Skip to content

Advertisement

You're viewing the new version of our site. Please leave us feedback.

Learn more

Molecular Pain

Open Access

Serotonin receptors are involved in the spinal mediation of descending facilitation of surgical incision-induced increase of Fos-like immunoreactivity in rats

  • João Walter S Silveira1,
  • Quintino M Dias1,
  • Elaine A Del Bel2 and
  • Wiliam A Prado1Email author
Molecular Pain20106:17

https://doi.org/10.1186/1744-8069-6-17

Received: 14 January 2010

Accepted: 23 March 2010

Published: 23 March 2010

Abstract

Background

Descending pronociceptive pathways may be implicated in states of persistent pain. Paw skin incision is a well-established postoperative pain model that causes behavioral nociceptive responses and enhanced excitability of spinal dorsal horn neurons. The number of spinal c-Fos positive neurons of rats treated intrathecally with serotonin, noradrenaline or acetylcholine antagonists where evaluated to study the descending pathways activated by a surgical paw incision.

Results

The number of c-Fos positive neurons in laminae I/II ipsilateral, lamina V bilateral to the incised paw, and in lamina X significantly increased after the incision. These changes: remained unchanged in phenoxybenzamine-treated rats; were increased in the contralateral lamina V of atropine-treated rats; were inhibited in the ipsilateral lamina I/II by 5-HT1/2B/2C (methysergide), 5-HT2A (ketanserin) or 5-HT1/2A/2C/5/6/7 (methiothepin) receptors antagonists, in the ipsilateral lamina V by methysergide or methiothepin, in the contralateral lamina V by all the serotonergic antagonists and in the lamina X by LY 278,584, ketanserin or methiothepin.

Conclusions

We conclude: (1) muscarinic cholinergic mechanisms reduce incision-induced response of spinal neurons inputs from the contralateral paw; (2) 5-HT1/2A/2C/3 receptors-mediate mechanisms increase the activity of descending pathways that facilitates the response of spinal neurons to noxious inputs from the contralateral paw; (3) 5-HT1/2A/2C and 5-HT1/2C receptors increases the descending facilitation mechanisms induced by incision in the ipsilateral paw; (4) 5-HT2A/3 receptors contribute to descending pronociceptive pathways conveyed by lamina X spinal neurons; (5) α-adrenergic receptors are unlikely to participate in the incision-induced facilitation of the spinal neurons.

Background

Bulbospinal pathways descend to the spinal cord to either inhibit (antinociceptive) or facilitate (pronociceptive) the transmission of nociceptive inputs (for review see [1, 2]). The contribution of supraspinal areas in the control of descending pronociceptive pathways was confirmed by several studies. As examples, the lesion or neural block of rostral ventromedial medulla (RVM) or periaqueductal gray (PAG) reduces the hyperalgesia induced by spinal nerve ligature [3, 4], or intraplantar injection of formalin [5, 6] or mustard oil [7]. Furthermore, low intensity electrical stimulation of, or low dose of glutamate into the RVM facilitates the response of spinal nociceptive neurons to noxious inputs, whereas high intensity electrical stimulation or high dose of glutamate produces the opposite effect [8].

Descending pronociceptive pathways may be implicated in states of persistent pain [9, 10] and elucidation of their spinal mediation may be useful for discovery of new antihyperalgesic drugs. Spinal serotonin produces antinociception but may be pronociceptive as well (for review see [11]). Also, spinal activation of α2-adrenergic receptors is antinociceptive whereas activation of α1-adrenergic receptors is pronociceptive [12, 13]. A spinal muscarinic cholinergic mechanism activated by descending noradrenergic inputs has also been proposed and it seems to be linked only with antinociception (for review see [14]).

Surgical incision of a rat paw causes primary and secondary punctate hyperalgesia [15] and increases the number of c-Fos positive neurons in the spinal cord [16], an immunohistochemical method that allows the identification of neurons activated by peripheral noxious stimulation [17]. Although being a poorly understood problem, very little effort has been dedicated toward research on the spinal mediation of descending mechanisms of post-incision pain, a model that may allow us to understand mechanisms of sensitization caused by surgery and investigate new therapies for postoperative pain.

The present study was therefore undertaken to examine the changes in the number of c-Fos positive neurons in the spinal cord of rats treated intrathecally with antagonists of serotonin, noradrenaline or acetylcholine, to evaluate whether they contribute in the spinal mediation of descending pronociceptive pathways activated by a surgical incision. The laminae I/II, V and X were systematically examined, since they are predominantly implicated in the reception, processing and rostral transmission of nociceptive information [11].

Results

Effects of intrathecal muscarinic cholinergic, α-adrenergic and serotonergic receptor antagonists on the number of Fos-immunoreactive neurons in the laminae I/II, and V of the rat spinal cord

The number of Fos-LI neurons was very low bilaterally in laminae I/II (Figure 1) and V (Figure 2) of non-incised and non-catheterized anesthetized rats (group A), and was slightly and non-significantly increased in non-incised anesthetized rats treated intrathecally with saline (group AS). The number of positive neurons was greater bilaterally in laminae I/II and V of incised rats treated intrathecally with saline (group ASI), the effect being significant at the ipsilateral laminae I/II and bilateral lamina V.
Figure 1

Effects of intrathecal atropine, phenoxybenzamine or methysergide in the incision-induced Fos-like immunoreactivity in lamina I/II. The experiments utilized 4-8 non-incised and non-catheterized anesthetized rats (A), non-incised and catheterized rats treated intrathecally with 5 μl of saline (AS), incised and catheterized rats treated intrathecally with 5 μl of saline (ASI), 30 μg/5 μl of atropine (ATR), 20 μg/5 μl of phenoxybenzamine (PBZ), or 30 μg/5 μl methysergide (MET). Surgical incision of the right hind paw was performed 3 h after PBZ or 15 min after the remaining antagonists. The number of Fos-like immunoreactive (Fos-LI) cells/0.2 mm2 are shown for lamina I/II of ipsilateral (a) or contralateral (b) spinal dorsal horn. Bars are mean ± S.E.M. of the number of Fos-LI cells/0.2 mm2 found in three sections taken from each rat. P < 0.05 compared to ASI (*) or AS (#).

Figure 2

Effects of intrathecal atropine, phenoxybenzamine or methysergide in the incision-induced Fos-like immunoreactivity in lamina V. The experiments utilized 4-8 non-incised and non-catheterized anesthetized rats (A), non-incised and catheterized rats treated intrathecally with 5 μl of saline (AS), incised and catheterized rats treated intrathecally with 5 μl of saline (ASI), 30 μg/5 μl of atropine (ATR), 20 μg/5 μl of phenoxybenzamine (PBZ), or 30 μg/5 μl methysergide (MET). Surgical incision of the right hind paw was performed 3 h after PBZ or 15 min after the remaining antagonists. The number of Fos-like immunoreactive (Fos-LI) cells/0.2 mm2 are shown for lamina V of ipsilateral (a) or contralateral (b) spinal dorsal horn. Bars are mean ± S.E.M. of the number of Fos-LI cells/0.2 mm2 found in three sections taken from each rat. P < 0.05 compared to A (+), ASI (*) or AS (#).

The incision-induced increase in the number of Fos-LI neurons in the ipsilateral laminae I/II of rats from the group ASI was significantly less intense following methysergide (30 μg/5 μl), and was not changed significantly by atropine (30 μg/5 μl) or phenoxybenzamine (20 μg/5 μl) (ANOVA: F5,26 = 24.64; P < 0.0001) (Figure 1a). Similar results were found in the contralateral laminae I/II, but the differences were not significant (ANOVA: F5,26 = 1.74; P > 0.05) (Figure 1b).

The incision-induced increase in the number of Fos-LI neurons in the ipsilateral lamina V of rats from the group ASI was less intense after methysergide (30 μg/5 μl), and was not changed by phenoxybenzamine (20 μg/5 μl) or atropine (30 μg/5 μl) (ANOVA: F5,26 = 9.94; P < 0.0001) (Figure 2a). Similar, but less intense effect occurred in the contralateral lamina V of rats treated with methysergide (30 μg/5 μl) or phenoxybenzamine (20 μg/5 μl) as compared with rats from the group ASI, but it was significantly more intense in the contralateral lamina V of rats treated with atropine (30 μg/5 μl) (ANOVA: F5,26 = 9.68; P < 0.0001) (Figure 2b).

Representative photomicrographs taken from sections of the spinal cords of control and test rats are given in Figure 3a and 3b for superficial and deep laminae, respectively.
Figure 3

Photomicrographs showing Fos-like immunoreactivity in rat spinal cord. Photomicrographs were taken from 40- μm thick sections and illustrate the expression of Fos-like immunoreactivity in laminae I-II (a), V (b), and X (c) of the spinal cord gray matter (L2/L3 level), 2 h after the plantar incision of the right hind paw. of non-catheterized anesthetized rats (A), non-incised and catheterized rats treated. intrathecally with saline (AS), and incised and catheterized rats treated intrathecally with saline (ASI), atropine (ATR = 30 μg/5 μl), phenoxybenzamine (PBZ = 20 μg/5. μl), methysergide (MET = 30 μg/5 μl), LY 278,584 (LY = 100 μg/5 μl), ketanserin. (KET = 30 μg/5 μl), or methiothepin (MEP = 1.5 μg/5 μl).

Effects of intrathecal muscarinic cholinergic, α-adrenergic and serotonergic receptor antagonists on the number of Fos-immunoreactive neurons in the lamina X of the rat spinal cord

The number of Fos-LI neurons in lamina X was very small in rats from group A and non-significantly higher in rats from group AS (Figure 4). The hind paw incision also induced a significant increase in the number of positive cells in lamina X, as compared to rats from group AS (ANOVA: F5,24 = 7.06; P < 0.0003). The hind paw incision-induced increase in the number of Fos-LI neurons in lamina X was slightly reduced by methysergide (30 μg/5 μl), and slightly increased by atropine (30 μg/5 μl) or phenoxybenzamine (20 μg/5 μl), but all changes occurred in a non significant manner. Representative photomicrographs taken from sections of the spinal cords of control and test rats are given in Figure 3c.
Figure 4

Effects of intrathecal atropine, phenoxybenzamine or methysergide in the incision-induced Fos-like immunoreactivity in lamina X. The experiments utilized 4-8 non-incised and non-catheterized anesthetized rats (A), non-incised and catheterized rats treated intrathecally with 5 μl of saline (AS), incised and catheterized rats treated intrathecally with 5 μl of saline (ASI), 30 μg/5 μl of atropine (ATR), 20 μg/5 μl of phenoxybenzamine (PBZ), or 30 μg/5 μl methysergide (MET). Surgical incision of the right hind paw was performed 3 h after PBZ or 15 min after the remaining antagonists. The number of Fos-like immunoreactive (Fos-LI) cells/0.2 mm2 are shown for lamina X. Bars are mean ± S.E.M. of the number of Fos-LI cells/0.2 mm2 found in three sections taken from each rat. P < 0.05 compared to AS (#).

Effects of intrathecal antagonists of 5-HT receptor subtypes on the number of Fos-immunoreactive neurons in the laminae I, II, and V of the rat spinal cord

The number of Fos-LI neurons was very low in laminae I/II (Figure 5) and V (Figure 6) of rats from group A, and was slightly but non-significantly increased in rats from group AS. The number of positive neurons was higher bilaterally in laminae I/II and V of incised rats treated with saline (group ASI), the effect being significant at the ipsilateral laminae I/II and bilateral lamina V.
Figure 5

Effects of intrathecal LY278,584, ketanserin or methiothepin in the incision-induced Fos-like immunoreactivity in lamina I/II. The experiments utilized 4-8 non-incised and non-catheterized anesthetized rats (A), non-incised and catheterized rats treated intrathecally with 5 μl of saline (AS), incised and catheterized rats treated intrathecally with 5 μl of saline (ASI), 100 μg/5 μl of LY 278,584 (LY), 30 μg/5 μl of ketanserin (KET) or 1,5 μg/5 μl of methiothepin (MEP). Surgical incision of the right hind paw was performed 15 min after each antagonist. The number of Fos-like immunoreactive (Fos-LI) cells/0.2 mm2 are shown for lamina I/II of ipsilateral (a) or contralateral (b) spinal dorsal horn. Bars are mean ± S.E.M. of the number of Fos-LI cells/0.2 mm2 found in three sections taken from each rat. P < 0.05 compared to ASI (*) or AS (#).

Figure 6

Effects of intrathecal LY278,584, ketanserin or methiothepin in the incision-induced Fos-like immunoreactivity in lamina V. The experiments utilized 4-8 non-incised and non-catheterized anesthetized rats (A), non-incised and catheterized rats treated intrathecally with 5 μl of saline (AS), incised and catheterized rats treated intrathecally with 5 μl of saline (ASI), 100 μg/5 μl of LY 278,584 (LY), 30 μg/5 μl of ketanserin (KET) or 1,5 μg/5 μl of methiothepin (MEP). Surgical incision of the right hind paw was performed 15 min after each antagonist. The number of Fos-like immunoreactive (Fos-LI) cells/0.2 mm2 is shown for lamina V of ipsilateral (a) or contralateral (b) spinal dorsal horn. Bars are mean ± S.E.M. of the number of Fos-LI cells/0.2 mm2 found in three sections taken from each rat. P < 0.05 compared to ASI (*) or AS (#).

The incision-induced increase in the number of Fos-LI neurons in the ipsilateral laminae I/II was significantly reduced by methiothepin (1,5 μg/5 μl) or ketanserin (30 μg/5 μl), but was not changed by LY 278,584 (100 μg/5 μl) (ANOVA: F5,30 = 24.77; P < 0.0001) (Figure 5a). However, no significant difference was demonstrated in the contralateral laminae I and II among the experimental groups (ANOVA: F5,30 = 2.02; P > 0.05) (Figure 5b).

The incision-induced increase in the number of Fos-LI neurons in the ipsilateral lamina V was also reduced by methiothepin (1,5 μg/5 μl), ketanserin (30 μg/5 μl) or LY 278,584 (100 μg/5 μl), but only the effect of methiothepin was significant (ANOVA: F5,26 = 9.94; P < 0.0001) (Figure 6a). In contrast, ketanserin, LY 278,584 and mainly methiothepin all reduced significantly the incision-induced increase in the number of positive cells in the contralateral lamina V (ANOVA: F5,30 = 7.27; P = 0.0001) (Figure 6b).

Representative photomicrographs taken from sections of the spinal cords of control and test rats are given in Figure 3a and 3b for superficial and deep laminae, respectively.

Effects of intrathecal antagonists of 5-HT receptor subtypes on the number of Fos-immunoreactive neurons in the lamina X of the rat spinal cord

The number of Fos-LI neurons in lamina X was very small in rats from group A and was non-significantly superior in rats from group AS (Figure 7). The hind paw incision also induced a significant increase in the number of positive cells in lamina X, as compared to rats from group AS, the effect being significantly reduced by LY 278,584 (100 μg/5 μl), ketanserin (30 μg/5 μl) or methiothepin (1,5 μg/5 μl) (ANOVA: F5,28 = 9.26; P < 0.0001). Representative photomicrographs taken from sections of the spinal cords of control and test rats are given in Figure 3c.
Figure 7

Effects of intrathecal LY278,584, ketanserin or methiothepin in the incision-induced Fos-like immunoreactivity in lamina X. The experiments utilized 4-8 non-incised and non-catheterized anesthetized rats (A), non-incised and catheterized rats treated intrathecally with 5 μl of saline (AS), incised and catheterized rats treated intrathecally with 5 μl of saline (ASI), 100 μg/5 μl of LY 278,584 (LY), 30 μg/5 μl of ketanserin (KET) or 1,5 μg/5 μl of methiothepin (MEP). The number of Fos-like immunoreactive (Fos-LI) cells/0.2 mm2 are shown for lamina X. Surgical incision of the right hind paw was performed 15 min after each antagonist. Bars are mean ± S.E.M. of the number of Fos-LI cells/0.2 mm2 found in three sections taken from each rat. P < 0.05 compared to ASI (*) or AS (#).

Discussion

Fos protein is a product of the proto-oncogene c-Fos, which is expressed in the neuronal nuclei few hours after an appropriate stimulus [18]. The increase in the expression of Fos-LI neurons in spinal cord has been used as a marker of the neuronal activity induced by noxious stimuli in various pain models [17]; for review see [19, 20]. Although being a poorly understood problem, very little effort has been dedicated toward research on the spinal mediation of descending mechanisms of post-incision pain, a model that may allow us to understand mechanisms of sensitization caused by surgery and investigate new therapies for postoperative pain.

The present study confirmed that a surgical incision in a rat hind paw increased significantly the number of Fos-LI neurons in the spinal dorsal horn laminae I/II ipsilateral, and lamina V bilateral to the incised paw as shown elsewhere [16, 21, 22].

Also, a significant increase in the number of Fos-LI neurons were also found in the lamina X of the spinal gray matter following the incision, as described elsewhere subsequent to peripheral or visceral nociceptive inputs [23].

Incision of a hind paw increases both the response of spinal wide dynamic range cells to mechanical stimulation [15] and the spontaneous activity of nociceptive primary afferents [24]. The model of incision pain differs from inflammatory pain models [25, 26], but the tissue reactions to incision are likely to involve some inflammatory responses to local injury [27].

Evidence that descending influences can affect spinal phenomena induced by a paw incision includes the demonstration that incision pain is further increased in rats with lesion of dorsolateral funiculus [28], that conveys descending pain inhibitory pathways [11], or electrolytic lesion of the anterior pretectal nucleus [29], that participates in the activation of descending mechanisms of pain control [30]; in the ipsilateral spinal cord, the incision-induced increase in the number of Fos-LI neurons was significantly reduced in the superficial lamina and significantly increased in the deep lamina of animals previously treated with bupivacaine in the contralateral anterior pretectal nucleus [16]; finally, descending pathways are known as the only sources of serotonin in the spinal cord [2, 11, 31].

Acetylcholine, noradrenaline and serotonin modulate noxious inputs processing in the spinal cord (for review see [11]. Atropine, a non selective muscarinic receptor antagonist was used here at 30 μg/rat, 3 fold superior to the dose known to be effective against the neostigmine-induced antinociception in rats [32].

Phenoxybenzamine, a non selective α-adrenergic receptor antagonist was used here at 20 μg/rat, 2.5 fold superior to the dose early shown to be effective against glutamate-induced analgesia from the RVM of rats [33]. Nonetheless, neither the atropine nor the phenoxybenzamine changed the incision-induced increase in the number of Fos-LI neurons in the laminae I/II, bilaterally, ipsilateral lamina V, or in lamina X. Consequently, muscarinic and α-adrenergic receptors are unlikely to participate in mechanisms that facilitate the response of spinal neurons to noxious inputs evoked by the incision. On the contrary, the increase in the number of Fos-LI neurons in the lamina V contralateral to the incised paw was significantly greater in atropine- than in saline-treated rats. In agreement to earlier studies [3436], we interpret the result as indirect evidence that a muscarinic cholinergic mechanism responds to a surgical incision reducing the response of spinal neurons to noxious inputs from the contralateral paw. A possibility remains that the depression of c-Fos labeling is due to toxic effects of the antagonists used in the study. However, as far as we know, there is no report showing that at the doses used in the study, the intrathecal antagonists induces toxic effects against spinal dorsal horn cells in rats.

Among the seven classes of serotonin receptors (5-HT1-7) currently known (for review see [37]), at least three (5-HT1/2/3/) were found in the spinal cord [38, 39] and are implicated in spinal pain processing [4043]. The antinociceptive effect of 5-HT may also occur via interaction with spinal 5-HT4 receptors [44, 45], but experiments involving pharmacological blockade of these receptors were not conducted in this study.

The increase in the number of Fos-LI neurons in the lamina I/II ipsilateral to the incised paw was significantly inhibited by antagonists for 5-HT1/2B/2C (methysergide), 5-HT2A (ketanserin) or 5-HT1/2A/2C/5/6/7 (methiothepin) receptors, but was not changed by a 5-HT3 antagonist (LY 278,584). The same effect was also significantly inhibited in the lamina V ipsilateral to the incised paw by methysergide or methiothepin, but remained unchanged following ketanserin or LY 278,584. Therefore, 5-HT1/2A/2C receptors in laminae I/II and 5-HT1/2Creceptors in lamina V, but not 5-HT3 receptors in either laminae I/II or V, are involved in the activity of a descending pathway that facilitates the response of spinal neurons to noxious inputs induced by a surgical incision in the ipsilateral paw.

The significant increase in the number of Fos-LI neurons in the contralateral lamina V was inhibited by all the serotonergic antagonists used in the study. Thus, 5-HT1/2A/2C/3 receptors contribute to the effect of serotonin in the activity of a descending pathway that facilitates the response of spinal neurons to noxious inputs induced by a surgical incision in the contralateral paw. Finally, the increase in the number of Fos-LI neurons in lamina X was significantly inhibited by LY 278,584, ketanserin or methiothepin, but not by methysergide. We then conclude that 5-HT2A/3 receptors contribute to the effects of serotonin in a descending pronociceptive pathway conveyed by lamina X spinal neurons.

Altogether, these results support the involvement of serotonin in descending mechanisms that facilitate the response of spinal neurons to nociceptive inputs in the spinal cord. Serotonergic nerve terminals found in the spinal cord originate from supraspinal sources [2, 11, 31] and, therefore, descending pathways utilizing serotonin somehow excite nociceptive cells in the spinal cord while a postoperative pain is in course. Bulbospinal influences from the RVM contribute to facilitation of noxious inputs and development of secondary hyperalgesia in persistent inflammatory, neuropathic, and visceral pain models[46]. Primary and secondary hyperalgesia observed after a surgical incision do not appear to be modulated by descending influences from the RVM, thus supporting the view that incision pain involves different mechanisms compared with inflammatory and neuropathic pain[47]. However, in this study Pogatzki et al., utilized rats 5 days after RVM lesion and, therefore, it is possible that a structure located more rostrally assumes the control of the primary and secondary hyperalgesia observed after a surgical incision. In fact, several other studies have shown the involvement of descending pain pathways in this model [29, 30, 4851].

The spinal actions of serotonin has long been associated to suppression of the responses to nociceptive inputs [5254], but evidence has accumulated questioning whether descending serotonergic pathways play an exclusive spinal suppressive effect against nociceptive inputs [53, 55]. The activation of spinal serotonin receptors has been associated with both pronociceptive and antinociceptive effects depending on algesimetric test, drug dosage, duration of the treatment and pathophysiological condition [5658].

The spinal dorsal horn contains high concentrations of 5-HT1A, and 5-HT1B receptors [39, 59], but the occurrence of 5-HT1D receptors is possible [60, 61]. The activation of spinal 5-HT1A may result analgesia [62, 63] (see also [64]), or hyperalgesia [65, 66], while the activation of spinal 5-HT1B receptors produces antinociception [67, 68].

The presence of spinal 5-HT2A [69, 70], and 5-HT2C receptors [71, 72] has already been demonstrated. Stimulation of spinal 5-HT2A receptors is pronociceptive [53, 73], but the presence of 5-HT2A receptors on spinal inhibitory interneurons supports an antinociceptive role for 5-HT [74, 75]. The activation of spinal 5-HT2C receptors excites neurons [55, 76, 77], and its distribution in the spinal cord is compatible with a pronociceptive role of 5-HT in the dorsal horn [12, 78]. Conversely, 5-HT2C sites on spinal inhibitory interneurons allow a potential antinociceptive role for serotonin [79].

Studies have demonstrated that 5-HT3 receptors are concentrated in superficial layers of the dorsal horn [59, 80] and a significant proportion is located on the terminals of C fibres [12, 81]. The activation of the 5-HT3 receptors depolarizes neurons in dorsal root ganglions and, therefore, is expected to increase the transmitter release from the primary afferent terminals into the spinal cord (see [64]). In line with the present results, the activation of spinal 5-HT3 receptors increases nociception [62, 82, 83], and blockade of spinal 5-HT3 receptor reduces the hypersensitivity of spinal dorsal horn neurons of nerve-ligated rats [84]. A pronociceptive role for 5-HT3 receptors in the spinal cord following activation of descending pronociceptive pathways has already been proposed [84, 85].

It is also noteworthy, that a direct facilitation by serotonin of glutamatergic synapses has been demonstrated in the spinal cord [86, 87] and, therefore, this mechanism may account for some of the excitatory effects of serotonin found here.

There is evidence that a peripheral damage simultaneously triggers both descending inhibition and facilitation onto both primary and secondary spinal hyperalgesic mechanisms, but the balance for primary hyperalgesia is different from the balance for secondary hyperalgesia (for review see [48]).

Behavioral nociceptive responses of our animals were not evaluated during this study but it may be assumed that the increased number of Fos-LI neurons in the dorsal horn ipsilateral to the incised paw essentially reflects primary hyperalgesia. By extension, a secondary hyperalgesia evoked by central sensitization may be assumed to occur when the effect was observed in the contralateral dorsal horn (see [88]). Therefore, our results show that incision-induced primary and secondary hyperalgesias seem to be spinally mediated by 5-HT1/2A/2C and 5-HT1/2A/2C/3 receptors, respectively.

Few studies have accessed which neurotransmitters are involved in the spinal processing of hyperalgesia in the mode of post-incision pain. They have shown that noradrenergic receptors are involved in the spinal mediation of descending inhibitory pathways in the primary hyperalgesia of postoperative pain [29, 49, 51]. Few reports are also available regarding secondary hyperalgesia after post-incision pain [47, 8994] but, as far we know, none of them have studied spinal neurotransmitters involved in descending mechanisms.

Conclusions

In conclusion (Table 1), 5-HT1/2A/2C receptors in laminae I/II and 5-HT1/2C receptors in lamina V contribute to the effects of serotonin in descending pathway that facilitates the response of spinal neurons to noxious inputs from the ipsilateral paw (primary hyperalgesia?); 5-HT1/2A/2C/3 receptors mediate the effects of a descending pathway that facilitates the response of spinal neurons to noxious inputs from the contralateral paw (secondary hyperalgesia?); and 5-HT2A/3 receptors contribute to the effects of serotonin in a descending pronociceptive pathway conveyed to lamina X spinal neurons. Finally, the study confirms that spinal muscarinic cholinergic mechanism responds to a surgical incision reducing the response of spinal neurons to noxious inputs from the contralateral paw.
Table 1

Effects of antagonists in the incision-induced increase of Fos-like immunoreactivity in the rat spinal cord

Antagonists

LI/LII

LV

LX

 

ipsi

contra

ipsi

contra

 

Atropine (muscarinic)

-

-

-

↑*

-

Phenoxybenzamine (α-adrenergic)

-

-

-

-

-

Methysergide (5-HT1/2B/2C)

↓*

-

↓#

↓#

↓#

LY 278,584 (5-HT3)

-

-

-

↓*

↓*

Ketanserin (5-HT2A)

↓*

-

-

↓*

↓*

Methiothepin (5-HT1/2A/2C/5/6/7)

↓*

-

↓*

↓*

↓*

Antagonists were injected intrathecally. Abbreviations: ipsi = ipsilateral and contra = contralateral to the incised hind paw; ↓ = decrease and ↑ = increase of the effect produced by the incision on the Fos-like immunoreactivity. (*) Significantly different from intrathecal saline-treated incised rats. (#) Abolished the significance of the incision-induced increase of Fos-like immunoreactivity but did not differ significantly from intrathecal saline-treated incised rats.

Methods

Subjects

Male Wistar rats (200-250 g) were used in this study. Animals were housed two to a cage under controlled temperature (22 ± 1°C) and on a 12-h light-dark cycle, with the dark cycle beginning at 07:00 h, and had free access to food and water. The experiments were approved by the Commission of Ethics in Animal Research, Faculty of Medicine of Ribeirão Preto, University of São Paulo (Number 009/2004). The guidelines of the Committee for Research and Ethical Issues of IASP [95] were followed throughout the experiments.

Surgery

Each rat was anesthetized with halothane via a loose-fitting, cone-shaped mask, and catheterization of the spinal subarachnoid space was performed as described elsewhere [96]. Briefly, a 20-gauge Weiss needle was introduced through the skin into the L5-L6 intervertebral space. The correct positioning of the needle was assured by a typical flick of the tail or hind paw. A 12-mm length of polyethylene tubing (PE tubing, o.d. = 0.4 mm, dead space = 10 μl) was then introduced through the needle to protrude 2.0 cm into the subarachnoid space in a cranial direction. The needle was then carefully removed and the tubing anchored to the back skin with a cotton thread suture. Drug or saline was injected intrathecally soon after the catheterization in a volume of 5 μl over a period of 60 s, followed by 5 μl of sterile saline at the same rate to flush the catheter. The plantar side of the right hind paw was prepared with a 10% povidone-iodine solution 15-min later. A 1-cm longitudinal incision was made with a surgical blade, through the skin and fascia of the plantar region, starting 0.5 cm from the proximal edge of the heel, as described elsewhere [97]. The plantaris muscle was elevated, but its origin and insertion were left intact. After hemostasia, the skin was apposed with one single suture of 5-0 nylon, and the animal was allowed to recover in the home cage for a period of 2 h. The positioning of the intrathecal catheter was verified when the spinal cord was removed for Fos immunohistochemistry.

Fos immunohistochemistry

The animals were sacrificed with an intraperitoneal overdose of sodium thiopental performed 2 h after the plantar incision, and perfused transcardially with saline followed by 4% paraformaldehyde in 0.1 M PBS, pH 7.4. The spinal cord was removed, fixed for 2 h in paraformaldehyde and stored for at least 48 h in 30% sucrose. The side ipsilateral to the incised paw was marked with a little knife cut. The samples were then frozen in Tissue Teck (Sakura®). Fos immunohistochemistry was processed, as described elsewhere [98], on 40 μm transverse sections obtained with a cryostat (Leica CM 1850) from L2-L3 spinal cord segments. The tissue sections were successively washed and incubated for 1 h in goat anti-rabbit biotinylated antibody (1:400 in PBS; Vector Laboratories, Burlingame, CA). They were then processed by the avidin-biotin immunoperoxidase method (Vectastain ABC kit, Vector Lab, Burlingame, CA, U.S.A.), and then Fos-like immunoreactivity (FLI) was revealed by the addition of chromogen diaminobenzidin (Sigma).

All reactions were performed at room temperature. Fos-like immunoreactivity was quantified using an image analysis system (Leica, Quantimet 500, Leica Microsystems Inc. Cambridge, UK) that identified and counted immunostained neurons according to a gray level that was empirically determined prior to analysis. The number of Fos-like immunoreactive (Fos-LI) neurons/section was calculated as the mean of the three sections examined for each rat. For analysis of the laminar distribution of Fos-LI neurons, the spinal cord gray matter was divided into three regions (laminae I-II, V, and X). Assessment of Fos-like immunoreactivity was conducted in a blind manner.

The number of Fos-LI neurons per region on both the ipsilateral and contralateral slides was counted in fixed area sizes (0.2 mm2 for spinal cord laminae I/II, V or X), using a software 9.0 image analysis system (W. Rasband, National Institute of Health). Only rats showing catheter tip positioned at the dorsal spinal cord were considered for data analysis.

Drugs

Atropine sulfate, a non selective muscarinic cholinergic antagonist, phenoxybenzamine hydrochloride, a non selective α-receptor antagonist, methysergide, a 5-HT1/2B/2C antagonist [99], ketanserin, a 5-HT2A antagonist [100], methiothepin, a 5-HT1/2A/2C/5/6/7 antagonist [101], and LY 278,584, a 5-HT3 antagonist [102, 103], were purchased from Sigma (St Louis, MO, USA) and diluted in sterile isotonic saline at the moment of the injection.

Experimental design

All animals were anesthetized and allocated to one of four experimental groups. A group for the overall control of the experiment had non-incised and non-catheterized anesthetized rats (group A), which were sacrificed 2 h after the beginning of anesthesia. A group for control of the effects of intrathecal catheterization had non-incised rats treated intrathecally with saline (5 μl) and were sacrificed 2 h later (group AS). A group for control of the effects of the hind paw incision had rats treated intrathecally with saline and submitted to the surgical incision of the right hind paw performed 15-min later (group ASI). Test groups had rats submitted to paw incision carried out 3 h after intrathecal phenoxybenzamine or 15-min after methysergide, atropine, LY 278,584, ketanserin or methiothepin. Animals from group ASI and test groups were sacrificed 2 h after the incision.

Statistics

The mean (± SEM) of the number Fos-LI cells/0.2 mm2 of 3 sections from the spinal cord of 4 - 8 animals per group was taken to allow comparisons among the different treatments. Comparisons of groups were made using one-way ANOVA followed by the Tukey's Multiple Comparison test. The level of significance was set at P < 0.05 in all cases.

Declarations

Acknowledgements

This study was supported by FAPESP. JWSS and QMD were the recipients of CNPq fellowships. We gratefully acknowledge MA Carvalho and PR Castania for skillful technical assistance. Dr A Leyva helped with English editing of the manuscript.

Authors’ Affiliations

(1)
Department of Pharmacology, Faculty of Medicine of Ribeirão Preto, University of Sao Paulo
(2)
Department of Morphology, Estomatology and Physiology, Faculty of Odontology of Ribeirão Preto, University of Sao Paulo

References

  1. Gebhart GF: Descending modulation of pain. Neurosci Biobehav Rev 2004, 27: 729–737. 10.1016/j.neubiorev.2003.11.008PubMedGoogle Scholar
  2. Fields HL, Heinricher MM, Mason P: Neurotransmitters in nociceptive modulatory circuits. Annu Rev Neurosci 1991, 14: 219–245. 10.1146/annurev.ne.14.030191.001251PubMedGoogle Scholar
  3. Kovelowski CJ, Ossipov MH, Sun H, Lai J, Malan TP, Porreca F: Supraspinal cholecystokinin may drive tonic descending facilitation mechanisms to maintain neuropathic pain in the rat. Pain 2000, 87: 265–273. 10.1016/S0304-3959(00)00290-6PubMedGoogle Scholar
  4. Pertovaara A, Wei H, Hamalainen MM: Lidocaine in the rostroventromedial medulla and the periaqueductal gray attenuates allodynia in neuropathic rats. Neurosci Lett 1996, 218: 127–130. 10.1016/S0304-3940(96)13136-0PubMedGoogle Scholar
  5. Vaccarino AL, Melzack R: Temporal processes of formalin pain: differential role of the cingulum bundle, fornix pathway and medial bulboreticular formation. Pain 1992, 49: 257–271. 10.1016/0304-3959(92)90150-APubMedGoogle Scholar
  6. Wiertelak EP, Roemer B, Maier SF, Watkins LR: Comparison of the effects of nucleus tractus solitarius and ventral medial medulla lesions on illness-induced and subcutaneous formalin-induced hyperalgesias. Brain Res 1997, 748: 143–150. 10.1016/S0006-8993(96)01289-9PubMedGoogle Scholar
  7. Pertovaara A: A neuronal correlate of secondary hyperalgesia in the rat spinal dorsal horn is submodality selective and facilitated by supraspinal influence. Exp Neurol 1998, 149: 193–202. 10.1006/exnr.1997.6688PubMedGoogle Scholar
  8. Zhuo M, Gebhart GF: Biphasic modulation of spinal nociceptive transmission from the medullary raphe nuclei in the rat. J Neurophysiol 1997, 78: 746–758.PubMedGoogle Scholar
  9. Calejesan AA, Ch'ang MH, Zhuo M: Spinal serotonergic receptors mediate facilitation of a nociceptive reflex by subcutaneous formalin injection into the hindpaw in rats. Brain Res 1998, 798: 46–54. 10.1016/S0006-8993(98)00394-1PubMedGoogle Scholar
  10. Wei F, Dubner R, Ren K: Nucleus reticularis gigantocellularis and nucleus raphe magnus in the brain stem exert opposite effects on behavioral hyperalgesia and spinal Fos protein expression after peripheral inflammation. Pain 1999, 80: 127–141. 10.1016/S0304-3959(98)00212-7PubMedGoogle Scholar
  11. Millan MJ: Descending control of pain. Prog Neurobiol 2002, 66: 355–474. 10.1016/S0301-0082(02)00009-6PubMedGoogle Scholar
  12. Millan MJ, Girardon S, Bervoets K: 8-OH-DPAT-induced spontaneous tail-flicks in the rat are facilitated by the selective serotonin (5-HT)2C agonist, RO 60–0175: blockade of its actions by the novel 5-HT2C receptor antagonist SB 206,553. Neuropharmacology 1997, 36: 743–745. 10.1016/S0028-3908(97)00071-3PubMedGoogle Scholar
  13. Stanfa LC, Dickenson AH: Enhanced alpha-2 adrenergic controls and spinal morphine potency in inflammation. Neuroreport 1994, 5: 469–472. 10.1097/00001756-199401120-00025PubMedGoogle Scholar
  14. Eisenach JC: Muscarinic-mediated analgesia. Life Sci 1999, 64: 549–554. 10.1016/S0024-3205(98)00600-6PubMedGoogle Scholar
  15. Zahn PK, Brennan TJ: Incision-induced changes in receptive field properties of rat dorsal horn neurons. Anesthesiology 1999, 91: 772–785. 10.1097/00000542-199909000-00030PubMedGoogle Scholar
  16. Villarreal CF, Del Bel EA, Prado WA: Involvement of the anterior pretectal nucleus in the control of persistent pain: a behavioral and c-Fos expression study in the rat. Pain 2003, 103: 163–174. 10.1016/S0304-3959(02)00449-9PubMedGoogle Scholar
  17. Hunt SP, Pini A, Evan G: Induction of c-fos-like protein in spinal cord neurons following sensory stimulation. Nature 1987, 328: 632–634. 10.1038/328632a0PubMedGoogle Scholar
  18. Hughes P, Dragunow M: Induction of immediate-early genes and the control of neurotransmitter-regulated gene expression within the nervous system. Pharmacol Rev 1995, 47: 133–178.PubMedGoogle Scholar
  19. Coggeshall RE: Fos, nociception and the dorsal horn. Prog Neurobiol 2005, 77: 299–352.PubMedGoogle Scholar
  20. Sandkuhler J: The organization and function of endogenous antinociceptive systems. Prog Neurobiol 1996, 50: 49–81.PubMedGoogle Scholar
  21. Sun X, Yokoyama M, Mizobuchi S, Kaku R, Nakatsuka H, Takahashi T, Morita K: The effects of pretreatment with lidocaine or bupivacaine on the spatial and temporal expression of c-Fos protein in the spinal cord caused by plantar incision in the rat. Anesth Analg 2004, 98: 1093–1098. 10.1213/01.ANE.0000104580.89717.A2PubMedGoogle Scholar
  22. Tsuda M, Koizumi S, Inoue K: Role of endogenous ATP at the incision area in a rat model of postoperative pain. Neuroreport 2001, 12: 1701–1704. 10.1097/00001756-200106130-00036PubMedGoogle Scholar
  23. Nicholas AP, Zhang X, Hokfelt T: An immunohistochemical investigation of the opioid cell column in lamina X of the male rat lumbosacral spinal cord. Neurosci Lett 1999, 270: 9–12. 10.1016/S0304-3940(99)00446-2PubMedGoogle Scholar
  24. Hamalainen MM, Gebhart GF, Brennan TJ: Acute effect of an incision on mechanosensitive afferents in the plantar rat hindpaw. J Neurophysiol 2002, 87: 712–720.PubMedGoogle Scholar
  25. Leonard PA, Arunkumar R, Brennan TJ: Bradykinin antagonists have no analgesic effect on incisional pain. Anesth Analg 2004, 99: 1166–1172. 10.1213/01.ANE.0000130348.85587.BEPubMedGoogle Scholar
  26. Pogatzki-Zahn EM, Shimizu I, Caterina M, Raja SN: Heat hyperalgesia after incision requires TRPV1 and is distinct from pure inflammatory pain. Pain 2005, 115: 296–307. 10.1016/j.pain.2005.03.010PubMedGoogle Scholar
  27. Wieseler-Frank J, Maier SF, Watkins LR: Central proinflammatory cytokines and pain enhancement. Neurosignals 2005, 14: 166–174. 10.1159/000087655PubMedGoogle Scholar
  28. Kina VA, Villarreal CF, Prado WA: The effects of intraspinal L-NOARG or SIN-1 on the control by descending pathways of incisional pain in rats. Life Sci 2005, 76: 1939–1951. 10.1016/j.lfs.2004.08.038PubMedGoogle Scholar
  29. Villarreal CF, Kina VA, Prado WA: Participation of brainstem nuclei in the pronociceptive effect of lesion or neural block of the anterior pretectal nucleus in a rat model of incisional pain. Neuropharmacology 2004, 47: 117–127. 10.1016/j.neuropharm.2004.03.002PubMedGoogle Scholar
  30. Villarreal CF, Prado WA: Modulation of persistent nociceptive inputs in the anterior pretectal nucleus of the rat. Pain 2007, 132: 42–52. 10.1016/j.pain.2007.01.021PubMedGoogle Scholar
  31. Stamford JA: Descending control of pain. Br J Anaesth 1995, 75: 217–227.PubMedGoogle Scholar
  32. Lavand'homme PM, Eisenach JC: Sex differences in cholinergic analgesia II: differing mechanisms in two models of allodynia. Anesthesiology 1999, 91: 1455–1461. 10.1097/00000542-199911000-00039PubMedGoogle Scholar
  33. Satoh M, Oku R, Akaike A: Analgesia produced by microinjection of L-glutamate into the rostral ventromedial bulbar nuclei of the rat and its inhibition by intrathecal alpha-adrenergic blocking agents. Brain Res 1983, 261: 361–364. 10.1016/0006-8993(83)90646-7PubMedGoogle Scholar
  34. Iwamoto ET, Marion L: Characterization of the antinociception produced by intrathecally administered muscarinic agonists in rats. J Pharmacol Exp Ther 1993, 266: 329–338.PubMedGoogle Scholar
  35. Li DP, Chen SR, Pan YZ, Levey AI, Pan HL: Role of presynaptic muscarinic and GABA(B) receptors in spinal glutamate release and cholinergic analgesia in rats. J Physiol 2002, 543: 807–818. 10.1113/jphysiol.2002.020644PubMed CentralPubMedGoogle Scholar
  36. Zhuo M, Gebhart GF: Tonic cholinergic inhibition of spinal mechanical transmission. Pain 1991, 46: 211–222. 10.1016/0304-3959(91)90078-CPubMedGoogle Scholar
  37. Hoyer D, Hannon JP, Martin GR: Molecular, pharmacological and functional diversity of 5-HT receptors. Pharmacol Biochem Behav 2002, 71: 533–554. 10.1016/S0091-3057(01)00746-8PubMedGoogle Scholar
  38. Hamon M, Gallissot MC, Menard F, Gozlan H, Bourgoin S, Verge D: 5-HT3 receptor binding sites are on capsaicin-sensitive fibres in the rat spinal cord. Eur J Pharmacol 1989, 164: 315–322. 10.1016/0014-2999(89)90472-XPubMedGoogle Scholar
  39. Thor KB, Nickolaus S, Helke CJ: Autoradiographic localization of 5-hydroxytryptamine1A, 5-hydroxytryptamine1B and 5-hydroxytryptamine1C/2 binding sites in the rat spinal cord. Neuroscience 1993, 55: 235–252. 10.1016/0306-4522(93)90469-VPubMedGoogle Scholar
  40. Danzebrink RM, Gebhart GF: Evidence that spinal 5-HT1, 5-HT2 and 5-HT3 receptor subtypes modulate responses to noxious colorectal distension in the rat. Brain Res 1991, 538: 64–75. 10.1016/0006-8993(91)90377-8PubMedGoogle Scholar
  41. Giordano J: Analgesic profile of centrally administered 2-methylserotonin against acute pain in rats. Eur J Pharmacol 1991, 199: 233–236. 10.1016/0014-2999(91)90462-YPubMedGoogle Scholar
  42. Radhakrishnan R, King EW, Dickman JK, Herold CA, Johnston NF, Spurgin ML, Sluka KA: Spinal 5-HT(2) and 5-HT(3) receptors mediate low, but not high, frequency TENS-induced antihyperalgesia in rats. Pain 2003, 105: 205–213. 10.1016/S0304-3959(03)00207-0PubMed CentralPubMedGoogle Scholar
  43. Zhuo M, Gebhart GF: Spinal serotonin receptors mediate descending facilitation of a nociceptive reflex from the nuclei reticularis gigantocellularis and gigantocellularis pars alpha in the rat. Brain Res 1991, 550: 35–48. 10.1016/0006-8993(91)90402-HPubMedGoogle Scholar
  44. Jeong CY, Choi JI, Yoon MH: Roles of serotonin receptor subtypes for the antinociception of 5-HT in the spinal cord of rats. Eur J Pharmacol 2004, 502: 205–211. 10.1016/j.ejphar.2004.08.048PubMedGoogle Scholar
  45. Liu FY, Xing GG, Qu XX, Xu IS, Han JS, Wan Y: Roles of 5-hydroxytryptamine (5-HT) receptor subtypes in the inhibitory effects of 5-HT on C-fiber responses of spinal wide dynamic range neurons in rats. J Pharmacol Exp Ther 2007, 321: 1046–1053. 10.1124/jpet.106.115204PubMedGoogle Scholar
  46. Urban MO, Gebhart GF: Supraspinal contributions to hyperalgesia. Proc Natl Acad Sci USA 1999, 96: 7687–7692. 10.1073/pnas.96.14.7687PubMed CentralPubMedGoogle Scholar
  47. Pogatzki EM, Urban MO, Brennan TJ, Gebhart GF: Role of the rostral medial medulla in the development of primary and secondary hyperalgesia after incision in the rat. Anesthesiology 2002, 96: 1153–1160. 10.1097/00000542-200205000-00019PubMedGoogle Scholar
  48. Vanegas H, Schaible HG: Descending control of persistent pain: inhibitory or facilitatory? Brain Res Brain Res Rev 2004, 46: 295–309. 10.1016/j.brainresrev.2004.07.004PubMedGoogle Scholar
  49. Wang Y, Feng C, Wu Z, Wu A, Yue Y: Activity of the descending noradrenergic pathway after surgery in rats. Acta Anaesthesiol Scand 2008, 52: 1336–1341. 10.1111/j.1399-6576.2007.01525.xPubMedGoogle Scholar
  50. Villarreal CF, Kina VA, Prado WA: Antinociception induced by stimulating the anterior pretectal nucleus in two models of pain in rats. Clin Exp Pharmacol Physiol 2004, 31: 608–613. 10.1111/j.1440-1681.2004.04057.xPubMedGoogle Scholar
  51. Hayashida K, DeGoes S, Curry R, Eisenach JC: Gabapentin activates spinal noradrenergic activity in rats and humans and reduces hypersensitivity after surgery. Anesthesiology 2007, 106: 557–562. 10.1097/00000542-200703000-00021PubMedGoogle Scholar
  52. Basbaum AI, Fields HL: Endogenous pain control mechanisms: review and hypothesis. Ann Neurol 1978, 4: 451–462. 10.1002/ana.410040511PubMedGoogle Scholar
  53. Eide PK, Hole K: The role of 5-hydroxytryptamine (5-HT) receptor subtypes and plasticity in the 5-HT systems in the regulation of nociceptive sensitivity. Cephalalgia 1993, 13: 75–85. 10.1046/j.1468-2982.1993.1302075.xPubMedGoogle Scholar
  54. Millan MJ: Endorphins and nociception: an overview. Methods Find Exp Clin Pharmacol 1982, 4: 445–462.PubMedGoogle Scholar
  55. Millan MJ: The induction of pain: an integrative review. Prog Neurobiol 1999, 57: 1–164. 10.1016/S0301-0082(98)00048-3PubMedGoogle Scholar
  56. Colpaert FC, Tarayre JP, Koek W, Pauwels PJ, Bardin L, Xu XJ, Wiesenfeld-Hallin Z, Cosi C, Carilla-Durand E, Assie MB, Vacher B: Large-amplitude 5-HT1A receptor activation: a new mechanism of profound, central analgesia. Neuropharmacology 2002, 43: 945–958. 10.1016/S0028-3908(02)00119-3PubMedGoogle Scholar
  57. Fasmer OB, Berge OG, Hole K: Changes in nociception after lesions of descending serotonergic pathways induced with 5,6-dihydroxytryptamine. Different effects in the formalin and tail-flick tests. Neuropharmacology 1985, 24: 729–734. 10.1016/0028-3908(85)90006-1PubMedGoogle Scholar
  58. Millan MJ, Seguin L, Honore P, Girardon S, Bervoets K: Pro- and antinociceptive actions of serotonin (5-HT)1A agonists and antagonists in rodents: relationship to algesiometric paradigm. Behav Brain Res 1996, 73: 69–77. 10.1016/0166-4328(96)00073-3PubMedGoogle Scholar
  59. Laporte AM, Doyen C, Nevo IT, Chauveau J, Hauw JJ, Hamon M: Autoradiographic mapping of serotonin 5-HT1A, 5-HT1D, 5-HT2A and 5-HT3 receptors in the aged human spinal cord. J Chem Neuroanat 1996, 11: 67–75. 10.1016/0891-0618(96)00130-5PubMedGoogle Scholar
  60. Bonaventure P, Voorn P, Luyten WH, Jurzak M, Schotte A, Leysen JE: Detailed mapping of serotonin 5-HT1B and 5-HT1D receptor messenger RNA and ligand binding sites in guinea-pig brain and trigeminal ganglion: clues for function. Neuroscience 1998, 82: 469–484. 10.1016/S0306-4522(97)00302-3PubMedGoogle Scholar
  61. Waeber C, Moskowitz MA: [3H]sumatriptan labels both 5-HT1D and 5-HT1F receptor binding sites in the guinea pig brain: an autoradiographic study. Naunyn Schmiedebergs Arch Pharmacol 1995, 352: 263–275. 10.1007/BF00168556PubMedGoogle Scholar
  62. Oyama T, Ueda M, Kuraishi Y, Akaike A, Satoh M: Dual effect of serotonin on formalin-induced nociception in the rat spinal cord. Neurosci Res 1996, 25: 129–135.PubMedGoogle Scholar
  63. Robles LI, Barrios M, Del Pozo E, Dordal A, Baeyens JM: Effects of K+ channel blockers and openers on antinociception induced by agonists of 5-HT1A receptors. Eur J Pharmacol 1996, 295: 181–188. 10.1016/0014-2999(95)00643-5PubMedGoogle Scholar
  64. Yoshimura M, Furue H: Mechanisms for the anti-nociceptive actions of the descending noradrenergic and serotonergic systems in the spinal cord. J Pharmacol Sci 2006, 101: 107–117. 10.1254/jphs.CRJ06008XPubMedGoogle Scholar
  65. Ali Z, Wu G, Kozlov A, Barasi S: The actions of 5-HT1 agonists and antagonists on nociceptive processing in the rat spinal cord: results from behavioural and electrophysiological studies. Brain Res 1994, 661: 83–90. 10.1016/0006-8993(94)91184-3PubMedGoogle Scholar
  66. Zhang Y, Yang Z, Gao X, Wu G: The role of 5-hydroxytryptamine1A and 5-hydroxytryptamine1B receptors in modulating spinal nociceptive transmission in normal and carrageenan-injected rats. Pain 2001, 92: 201–211. 10.1016/S0304-3959(01)00259-7PubMedGoogle Scholar
  67. Alhaider AA, Wilcox GL: Differential roles of 5-hydroxytryptamine1A and 5-hydroxytryptamine1B receptor subtypes in modulating spinal nociceptive transmission in mice. J Pharmacol Exp Ther 1993, 265: 378–385.PubMedGoogle Scholar
  68. Xu W, Qiu XC, Han JS: Serotonin receptor subtypes in spinal antinociception in the rat. J Pharmacol Exp Ther 1994, 269: 1182–1189.PubMedGoogle Scholar
  69. Cornea-Hebert V, Riad M, Wu C, Singh SK, Descarries L: Cellular and subcellular distribution of the serotonin 5-HT2A receptor in the central nervous system of adult rat. J Comp Neurol 1999, 409: 187–209. 10.1002/(SICI)1096-9861(19990628)409:2<187::AID-CNE2>3.0.CO;2-PPubMedGoogle Scholar
  70. Maeshima T, Ito R, Hamada S, Senzaki K, Hamaguchi-Hamada K, Shutoh F, Okado N: The cellular localization of 5-HT2A receptors in the spinal cord and spinal ganglia of the adult rat. Brain Res 1998, 797: 118–124. 10.1016/S0006-8993(98)00360-6PubMedGoogle Scholar
  71. Fonseca MI, Ni YG, Dunning DD, Miledi R: Distribution of serotonin 2A, 2C and 3 receptor mRNA in spinal cord and medulla oblongata. Brain Res Mol Brain Res 2001, 89: 11–19. 10.1016/S0169-328X(01)00049-3PubMedGoogle Scholar
  72. Wu S, Zhu M, Wang W, Wang Y, Li Y, Yew DT: Changes of the expression of 5-HT receptor subtype mRNAs in rat dorsal root ganglion by complete Freund's adjuvant-induced inflammation. Neurosci Lett 2001, 307: 183–186. 10.1016/S0304-3940(01)01946-2PubMedGoogle Scholar
  73. Kjorsvik A, Tjolsen A, Hole K: Activation of spinal serotonin(2A/2C) receptors augments nociceptive responses in the rat. Brain Res 2001, 910: 179–181. 10.1016/S0006-8993(01)02652-XPubMedGoogle Scholar
  74. Obata H, Saito S, Sasaki M, Ishizaki K, Goto F: Antiallodynic effect of intrathecally administered 5-HT(2) agonists in rats with nerve ligation. Pain 2001, 90: 173–179. 10.1016/S0304-3959(00)00401-2PubMedGoogle Scholar
  75. Sasaki M, Ishizaki K, Obata H, Goto F: Effects of 5-HT2 and 5-HT3 receptors on the modulation of nociceptive transmission in rat spinal cord according to the formalin test. Eur J Pharmacol 2001, 424: 45–52. 10.1016/S0014-2999(01)01117-7PubMedGoogle Scholar
  76. Cardenas CG, Del Mar LP, Scroggs RS: Two parallel signaling pathways couple 5HT1A receptors to N- and L-type calcium channels in C-like rat dorsal root ganglion cells. J Neurophysiol 1997, 77: 3284–3296.PubMedGoogle Scholar
  77. Cardenas CG, Mar LP, Vysokanov AV, Arnold PB, Cardenas LM, Surmeier DJ, Scroggs RS: Serotonergic modulation of hyperpolarization-activated current in acutely isolated rat dorsal root ganglion neurons. J Physiol 1999,518(Pt 2):507–523. 10.1111/j.1469-7793.1999.0507p.xPubMed CentralPubMedGoogle Scholar
  78. Bervoets K, Millan MJ, Colpaert FC: Agonist action at 5-HT1C receptors facilitates 5-HT1A receptor-mediated spontaneous tail-flicks in the rat. Eur J Pharmacol 1990, 191: 185–195. 10.1016/0014-2999(90)94146-OPubMedGoogle Scholar
  79. Bardin L, Schmidt J, Alloui A, Eschalier A: Effect of intrathecal administration of serotonin in chronic pain models in rats. Eur J Pharmacol 2000, 409: 37–43. 10.1016/S0014-2999(00)00796-2PubMedGoogle Scholar
  80. Laporte AM, Koscielniak T, Ponchant M, Verge D, Hamon M, Gozlan H: Quantitative autoradiographic mapping of 5-HT3 receptors in the rat CNS using [125I]iodo-zacopride and [3H]zacopride as radioligands. Synapse 1992, 10: 271–281. 10.1002/syn.890100402PubMedGoogle Scholar
  81. Morales M, McCollum N, Kirkness EF: 5-HT(3)-receptor subunits A and B are co-expressed in neurons of the dorsal root ganglion. J Comp Neurol 2001, 438: 163–172. 10.1002/cne.1307PubMedGoogle Scholar
  82. Ali Z, Wu G, Kozlov A, Barasi S: The role of 5HT3 in nociceptive processing in the rat spinal cord: results from behavioural and electrophysiological studies. Neurosci Lett 1996, 208: 203–207. 10.1016/0304-3940(95)12600-7PubMedGoogle Scholar
  83. Garraway SM, Hochman S: Serotonin increases the incidence of primary afferent-evoked long-term depression in rat deep dorsal horn neurons. J Neurophysiol 2001, 85: 1864–1872.PubMedGoogle Scholar
  84. Suzuki R, Rahman W, Hunt SP, Dickenson AH: Descending facilitatory control of mechanically evoked responses is enhanced in deep dorsal horn neurones following peripheral nerve injury. Brain Res 2004, 1019: 68–76. 10.1016/j.brainres.2004.05.108PubMedGoogle Scholar
  85. Green GM, Scarth J, Dickenson A: An excitatory role for 5-HT in spinal inflammatory nociceptive transmission; state-dependent actions via dorsal horn 5-HT(3) receptors in the anaesthetized rat. Pain 2000, 89: 81–88. 10.1016/S0304-3959(00)00346-8PubMedGoogle Scholar
  86. Li P, Kerchner GA, Sala C, Wei F, Huettner JE, Sheng M, Zhuo M: AMPA receptor-PDZ interactions in facilitation of spinal sensory synapses. Nat Neurosci 1999, 2: 972–977. 10.1038/14771PubMedGoogle Scholar
  87. Li P, Zhuo M: Silent glutamatergic synapses and nociception in mammalian spinal cord. Nature 1998, 393: 695–698. 10.1038/31496PubMedGoogle Scholar
  88. Fu KY, Light AR, Maixner W: Long-lasting inflammation and long-term hyperalgesia after subcutaneous formalin injection into the rat hindpaw. J Pain 2001, 2: 2–11. 10.1054/jpai.2001.9804PubMedGoogle Scholar
  89. Curtin LI, Grakowsky JA, Suarez M, Thompson AC, DiPirro JM, Martin LB, Kristal MB: Evaluation of buprenorphine in a postoperative pain model in rats. Comp Med 2009, 59: 60–71.PubMed CentralPubMedGoogle Scholar
  90. Pogatzki EM, Niemeier JS, Brennan TJ: Persistent secondary hyperalgesia after gastrocnemius incision in the rat. Eur J Pain 2002, 6: 295–305. 10.1053/eujp.2002.0339PubMedGoogle Scholar
  91. Zahn PK, Brennan TJ: Primary and secondary hyperalgesia in a rat model for human postoperative pain. Anesthesiology 1999, 90: 863–872. 10.1097/00000542-199903000-00030PubMedGoogle Scholar
  92. Jones TL, Lustig AC, Sorkin LS: Secondary hyperalgesia in the postoperative pain model is dependent on spinal calcium/calmodulin-dependent protein kinase II alpha activation. Anesth Analg 2007, 105: 1650–1656. 10.1213/01.ane.0000287644.00420.49PubMedGoogle Scholar
  93. Pogatzki EM, Niemeier JS, Sorkin LS, Brennan TJ: Spinal glutamate receptor antagonists differentiate primary and secondary mechanical hyperalgesia caused by incision. Pain 2003, 105: 97–107. 10.1016/S0304-3959(03)00169-6PubMedGoogle Scholar
  94. Pogatzki-Zahn EM, Niemeier JS, Sorkin LS, Brennan TJ: [Spinal glutamate receptor antagonists differentiate primary and secondary mechanical hyperalgesia caused by incision]. Schmerz 2006, 20: 245–253. 10.1007/s00482-006-0481-8PubMedGoogle Scholar
  95. Zimmermann M: Ethical guidelines for investigations of experimental pain in conscious animals. Pain 1983, 16: 109–110. 10.1016/0304-3959(83)90201-4PubMedGoogle Scholar
  96. Prado WA: Antinociceptive potency of intrathecal morphine in the rat tail flick test: a comparative study using acute lumbar catheter in rats with or without a chronic atlanto-occipital catheter. J Neurosci Methods 2003, 129: 33–39. 10.1016/S0165-0270(03)00197-3PubMedGoogle Scholar
  97. Brennan TJ, Vandermeulen EP, Gebhart GF: Characterization of a rat model of incisional pain. Pain 1996, 64: 493–501. 10.1016/0304-3959(95)01441-1PubMedGoogle Scholar
  98. Lino-de-Oliveira C, Sales AJ, Del Bel EA, Silveira MC, Guimaraes FS: Effects of acute and chronic fluoxetine treatments on restraint stress-induced Fos expression. Brain Res Bull 2001, 55: 747–754. 10.1016/S0361-9230(01)00566-4PubMedGoogle Scholar
  99. Silberstein SD: Methysergide. Cephalalgia 1998, 18: 421–435. 10.1046/j.1468-2982.1998.1807421.xPubMedGoogle Scholar
  100. Glennon RA, Metwally K, Dukat M, Ismaiel AM, De los Angeles J, Herndon J, Teitler M, Khorana N: Ketanserin and spiperone as templates for novel serotonin 5-HT(2A) antagonists. Curr Top Med Chem 2002, 2: 539–558. 10.2174/1568026023393787PubMedGoogle Scholar
  101. Hoyer D, Clarke DE, Fozard JR, Hartig PR, Martin GR, Mylecharane EJ, Saxena PR, Humphrey PP: International Union of Pharmacology classification of receptors for 5-hydroxytryptamine (Serotonin). Pharmacol Rev 1994, 46: 157–203.PubMedGoogle Scholar
  102. Miller K, Weisberg E, Fletcher PW, Teitler M: Membrane-bound and solubilized brain 5HT3 receptors: improved radioligand binding assays using bovine area postrema or rat cortex and the radioligands 3H-GR65630, 3H-BRL43694, and 3H-LY278584. Synapse 1992, 11: 58–66. 10.1002/syn.890110108PubMedGoogle Scholar
  103. Wong DT, Robertson DW, Reid LR: Specific [3H]LY278584 binding to 5-HT3 recognition sites in rat cerebral cortex. Eur J Pharmacol 1989, 166: 107–110. 10.1016/0014-2999(89)90689-4PubMedGoogle Scholar

Copyright

© Silveira et al; licensee BioMed Central Ltd. 2010

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Advertisement