Differential Contribution of 5-HT₄, 5-HT₅, and 5-HT₆ Receptors to Acute Pruriceptive Processing Induced by Chloroquine and Histamine in Mice

Abstract

The involvement of serotonin (5-HT) and/or noradrenaline in acute pruriceptive processing in the central nervous system (CNS) has been reported using antidepressants, such as milnacipran, a serotonin and noradrenaline reuptake inhibitor, and mirtazapine, a noradrenergic and specific serotonergic antidepressant; however, the roles of 5-HT receptor family in acute pruriceptive processing have not been fully elucidated in the CNS. In the present study, scratching behavior induced by chloroquine (CQ) was ameliorated by milnacipran or mirtazapine, and these effects were reversed by SB207266, a 5-HT₄ antagonist, or SB258585, a 5-HT₆ antagonist, but not by SB258585, a 5-HT₅ antagonist. Moreover, CQ-induced scratches were mitigated by intrathecal injection of 5-HT₄ agonists, such as BIMU8 and ML10302, and the 5-HT₆ agonist, WAY208466. Conversely, histamine-induced scratches were not affected by the 5-HT₄ agonists or a 5-HT₆ agonist. Similarly, the amelioration of histamine-induced scratches by these antidepressants was not reversed by the 5-HT₄, 5-HT₅, or 5-HT₆ receptor antagonist. Therefore, 5-HT is involved in the amelioration of CQ-induced scratches by milnacipran and mirtazapine, and 5-HT₄, 5-HT₅, and 5-HT₆ receptors play differential roles in acute pruriceptive processing after administration of CQ or histamine.

INTRODUCTION

Itch (pruritus) is defined as an irritating and unpleasant sensation that evokes a desire to scratch. The itch-scratch cycle in chronic pruritus is harmful and affects the patients with itch caused by various diseases. However, the underlying neural mechanisms are poorly understood. Itch has been generally divided into two types; histamine-dependent and histamine-independent itch.¹⁾

Serotonin (5-HT) and noradrenaline affect itch processing via descending neuronal pathways in the central nervous system (CNS).^2,3) The intrathecal treatment of milnacipran, a serotonin and noradrenaline reuptake inhibitor (SNRI), or mirtazapine, a noradrenergic and specific serotonergic antidepressant (NaSSA), both of which are generally considered to increase the concentrations of 5-HT in the synaptic cleft, mitigated the biting behavior and scratches, which are representative behavioral responses specific to pruritus.^4–7)

Noradrenaline signals in the spinal cord play pivotal roles in acute itch. A pretreatment with α₁- and α₂-adrenoceptor agonists attenuated 5-HT-induced biting⁴⁾ and the effects were reversed by α-adrenoceptor antagonists.^8,9) Collectively, α-adrenoceptors contribute to the modulation of acute itch processing.

By contrast, the contribution of 5-HT to acute itch remains poorly understood. Scratching behavior was reduced in mice lacking 5-HT or 5-HT-expressing neurons in the CNS,^10,11) indicating that decreased 5-HT ameliorates itch sensation. Meanwhile, the intrathecal administration of 5-HT attenuated the induction of itch-related behavior by chloroquine (CQ),⁷⁾ suggesting that increased 5-HT reduces the itch-related signals in the spinal cord. These contradictory results for the functions of 5-HT propose the involvement of 5-HT receptors in acute itch processing; however, the subtypes of 5-HT receptors involved in this process remain unclarified.

5-HT receptors can be divided into 7 families and 15 subfamilies. The serotonergic system via 5-HT_1A signaling plays a crucial role in the descending control of itch in the CNS.¹¹⁾ A recent study demonstrated that 5-HT₃ and 5-HT₇ receptors had differential roles in acute itch by CQ or histamine.⁷⁾ Focusing on G-proteins coupling to 5-HT receptors, 5-HT_1A receptors are coupled to Gi/o-proteins, while 5-HT₇ receptors are coupled to Gs-proteins. 5-HT₇ receptor agonists attenuated pruritus induced by CQ, but not histamine.⁷⁾ However, the roles of other 5-HT receptors in itch processing have not yet been investigated. Thus, to confirm whether G-proteins coupling to 5-HT receptors are associated with the effects of agonists and antagonists of 5-HT receptors on pruritogen-induced scratching, 5-HT₄/5-HT₆ receptors and 5-HT₅ receptors coupling to Gs and Gi/o, respectively, were considered as the targets of this research, and the roles of 5-HT₄, 5-HT₅, and 5-HT₆ receptors in itch induced by the administration of CQ or histamine were explored.

MATERIALS AND METHODS

Mice

All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Miyazaki (Approval Number: 2015-523), and adhered to ethical consideration in animal research.¹²⁾

Male C57BL/6J mice were obtained from Charles River Japan (Kanagawa, Japan) and were maintained on a reversed 12-h dark–light cycle with food and water provided ad libitum for at least 1 week habituation before experimentation. Medications were treated across 1–2 weeks for recovery. Briefly, under 3% isoflurane anesthesia (Abbott Laboratories, Chicago, IL, U.S.A.), various concentrations of drugs were treated with intrathecal injection in a volume of 5 µL through lumbar puncture 5 min before an intradermal injection of pruritogens in a volume of 50 µL into the nape of the neck. Scratches were counted for 15 min after the injection of CQ or histamine as pruritogens. If mice died or were sick during or before the experiment, it was not possible to validate their recording positions, and, thus, they were removed from the data analysis.

Drugs

CQ and histamine, which were given as pruritogens, have been used as histaminergic and non-histaminergic drugs, respectively.^13–16) Histamine and chloroquine diphosphate were obtained from Tokyo Chemical Industry (Tokyo, Japan).

Milnacipran hydrochloride, an SNRI, was a gift from Asahi Kasei Pharma (Tokyo, Japan) and mirtazapine hydrochloride, a NaSSA, was obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). These drugs were dissolved in saline, and stored at −30 °C until use.

SB207266, SB699551, and SB258585 are antagonists of the 5-HT₄ receptor, 5-HT₅ receptor, and 5-HT₆ receptor, respectively. BIMU8 and ML10302 are 5-HT₄ receptor agonists, and WAY208466 is a 5-HT₆ receptor agonist. These antagonists and agonists were obtained from Sigma-Aldrich, dissolved in saline, and stored at −30 °C until use.

Scratching Behavior

Scratches were used to evaluate the induction of pruritus as previously described.^6,7) Scratching behavior is defined as the rapid, repetitive, and back-and-forth movement of the hind paw toward the injection site. Mice were treated with 10 µg of saline, milnacipran, or mirtazapine, followed by the treatment of 500 µg of histamine or 200 µg of CQ in a volume of 50 µL. CQ- or histamine-induced pruritus was then assessed by counting scratching events with the hind paw. The investigators who counted the scratching behaviors were blinded to the mouse information.

The milnacipran or mirtazapine attenuated the induction of scratching events by CQ or histamine.⁶⁾ A previous study showed the involvement of 5-HT₃ and 5-HT₇ receptors in scratching events induced by CQ or histamine.⁷⁾ Therefore, to examine whether 5-HT₄, 5-HT₅, and 5-HT₆ receptors are involved in the effects of these antidepressants on either scratching behavior, various doses of SB207266, SB699551, or SB258585 were intrathecally injected, CQ or histamine was injected 5 min later, and the induction of scratches were then assessed for 15 min. Furthermore, to reconfirm whether the 5-HT₄ or 5-HT₆ receptors contribute to the effects of these antidepressants, various concentrations of BIMU8, ML10302, or WAY208466 were administered before the intradermal injection of CQ or histamine, and their effects were evaluated based on changes in the numbers of scratching events for 15 min.

Statistical Analysis

Scratching events are shown in numbers. Data analyses of scratches were conducted using the Mann–Whitney U test and an ANOVA with repeated measures, followed by Fisher’s protected least significant difference test. All data are presented as means ± standard error of the mean (S.E.M.).

RESULTS

Effects of SB207266, a 5-HT₄ Receptor Antagonist, on the Attenuation of Scratches by Milnacipran or Mirtazapine

The numbers of scratches for 15 min after intradermal injection of saline were 7 ± 2. On the other hand, the treatment of CQ or histamine strongly induced scratching behavior in mice pretreated with saline. The numbers of scratches after treatment of 200 µg of CQ and 500 µg of histamine were 24 ± 3 and 25 ± 2, respectively (Figs. 1A, B, Control).

Fig. 1. Effects of SB207266, a 5-HT₄ Receptor Antagonist, on the Attenuation of Scratching Events Induced by CQ (A) or Histamine (B) in Mice Pretreated with Milnacipran or Mirtazapine

The numbers of scratching events for 15 min after the injection of 200 µg of CQ in a volume of 50 µL into the nape of the neck 5 min after the intrathecal administration of 5 µL saline (n = 5, Control) in control mice, and saline (n = 6, Baseline), 0.01 µg (n = 5), 0.1 µg (n = 6), 1 µg (n = 6), or 10 µg (n = 5) of SB207266 (Chloroquine), and after the injection of 500 µg of histamine following the intrathecal administration of 5 µL saline (n = 6, Control) in control mice, and 5 µL saline (n = 5, Baseline), 10 µg (n = 5), 100 µg (n = 6), 1000 µg (n = 5), or 10000 µg (n = 5) of SB207266 (Histamine) in a volume of 5 µL in mice pretreated with 10 µg of milnacipran or mirtazapine. All points represent the number of scratching events ± S.E.M. The significance of differences between the Control and Baseline was analyzed by the Mann–Whitney U test, and was represented as ^# p < 0.05. The significance of differences between Baseline and the various concentrations of SB207266 was analyzed by ANOVA with repeated measures, and was represented as * p < 0.05.

To examine the effects of antidepressants, milnacipran or mirtazapine, on CQ- or histamine-induced scratches, 10 µg of either antidepressant in a volume of 5 µL was intrathecally administered 5 min before the administration of CQ or histamine. The number of CQ- or histamine-induced scratching events was significantly lower in mice pretreated with these antidepressants than in control mice (Figs. 1A, B, Baseline). This numbers of scratches were considered as the basal level for assessing the effects of SB207266, an antagonist of the 5-HT₄ receptor, on the induction of scratches by CQ or histamine in mice pretreated with these antidepressants. The numbers of CQ-induced scratches in mice pretreated with milnacipran and mirtazapine were 9 ± 3 and 10 ± 1, respectively, while those of histamine-induced scratches were 11 ± 2 and 10 ± 1, respectively (Figs. 1A, B, Baseline).

The ameliorating effects of these antidepressants on CQ- or histamine-induced scratching behavior appear to be due to the involvement of 5-HT receptors, including 5-HT₄ receptors, as well as adrenoceptors in acute itch processing. Therefore, to confirm the role of 5-HT₄ receptors in acute itch processing, various concentrations of SB207266 were injected 5 min before the administration of 10 µg of either antidepressant, and the effects of SB207266 were assessed by the number of pruritogen-induced scratches in mice pretreated with these antidepressants. The administration of 1 and 10 µg, but not 0.01 or 0.1 µg, of SB207266 significantly reversed the decreases in the number of CQ-induced scratches in mice pretreated with these antidepressants, and the numbers of scratches were similar in mice administered by these doses of SB207266 and in control mice (Fig. 1A, Chloroquine). In contrast, the decreased numbers of histamine-induced scratches in mice pretreated with these antidepressants were not reversed by the intrathecal injection of 10, 100, 1000, and 10000 µg of SB207266 (Fig. 1B, Histamine).

Effects of BIMU8 and ML10302, 5-HT₄ Receptor Agonists, on Scratching Behavior Induced by CQ or Histamine

The induction of scratching events by CQ, but not histamine, was reversed by SB207266, as shown in Fig. 1, indicating the involvement of 5-HT₄ receptors in pruritic processing after CQ administration. To confirm the role of 5-HT₄ receptors in pruritogen-induced scratching behavior, the effects of BIMU8 and ML10302, 5-HT₄ receptor agonists, were evaluated. The numbers of CQ- and histamine-induced scratches in mice with saline were used as the basal level for assessing the effects of BIMU8 and ML10302, and were 20 ± 2 and 23 ± 2, respectively (Figs. 2A, B, Control).

Fig. 2. Effects of BIMU8 and ML10302, 5-HT₄ Receptor Agonists, on Scratching Events Induced by CQ (A) or Histamine (B)

The numbers of scratching events for 15 min after the injection of 200 µg of CQ in a volume of 50 µL into the nape of the neck 5 min after the intrathecal administration of saline (n = 5, Control), 0.01 µg (n = 6), 0.1 µg (n = 5), 1 µg (n = 6), or 10 µg (n = 5) of BIMU8 or ML10302 (Chloroquine), and after the injection of 500 µg of histamine following the intrathecal administration of saline (n = 6, Control), 10 µg (n = 5), 100 µg (n = 6), 1000 µg (n = 5), or 10000 µg (n = 5) of BIMU8 or ML10302 in a volume of 5 µL (Histamine). All points represent the number of scratching events ± S.E.M. The significance of differences from the Control was statistically analyzed by ANOVA with repeated measures, and was represented as * p < 0.05.

The number of CQ-induced scratches was dose-dependently decreased by BIMU8 or ML10302. One and 10 µg of BIMU8 or ML10302 significantly decreased the number of scratches, whereas the pretreatment with 0.01 or 0.1 µg of BIMU8 or ML10302 did not (Fig. 2A, Chloroquine). In contrast, there was no significant change in the numbers of histamine-induced scratching events in mice pretreated with 10, 100, 1000, and 10000 µg of BIMU8 or ML10302 (Fig. 2B, Histamine).

Effects of SB699551, a 5-HT₅ Receptor Antagonist, on the Attenuation of Scratches by Milnacipran or Mirtazapine

Scratching behavior was conspicuously induced by an injection of CQ or histamine into the mice pretreated with saline, and the numbers were 23 ± 3 and 28 ± 2, respectively (Figs. 3A, B, Control), while the saline injection scarcely induced scratching events (8 ± 1).

Fig. 3. Effects of SB699551, a 5-HT₅ Receptor Antagonist, on the Attenuation of Scratching Events Induced by CQ (A) or Histamine (B) in Mice Pretreated with Milnacipran or Mirtazapine

The numbers of scratching events for 15 min after the injection of 200 µg of CQ in a volume of 50 µL into the nape of the neck 5 min after the intrathecal administration of 5 µL saline (n = 6, Control) in control mice, and saline (n = 5, Baseline), 10 µg (n = 5), 100 µg (n = 6), 1000 µg (n = 6), or 10000 µg (n = 5) of SB699551 (Chloroquine), or after the injection of 500 µg of histamine following the intrathecal administration of 5 µL saline (n = 5, Control) in control mice, and saline (n = 6, Baseline), 10 µg (n = 5), 100 µg (n = 6), 1000 µg (n = 6), or 10000 µg (n = 5) of SB699551 (Histamine) in a volume of 5 µL in mice pretreated with 10 µg of milnacipran or mirtazapine. All points represent the number of scratching events ± S.E.M. The significance of differences between the Control and Baseline was analyzed by the Mann–Whitney U test, and was represented as ^# p < 0.05. There were no significant differences between Baseline and the other concentrations of SB699551 as determined by ANOVA with repeated measures.

To clarify the role of 5-HT₅ receptors in the effects of pretreatment with milnacipran and mirtazapine, the changes in the numbers of pruritogen-induced scratches were examined after the pretreatment with these antidepressants. The numbers of scratching events induced by CQ and histamine in mice pretreated with these antidepressants were significantly decreased, and were used as the basal level to assess the effects of SB699551, a 5-HT₅ receptor antagonist (Figs. 3A, B, Baseline). Various concentrations of SB699551 were injected 5 min before the administration of these antidepressants, and the effects of this antagonist were evaluated. The decreased numbers of pruritogen-induced scratches by these antidepressants-were not significantly changed in mice pretreated with 10, 100, 1000, or 10000 µg of SB699551 (Figs. 3A, B, SB699551).

Effects of SB258585, a 5-HT₆ Receptor Antagonist, on the Attenuation of Scratches by Milnacipran or Mirtazapine

The numbers of scratching behavior induced by intradermal injection of saline, CQ and histamine were 9 ± 2, 22 ± 2 and 27 ± 2, respectively, and were basically similar to those of 5-HT₄ receptor experimental group (Figs. 4A, B, Control). In addition, the numbers of scratching events induced by CQ or histamine were significantly attenuated by the treatment with milnacipran or mirtazapine, and these decreased numbers of scratches were considered as the basal level (Figs. 4A, B, Baseline).

Fig. 4. Effects of SB258585, a 5-HT₆ Receptor Antagonist, on the Attenuation of Scratching Events Induced by CQ (A) or Histamine (B) in Mice Pretreated with Milnacipran or Mirtazapine

The numbers of scratching events for 15 min after the injection of 200 µg of CQ in a volume of 50 µL into the nape of the neck 5 min after the intrathecal administration of 5 µL saline (n = 5, Control) in control mice, and saline (n = 6, Baseline), 0.01 µg (n = 6), 0.1 µg (n = 6), 1 µg (n = 6), or 10 µg (n = 5) of SB258585 (Chloroquine), or after the injection of 500 µg of histamine following the intrathecal administration of saline (n = 6, Control) in control mice, and saline (n = 5, Baseline), 10 µg (n = 5), 100 µg (n = 5), 1000 µg (n = 6), or 10000 µg (n = 6) of SB258585 (Histamine) in mice pretreated with 10 µg of milnacipran or mirtazapine. All points represent the number of scratching events ± S.E.M. The significance of differences between the Control and Baseline was analyzed by the Mann–Whitney U test, and was represented as ^# p < 0.05. The significance of differences between Baseline and the other concentrations of SB258585 was analyzed by ANOVA with repeated measures, and was represented as * p < 0.05.

To examine whether 5-HT₆ receptors are involved in the attenuating effects of milnacipran or mirtazapine on scratching events induced by CQ or histamine, various concentrations of SB258585, an antagonist of 5-HT₆ receptors, were administered 5 min before the injection of 10 µg of these antidepressants, and its effects were assessed by counting the numbers of pruritogen-induced scratches. The numbers of CQ-induced scratches were significantly reduced in mice pretreated with these antidepressants, and the reduced numbers of scratching events were reversed by the injection of 0.01, 0.1, 1, and 10 µg of SB258585, and the numbers of scratches in mice administered by 1 and 10 µg of SB258585 and in control mice were similar each other (Fig. 4A, Chloroquine). The injection of these antidepressants also decreased the numbers of histamine-induced scratches. However, the administration of various concentrations of SB258585 did not reverse the decreased numbers of scratching by the treatment with these antidepressants (Fig. 4B, Histamine).

Effects of the Injection of WAY208466, a 5-HT₆ Receptor Agonist, on Scratches by CQ or Histamine

The attenuating effects of these antidepressants on scratches induced by CQ, but not histamine, were reversed by SB258585 as shown in Fig. 4, indicating that 5-HT₆ receptors are involved in the attenuation of CQ-induced scratches by these antidepressants. To confirm the role of 5-HT₆ receptors in pruritogen-induced scratching behavior, the effects of WAY208466, a 5-HT₆ receptor agonist, were investigated. The numbers of CQ- and histamine-induced scratches in mice pretreated with saline were used as the basal level to assess the effects of WAY208466, and were 23 ± 2 and 22 ± 2, respectively (Figs. 5A, B, Control). The numbers of CQ-induced scratches were dose-dependently decreased by the injection of various concentrations of WAY208466. Indeed, the injection of 1 and 10 µg of WAY208466 significantly reduced the numbers of scratches, whereas the effects of 0.01 or 0.1 µg of WAY208466 did not reach the significant level (Fig. 5A, Chloroquine). On the other hand, no significant change in the numbers of histamine-induced scratches was observed in mice pretreated with various concentration of WAY208466 (Fig. 5B, Histamine).

Fig. 5. Effects of the Intrathecal Administration of WAY208466, an Agonist of 5-HT₆ Receptors, on Scratching Events Induced by CQ (A) or Histamine (B)

The numbers of scratching events for 15 min after the injection of 200 µg of CQ in a volume of 50 µL into the nape of the neck 5 min after the intrathecal administration of saline (n = 6, Control), 0.01 µg (n = 5), 0.1 µg (n = 5), 1 µg (n = 6), or 10 µg (n = 6) of WAY208466 (Chloroquine), or after the injection of 500 µg of histamine following the intrathecal administration of saline (n = 6, Control), 10 µg (n = 5), 100 µg (n = 6), 1000 µg (n = 5), or 10000 µg (n = 5) of WAY208466 in a volume of 5 µL (Histamine). All points represent the number of scratching events ± S.E.M. The significance of differences from the Control was analyzed by ANOVA with repeated measures, and was represented as * p < 0.05.

DISCUSSION

The effects of 5-HT on itch processing in the CNS remain inconsistent. The induction of scratches by a pruritogen was attenuated in mice lacking 5-HT neurons in the rostral ventromedial medulla. Similarly, the pharmacological ablation of bulbospinal serotonergic fibers significantly reduced the number of scratching events elicited by CQ. Furthermore, enhancements in the tone of bulbospinal serotonergic fibers were shown to facilitate the induction of scratching behavior by augmenting gastrin-releasing peptide/gastrin-releasing peptide receptor signals via the 5-HT_1A receptor,¹¹⁾ indicating that an incremental or decremental level of 5-HT increased or decreased the number of scratching events induced by pruritogens, respectively. In contrast, the intrathecal injection of 5-HT dose-dependently reduced CQ-induced scratches.⁷⁾ Similarly, pruritogen-induced scratches were mitigated by the administration of 5-HT,¹⁷⁾ indicating that scratches induced by pruritogens are ameliorated by an increase in the concentration of 5-HT. Therefore, the relationship between the amount of 5-HT and its effects on pruritogen-evoked scratches remains controversial.

5-HT binds to any one of the 15 subtypes of 5-HT receptors,^18,19) and the effects of an intrathecal administration of 5-HT on scratching events induced by pruritogens can be explained by summing up the effects of each subtype of 5-HT receptors, including positive and negative effects, indicating that investigating contribution of each 5-HT receptor to itch processing is essential for identifying the net role of 5-HT. Previous findings and the present results demonstrated the effects of antagonists and/or agonists of 5-HT₃/5-HT₇ receptors⁷⁾ and 5-HT₄/5-HT₅/5-HT₆ receptors on the induction of scratching events by pruritogens, respectively. Among 5-HT receptors, four receptors, 5-HT₃, 5-HT₄, 5-HT₆, and 5-HT₇, appear to contribute to itch processing, judging from the results showing that these four 5-HT receptors are involved in the attenuation of CQ-induced scratching events. The 5-HT₃, 5-HT₄, 5-HT₆, and 5-HT₇ receptors have been identified as excitatory receptors in the spinal dorsal horn,²⁰⁾ in which the 5-HT₃ receptors are ligand-gated ion channel receptors that trigger rapid depolarization though the opening of non-selective cation channels, such as Na⁺, Ca²⁺ influx and K⁺ efflux. The 5-HT₄, 5-HT₆, and 5-HT₇ receptors are Gs-protein coupled receptors and promote the formation of cAMP by activating adenylate cyclase. cAMP as an intracellular messenger phosphorylates protein kinase A, which modulates Ca²⁺ influx and membrane excitability.¹⁹⁾ Considering the excitatory roles of these three 5-HT receptors, serotonergic axons terminating on inhibitory neurons expressing these 5-HT receptors may exert the attenuating effects on itch processing in the CNS.

Taken together, our previous⁷⁾ and present study showed that CQ-induced scratching events were attenuated by the agonists of 5-HT₃, 5-HT₄, 5-HT₆, and 5-HT₇ receptors, and were attenuated by the pretreatment with SNRI or NaSSA, which are generally considered to increase the concentrations of 5-HT in the synaptic cleft. Collectively, these findings indicate that these 5-HT receptors play a crucial role in the attenuation of CQ-induced scratching events. Therefore, a previous finding in which CQ-induced scratches were attenuated by intrathecal administration of 5-HT⁷⁾ may be explained by the activation of these 5-HT receptors. Contrary to the effects of the 5-HT receptors on CQ-induced scratching events, histamine-induced scratches were scarcely affected by 5-HT or agonists of 5-HT receptors, including 5-HT₄, 5-HT₆, and 5-HT₇ receptors. Therefore, a part of 5-HT receptors appears to have no significant effects on scratching events induced by histamine, and 5-HT may not modulate itch signals derived from primary sensory neurons expressing histamine receptors, indicating that pruriceptive processing in the CNS atter intradermal administration of CQ or histamine can be differentially modified by 5-HT receptors. CQ-induced itch signals are modified by Gs-coupled 5-HT receptors, 5-HT₄, 5-HT₆, and 5-HT₇ receptors, which may be linked to excitatory functions, while pruritus processing induced by histamine remains unchanged by these 5-HT receptors.

On the other hand, the role of 5-HT₅ receptors in the itch system may differ from those of 5-HT₃, 5-HT₄, 5-HT₆, and 5-HT₇ receptors because a treatment with the antagonist of 5-HT₅ receptors in mice pretreated with milnacipran or mirtazapine did not affect the numbers of scratching events induced by CQ or histamine as shown in the present study. The reasons that 5-HT₅ receptors were not effective on pruritogen-induced scratching remain uncertain. Considering that 5-HT₅ receptors are coupled to Gi/o-proteins,²⁰⁾ 5-HT receptors coupling to Gi/o may not exert attenuating effects on acute itch processing. The roles of 5-HT₅ receptors in pruriceptive processing appear to differ from those in nociceptive processing. The expression of 5-HT₅ receptors was previously localized in the superficial layers of the spinal dorsal horn,²¹⁾ and acute and neuropathic pain were ameliorated by a treatment with a 5-HT₅ antagonist, SB699551.^22–24) The administration of the same antagonist did not reverse the amelioration of CQ- or histamine-evoked scratches by the pretreatment with these antidepressants, suggesting that 5-HT₅ receptors may not be involved in acute itch induced by CQ or histamine. The differential effects of a 5-HT₅ receptor antagonist on pain and itch signals may derive from the different roles of 5-HT₅ receptors in pruriceptive and nociceptive processing. It will be important to investigate further the precise role of other 5-HT receptors, including 5-HT_1A, to elucidate the net effect of 5-HT receptors on acute pruriceptive processing regarding the subtypes of G-proteins.

Compared to the roles of 5-HT receptors in nociceptive and pruriceptive processing, the contribution of 5-HT receptors to nociceptive processing has been widely investigated by the intrathecal administration of agonists and/or antagonists of 5-HT receptors.^25–27) The roles of 5-HT₃, 5-HT₄, and 5-HT₇ receptors in nociceptive and pruriceptive processing appear to be similar. An agonist of the 5-HT₃ receptor exerted antinociceptive effects in pain-related behavior, and these effects were antagonized by an antagonist of this receptor.^28,29) 5-HT₄ receptor agonists also exerted antinociceptive effects, which were prevented by their antagonists.^30–32) Furthermore, the antinociceptive effects of 5-HT were modified by antagonists or agonists of 5-HT₃ and 5-HT₄ receptors.^26,33) Similarly, an agonist of 5-HT₇ receptors inhibited algogen-induced behavior,^34,35) and their antagonists blocked the antinociceptive effects.³⁶⁾ Meanwhile, the effects of 5-HT₆ receptor antagonists on nociceptive behavior remain controversial,^37,38) while CQ-induced scratches were ameliorated by a 5-HT₆ receptor agonist. Collectively, the agonists and antagonists of 5-HT₃, 5-HT₄, and 5-HT₇ receptors, but not 5-HT₆ receptors, exerted the similar effects on nociceptive and pruriceptive process, suggesting that these receptors are, at least in part, involved in the inhibitory system common to nociceptive and pruriceptive processing in the CNS,

The majority of CQ- and histamine-responsive primary afferent neurons only responded to one pruritogen,¹⁶⁾ indicating that CQ and histamine activate separate primary afferent neurons. CQ and histamine were previously shown to bind to histamine receptors and Mas-related G protein-coupled receptor A3, respectively.³⁹⁾ Additionally, the percentages of primary afferent neurons responding to CQ was significantly lower in primary sensory neurons isolated from TRPA1-deficient mice than wild-type mice, while those of histamine-responding primary afferent neurons in these two mouse strains were similar to each other.¹³⁾ In humans, a pretreatment with an antihistamine also blocked itch induced by histamine, but not cowhage,⁴⁰⁾ and cowhage activated a population of C-fiber afferents that differed from histamine-responsive afferents,⁴¹⁾ indicating that the pathway of cowhage-mediated pruritus is different from that of histamine-mediated pruritus. Similarly, a previous study demonstrated that the induction of scratching events by CQ, but not histamine, was attenuated by the intrathecal administration of an α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor antagonist, perampanel, while histamine-induced scratches were reduced by administration of kainate receptor antagonists.^42,43) These findings indicate that CQ and histamine generate itch signals in different primary afferents, and pruriceptive signals evoked by CQ and histamine are then transmitted to secondary cells expressing AMPA receptors and kainate receptors in the dorsal horn, respectively.

CONCLUSION

Scratching behavior induced by CQ was mitigated by the pretreatment with milnacipran or mirtazapine, and reversed by antagonizing 5-HT₄ and 5-HT₆ receptors, but not a 5-HT₅ receptor. CQ-induced scratches were also attenuated by 5-HT₄ and 5-HT₆ agonists, whereas histamine-induced scratches were not modified by 5-HT₄ or 5-HT₆ agonists or reversed by 5-HT₄, 5-HT₅, or 5-HT₆ receptor antagonists. Collectively, the present results indicate that 5-HT₄, 5-HT₅, and 5-HT₆ receptors play different roles in the pruriceptive processing induced by acute pruritus.

Acknowledgments

This work was supported in part by JSPS KAKENHI (Grants-in-Aid for Scientific Research) Grant Nos. 22K15767 (Y.M.), 20K07760 (H.F.), 23K14301 (A.H-T.), and 21K07503 (Y.I.).

Conflict of Interest

The authors declare no conflict of interest.

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