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Antinociceptive effects of galanin in the central nucleus of amygdala of rats, an involvement of opioid receptors
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available at www.sciencedirect.com
www.elsevier.com/locate/brainres
Research Report
Antinociceptive effects of galanin in the central nucleus of amygdala of rats, an involvement of opioid receptors Wu-Yang Jin, Zhuo Liu, Dong Liu, Long-Chuan Yu⁎ Neurobiology Laboratory and State Key Laboratory of Biomembrane and Membrane Biotechnology, College of Life Sciences, Peking University, Beijing 100871, P.R. China
A R T I C LE I N FO
AB S T R A C T
Article history:
The central nucleus of amygdala (CeA) is a very important brain structure involved in
Accepted 21 December 2009
multiple physiological functions, especially in pain modulation. There are high densities of
Available online 4 January 2010
galanin and galanin receptors found in the CeA. The present study was performed to explore the antinociceptive effects of galanin in the CeA of rats, and possible involvements of opioid
Keywords:
receptors in the galanin-induced antinociception. Intra-CeA injection of galanin induced
Antinociceptive effect
dose-dependent increases in hindpaw withdrawal latencies (HWLs) to noxious thermal and
Central nucleus of amygdala (CeA)
mechanical stimulations in rats. Interestingly, the amtinociceptive effect induced by intra-
Galanin
CeA injection of galanin was blocked by intra-CeA injection of naloxone, a common opioid
Hindpaw withdrawal latency
receptor antagonist, indicating an involvement of opioid receptors in the galanin-induced
Mu-Opioid receptor
antinociception in the CeA of rats. Moreover, intra-CeA injection of either selective mu-
Delta-Opioid receptor
opioid receptor antagonist β-funaltrexamine (β-FNA) or delta-opioid receptor antagonist naltrindole, but not kappa-opioid receptor antagonist nor-binaltorphimine (nor-BNI), significantly attenuated the galanin-induced increases in HWLs in the CeA of rats. Taken together, the results demonstrate that galanin induces antinociceptive effects in the CeA of rats, and both mu- and delta-opioid receptors are involved in the galanin-induced antinociception. © 2009 Elsevier B.V. All rights reserved.
1.
Introduction
Galanin, a 29 (30 in human) amino acid residue neuropeptide, is widely distributed in the central nervous system and peripheral tissues (Melander et al., 1986; Merchenthaler et al., 1993; Robinson and Brewer, 2008). Galanin has been shown to participate in a variety of physiological functions, such as reproduction, memory and food intake (Counts et al., 2008; Hobson et al., 2008; Merchenthaler et al., 1993; Rajendren, 2002; Taylor et al., 2009). There are three galanin receptors subtypes, GalR1, GalR2 and GalR3, and all the galanin
receptors are G protein coupled receptors (Branchek et al., 2000; Robinson and Brewer, 2008). Numerous studies have demonstrated that galanin and its receptors are involved in the transmission and modulation of nociceptive information at spinal levels (Hua et al., 2004; Liu and Hokfelt, 2002; Wiesenfeld-Hallin et al., 2005; Xu et al., 2008; Zhang et al., 2000). In the brain, studies have demonstrated that galanin plays an antinociceptive role in the hypothalamic arcuate nucleus in intact rats, in rats with inflammation and in rats with chronic neuropathic pain (Gu et al., 2007; Sun et al., 2003), indicating that galanin and
⁎ Corresponding author. Fax: +86 10 6275 1526. E-mail address: [email protected] (L.-C. Yu). 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.12.060
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galanin receptors play an important role in the antinociception modulation in the central nervous system. Wang et al. also found that galanin and galanin receptors are involved in pain modulation in periaqueductal grey (PAG) of rats (Sun and Yu, 2005; Wang et al., 1999). Interestingly, previous studies also demonstrated an interaction between galanin and opioids in nociceptive modulation in the spinal cord (Zhang et al., 2000), as well as in the brain (Sun and Yu, 2005; Wang et al., 1999). The central nucleus of amygdala (CeA) is a very important brain structure involved in multiple physiological functions (Ahn and Phillips, 2002; Beckman et al., 2009; Goncalves et al., 2008; Han and Yu, 2009). Bie et al. investigated the potential adaptive function of delta opioid receptor in neurons of the CeA and found that in rats displaying morphine-induced behavior of conditioned place preference the overall synaptic strength of glutamate synapses in CeA neurons was significantly enhanced, implying an involvement of delta opioid receptor in opioid reward and addiction in the CeA (Bie et al., 2009). Recently, Beckman et al. (2009) reported that opioid activity in the CeA can modulate the feeding inhibition of melanocortin stimulation of the hypothalamic paraventricular nucleus. Studies have demonstrated that the CeA is involved in pain modulation in the central nervous system (Goncalves et al., 2008; Han and Yu, 2009; Neugebauer et al., 2004; Xu et al., 2003). Both high densities of galanin and galanin receptors are found in the CeA (Melander et al., 1986; Merchenthaler et al., 1993; Mennicken et al., 2002). These reports strongly suggest a nociceptive modulation role of galanin in the CeA. Therefore, the present study was performed to explore the antinociceptive effects of galanin in the CeA of rats, and possible involvements of opioid receptors in the galanin-induced antinociception.
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2.
Results
2.1.
Antinociceptive effects of galanin in the CeA of rats
Four groups of rats received intra-CeA injection of 0.1 (n=8), 0.5 (n=7) or 1 nmol (n=8) of galanin, or 1 μl of 0.9% saline as a control (n=7). As shown in Fig. 1, the HWLs to thermal and mechanical stimulations increased significantly in a dose-dependent manner after intra-CeA injection of 0.1 nmol of galanin (Hot-plate Test: Fleft/left (1,13)=10.62, P<0.01; Fright/right (1,13)=14.30, P<0.01; Randall Selitto Test: Fleft/left (1,13) =10.00, P<0.01; Fright/right (1,13)=5.05, P<0.05), 0.5 nmol of galanin (Hot-plate Test: Fleft/left (1,12) =38.32, P<0.001; Fright/right (1,12)=29.66, P<0.001; Randall Selitto Test: Fleft/left (1,12)=18.78, P<0.01; Fright/right (1,12)=8.27, P<0.05) and 1 nmol of galanin (Hot-plate Test: Fleft/left (1,13)=28.67, P<0.001; Fright/right (1,13) =26.13, P< 0.001; Randall Selitto Test: Fleft/left (1,13)=21.54, P<0.001; Fright/right (1,13)=12.97, P<0.01) compared with the control group. The results demonstrated that intra-CeA injection of galanin induced significant antinociceptive effects in rats.
2.2. Influence of intra-CeA injection of naloxone on the galanin-induced antinociception Two groups of rats received intra-CeA injection of 1 nmol of galanin, followed 5 min later by intra-CeA injection of 1 μg of the common opioid receptor antagonist naloxone (n = 6), or 1 μl of 0.9% saline as a control (n = 7). As shown in Fig. 2, the galanininduced increases in HWLs were significantly attenuated after intra-CeA injection of naloxone (Hot-plate Test: Fleft/left = (1,11) 142.99, P < 0.001; Fright/right (1,11) = 24.90, P < 0.001; Randall Selitto Test: Fleft/left (1,11) = 431.07, P < 0.001; Fright/right (1,11) = 147.51,
Fig. 1 – Effects of intra-CeA injection of galanin on the HWLs to thermal (A and B) and mechanical stimulation (C and D) in rats. Left HWL: A and C; right HWL: B and D. Intra-CeA injection of 1 μl of 0.9% saline is as the control group. Data are presented as mean ± S.E.M. The statistical difference between groups is determined by two-way ANOVA. CeA, the central nucleus of amygdala; HWL, hindpaw withdrawal latency. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the control group.
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Fig. 2 – Effects of intra-CeA injection of naloxone on the galanin-induced increases in HWLs to thermal (A and B) and mechanical stimulation (C and D) in rats. Left HWL: A and C; right HWL: B and D. Time = 0 min: intra-CeA injection of 1 nmol of galanin; time = 5 min: intra-CeA injection of 1 nmol of naloxone or 1 μl of 0.9% saline as the control group. Data are presented as mean ± S.E.M. The statistical difference between groups is determined by two-way ANOVA. CeA, the central nucleus of amygdala; HWL, hindpaw withdrawal latency. ***P < 0.001 compared with the control group.
P < 0.001) compared with the saline group, indicating that opioid receptors are involved in the galanin-induced antinociception in the CeA of rats.
Another group of rats (n = 6) received intra-CeA injection of 1 μl of 0.9% saline, followed 5 min later by intra-CeA injection of 1 μg of naloxone. As shown in Fig. 2, there were
Fig. 3 – Effects of intra-CeA injection of selective opioid receptor antagonists on the galanin-induced increases in HWLs to thermal (A and B) and mechanical stimulation (C and D) in rats. Left HWL: A and C; right HWL: B and D. Time = 0 min: intra-CeA injection of 1 nmol of galanin; time = 5 min: intra-CeA injection of 1 nmol of β-FNA, naltrindole or nor-BNI, or 1 μl of 0.9% saline as the control group. Data are presented as mean ± S.E.M. The statistical difference between groups is determined by two-way ANOVA. CeA, the central nucleus of amygdala; HWL, hindpaw withdrawal latency; β-FNA, β-funaltrexamine; nor-BNI, nor-binaltorphimine. *P < 0.05 and ***P < 0.001 are compared with the control group.
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no marked changes of HWLs after intra-CeA injection of naloxone in rats.
2.3. Involvements of different opioid receptors in the galanin-induced antinociception Four groups of rats received intra-CeA injection of 1 nmol of galanin, followed 5 min later by intra-CeA injection of 1 nmol of nor-BNI (n= 6), 1 nmol of β-FNA (n= 7), 1 nmol of naltrindole (n =7), or 1 μl of 0.9% saline as a control (n= 7). The results were shown in Fig. 3. The HWLs to thermal and mechanical stimulations increased markedly after intra-CeA injection of 1 nmol of galanin. Compared with the control group, the galanin-induced increases in HWLs were attenuated significantly after administration of 1 nmol of β-FNA (Hot-plate Test: Fleft/left (1,12) =50.64, P <0.001; Fright/right (1,12) =28.16, P <0.001; Randall Selitto Test: Fleft/left (1,12) =942.15, P< 0.001; Fright/right (1,12) =368.91, P <0.001) or 1 nmol of naltrindole (Hot-plate Test: Fleft/left (1,12) = 5.48, P <0.05; Fright/right (1,12) = 7.96, P <0.05; Randall Selitto Test: Fleft/left (1,12) =39.87, P <0.001; Fright/right =48.94, P <0.001), but not 1 nmol of nor-BNI (Hot-plate Test: Fleft/left (1,11) = 1.45, P = 0.26; Fright/right (1,11)= 1.10, P = 0.32; Randall Selitto Test: Fleft/left (1,11)= 1.63, P =0.23; Fright/right (1,11) = 3.12, P =0.12), as shown in Fig. 3. The results suggest that mu- and delta-opioid receptors, but not kappa-opioid receptors, are involved in the galanin-induced antinociception in the CeA of rats. Two groups of rats received intra-CeA injection of 1 μl of 0.9% saline, followed 5 min later by 1 nmol of beta-FNA (n = 6) or 1 nmol of nor-BNI (n = 6). There were no marked changes in HWLs during 60 min after the injection, as shown in Fig. 3.
Fig. 4 – Illustration of the location of the tips of the injection needle.
3.
19
Discussion
Lots of studies have demonstrated that galanin and its receptors play important roles in the transmission and modulation of nociceptive information in the spinal cord (Hua et al., 2004; Liu and Hokfelt, 2002; Wiesenfeld-Hallin et al., 2005; Xu et al., 2008; Zhang et al., 2000). There are very few reports related to the roles of galanin in pain modulation at supraspinal levels. Previous studies in our laboratory demonstrated that galanin and galanin receptors are involved in the pain modulation in the brain (Gu et al., 2007; Sun et al., 2003). Periaqueductal grey (PAG) is an important brain structure in the transmission and/or modulation of nociceptive information, and as a key station in the descending pathway of analgesia from brainstem to the spinal cord (Millan, 2002). Wang et al. found that administration of galanin to PAG induced dose-dependent increases in the nociceptive response latencies in rats; the effect of galanin was attenuated by following injection of naloxone into PAG. The results indicate an antinociceptive role of galanin, and a possible interaction between galanin and opioid peptides in the PAG of rats (Wang et al., 2000; Wang et al., 1999). The CeA is a very important brain region involved in pain modulation in the central nervous system (Goncalves et al., 2008; Han and Yu, 2009; Neugebauer et al., 2004; Xu et al., 2003). The present study demonstrated that intra-CeA injection of galanin induced dose-dependent increases in HWLs to noxious thermal and mechanical stimulations in rats, suggesting that galanin induces inhibitory effects on the transmission of nociceptive information mediated by the galanin receptors in the central nervous system. The results clearly showed that galanin plays important roles in pain modulation in the CeA of rats. It has been well documented that opioid peptides play a key role in pain modulation in the central nervous system (Fields and Basbaum, 1999; Millan 2002). There are opioid peptides and opioid receptors distributed in the CeA (Mansour et al., 1994; Mansour et al., 1988). Our results demonstrated that the antinociceptive effects induced by intra-CeA injection of galanin could also be blocked by naloxone, indicating an involvement of opioid receptors in the galanin-induced antinociception. It is known that there are three types of opioid receptors, mu-, delta- and kappa-receptors. Further results showed that intra-CeA injection of the selective mu-opioid receptor antagonist β-FNA or the selective delta-opioid receptor antagonist naltrindole, but not the selective kappa-opioid receptor antagonist nor-BNI, could attenuate the galanin-induced antinociception, indicating that mu- and delta-opioid receptors, not kappa-opioid receptors, are involved in the galanin-induced antinociception in the CeA. All the doses of the antagonists used in this study are based on the results in previous studies of our laboratory (Sun et al., 2003; Sun and Yu, 2005). Thus, the doses used should be effective for the receptor that the antagonists are blocking. In summary, the present study demonstrated that intraCeA injection of galanin induced dose-dependent increases in HWLs to noxious thermal and mechanical stimulations in rats. Furthermore, the antinociception induced by intra-CeA injection of galanin was blocked by intra-CeA injection of the
20
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selective mu-opioid receptor antagonist β-FNA or delta-opioid receptor antagonist naltrindole, but not kappa-opioid receptor antagonist nor-BNI, indicating the involvement of mu- and delta-opioid receptors, but not kappa-opioid receptors, in the galanin-induced antinociception in the CeA.
4.
Experimental procedures
4.1.
Animals
All experiments were carried out on freely moving male Wister rats weighing between 200 and 250 g (Experimental Animal Center, Academy of Military Medical Sciences, Beijing, China). The rats were housed in cages with free access to food and water, and maintained in a room temperature of 24 ± 2 °C with a normal day/night cycle. All experiments were performed according to the guidelines of the International Association for the Study of Pain (Zimmermann, 1983) and every effort was made to minimize both the animal suffering and the number of animals used.
4.2.
Chemicals
Solutions for intra-CeA injection were prepared with 0.9% sterilized saline, each with a volume of 1 μl containing: (1) 0.1, 0.5 or 1 nmol of galanin (galanin, Tocris, UK); (2) 1 μg of naloxone (naloxone hydrochloride, Sigma, St. Louis, MO); (3) 1 nmol of β-funaltrexamine (β-FNA; Tocris Cookson, Bristol, UK); (4) 1 nmol of nor-binaltorphimine (nor-BNI; Tocris Cookson); (5) 1 nmol of naltrindole (naltrindole hydrochloride; Tocris Cookson).
4.3.
Nociceptive tests
Rats were accustomed to the test condition for 5 days before the experiment to minimize the stress induced by handling and measurements. The hindpaw withdrawal latencies (HWLs) during thermal and mechanical stimulation were measured as described previously (Li et al., 2005; Zhou et al., 2003). Briefly, the entire ventral surface of the rat hindpaw was placed manually on a hot plate, which was maintained at a temperature of 52± 2 °C. The time to hindpaw withdrawal was measured in seconds and referred to as the HWL to thermal stimulation. The Randall Selitto Test (Ugo Basile, Type 7200, Italy) was used to assess the HWL to mechanical stimulation. A wedge-shaped pusher at a loading rate of 30 g/s was applied to the dorsal surface of the hindpaw. The latency required to initiate the withdrawal response was assessed and expressed in seconds. Before intraCeA injection, the HWLs was tested three times and regarded as the basal HWLs. The HWLs recorded during subsequent experiments were expressed as percentage changes of the basal level for each rat (% changes of the HWL). Each rat was tested by both types of stimulation. Every measurement of the HWL to both thermal and mechanical stimulation was finished within 2 min. A cut-off limit of 15 s was set up to avoid tissue damage.
4.4.
Surgery and Intra-CeA injection
The rats were anesthetized by intraperitoneal chloral hydrate (400 mg/kg) and were placed on a stereotaxic instrument. A
stainless steel guide cannula of 0.8 mm outer-diameter was directed into the CeA (B, −2.2 mm; LR, 4.0 mm; V, 8.0 mm. B, anterior (+) or posterior (−) to Bregma; L or R, left or right to midline; V, ventral to the surface of skull) according to Paxinos and Watson (1998) and was fixed to the skull by dental acrylic (Paxinos and Watson, 1998). There were more than 3 days for rats to recover from the operation. On the day of experiment, a stainless steel needle with 0.4 mm diameter was directly inserted into the guide cannula with 1.5 mm beyond the tip of the latter. One microliter volume of solution was thereafter infused into the CeA over 1 min.
4.5.
Statistics
At the end of the experiments, the location of the tip of the injection needle was verified and the injection points are shown in Fig. 4. Only the results from nociceptive tests that the tips of the injection needle were within the CeA were used for statistical analysis. Data from the experiment were expressed as mean ± S.E.M. Statistical difference between groups was determined by two-way analysis of variance (ANOVA) for repeated measurements (Fleft/left is the F value of the two groups: the left HWL of the first group compared with the left HWL of the second group). ⁎P < 0.05, ⁎⁎P < 0.01 and ⁎⁎⁎P < 0.001 were considered as significant differences.
Acknowledgments The work is supported by funds from the National Natural Science Foundation of China (NSFC 30470542, 30870802), National Ministry of Education grant (20040001057), National Undergraduate Innovative Test Program Research Endowment sponsored by the National Ministry of Education and National Program of Basic Research sponsored by the Ministry of Science and Technology of China (2006CB500706, 2009CB522002).
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available at www.sciencedirect.com
www.elsevier.com/locate/brainres
Research Report
Antinociceptive effects of galanin in the central nucleus of amygdala of rats, an involvement of opioid receptors Wu-Yang Jin, Zhuo Liu, Dong Liu, Long-Chuan Yu⁎ Neurobiology Laboratory and State Key Laboratory of Biomembrane and Membrane Biotechnology, College of Life Sciences, Peking University, Beijing 100871, P.R. China
A R T I C LE I N FO
AB S T R A C T
Article history:
The central nucleus of amygdala (CeA) is a very important brain structure involved in
Accepted 21 December 2009
multiple physiological functions, especially in pain modulation. There are high densities of
Available online 4 January 2010
galanin and galanin receptors found in the CeA. The present study was performed to explore the antinociceptive effects of galanin in the CeA of rats, and possible involvements of opioid
Keywords:
receptors in the galanin-induced antinociception. Intra-CeA injection of galanin induced
Antinociceptive effect
dose-dependent increases in hindpaw withdrawal latencies (HWLs) to noxious thermal and
Central nucleus of amygdala (CeA)
mechanical stimulations in rats. Interestingly, the amtinociceptive effect induced by intra-
Galanin
CeA injection of galanin was blocked by intra-CeA injection of naloxone, a common opioid
Hindpaw withdrawal latency
receptor antagonist, indicating an involvement of opioid receptors in the galanin-induced
Mu-Opioid receptor
antinociception in the CeA of rats. Moreover, intra-CeA injection of either selective mu-
Delta-Opioid receptor
opioid receptor antagonist β-funaltrexamine (β-FNA) or delta-opioid receptor antagonist naltrindole, but not kappa-opioid receptor antagonist nor-binaltorphimine (nor-BNI), significantly attenuated the galanin-induced increases in HWLs in the CeA of rats. Taken together, the results demonstrate that galanin induces antinociceptive effects in the CeA of rats, and both mu- and delta-opioid receptors are involved in the galanin-induced antinociception. © 2009 Elsevier B.V. All rights reserved.
1.
Introduction
Galanin, a 29 (30 in human) amino acid residue neuropeptide, is widely distributed in the central nervous system and peripheral tissues (Melander et al., 1986; Merchenthaler et al., 1993; Robinson and Brewer, 2008). Galanin has been shown to participate in a variety of physiological functions, such as reproduction, memory and food intake (Counts et al., 2008; Hobson et al., 2008; Merchenthaler et al., 1993; Rajendren, 2002; Taylor et al., 2009). There are three galanin receptors subtypes, GalR1, GalR2 and GalR3, and all the galanin
receptors are G protein coupled receptors (Branchek et al., 2000; Robinson and Brewer, 2008). Numerous studies have demonstrated that galanin and its receptors are involved in the transmission and modulation of nociceptive information at spinal levels (Hua et al., 2004; Liu and Hokfelt, 2002; Wiesenfeld-Hallin et al., 2005; Xu et al., 2008; Zhang et al., 2000). In the brain, studies have demonstrated that galanin plays an antinociceptive role in the hypothalamic arcuate nucleus in intact rats, in rats with inflammation and in rats with chronic neuropathic pain (Gu et al., 2007; Sun et al., 2003), indicating that galanin and
⁎ Corresponding author. Fax: +86 10 6275 1526. E-mail address: [email protected] (L.-C. Yu). 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.12.060
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galanin receptors play an important role in the antinociception modulation in the central nervous system. Wang et al. also found that galanin and galanin receptors are involved in pain modulation in periaqueductal grey (PAG) of rats (Sun and Yu, 2005; Wang et al., 1999). Interestingly, previous studies also demonstrated an interaction between galanin and opioids in nociceptive modulation in the spinal cord (Zhang et al., 2000), as well as in the brain (Sun and Yu, 2005; Wang et al., 1999). The central nucleus of amygdala (CeA) is a very important brain structure involved in multiple physiological functions (Ahn and Phillips, 2002; Beckman et al., 2009; Goncalves et al., 2008; Han and Yu, 2009). Bie et al. investigated the potential adaptive function of delta opioid receptor in neurons of the CeA and found that in rats displaying morphine-induced behavior of conditioned place preference the overall synaptic strength of glutamate synapses in CeA neurons was significantly enhanced, implying an involvement of delta opioid receptor in opioid reward and addiction in the CeA (Bie et al., 2009). Recently, Beckman et al. (2009) reported that opioid activity in the CeA can modulate the feeding inhibition of melanocortin stimulation of the hypothalamic paraventricular nucleus. Studies have demonstrated that the CeA is involved in pain modulation in the central nervous system (Goncalves et al., 2008; Han and Yu, 2009; Neugebauer et al., 2004; Xu et al., 2003). Both high densities of galanin and galanin receptors are found in the CeA (Melander et al., 1986; Merchenthaler et al., 1993; Mennicken et al., 2002). These reports strongly suggest a nociceptive modulation role of galanin in the CeA. Therefore, the present study was performed to explore the antinociceptive effects of galanin in the CeA of rats, and possible involvements of opioid receptors in the galanin-induced antinociception.
17
2.
Results
2.1.
Antinociceptive effects of galanin in the CeA of rats
Four groups of rats received intra-CeA injection of 0.1 (n=8), 0.5 (n=7) or 1 nmol (n=8) of galanin, or 1 μl of 0.9% saline as a control (n=7). As shown in Fig. 1, the HWLs to thermal and mechanical stimulations increased significantly in a dose-dependent manner after intra-CeA injection of 0.1 nmol of galanin (Hot-plate Test: Fleft/left (1,13)=10.62, P<0.01; Fright/right (1,13)=14.30, P<0.01; Randall Selitto Test: Fleft/left (1,13) =10.00, P<0.01; Fright/right (1,13)=5.05, P<0.05), 0.5 nmol of galanin (Hot-plate Test: Fleft/left (1,12) =38.32, P<0.001; Fright/right (1,12)=29.66, P<0.001; Randall Selitto Test: Fleft/left (1,12)=18.78, P<0.01; Fright/right (1,12)=8.27, P<0.05) and 1 nmol of galanin (Hot-plate Test: Fleft/left (1,13)=28.67, P<0.001; Fright/right (1,13) =26.13, P< 0.001; Randall Selitto Test: Fleft/left (1,13)=21.54, P<0.001; Fright/right (1,13)=12.97, P<0.01) compared with the control group. The results demonstrated that intra-CeA injection of galanin induced significant antinociceptive effects in rats.
2.2. Influence of intra-CeA injection of naloxone on the galanin-induced antinociception Two groups of rats received intra-CeA injection of 1 nmol of galanin, followed 5 min later by intra-CeA injection of 1 μg of the common opioid receptor antagonist naloxone (n = 6), or 1 μl of 0.9% saline as a control (n = 7). As shown in Fig. 2, the galanininduced increases in HWLs were significantly attenuated after intra-CeA injection of naloxone (Hot-plate Test: Fleft/left = (1,11) 142.99, P < 0.001; Fright/right (1,11) = 24.90, P < 0.001; Randall Selitto Test: Fleft/left (1,11) = 431.07, P < 0.001; Fright/right (1,11) = 147.51,
Fig. 1 – Effects of intra-CeA injection of galanin on the HWLs to thermal (A and B) and mechanical stimulation (C and D) in rats. Left HWL: A and C; right HWL: B and D. Intra-CeA injection of 1 μl of 0.9% saline is as the control group. Data are presented as mean ± S.E.M. The statistical difference between groups is determined by two-way ANOVA. CeA, the central nucleus of amygdala; HWL, hindpaw withdrawal latency. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the control group.
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Fig. 2 – Effects of intra-CeA injection of naloxone on the galanin-induced increases in HWLs to thermal (A and B) and mechanical stimulation (C and D) in rats. Left HWL: A and C; right HWL: B and D. Time = 0 min: intra-CeA injection of 1 nmol of galanin; time = 5 min: intra-CeA injection of 1 nmol of naloxone or 1 μl of 0.9% saline as the control group. Data are presented as mean ± S.E.M. The statistical difference between groups is determined by two-way ANOVA. CeA, the central nucleus of amygdala; HWL, hindpaw withdrawal latency. ***P < 0.001 compared with the control group.
P < 0.001) compared with the saline group, indicating that opioid receptors are involved in the galanin-induced antinociception in the CeA of rats.
Another group of rats (n = 6) received intra-CeA injection of 1 μl of 0.9% saline, followed 5 min later by intra-CeA injection of 1 μg of naloxone. As shown in Fig. 2, there were
Fig. 3 – Effects of intra-CeA injection of selective opioid receptor antagonists on the galanin-induced increases in HWLs to thermal (A and B) and mechanical stimulation (C and D) in rats. Left HWL: A and C; right HWL: B and D. Time = 0 min: intra-CeA injection of 1 nmol of galanin; time = 5 min: intra-CeA injection of 1 nmol of β-FNA, naltrindole or nor-BNI, or 1 μl of 0.9% saline as the control group. Data are presented as mean ± S.E.M. The statistical difference between groups is determined by two-way ANOVA. CeA, the central nucleus of amygdala; HWL, hindpaw withdrawal latency; β-FNA, β-funaltrexamine; nor-BNI, nor-binaltorphimine. *P < 0.05 and ***P < 0.001 are compared with the control group.
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no marked changes of HWLs after intra-CeA injection of naloxone in rats.
2.3. Involvements of different opioid receptors in the galanin-induced antinociception Four groups of rats received intra-CeA injection of 1 nmol of galanin, followed 5 min later by intra-CeA injection of 1 nmol of nor-BNI (n= 6), 1 nmol of β-FNA (n= 7), 1 nmol of naltrindole (n =7), or 1 μl of 0.9% saline as a control (n= 7). The results were shown in Fig. 3. The HWLs to thermal and mechanical stimulations increased markedly after intra-CeA injection of 1 nmol of galanin. Compared with the control group, the galanin-induced increases in HWLs were attenuated significantly after administration of 1 nmol of β-FNA (Hot-plate Test: Fleft/left (1,12) =50.64, P <0.001; Fright/right (1,12) =28.16, P <0.001; Randall Selitto Test: Fleft/left (1,12) =942.15, P< 0.001; Fright/right (1,12) =368.91, P <0.001) or 1 nmol of naltrindole (Hot-plate Test: Fleft/left (1,12) = 5.48, P <0.05; Fright/right (1,12) = 7.96, P <0.05; Randall Selitto Test: Fleft/left (1,12) =39.87, P <0.001; Fright/right =48.94, P <0.001), but not 1 nmol of nor-BNI (Hot-plate Test: Fleft/left (1,11) = 1.45, P = 0.26; Fright/right (1,11)= 1.10, P = 0.32; Randall Selitto Test: Fleft/left (1,11)= 1.63, P =0.23; Fright/right (1,11) = 3.12, P =0.12), as shown in Fig. 3. The results suggest that mu- and delta-opioid receptors, but not kappa-opioid receptors, are involved in the galanin-induced antinociception in the CeA of rats. Two groups of rats received intra-CeA injection of 1 μl of 0.9% saline, followed 5 min later by 1 nmol of beta-FNA (n = 6) or 1 nmol of nor-BNI (n = 6). There were no marked changes in HWLs during 60 min after the injection, as shown in Fig. 3.
Fig. 4 – Illustration of the location of the tips of the injection needle.
3.
19
Discussion
Lots of studies have demonstrated that galanin and its receptors play important roles in the transmission and modulation of nociceptive information in the spinal cord (Hua et al., 2004; Liu and Hokfelt, 2002; Wiesenfeld-Hallin et al., 2005; Xu et al., 2008; Zhang et al., 2000). There are very few reports related to the roles of galanin in pain modulation at supraspinal levels. Previous studies in our laboratory demonstrated that galanin and galanin receptors are involved in the pain modulation in the brain (Gu et al., 2007; Sun et al., 2003). Periaqueductal grey (PAG) is an important brain structure in the transmission and/or modulation of nociceptive information, and as a key station in the descending pathway of analgesia from brainstem to the spinal cord (Millan, 2002). Wang et al. found that administration of galanin to PAG induced dose-dependent increases in the nociceptive response latencies in rats; the effect of galanin was attenuated by following injection of naloxone into PAG. The results indicate an antinociceptive role of galanin, and a possible interaction between galanin and opioid peptides in the PAG of rats (Wang et al., 2000; Wang et al., 1999). The CeA is a very important brain region involved in pain modulation in the central nervous system (Goncalves et al., 2008; Han and Yu, 2009; Neugebauer et al., 2004; Xu et al., 2003). The present study demonstrated that intra-CeA injection of galanin induced dose-dependent increases in HWLs to noxious thermal and mechanical stimulations in rats, suggesting that galanin induces inhibitory effects on the transmission of nociceptive information mediated by the galanin receptors in the central nervous system. The results clearly showed that galanin plays important roles in pain modulation in the CeA of rats. It has been well documented that opioid peptides play a key role in pain modulation in the central nervous system (Fields and Basbaum, 1999; Millan 2002). There are opioid peptides and opioid receptors distributed in the CeA (Mansour et al., 1994; Mansour et al., 1988). Our results demonstrated that the antinociceptive effects induced by intra-CeA injection of galanin could also be blocked by naloxone, indicating an involvement of opioid receptors in the galanin-induced antinociception. It is known that there are three types of opioid receptors, mu-, delta- and kappa-receptors. Further results showed that intra-CeA injection of the selective mu-opioid receptor antagonist β-FNA or the selective delta-opioid receptor antagonist naltrindole, but not the selective kappa-opioid receptor antagonist nor-BNI, could attenuate the galanin-induced antinociception, indicating that mu- and delta-opioid receptors, not kappa-opioid receptors, are involved in the galanin-induced antinociception in the CeA. All the doses of the antagonists used in this study are based on the results in previous studies of our laboratory (Sun et al., 2003; Sun and Yu, 2005). Thus, the doses used should be effective for the receptor that the antagonists are blocking. In summary, the present study demonstrated that intraCeA injection of galanin induced dose-dependent increases in HWLs to noxious thermal and mechanical stimulations in rats. Furthermore, the antinociception induced by intra-CeA injection of galanin was blocked by intra-CeA injection of the
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selective mu-opioid receptor antagonist β-FNA or delta-opioid receptor antagonist naltrindole, but not kappa-opioid receptor antagonist nor-BNI, indicating the involvement of mu- and delta-opioid receptors, but not kappa-opioid receptors, in the galanin-induced antinociception in the CeA.
4.
Experimental procedures
4.1.
Animals
All experiments were carried out on freely moving male Wister rats weighing between 200 and 250 g (Experimental Animal Center, Academy of Military Medical Sciences, Beijing, China). The rats were housed in cages with free access to food and water, and maintained in a room temperature of 24 ± 2 °C with a normal day/night cycle. All experiments were performed according to the guidelines of the International Association for the Study of Pain (Zimmermann, 1983) and every effort was made to minimize both the animal suffering and the number of animals used.
4.2.
Chemicals
Solutions for intra-CeA injection were prepared with 0.9% sterilized saline, each with a volume of 1 μl containing: (1) 0.1, 0.5 or 1 nmol of galanin (galanin, Tocris, UK); (2) 1 μg of naloxone (naloxone hydrochloride, Sigma, St. Louis, MO); (3) 1 nmol of β-funaltrexamine (β-FNA; Tocris Cookson, Bristol, UK); (4) 1 nmol of nor-binaltorphimine (nor-BNI; Tocris Cookson); (5) 1 nmol of naltrindole (naltrindole hydrochloride; Tocris Cookson).
4.3.
Nociceptive tests
Rats were accustomed to the test condition for 5 days before the experiment to minimize the stress induced by handling and measurements. The hindpaw withdrawal latencies (HWLs) during thermal and mechanical stimulation were measured as described previously (Li et al., 2005; Zhou et al., 2003). Briefly, the entire ventral surface of the rat hindpaw was placed manually on a hot plate, which was maintained at a temperature of 52± 2 °C. The time to hindpaw withdrawal was measured in seconds and referred to as the HWL to thermal stimulation. The Randall Selitto Test (Ugo Basile, Type 7200, Italy) was used to assess the HWL to mechanical stimulation. A wedge-shaped pusher at a loading rate of 30 g/s was applied to the dorsal surface of the hindpaw. The latency required to initiate the withdrawal response was assessed and expressed in seconds. Before intraCeA injection, the HWLs was tested three times and regarded as the basal HWLs. The HWLs recorded during subsequent experiments were expressed as percentage changes of the basal level for each rat (% changes of the HWL). Each rat was tested by both types of stimulation. Every measurement of the HWL to both thermal and mechanical stimulation was finished within 2 min. A cut-off limit of 15 s was set up to avoid tissue damage.
4.4.
Surgery and Intra-CeA injection
The rats were anesthetized by intraperitoneal chloral hydrate (400 mg/kg) and were placed on a stereotaxic instrument. A
stainless steel guide cannula of 0.8 mm outer-diameter was directed into the CeA (B, −2.2 mm; LR, 4.0 mm; V, 8.0 mm. B, anterior (+) or posterior (−) to Bregma; L or R, left or right to midline; V, ventral to the surface of skull) according to Paxinos and Watson (1998) and was fixed to the skull by dental acrylic (Paxinos and Watson, 1998). There were more than 3 days for rats to recover from the operation. On the day of experiment, a stainless steel needle with 0.4 mm diameter was directly inserted into the guide cannula with 1.5 mm beyond the tip of the latter. One microliter volume of solution was thereafter infused into the CeA over 1 min.
4.5.
Statistics
At the end of the experiments, the location of the tip of the injection needle was verified and the injection points are shown in Fig. 4. Only the results from nociceptive tests that the tips of the injection needle were within the CeA were used for statistical analysis. Data from the experiment were expressed as mean ± S.E.M. Statistical difference between groups was determined by two-way analysis of variance (ANOVA) for repeated measurements (Fleft/left is the F value of the two groups: the left HWL of the first group compared with the left HWL of the second group). ⁎P < 0.05, ⁎⁎P < 0.01 and ⁎⁎⁎P < 0.001 were considered as significant differences.
Acknowledgments The work is supported by funds from the National Natural Science Foundation of China (NSFC 30470542, 30870802), National Ministry of Education grant (20040001057), National Undergraduate Innovative Test Program Research Endowment sponsored by the National Ministry of Education and National Program of Basic Research sponsored by the Ministry of Science and Technology of China (2006CB500706, 2009CB522002).
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