brain research 1543 (2014) 73–82

Available online at www.sciencedirect.com

www.elsevier.com/locate/brainres

Research Report

Functional expression of 5-HT7 receptor on the substantia gelatinosa neurons of the trigeminal subnucleus caudalis in mice Eun Ju Yang, Seong Kyu Han, Soo Joung Parkn Department of Oral Physiology and Institute of Oral Bioscience, School of Dentistry, Chonbuk National University, Jeonju, 664-14, 1 Ga, Deokjin-Dong, Jeonbuk 561-756, Republic of Korea

art i cle i nfo

ab st rac t

Article history:

The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc; medullary dorsal

Accepted 21 October 2013

horn) receives and processes orofacial nociceptive inputs, and serotonergic fibers involved

Available online 26 October 2013

in the descending modulation of nociception are more densely distributed in the superficial laminae of the Vc. This study investigated the direct effects of 5-HT1A/7 receptor

Keywords:

agonist 8-OH-DPAT on SG neurons of the Vc to assess functional expression of the 5-HT7

5-HT7 receptor Trigeminal subnucleus caudalis Substantia gelatinosa Gramicidin-perforated patch clamp Single-cell RT-PCR

receptor using gramicidin-perforated patch-clamp in postnatal day (PND) 5-84 male mice. Of the 70 SG neurons tested, bath application of 8-OH-DPAT (30 μM) induced depolarization (n ¼33), hyperpolarization (n ¼16) or no response (n ¼21). In another 10 SG neurons, 8-OHDPAT in the presence of 5-HT1A receptor antagonist WAY-100635 (1 μM) elicited either depolarization (n ¼6) or no response (n¼ 4); hyperpolarization was not observed. The 8-OHDPAT-induced depolarization was significantly blocked by the selective 5-HT7 receptor antagonist SB-269970 (10 μM; n ¼8), but not by WAY-100635 (1 μM; n ¼5). The depolarizing effect of 8-OH-DPAT was maintained in the presence of TTX, CNQX, AP5, picrotoxin, and strychnine, indicating direct postsynaptic action of 8-OH-DPAT on SG neurons (n ¼6). 5-HT7 receptor mRNA was also detected in five of 21 SG neurons by single-cell RT-PCR. The mean amplitude of 8-OH-DPAT-induced depolarization in PND 5-21 mice (n ¼21) was significantly larger than that in PND 22-84 mice (n ¼12), although the proportion of SG neurons responding to 8-OH-DPAT by depolarization did not differ significantly between two age groups of mice. These results indicate that 5-HT7 receptors are functionally expressed in a subpopulation of SG neurons of the Vc and activation of 5-HT7 receptors plays an important role in modulating orofacial nociceptive processing in the SG neurons of the Vc. & 2013 Elsevier B.V. All rights reserved.

Abbreviations: 5-HT, cerebrospinal fluid; AP5, GAPDH,

5-hydroxytryptamine; 8-OH-DPAT, (7)-8-hydroxy-2-(di-n-propylamino) tetralin; ACSF, D,L-2-amino-5-phosphonopentanoic acid; CNQX,

glyceraldehyde 3-phosphate dehydrogenase; PND,

transcriptase-polymerase chain reaction; SB-269970, hydrochloride; SG,

substantia gelatinosa; TTX,

artificial

6-cyano-7-nitroquinoxaline-2,3-dione;

postnatal day; RMP,

resting membrane potential; RT-PCR,

reverse

(2R)-1-[(3-Hydroxyphenyl)sulfonyl]-2-[2-(4-methyl-1-piperidinyl)ethyl]pyrrolidine

tetrodotoxin citrate; Vc,

trigeminal subnucleus caudalis; WAY-100635,

methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide maleate n Corresponding author: Fax: þ82 63 270 4004. E-mail address: [email protected] (S.J. Park). 0006-8993/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.brainres.2013.10.041

N-[2-[4-(2-

74

1.

brain research 1543 (2014) 73–82

Introduction

The trigeminal subnucleus caudalis (Vc; also called the medullary dorsal horn) is the first relay site in nociceptive transmission from the orofacial region, and is structurally and functionally similar to the spinal dorsal horn (Sessle, 2000). The substantia gelatinosa (SG, lamina II) of the Vc and spinal dorsal horn receives Aδ- and C-primary afferent inputs and has been considered a critical region in the processing and modulation of nociceptive information conveyed to the higher brain centers (Cervero and Iggo, 1980; Todd, 2010). The serotonergic pathways originating from the rostroventral medulla, including the nucleus raphe magnus, play a major role in the descending modulation of nociception in the Vc (Chiang et al., 1994; Seo et al., 2002; Okamoto et al., 2005, 2007) and the spinal dorsal horn (Bardin, 2011; Bardin et al., 2000; Dogrul et al., 2009; Jeong et al., 2004). Descending serotonergic fibers are more densely distributed in the superficial laminae (i.e. laminae I and II) than in the deeper laminae of the Vc or the spinal dorsal horn, in which serotonin (5-hydroxytryptamine, 5-HT) can either inhibit or facilitate nociceptive transmission depending on the 5-HT receptor subtype activated (Bardin, 2011; Cropper et al., 1984; Li et al., 1997; Millan, 2002). The 5-HT receptors are divided into seven classes (5-HT1-7; Hoyer et al., 2002). Our previous study demonstrated that activation of 5-HT1A and 5-HT3 receptors induced hyperpolarization and depolarization, respectively, and activation of 5-HT2 and 5-HT4 receptors produced either hyperpolarization or depolarization in the SG neurons of the Vc (Yin et al., 2011). The 5-HT7 receptor is a G-protein coupled receptor that is the most recently identified 5-HT receptor subtype (Ruat et al., 1993), and has been implicated in pain modulation. For instance, systemically administered 5-HT7 receptor agonists exert dose-dependent inhibition of capsaicin- or nerve injuryinduced mechanical hypersensitivity and/or thermal hyperalgesia, which are blocked by systemic co-administration of the selective 5-HT7 receptor antagonist in mice (Brenchat et al., 2009, 2010). Immunocytochemical study of 5-HT7 receptor distribution in the spinal cord has revealed that the 5-HT7 receptor is mainly localized in the spinal dorsal horn, particularly in the two superficial laminae, and is expressed on primary afferent fibers, intrinsic dorsal horn neurons, and glial cells (Doly et al., 2005; Meuser et al., 2002). Several behavioral studies employing intrathecal administration of 5-HT7 receptor antagonists or agonists have suggested an antinociceptive role for spinal 5-HT7 receptors (Brenchat et al., 2012a; Dogrul and Seyrek, 2006; Dogrul et al., 2009; Viguier et al., 2012). In contrast, a pronociceptive role of spinal 5-HT7 receptors has also been proposed by other studies (Amaya-Castellanos et al., 2011; Godínez-Chaparro et al., 2012; Rocha-González et al., 2005). Since selective 5-HT7 receptor agonists are not readily available, the function and mechanism of action of 5-HT7 receptors in nociceptive processing at the medullary and spinal dorsal horns have not yet been fully elucidated. Garraway and Hochman (2001) reported that 5-HT7 but not 5-HT1A receptor activation appears to contribute to facilitatory action of (7)-8hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) on the dorsal root-evoked synaptic responses of deep dorsal horn neurons in

the neonatal rat. Costa et al. (2012) demonstrated that 8-OHDPAT enhances AMPA receptor-mediated synaptic transmission via postsynaptic 5-HT7 receptors in the hippocampus. In this study, we examined the effects of 5-HT1A/7 receptor agonist 8-OH-DPAT and selective 5-HT7 receptor antagonist (2R)-1[(3-Hydroxyphenyl)sulfonyl]-2-[2-(4-methyl-1-piperidinyl)ethyl] pyrrolidine hydrochloride (SB-269970; Hagan et al., 2000; Lovell et al., 2000) on SG neurons of the Vc to assess functional expression of the 5-HT7 receptor using the gramicidinperforated patch-clamp technique in male mice.

2.

Results

Using a gramicidin-perforated patch technique, a total of 80 SG neurons were recorded from the Vc of 65 male mice. Of the 70 SG neurons tested, the bath application of 30 μM 8OH-DPAT alone produced depolarization in 33 (47%) neurons, hyperpolarization in 16 (23%) neurons and no response in 21 (30%) neurons (Fig. 1). The mean resting membrane potential (RMP) and the mean amplitude of the membrane potential change was  60.971.3 mV and 5.770.5 mV (n¼ 33), respectively, in neurons showing a depolarizing response to 8-OH-DPAT,

Fig. 1 – Responses of three different substantia gelatinosa (SG) neurons of the trigeminal subnucleus caudalis (Vc) to 8-OH-DPAT in gramicidin-perforated patch recordings under current-clamp mode. Application of 30 μM 8-OH-DPAT alone produced depolarization (A), no response (B), or hyperpolarization (C), respectively. Bars indicate the duration of 8-OH-DPAT application.

brain research 1543 (2014) 73–82

and 62.872.1 mV and  7.671.2 mV (n¼16), respectively, in neurons showing a hyperpolarizing response to 8-OH-DPAT. The mean RMP of neurons that were not affected by 8-OH-DPAT was  61.571.9 mV (n¼ 21). There was no significant difference in the RMPs among the various responding groups that were examined (P40.05, one-way ANOVA). In another 10 SG neurons, 30 μM 8-OH-DPAT in the presence of 5-HT1A receptor antagonist N-[2[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide maleate (WAY-100635, 1 μM) resulted in either depolarization (6.971.6 mV) or no response, which occurred in 6 neurons (60%; RMP¼ 61.974.9 mV) and 4 neurons (40%; RMP¼  61.372.4 mV), respectively. However, 8-OH-DPATinduced hyperpolarization did not occur in the presence of WAY100635. When another treatment of 8-OH-DPAT was applied consecutively to the SG neurons that exhibited depolarization to the primary 8-OH-DPAT treatment, the second application of 8-OH-DPAT generated a similar depolarization amplitude to that of the first application in the SG neurons tested (Fig. 2A). No significant difference was found in the mean amplitude of 8-OH-DPAT-induced depolarization between the first (5.37 1.0 mV) and second (6.371.3 mV) applications of 8-OH-DPAT (n¼ 6, P40.05, Wilcoxon signed-rank test; Fig. 2B). To confirm that the 5-HT7 receptor was responsible for the 8-OH-DPAT-induced depolarization, we used the selective 5-HT7 receptor antagonist SB-269970. Of the 8 SG neurons showing a depolarizing response to 8-OH-DPAT, depolarization was completely blocked (n ¼5) or partially blocked (n ¼3) in the presence of 10 μM SB-269970 (Fig. 3A). However, the 5-HT1A receptor antagonist 1 μM WAY-100635 did not affect 8-OH-DPAT-induced depolarization in any of the 5 neurons

Fig. 2 – Responses to repeated application of 30 μM 8-OHDPAT in SG neurons of the Vc. (A) A second application of 8-OH-DPAT produced a similar depolarization amplitude to that produced by the first application. Bars indicate the duration of 8-OH-DPAT application. (B) There was no significant difference in the mean depolarization amplitude between the first and the second application of 8-OH-DPAT (n ¼6, P40.05). NS, not significant.

75

tested (Fig. 3B). The addition of 10 μM SB-269970 significantly reduced the mean amplitude of 8-OH-DPAT-induced depolarization from 6.071.3 mV to 1.270.6 mV (n¼ 8, Po0.05, Wilcoxon signed-rank test; Fig. 3D). On the other hand, this mean amplitude (6.371.3 mV) was not affected by the addition of 1 μM WAY-100635 (n¼5, P40.05, Wilcoxon signed-rank test; Fig. 3E). In addition, 8-OH-DPAT-induced hyperpolarization was completely blocked by 1 μM WAY100635 in one SG neuron (data not shown). Interestingly, the 8-OH-DPAT-induced depolarizing response was unmasked when 8-OH-DPAT was applied in the presence of 1 μM WAY100635 in one SG neuron hyperpolarized by 8-OH-DPAT (Fig. 3C) and in two SG neurons showing no response to 8-OH-DPAT (data not shown). These results indicate that 8OH-DPAT-induced depolarization was mediated by the 5-HT7 receptor, but not by 5-HT1A receptor. To determine whether 8-OH-DPAT can act directly on postsynaptic membranes to depolarize SG neurons, we examined 8-OH-DPAT-induced depolarization in the presence of the Naþ channel blocker tetrodotoxin citrate (TTX), NMDA glutamate receptor antagonist D,L-2-amino-5-phosphonopentanoic acid (AP5), non-NMDA glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), GABAA receptor antagonist picrotoxin, and glycine receptor antagonist strychnine to exclude effect due to presynaptic neurotransmitter release by action potential or presynaptic 5-HT7 receptors. 8-OH-DPAT-induced depolarization persisted in the presence of 0.5 μM TTX, 20 μM AP5, 10 μM CNQX, 50 μM picrotoxin, and 2 μM strychnine in 7 SG neurons (Fig. 4A), indicating direct postsynaptic action of 8-OH-DPAT on SG neuron tested. No significant difference was found between the mean depolarization amplitude produced by 8-OH-DPAT alone (5.170.9 mV) and that produced by 8-OH-DPAT in the presence of the above-mentioned blockers (5.071.0 mV; n¼ 6, P40.05, Wilcoxon signed-rank test; Fig. 4B). To confirm 5-HT7 receptor mRNA expression in the SG neurons, single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) was performed. Consistent with electrophysiological data indicating the direct depolarizing action of 8-OH-DPAT on SG neurons through postsynaptic 5-HT7 receptors, single-cell RT-PCR revealed the presence of mRNA for 5-HT7 receptor subtypes in the SG neurons (Fig. 5). Five (24%) of 21 SG neurons that showed the presence of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expressed 5-HT7 receptor mRNA among 3 male mice. In mice, postnatal age of three weeks is a critical period for changes in 5-HT-induced hyperpolarizing responses (Park et al., 2012). To examine age-related changes in the effects of 8-OHDPAT on SG neurons, a total of 58 male mice, with 70 8-OH DPAT tested SG neurons (Fig. 1), were divided into postnatal day (PND) 5-21 and PND 22-84 groups. The mean RMPs of SG neurons in PND 5-21 and PND 22-84 mice were 61.571.1 mV (n¼ 47) and 61.571.9 mV (n¼ 23), respectively, and there was no significant difference between them (P40.05, Wilcoxon rank-sum test). There was no significant difference in the proportion of SG neurons responding to 8-OH-DPAT by depolarization or hyperpolarization, or showing no response between PND 5-21 and PND 22-84 mice (P40.05, χ2 test; Fig. 6A). However, there was a significant difference in the mean amplitude of the 8-OH-DPAT-induced depolarization of

76

brain research 1543 (2014) 73–82

Fig. 3 – Effects of 5-HT1A and 5-HT7 receptor antagonists on responses of SG neurons of the Vc to 30 μM 8-OH-DPAT. (A) 5-HT7 receptor antagonist SB-269970 (10 μM) abolished 8-OH-DPAT-induced depolarization. (B) 5-HT1A receptor antagonist WAY100635 (1 μM) did not affect 8-OH-DPAT-induced depolarization. (C) When the 8-OH-DPAT-induced hyperpolarization was blocked by WAY-100635 (1 μM), depolarizing response to 8-OH-DPAT was appeared. (D) There was a significant difference between the mean depolarization amplitude produced by 8-OH-DPAT alone and that produced by 8-OH-DPAT in the presence of SB-269970 (n ¼ 8, Po0.05). (E) There was no significant difference between the mean depolarization amplitude produced by 8-OH-DPAT alone and that produced by 8-OH-DPAT in the presence of WAY-100635 (n ¼5, P40.05). NS, not significant. SG neurons between the two age groups. The mean amplitude of 8-OH-DPAT-induced depolarization in PND 5-21 mice (6.570.6 mV, n¼21) was significantly larger than that in PND 22-84 mice (4.270.7 mV, n¼ 12; Po0.05, Wilcoxon rank-sum test; Fig. 6B).

3.

Discussion

In this study, 8-OH-DPAT induced depolarization, hyperpolarization or no response in SG neurons of the Vc, while

8-OH-DPAT in the presence of WAY-100635 elicited either depolarization or no response. 8-OH-DPAT-induced depolarization was significantly blocked by SB-269970, but not by WAY-100635, and was maintained in the presence of TTX, CNQX, AP5, picrotoxin, and strychnine. 5-HT7 receptor mRNA was also detected in the SG neurons. Well-characterized 5-HT7 receptor selective agonists are not yet commercially available and putative selective 5-HT7 receptor agonists also have an affinity for other receptors (Bosker et al., 2009; Brenchat et al., 2012b). Recent development of the selective 5-HT7 receptor antagonist SB-269970 (Hagan et al.,

brain research 1543 (2014) 73–82

Fig. 4 – Direct depolarizing action of 8-OH-DPAT (30 μM) on SG neurons of the Vc. (A) 8-OH-DPAT-induced depolarization remained when 0.5 μM TTX, 20 μM AP5, 10 μM CNQX, 50 μM picrotoxin, and 2 μM strychnine were present. Bars indicate the duration of application of 8-OHDPAT and above-mentioned blockers, respectively. (B) There was no significant difference between the mean depolarization amplitude produced by 8-OH-DPAT alone and that produced by 8-OH-DPAT in the presence of blockers (n ¼7, P40.05). NS, not significant.

Fig. 5 – Single-cell RT-PCR mRNA analysis run for 5-HT7 receptor and housekeeping gene GAPDH on SG neurons of the Vc. 5-HT7 receptor mRNA was detected in 2 of the 6 neurons examined. The sequences of RT-PCR primers used for mRNA detection are given. L, 100 bp ladder; 1–6, harvested single SG neurons; NC, negative control (only touching the brainstem slice surface); PC, positive control (whole brain tissue).

2000; Lovell et al., 2000) has led to a better understanding of the role of 5-HT7 receptors in nociception. However, the role of the 5-HT7 receptor in nociceptive transmission at the spinal cord

77

level is controversial, because recent behavioral studies on spinal 5-HT7 receptors have reported conflicting results. Some behavioral studies using inflammatory or neuropathic pain models suggested that activation of spinal 5-HT7 receptors produces antinociception. For example, the mechanical allodynia induced by intradermal capsaicin and/or nerve injury was reversed by intrathecal administration of the 5-HT7 receptor agonist in adult male rats, and this antinociceptive effect of 5-HT7 receptor agonists was completely blocked by SB-269970 given intrathecally (Brenchat et al., 2012a; Viguier et al., 2012). In addition, spinal 5-HT7 receptors have been shown to be involved in μ-opioid receptor- and cannabinoid receptor type 1mediated antinociception, as intrathecal administration of the 5-HT7 receptor antagonist SB-269970 blocks both systemically-administered opioids and cannabinoids in adult male mice (Dogrul and Seyrek, 2006; Seyrek et al., 2010; Yanarates et al., 2010), as well as morphine microinjected into the rostroventral medulla in adult male rats (Dogrul et al., 2009). On the other hand, other behavioral studies suggest a pronociceptive role for 5-HT7 receptors in the spinal cord. Intrathecal administration of 5-HT1A/7 receptor agonist 5-CT increased formalin-induced acute nociceptive behavior at lower doses and this pronociceptive effect of 5-CT was blocked by spinal administration of SB-269970, but not by WAY-100635 in adult female rats (Rocha-González et al., 2005). Activation of spinal 5-HT7 receptors promotes development and maintenance of formalin-induced secondary mechanical allodynia and hyperalgesia in adult female rats (Godínez-Chaparro et al., 2012). Intrathecal application of SB-269970 significantly reduced nerve injury-induced mechanical allodynia in adult female rats (Amaya-Castellanos et al., 2011) According to the aforementioned studies, activation of the 5-HT7 receptor produces antinociceptive effects in male animals and pronociceptive effects in female animals. We used only male mice in this study to rule out the potential influences of sex hormones or the menstrual cycle, since gender might affect the role of 5-HT7 receptors in nociceptive processing at the level of the Vc or the spinal dorsal horn (Lei et al., 2011). 5-HT7 receptors are predominantly expressed in the two superficial laminae of the dorsal horn in the spinal cord (Doly et al., 2005; Meuser et al., 2002). The 5-HT7 receptor is positively coupled to adenylate cyclase activity, increasing cAMP levels, and is expected to exert excitatory effects (Ruat et al., 1993; Vanhoenacker et al., 2000). In this study, applying 5-HT1A/7 receptor agonist 8-OH-DPAT depolarized 47% of SG neurons, and this depolarizing action was blocked by the selective 5-HT7 receptor antagonist SB-269970. 8-OH-DPAT also induced hyperpolarization in 23% of SG neurons, which could be blocked by 5-HT1A receptor antagonist WAY-100635. When 8-OH-DPAT was applied in the presence of WAY100635, 60% of SG neurons were depolarized and no neurons were hyperpolarized. In previous studies, 8-OH-DPAT only induced hyperpolarization or outward current through the 5-HT1A receptor in a very small number of SG neurons at the Vc in mice (Yin et al., 2011) and guinea pigs (Grudt et al., 1995) and at the spinal dorsal horn in rats (Abe et al., 2009; Ito et al., 2000). Examination of a broader SG neuronal population might, in part, explain this discrepancy, since we have recorded a large number of SG neurons regardless of 5-HT response, rather than selecting only those SG neurons

78

brain research 1543 (2014) 73–82

Fig. 6 – Effects of age on depolarizing responses of SG neurons of the Vc to 30 μM 8-OH-DPAT. (A) Summary of the effects of 8-OH-DPAT on SG neurons in PND 5-21 and PND 22-84 groups. There was no significant difference in the proportion of SG neurons depolarized by 8-OH-DPAT between PND 5-21 and PND 22-84 mice (P40.05, χ2 test). The numbers in the columns represent the number of neurons responding. (B) Comparison of the mean amplitude of 8-OH-DPAT-induced depolarization of SG neurons between two age groups. The mean amplitude of 8-OH-DPAT-induced depolarization was significantly higher in PND 5-21 group (n¼ 21) than in PND 22-84 group (n ¼ 12) (nPo0.05).

which showed hyperpolarization to 5-HT. In addition, the gramicidin-perforated patch clamp technique allows for preservation of the intracellular second messenger systems required for 5-HT7 receptor-induced depolarization. The inhibitory response of 8-OH-DPAT induced through the 5-HT1A receptor in whole cell patch clamp recordings (Abe et al., 2009; Grudt et al., 1995; Ito et al., 2000) may be mediated by the membrane-delimited coupling of G proteins to Kþ channels without involvement of the cAMP second messenger system (Muraki et al., 2004; Penington et al., 1993). AMPA receptor-mediated excitatory postsynaptic currents are either enhanced via the 5-HT7 receptor or reduced via the 5-HT1A receptor by 8-OH-DPAT in the hippocampus (Costa et al., 2012). Similarly, 8-OH-DPAT facilitates dorsal root-evoked synaptic responses in the majority of the deep dorsal horn neurons, probably due to activation of the 5-HT7 receptor, but not the 5-HT1A receptor, in the rat spinal cord (Garraway and Hochman, 2001). These results suggest that 8-OH-induced depolarization is mediated by activation of the 5-HT7 receptor in SG neurons of the Vc. 5-HT7 receptor activation increases hyperpolarizationactivated nonselective cation currents in the dorsal root ganglia and the anterodorsal thalamus (Cardenas et al., 1999; Chapin and Andrade, 2001) and inhibits the calciumactivated potassium channel in the intralaminar and midline thalamus and hippocampus (Gill et al., 2002; Goaillard and Vincent, 2002). Further studies are needed to clarify the mechanism of 5-HT7 receptor-induced depolarization in the SG neurons of the Vc. Prolonged exposure to agonists results in desensitization of 5-HT7 receptor-mediated cyclic AMP formation in frontocortical astrocytes (Shimizu et al., 1998). Single or repeated systemic administration of 5-HT7 receptor antagonist SB 269970 decreases 5-HT7 receptor-mediated enhancement of the bursting activity in hippocampal slices (Tokarski et al., 2012). In our study, the amplitude of

8-OH-DPAT-induced depolarization was maintained after a second successive application, suggesting that there was no rapid desensitization to the 5-HT7 receptor-induced depolarizing effect of 8-OH-DPAT. In the present study, 8-OH-DPAT had direct postsynaptic depolarizing action on SG neurons of the Vc because 8-OHDPAT-induced depolarization was preserved in the presence of TTX, CNQX, AP5, picrotoxin, and strychnine. Consistent with this postsynaptic depolarizing effect of 8-OH-DPAT, single-cell RT-PCR also demonstrated that 24% of SG neurons expressed 5-HT7 receptor mRNA. The percentage of SG neurons expressing 5-HT7 receptor mRNA was lower than that showing a depolarizing response to 8-OH-DPAT in a gramicidin-perforated patch-clamp recording. It is likely that 5-HT7 receptor mRNA extracted from the cytosol of each SG neuron may not be enough for RT-PCR detection and degradation may be occurring during harvest (Hill et al., 2007). Costa et al. (2012) demonstrated that AMPA receptormediated transmission enhancement by 5-HT7 receptors involves only a postsynaptic mechanism while inhibition by 5-HT1 receptors involves both pre- and postsynaptic mechanisms in the hippocampus. Anatomical study also has revealed substantial expression of 5-HT7 receptors in postsynaptic SG neurons of the spinal dorsal horn, with 41% of the total 5-HT7 receptor immunoreactivity in cell bodies and dendrites of intrinsic neurons (postsynaptic labeling), 39% in axons and axon terminals of both primary afferent fibers and intrinsic neurons (presynaptic labeling), and the remaining 20% observed in glial cells (Doly et al., 2005). The SG mainly consists of glutamatergic excitatory interneurons and GABAergic inhibitory interneurons (Zeilhofer et al., 2012), and, in this study, activation of 5-HT7 receptors on SG neurons of the Vc induced depolarization. Intrathecal application of the GABAA receptor antagonist bicuculline significantly reduced the antinociceptive effects of 5-HT7 receptor agonists in

brain research 1543 (2014) 73–82

nerve-injured rats, unlike administration of the GABAB receptor antagonist phaclofen or the opioid receptor antagonist naloxone (Viguier et al., 2012). Because 8-OH-DPAT-induced depolarization in SG neurons of the Vc was mediated by direct action on postsynaptic 5-HT7 receptors, the antinociceptive effect of 5-HT7 receptor activation in the Vc and the spinal dorsal horn may result from activation of the inhibitory GABAergic SG neurons through postsynaptic 5-HT7 receptors, release of GABA, and subsequent inhibition of nociceptive transmission (Bardin, 2011). The pronociceptive effect of 5-HT7 receptors could also result from 5-HT7 receptor-induced depolarization of glutamatergic excitatory interneurons in the SG of the Vc. Somatostatin elicits outward currents only in inhibitory neurons, but has no effect on excitatory neurons in the SG of the adult rat spinal dorsal horn (Yasaka et al., 2010). Further studies are necessary to determine whether SG neurons showing 5-HT7 receptor-mediated depolarization are inhibitory or excitatory interneurons. The mean amplitude of 8-OH-DPAT-induced depolarization was greater in PND 5-21 mice, although the proportion of depolarized neurons was not significantly different between PND 5-21 and PND 22-84 mice. Expression of 5-HT7 receptor mRNA progressively diminished during postnatal development, with a dramatic decrease during the second and third postnatal weeks in the rat prefrontal cortex (Béïque et al., 2004) and the mouse hippocampus (Renner et al., 2012). The level of 3H-8-OH-DPAT binding in the SG of the piglet Vc demonstrated age-related changes, reaching its peak on PND 12 and then gradually decreasing until PND 60 (Niblock et al., 2004), which might explain the larger depolarizing responses to 8-OHDPAT observed in PND 5-21 mice compared to PND 22-84 mice in this study. After PND 21, abrupt changes occur in the descending modulatory effect of the rostroventral medulla or the dorsolateral funiculus stimulation on spinal dorsal horn neurons (Fitzgerald and Koltzenburg, 1986; Hathway et al., 2009). These results indicate that 5-HT7 receptor-induced depolarization might undergo postnatal developmental change in SG neurons of the Vc. Taken together, these results indicate that 8-OH-DPAT exerts a direct postsynaptic depolarizing action on a subpopulation of SG neurons of the Vc by activating 5-HT7 receptors, suggesting that the 5-HT7 receptor is involved in modulating orofacial nociceptive processing in SG neurons of the Vc and should be considered a potential target in developing therapeutic agents to treat orofacial pain.

cerebrospinal fluid (ACSF) containing (in mM) 126 NaCl, 2.5 KCl, 2.4 CaCl2, 1.2 MgCl2, 11 D‐glucose, 1.4 NaH2PO4 and 25 NaHCO3 (pH 7.4, bubbled with 95% O2 and 5% CO2). The slices were allowed to recover in oxygenated ACSF for at least 1 h at room temperature.

4.2.

Experimental procedures

4.1.

Animals and brainstem slice preparation

This study was approved by the Experimental Animal Care and Ethics Committee of Chonbuk National University (20111-0004). Male ICR mice (Damul Science, Suwon, Korea) PND 5-84 were used. Animals were housed under 12-hour light/ dark cycles with free access to water and food, and were decapitated between 10:00 and 12:00. The brains were rapidly removed and coronal brainstem slices (150200 mm in thickness) containing the Vc were prepared with a vibratome (Microm, Walldorf, Germany) in ice-cold artificial

Electrophysiology

One brainstem slice was transferred to the recording chamber, submerged, and continuously superfused with oxygenated ACSF at a flow rate of 4–5 ml/min. The slice was viewed on the stage of an upright microscope (BX51WI, Olympus, Tokyo, Japan) equipped with differential interference contrast optics. Patch pipettes were pulled from borosilicate glass capillaries (PG52151-4, WPI, Sarasota, FL, USA) on a Flaming/ Brown micropipette puller (P-97; Sutter Instruments Co., Novato, CA, USA), and filled with solution containing (in mM) 130 KCl, 5 NaCl, 0.4 CaCl2, 1 MgCl2, 10 HEPES and 1.1 EGTA (pH 7.3 adjusted with KOH). For perforated-patch recordings, gramicidin (Sigma, St. Louis, MO, USA) was dissolved in dimethylsulfoxide (Sigma) to a concentration of 2.5–5 mg/ml and then diluted in the pipette solution to a final concentration of 2.5–5 mg/ml just before use. The tip of the electrode was loaded with a small volume of gramicidin-free pipette solution and the electrode was backfilled with the gramicidin-containing solution. The tip resistances of the recording electrodes were between 5 and 8 MΩ. Offset potentials were compensated directly before seal formation. After cell attachment, access resistance was monitored and experiments began when in the range of 40–100 MΩ. Recordings began 15–20 min after giga-seal formation when the RMP of the neuron reached a stable level below  50 mV. Signals were amplified with an Axopatch 200B (Axon Instruments, San Francisco, CA, USA), filtered with a Bessel filter at 2 kHz, and digitized with the Digidata 1440A (Axon Instruments) at 1 kHz. Data were collected and analyzed with the Clampex 10.2 software (Axon Instruments). All recordings were made at room temperature. The SG of the Vc was clearly visible as a translucent band just medial to the spinal trigeminal tract, which travels along the slice's lateral edge. The gramicidin-perforated patch recordings were made from visually identified SG neurons in current-clamp mode. Any SG neurons that displayed a shift in membrane potential Z2.0 mV were considered to have responded.

4.3.

4.

79

Drugs

The drugs used were as follows: 8-OH-DPAT (30 μM), WAY100635 (1 μM), SB-269970 (10 μM), TTX (0.5 μM), CNQX (10 μM), AP5 (20 μM), picrotoxin (50 μM), and strychnine hydrochloride (2 μM). SB-269970 was purchased from Tocris Bioscience (Ellisville, MO, USA), and all other drugs were purchased from Sigma. Stock solutions of all drugs were dissolved in distilled water, except picrotoxin, which was dissolved in dimethylsulfoxide. Stock solutions were diluted to working concentrations using ACSF immediately before use and applied by superfusion. We applied 8-OH-DPAT for 2–3 min and allowed a washout/recovery period of 10–20 min between subsequent

80

brain research 1543 (2014) 73–82

applications. Pretreatment drugs were applied 3 min before application of 8-OH-DPAT.

4.4.

Single-cell RT-PCR

Patch electrodes used for harvesting were baked at 250 1C for 6 h before being pulled. The cytoplasmic content was harvested under visual control using a patch electrode filled with 8 ml autoclaved solution containing (in mM) 140 KCl, 10 EGTA, 1 MgCl2, 1 CaCl2, and 10 HEPES (pH 7.3). To control for the debris that might enter the pipette and be amplified, mock harvest was conducted, in which the patch electrode was lowered to the surface of the slice but cellular content was not aspirated. Immediately after harvesting, the individual cellular content was expelled from the patch electrode into a PCR tube containing RT pre-mixture consisting of 0.7 ml of 3 μg/μl random primers, 0.7 ml of 40 U/μl RNase OUT™ (Invitrogen, Carlsbad, CA, USA) and distilled water to a final volume of 5 ml. The cytoplasmic content and RT pre-mixture were mixed and then briefly centrifuged. The sample was incubated at 65 1C for 5 min, then on ice for at least 1 min. After the first reaction, RT reaction solution containing 4 μl of 5  RT buffer, 2 μl of 0.1 M dithiothreitol, 1 μl of 10 mM dNTP mix, 0.5 μl of 200 U/μl Superscript III (Invitrogen) and 0.5 μl of distilled water was added. The cDNA synthesis was performed at 25 1C for 5 min, then 50 1C for 1 h and finally at 70 1C for 15 min. The RT products were then kept in a freezer at 20 1C. The RT-PCR primer sequences for 5-HT7 receptor are presented in Fig. 5. PCR was performed through 50 cycles of amplification using the following conditions: denaturation at 94 1C for 30 s, annealing at 62 1C for 30 s, and elongation at 72 1C for 45 s. This was followed by a final extension at 72 1C for 10 min. The PCR products were electrophoresed on a 1.5% agarose gel (Promega, Madison, WI, USA) and then analyzed using a Lab Systems analyzer (Bio-Rad, Milano, Italy).

4.5.

Statistical analysis

The data were expressed as the mean7SEM. For statistical analysis, Wilcoxon signed-rank test, Wilcoxon rank-sum test and χ2 test were used where appropriate. The level of significance was set at Po0.05.

Acknowledgment This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-0070100).

references

Abe, K., Kato, G., Katafuchi, T., Tamae, A., Furue, H., Yoshimura, M., 2009. Responses to 5-HT in morphologically identified neurons in the rat substantia gelatinosa in vitro. Neuroscience 159, 316–324. Amaya-Castellanos, E., Pineda-Farias, J.B., Castañeda-Corral, G., Vidal-Cantú, G.C., Murbartián, J., Rocha-González, H.I., Granados-Soto, V., 2011. Blockade of 5-HT7 receptors reduces

tactile allodynia in the rat. Pharmacol. Biochem. Behav. 99, 591–597. Bardin, L., 2011. The complex role of serotonin and 5-HT receptors in chronic pain. Behav. Pharmacol. 22, 390–404. Bardin, L., Lavarenne, J., Eschalier, A., 2000. Serotonin receptor subtypes involved in the spinal antinociceptive effect of 5-HT in rats. Pain 86, 11–18. Béïque, J.C., Campbell, B., Perring, P., Hamblin, M.W., Walker, P., Mladenovic, L., Andrade, R., 2004. Serotonergic regulation of membrane potential in developing rat prefrontal cortex: coordinated expression of 5-hydroxytryptamine (5-HT)1A, 5HT2A, and 5-HT7 receptors. J. Neurosci. 24, 4807–4817. Bosker, F.J., Folgering, J.H., Gladkevich, A.V., Schmidt, A., van der Hart, M.C., Sprouse, J., den Boer, J.A., Westerink, B.H., Cremers, T.I., 2009. Antagonism of 5-HT1A receptors uncovers an excitatory effect of SSRIs on 5-HT neuronal activity, an action probably mediated by 5-HT7 receptors. J. Neurochem. 108, 1126–1135. Brenchat, A., Romero, L., García, M., Pujol, M., Burgueño, J., Torrens, A., Hamon, M., Baeyens, J.M., Buschmann, H., Zamanillo, D., Vela, J.M., 2009. 5-HT7 receptor activation inhibits mechanical hypersensitivity secondary to capsaicin sensitization in mice. Pain 141, 239–247. Brenchat, A., Nadal, X., Romero, L., Ovalle, S., Muro, A., SánchezArroyos, R., Portillo-Salido, E., Pujol, M., Montero, A., Codony, X., Burgueño, J., Zamanillo, D., Hamon, M., Maldonado, R., Vela, J.M., 2010. Pharmacological activation of 5-HT7 receptors reduces nerve injury-induced mechanical and thermal hypersensitivity. Pain 149, 483–494. Brenchat, A., Zamanillo, D., Hamon, M., Romero, L., Vela, J.M., 2012a. Role of peripheral versus spinal 5-HT7 receptors in the modulation of pain undersensitizing conditions. Eur. J. Pain 16, 72–81. Brenchat, A., Rocasalbas, M., Zamanillo, D., Hamon, M., Vela, J.M., Romero, L., 2012b. Assessment of 5-HT7 receptor agonists selectivity using nociceptive and thermoregulation tests in knockout versus wild-type mice. Adv. Pharmacol. Sci. http://dxdoi.org/10.1155/2012/312041 (Article ID 312041, 9 p.). Cardenas, C.G., Del Mar, L.P., Vysokanov, A.V., Arnold, P.B., Cardenas, L.M., Surmeier, D.J., Scroggs, R.S., 1999. Serotonergic modulation of hyperpolarization-activated current in acutely isolated rat dorsal root ganglion neurons. J. Physiol. 518, 507–523. Cervero, F., Iggo, A., 1980. The substantia gelatinosa of the spinal cord: a critical review. Brain 103, 717–772. Chapin, E.M., Andrade, R., 2001. A 5-HT7 receptor-mediated depolarization in the anterodorsal thalamus. II. Involvement of the hyperpolarization-activated current Ih. J. Pharmacol. Exp. Ther. 297, 403–409. Chiang, C.Y., Hu, J.W., Sessle, B.J., 1994. Parabrachial area and nucleus raphe magnus-induced modulation of nociceptive and nonnociceptive trigeminal subnucleus caudalis neurons activated by cutaneous or deep inputs. J. Neurophysiol. 71, 2430–2445. Costa, L., Trovato, C., Musumeci, S.A., Catania, M.V., Ciranna, L., 2012. 5-HT1A and 5-HT7 receptors differently modulate AMPA receptor-mediated hippocampal synaptic transmission. Hippocampus 22, 790–801. Cropper, E.C., Eisenman, J.S., Azmitia, E.C., 1984. 5-HTImmunoreactive fibers in the trigeminal nuclear complex of the rat. Exp. Brain Res. 55, 515–522. Dogrul, A., Seyrek, M., 2006. Systemic morphine produces antinociception mediated by spinal 5-HT7, but not 5-HT1A and 5-HT2 receptors in the spinal cord. Br. J. Pharmacol. 149, 498–505. Dogrul, A., Ossipov, M.H., Porreca, F., 2009. Differential mediation of descending pain facilitation and inhibition by spinal 5HT-3 and 5HT-7 receptors. Brain Res. 1280, 52–59.

brain research 1543 (2014) 73–82

Doly, S., Fischer, J., Brisorgueil, M.J., Vergé, D., Conrath, M., 2005. Pre- and postsynaptic localization of the 5-HT7 receptor in rat dorsal spinal cord: immunocytochemical evidence. J. Comp. Neurol. 490, 256–269. Fitzgerald, M., Koltzenburg, M., 1986. The functional development of descending inhibitory pathways in the dorsolateral funiculus of the newborn rat spinal cord. Brain Res. 389, 261–270. Garraway, S.M., Hochman, S., 2001. Pharmacological characterization of serotonin receptor subtypes modulating primary afferent input to deep dorsal horn neurons in the neonatal rat. Br. J. Pharmacol. 132, 1789–1798. Gill, C.H., Soffin, E.M., Hagan, J.J., Davies, C.H., 2002. 5-HT7 receptors modulate synchronized network activity in rat hippocampus. Neuropharmacology 42, 82–92. Godínez-Chaparro, B., López-Santillán, F.J., Orduña, P., GranadosSoto, V., 2012. Secondary mechanical allodynia and hyperalgesia depend on descending facilitation mediated by spinal 5-HT4, 5-HT6 and 5-HT7 receptors. Neuroscience 222, 379–391. Goaillard, J.M., Vincent, P., 2002. Serotonin suppresses the slow afterhyperpolarization in rat intralaminar and midline thalamic neurones by activating 5-HT7 receptors. J. Physiol. 541, 453–465. Grudt, T.J., Williams, J.T., Travagli, R.A., 1995. Inhibition by 5hydroxytryptamine and noradrenaline in substantia gelatinosa of guinea-pig spinal trigeminal nucleus. J. Physiol. 485, 113–120. Hagan, J.J., Price, G.W., Jeffrey, P., Deeks, N.J., Stean, T., Piper, D., Smith, M.I., Upton, N., Medhurst, A.D., Middlemiss, D.N., Riley, G.J., Lovell, P.J., Bromidge, S.M., Thomas, D.R., 2000. Characterization of SB-269970-A, a selective 5-HT7 receptor antagonist. Br. J. Pharmacol. 130, 539–548. Hathway, G.J., Koch, S., Low, L., Fitzgerald, M., 2009. The changing balance of brainstem–spinal cord modulation of pain processing over the first weeks of rat postnatal life. J. Physiol. 587, 2927–2935. Hill, E.L., Gallopin, T., Férézou, I., Cauli, B., Rossier, J., Schweitzer, P., Lambolez, B., 2007. Functional CB1 receptors are broadly expressed in neocortical GABAergic and glutamatergic neurons. J. Neurophysiol. 97, 2580–2589. Hoyer, D., Hannon, J.P., Martin, G.R., 2002. Molecular, pharmacological and functional diversity of 5-HT receptors. Pharmacol. Biochem. Behav. 71, 533–554. Ito, A., Kumamoto, E., Takeda, M., Shibata, K., Sagai, H., Yoshimura, M., 2000. Mechanisms for ovariectomy-induced hyperalgesia and its relief by calcitonin: participation of 5-HT1A-like receptor on C-afferent terminals in substantia gelatinosa of the rat spinal cord. J. Neurosci. 20, 6302–6308. Jeong, C.Y., Choi, J.I., Yoon, M.H., 2004. Roles of serotonin receptor subtypes for the antinociception of 5-HT in the spinal cord of rats. Eur. J. Pharmacol. 502, 205–211. Lei, J., Jin, L., Zhao, Y., Sui, M.Y., Huang, L., Tan, Y.X., Chen, Y.K., You, H.J., 2011. Sex-related differences in descending norepinephrine and serotonin controls of spinal withdrawal reflex during intramuscular saline induced muscle nociception in rats. Exp. Neurol. 228, 206–214. Li, J.L., Kaneko, T., Nomura, S., Li, Y.Q., Mizuno, N., 1997. Association of serotonin-like immunoreactive axons with nociceptive projection neurons in the caudal spinal trigeminal nucleus of the rat. J. Comp. Neurol. 384, 127–141. Lovell, P.J., Bromidge, S.M., Dabbs, S., Duckworth, D.M., Forbes, I.T., Jennings, A.J., King, F.D., Middlemiss, D.N., Rahman, S.K., Saunders, D.V., Collin, L.L., Hagan, J.J., Riley, G.J., Thomas, D.R., 2000. A novel, potent, and selective 5-HT7 antagonist: (R)-3-(2-(2-(4-methylpiperidin-1-yl)ethyl) pyrrolidine-1-sulfonyl) phenol (SB-269970). J. Med. Chem. 43, 342–345.

81

Meuser, T., Pietruck, C., Gabriel, A., Xie, G., Lim, K., Pierce, P.P., 2002. 5-HT7 receptors are involved in mediating 5-HT-induced activation of rat primary afferent neurons. Life Sci. 71, 2279–2289. Millan, M.J., 2002. Descending control of pain. Prog. Neurobiol. 66, 355–474. Muraki, Y., Yamanaka, A., Tsujino, N., Kilduff, T.S., Goto, K., Sakurai, T., 2004. Serotonergic regulation of the orexin/ hypocretin neurons through the 5-HT1A receptor. J. Neurosci. 24, 7159–7166. Niblock, M.M., Kinney, H.C., Luce, C.J., Belliveau, R.A., Filiano, J.J., 2004. The development of the medullary serotonergic system in the piglet. Auton. Neurosci. 110, 65–80. Okamoto, K., Kimura, A., Donishi, T., Imbe, H., Senba, E., Tamai, Y., 2005. Central serotonin 3 receptors play an important role in the modulation of nociceptive neural activity of trigeminal subnucleus caudalis and nocifensive orofacial behavior in rats with persistent temporomandibular joint inflammation. Neuroscience 135, 569–581. Okamoto, K., Imbe, H., Kimura, A., Donishi, T., Tamai, Y., Senba, E., 2007. Activation of central 5HT2A receptors reduces the craniofacial nociception of rats. Neuroscience 147, 1090–1102. Park, S.A., Yang, E.J., Han, S.K., Park, S.J., 2012. Age-related changes in the effects of 5-hydroxytryptamine on substantia gelatinosa neurons of the trigeminal subnucleus caudalis. Neurosci. Lett. 510, 78–81. Penington, N.J., Kelly, J.S., Fox, A.P., 1993. Whole-cell recordings of inwardly rectifying Kþ currents activated by 5-HT1A receptors on dorsal raphe neurones of the adult rat. J. Physiol. 469, 387–405. Renner, U., Zeug, A., Woehler, A., Niebert, M., Dityatev, A., Dityateva, G., Gorinski, N., Guseva, D., Abdel-Galil, D., Fröhlich, M., Döring, F., Wischmeyer, E., Richter, D.W., Neher, E., Ponimaskin, E.G., 2012. Heterodimerization of serotonin receptors 5-HT1A and 5-HT7 differentially regulates receptor signalling and trafficking. J. Cell Sci. 125, 2486–2499. Rocha-González, H.I., Meneses, A., Carlton, S.M., Granados-Soto, V., 2005. Pronociceptive role of peripheral and spinal 5-HT7 receptors in the formalin test. Pain 117, 182–192. Ruat, M., Traiffort, E., Leurs, R., Tardivel-Lacombe, J., Diaz, J., Arrang, J.M., Schwartz, J.C., 1993. Molecular cloning, characterization, and localization of a high-affinity serotonin receptor (5-HT7) activating cAMP formation. Proc. Natl. Acad. Sci. USA 90, 8547–8551. Seo, K., Fujiwara, N., Hu, J.W., Cairns, B.E., Someya, G., 2002. Intrathecal administration of 5-HT3 receptor agonist modulates jaw muscle activity evoked by injection of mustard oil into the temporomandibular joint in the rat. Brain Res. 934, 157–161. Sessle, B.J., 2000. Acute and chronic craniofacial pain: brainstem mechanisms of nociceptive transmission and neuroplasticity, and their clinical correlates. Crit. Rev. Oral Biol. Med. 11, 57–91. Seyrek, M., Kahraman, S., Deveci, M.S., Yesilyurt, O., Dogrul, A., 2010. Systemic cannabinoids produce CB1-mediated antinociception by activation of descending serotonergic pathways that act upon spinal 5-HT7 and 5-HT2A receptors. Eur. J. Pharmacol. 649, 183–194. Shimizu, M., Nishida, A., Zensho, H., Miyata, M., Yamawaki, S., 1998. Agonist-induced desensitization of adenylyl cyclase activity mediated by 5-hydroxytryptamine7 receptors in rat frontocortical astrocytes. Brain Res. 784, 57–62. Todd, A.J., 2010. Neuronal circuitry for pain processing in the dorsal horn. Nat. Rev. Neurosci. 11, 823–836. Tokarski, K., Zelek-Molik, A., Duszyńska, B., Satała, G., Bobula, B., Kusek, M., Chmielarz, P., Nalepa, I., Hess, G., 2012. Acute and repeated treatment with the 5-HT7 receptor antagonist SB 269970 induces functional desensitization of 5-HT7 receptors in rat hippocampus. Pharmacol. Rep. 64, 256–265.

82

brain research 1543 (2014) 73–82

Vanhoenacker, P., Haegeman, G., Leysen, J.E., 2000. 5-HT7 receptors: current knowledge and future prospects. Trends Pharmacol Sci. 21, 70–77. Viguier, F., Michot, B., Kayser, V., Bernard, J.F., Vela, J.M., Hamon, M., Bourgoin, S., 2012. GABA, but not opioids, mediates the antihyperalgesic effects of 5-HT7 receptor activation in rats suffering from neuropathic pain. Neuropharmacology 63, 1093–1106. Yanarates, O., Dogrul, A., Yildirim, V., Sahin, A., Sizlan, A., Seyrek, M., Akgül, O., Kozak, O., Kurt, E., Aypar, U., 2010. Spinal 5-HT7 receptors play an important role in the antinociceptive and antihyperalgesic effects of tramadol and its metabolite, Odesmethyltramadol, via activation of descending serotonergic pathways. Anesthesiology 112, 696–710.

Yasaka, T., Tiong, S.Y., Hughes, D.I., Riddell, J.S., Todd, A.J., 2010. Populations of inhibitory and excitatory interneurons in lamina II of the adult rat spinal dorsal horn revealed by a combined electrophysiological and anatomical approach. Pain 151, 475–488. Yin, H., Park, S.A., Han, S.K., Park, S.J., 2011. Effects of 5hydroxytryptamine on substantia gelatinosa neurons of the trigeminal subnucleus caudalis in immature mice. Brain Res. 1368, 91–101. Zeilhofer, H.U., Wildner, H., Yévenes, G.E., 2012. Fast synaptic inhibition in spinal sensory processing and pain control. Physiol. Rev. 92, 193–235.