses of i.t.-administered S-(+)-fenfluramine in mice. The duration of scratching, biting and licking induced by S-(+)-fenfluramine or Ringer was determined during a 20min period starting immediately after injection. (b) Time-courses of the scratching, biting and licking response induced by i.t. injection of S-(+)-fenfluramine (0.1nmol) or Ringer alone. The ordinate shows the total time of scratching, biting and licking response which occurred during each 5min of measurement. These data are given as the means and SEM for groups of 10 mice. ∗∗P<0.01 when compared with Ringer controls.

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Fig. 2. Effect of morphine on S-(+)-fenfluramine-induced scratching, biting and licking response in mice. Morphine was administered i.p. 5min prior to injection of S-(+)-fenfluramine (0.1nmol). The duration of scratching, biting and licking induced by S-(+)-fenfluramine was determined during a 20min period starting immediately after i.t. injection. These data are given as the means and SEM for groups of 10 mice. ∗∗P<0.01 when compared with S-(+)-fenfluramine alone.

3.2. Effects of 5-HT antiserum and 5-HT receptor antagonists on S-(+)-fenfluramine-induced nociceptive behavior 

To test whether 5-HT release is involved in the effects of S-(+)-fenfluramine, the effect of pretreatment with 5-HT antiserum was examined. When administered i.t. 5min before the injection of S-(+)-fenfluramine, 5-HT antiserum (1:1250–1:50) reduced S-(+)-fenfluramine-induced nociceptive behavior in a dilution-related manner (Fig. 3a). Ketanserin (0.3–3.0nmol), a selective 5-HT2 receptor antagonist, co-administered i.t. with S-(+)-fenfluramine also caused a dose-dependent inhibition of S-(+)-fenfluramine-induced nociceptive behavior with an ID50 value of 0.68 (0.60–0.77)nmol (Fig. 3b).

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Fig. 3. Effect of 5-HT antiserum and ketanserin on S-(+)-fenfluramine-induced nociceptive behavior in mice. (a) The antiserum against 5-HT or Ringer was administered i.t. 5min prior to injection of S-(+)-fenfluramine (0.1nmol). The duration of scratching, biting and licking induced by S-(+)-fenfluramine was determined during a 20min period starting immediately after i.t. injection of S-(+)-fenfluramine. (b) Ketanserin was co-administered i.t. with S-(+)-fenfluramine (0.1nmol). The duration of scratching, biting and licking induced by S-(+)-fenfluramine was determined during a 20min period starting immediately after injection. These data are given as the means and SEM for groups of 10 mice. (a) ∗∗P<0.01 when compared with Ringer plus S-(+)-fenfluramine-treated group. (b) ∗∗P<0.01 when compared with S-(+)-fenfluramine alone.

A single administration of 1.0nmol, but not 0.5nmol of WAY-100635, a selective 5-HT1A receptor antagonist, caused a significant nociceptive behavior under conditions tested (data not shown). Therefore, 0.5 nmol of WAY-100635 was applied in combination with S-(+)-fenfluramine. As shown in Table 1, WAY-100635 did not inhibit S-(+)-fenfluramine-induced nociceptive behavior. Moreover, ramosetron, a selective 5-HT3 receptor antagonist, even at a comparatively high dose of 4nmol had no effect on S-(+)-fenfluramine-induced nociceptive behavior (Table 1).

Table 1. Effects of WAY-100635 and ramosetron on S-(+)-fenfluramine-induced nociceptive behavior in mice

	Agents	Behavioral response (s/20min)
	Ringer	41.6±4.1
	WAY-100635 (0.5nmol)	43.3±3.1
	Ramosetron (4.0nmol)	37.1±3.7

Each agent was co-administered i.t. with S-(+)-fenfluramine (0.1nmol). The duration of scratching, biting and licking induced by S-(+)-fenfluramine was determined during a 20min period starting immediately after i.t. injection. These data are given as the means±SEM for groups of 10 mice.

3.3. Effects of SP antiserum and RP67580 on S-(+)-fenfluramine-induced nociceptive behavior 

As shown in Fig. 4a, pretreatment with SP antiserum (1:4096–1:1024) reduced S-(+)-fenfluramine-induced nociceptive behavior in a dilution-related manner, similar to that of 5-HT antiserum. In addition, RP67580 (0.5–8nmol), a selective NK1 receptor antagonist, produced a dose-dependent inhibition of S-(+)-fenfluramine-induced nociceptive behavior with an ID50 value of 5.12 (4.89–5.35)nmol (Fig. 4b).

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Fig. 4. Effect of SP antiserum and RP67580 on S-(+)-fenfluramine-induced nociceptive behavior in mice. (a) The antiserum against SP or Ringer was administered i.t. 5min prior to injection of S-(+)-fenfluramine (0.1nmol). The duration of scratching, biting and licking induced by S-(+)-fenfluramine was determined during a 20min period starting immediately after i.t. injection of S-(+)-fenfluramine. (b) RP67580 was co-administered i.t. with S-(+)-fenfluramine (0.1nmol). The duration of scratching, biting and licking induced by S-(+)-fenfluramine was determined during a 20min period starting immediately after injection. These data are given as the means and SEM for groups of 10 mice. (a) ∗∗P<0.01 when compared with Ringer plus S-(+)-fenfluramine-treated group. (b) ∗∗P<0.01 when compared with S-(+)-fenfluramine alone.

4. Discussion 

The present data clearly show that i.t.-administered S-(+)-fenfluramine (0.01–0.1nmol) produced nociceptive behavior consisting of scratching, biting and licking, which peaked at 5–15min and almost disappeared at 20min after injection. S-(+)-Fenfluramine (0.1nmol)-induced nociceptive behavior was dose-dependently reduced by quite small doses of morphine (0.02–0.5mg/kg, i.p.). These results are similar to our previously reported findings in which i.t. administration of SP in mice produced nociceptive behavior (Takahashi et al., 1987), though the peak time effect of S-(+)-fenfluramine was much later than that of SP-induced nociceptive behavior, which peaked at 0–5min following i.t. administration.

It is known that the spinal 5-HT system is involved in both inhibition and facilitation of nociceptive transmission (for review, see Eide and Hole, 1993). Electrophysiological studies have provided evidence that 5-HT1A and 5-HT2 receptors mediate inhibitory and excitatory effects of 5-HT on neuronal excitation, respectively (El-Yassir et al., 1988, Araneda and Andrade, 1991, Brandao et al., 1991). The different responses after stimulation of 5-HT1A and 5-HT2 receptors have been explained by the fact that these receptors couple to different intracellular signaling systems. The 5-HT1A receptors inhibit adenylate cyclase activity (De Vivo and Maayani, 1986), whereas the 5-HT2 receptors activate phospholipase C and the inositol triphosphate (IP3) pathway, leading to a release of intracellular calcium and subsequent neuronal excitation (for review, see Fozard, 1987). These facts indicate that the divergence of the 5-HT system on the spinal regulation in nociceptive transmission may be also explained by the difference of 5-HT receptors; activation of 5-HT1A and 5-HT2 receptors produces inhibition and facilitation in the spinal nociceptive transmission, respectively. Dual actions in facilitating and inhibiting the spinal nociceptive transmission are not limited to 5-HT; they are also observed in spermine (Tan-No et al., 2000, Tan-No et al., 2003), dynorphins (Tan-No et al., 1996, Tan-No et al., 2002, Tan-No et al., 2005a, Tan-No et al., 2005b) and nociceptin (Sakurada et al., 1999). In the present study, S-(+)-fenfluramine-induced nociceptive behavior was significantly reduced by pretreatment with 5-HT antiserum and co-administration of ketanserin but not with WAY-100635 or ramosetron. In mice, i.t. administration of (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), a selective 5-HT2 receptor agonist, produces nociceptive behavior (Eide and Hole, 1991b, Mjellem et al., 1993) which is similar to that seen after i.t. administration of S-(+)-fenfluramine and SP. Taken together with these previous reports, the present results suggest that S-(+)-fenfluramine produces nociceptive behavior through the activation of 5-HT2 receptors following 5-HT release. The present study also showed that the dose–response for S-(+)-fenfluramine-induced nociceptive behavior was a bell-shaped pattern. It has been reported that i.t. administration of (+)-8-hydroxy-2-(di-n-propylamino)-tetralin ((+)-8-OH-DPAT), a selective 5-HT1A receptor agonist, produces an antinociceptive effect in the mouse tail-flick test (Eide and Hole, 1991a, Eide and Hole, 1991b), and inhibits nociceptive behavior elicited by N-methyl-d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole-proprionic acid (AMPA) (Mjellem et al., 1993). One possible explanation for the decreased effect of S-(+)-fenfluramine at a higher dose (0.3nmol) is that activation for 5-HT1A receptors through release of 5-HT by i.t. administration of S-(+)-fenfluramine at a higher dose has an inhibitory effect on S-(+)-fenfluramine-induced nociceptive behavior.

We also demonstrated that S-(+)-fenfluramine-induced nociceptive behavior was significantly inhibited by RP67580. It has recently been reported that stimulation of spinal 5-HT2A/2C receptors by DOI potentiates the capsaicin-induced release of SP-like immunoreactivity in the rat dorsal horn in an experiment using microdialysis (Bertelsen et al., 2003). It is, therefore, reasonable to presume that S-(+)-fenfluramine-induced nociceptive behavior may be due to stimulation of NK1 receptors indirectly, possibly through SP release. This interpretation is supported by an additional data that pretreatment with SP antiserum resulted in a significant reduction of the S-(+)-fenfluramine-induced nociceptive behavior. Similar observations have been obtained by using SP antiserum in nociceptive behavior elicited by i.t. injection of pilocarpine (Sakurada et al., 1993), high-dose morphine (Sakurada et al., 1996) and extremely low-dose nociceptin (Sakurada et al., 1999).

In conclusion, we have presented evidence that i.t.-administered S-(+)-fenfluramine produces nociceptive behavior. The mechanism seems to be that S-(+)-fenfluramine activates 5-HT2 receptors following 5-HT release and facilitates SP release, which subsequently acts on NK1 receptors to produce the spinal cord-mediated nociceptive behavior.