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Peripheral nerve injury decreases the expression of metabolic glutamate receptor 7 in dorsal root ganglion neurons
Neuroscience Letters 531 (2012) 52–56
Contents lists available at SciVerse ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Peripheral nerve injury decreases the expression of metabolic glutamate receptor 7 in dorsal root ganglion neurons Jian-You Li a,1 , Xiang Wang b,1 , Peng-Tian Ji b , Xiong-Feng Li a , Guo-Hua Guan a , Xue-Sheng Jiang a , Guo-shun Zhou a , Feng Hua b , Nan Wang c,∗ a
Orthopedics Department, Huzhou Central Hospital, 198 Hongqi Road, Huzhou 313000, People’s Republic of China Huzhou Key Laboratory of Molecular Medicine, Huzhou Central Hospital, 198 Hongqi Road, Huzhou 313000, People’s Republic of China c Medical Department, Huzhou Traditional Chinese Medicine Hospital, 82 Nanjie Road, Huzhou 313000, People’s Republic of China b
h i g h l i g h t s mGluR7 is expressed in peptidergic and large DRG neurons but not in IB4+ neurons. mGluR7 is anterogradely transported from DRG cell body to the peripheral site. After peripheral nerve injury, the expression of mGluR7 is down-regulated.
a r t i c l e
i n f o
Article history: Received 4 September 2012 Accepted 9 October 2012 Keywords: mGluR7 Dorsal root ganglion Axotomy Neuropathic pain
a b s t r a c t Group II and III metabolic glutamate receptors (mGluRs) are responsible for the glutamate-mediated postsynaptic excitation of neurons. Previous pharmacological evidences show that activation of mGluR7 could inhibit nociceptive reception. However, the distribution and expression patterns of mGluR7 after peripheral injury remain unclear. Herein we found that mGluR7 was expressed in the rat peptidergic dorsal root ganglion (DRG) neurons and large neurons, but rarely in isolectin B4 positive neurons. Sciatic nerve ligation experiment showed that mGluR7 was anterogradely transported from cell body to the peripheral site. Furthermore, after peripheral nerve injury, mGluR7 expression was down-regulated in both peptidergic and large DRG neurons. Our work suggests that mGluR7 might be involved in the regulation of pathological pain after peripheral nerve injury. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Glutamate is one of the most prominent excitatory transmitters in the nervous system. According to the mechanisms by which their activation gives rise to the postsynaptic current, glutamate receptors could be divided into two different groups, ionotropic glutamate receptors (iGluRs) and metabolic glutamate receptors (mGluRs) [17]. Different from the iGluRs, mGluRs belong to the seven transmembrane G-protein coupled receptors and have no channel activity. They could activate the downstream ion channels through a G-protein mediated signaling pathway [5]. Based on their signal transduction pathways and pharmacological profiles, mGluRs could be divided into three groups, groups I–III [2]. Both iGluRs and mGluRs have been shown to modulate the synaptic plasticity [4].
∗ Corresponding author. Tel.: +86 572 2021757; fax: +86 572 2023599. E-mail address: [email protected] (N. Wang). 1 These authors contribute equally to this work. 0304-3940/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2012.10.014
More and more evidences suggest that glutamate pathway is involved in the nociceptive perception and chronic pain. Vesicular glutamate transporter 2 (VGLUT2) has been proved to be involved in the TRPV1 thermal nociception and normal itch response [9,12]. VGLUT3, expressed in unmyelinated TRPV1-negative neurons, is critical for mechanical hypersensitivity caused by injury [20]. After sciatic nerve transection, the expression level of VGLUT1 and VGLUT2 is both down-regulated [1]. Pharmacological evidence shows that inhibition of the NMDA and AMPA, two major iGluRs, could significantly relief neuropathic pain and enhance the antinociceptive effects of the electroacupuncture [11,26]. Blocking the peripheral mGluR activity could increase the capsaicin-induced nociceptive behaviors and nociceptor activity [3]. Administration of AMN082, agonist of mGluR7, could significantly attenuate neuropathic pain and inflammatory pain, and enhance the analgesic effects of morphine [6,16]. Previous studies have shown that mGluR7 is widely expressed in central nervous system and peripheral nervous system [8,10], yet the expression profile of mGluR7 in the DRG neurons after peripheral nerve injury is still unclear. In this paper, our study shows that mGluR7 is expressed in peptidergic and large DRG neurons, but
J.-Y. Li et al. / Neuroscience Letters 531 (2012) 52–56
rarely in isolectin B4 (IB4) positive neurons. Sciatic nerve ligation experiment indicates that mGluR7 could be delivered to the peripheral terminal. The expression of mGluR7 was down-regulated after axotomy. The above evidences suggest the involvement of mGluR7 in regulation of pathological pain after peripheral nerve injury. 2. Materials and methods 2.1. Animals and surgery Animal care was performed in accordance with the policy of Society for Neuroscience (USA) for neuroscience research. The experimental protocol was approved by the Animal Care and Use Committee of Huzhou Central Hospital. Male Sprague-Dawley (SD) rats (∼200 g, Animal Center of Zhejiang Academy of Medical Sciences) were housed in the 12:12 h light–dark cycle room. To generate the neuropathic rat model, sciatic nerve was axotomized according to the literature [24]. In brief, after SD rats were anesthetized, 3–5 mm sciatic nerve from the left limb was transected. Rats were used for further experiments on the 2nd and 7th day after surgery. For the sciatic nerve ligation, sciatic nerve was exposed and ligated after SD rats were anesthetized. Rats were used for further experiments on the 1st day after surgery. 2.2. Immunohistochemistry After deep anesthesia, the L4–L5 DRGs were dissected and fixed in 4% paraformaldehyde for 4 h, following by cryoprotection in 20% sucrose in PBS. Samples were then sectioned on a cryostat at 10 m and mounted onto microscope slides (Fisher Scientific) for staining. Tissue was incubated in PBS with 0.3% Triton X-100 plus 5% bovine serum albumin (BSA, Sigma) for 1 h at room temperature. Primary antibodies diluted in PBS with 0.3% Triton X-100 and 1% BSA were added and incubated overnight at 4 ◦ C. After washed three times using PBS for 30 min, secondary antibodies were added and incubated with samples for 2 h at room temperature. After the slides were mounted, images were acquired with confocal microscope. The following primary antibodies were used: mGluR7 (1:500, Millipore-Chemicon), calcitonin gene-related peptide (CGRP; 1:500, Sigma), Substance P (SP; 1:500, Neuromics), IB4 (1:500, Sigma), neurofilament 200 (NF200; 1:1000, Sigma) and Vanilloid receptor (TrpV1; 1:100, Santa Cruz). 2.3. Western blot L4–L5 DRGs from normal and axotomized rats were dissected after rats were deep anesthetized. Total proteins were extracted using the RIPA buffer (Cell Signaling). Samples were then separated by SDS-PAGE, transferred and probed with primary antibodies overnight at 4 ◦ C. After reacted with secondary antibodies for 1 h at room temperature, samples were visualized with chemiluminescence (GE Healthcare Life Sciences). Primary antibodies against mGluR7 (1:500, Millipore-Chemicon) and actin (1:5000, Abcam) were used. 2.4. RT-PCR After deep anesthesia, L4–L5 DRGs were dissected and stored in −80 ◦ C. RNA from normal and axotomized rat DRGs was extracted with Trizol (Invitrogen) according to the protocol. Reverse transcript was then performed using M-MLV reverse transcriptase (Promega). The following primers were used for PCR to detect the mRNA expression: for mGluR7, 5 -TCAGTGGCGCTGGGAATGCT-3 and 5 -GCCAGGCTGGGTGACAGAAT-3 ; for GAPDH, 5 -ACAGCAAC AGGGTGGTGGAC-3 and 5 -TTTGAGGGTGCAGCGAACTT-3 .
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3. Results Previous studies have shown that mGluR7 is widely expressed in central nervous system and peripheral nervous system [8,10]. However, the detailed expression profile of mGluR7 in dorsal root ganglion neurons is still not quite clear. Immunohistochemistry experiment shows that 85.11% ± 2.19% DRG neurons could express mGluR7. To identify the subpopulation of mGluR7 positive DRG neurons, we analyzed co-expression of mGluR7 with SP, CGRP, IB4, TRPV1 and NF200. Double immunolabeling shows that 86.08% ± 3.35% SP positive neurons and 91.78% ± 2.96% CGRP positive neurons could express mGluR7. Almost all the NF200 positive large DRG neurons are mGluR7 positive. In contrast, only 21.77% ± 3.51% IB4 positive neurons could express mGluR7 (Fig. 1). These data suggest that mGluR7 is specifically expressed in the peptidergic neurons and large DRG neurons. Recent studies suggested that group II/III mGluRs could exert endogenous activitydependent modulation of TRPV1 receptors on peripheral nociceptors [3]. It has been shown that TRPV1 exclusively expressed in peptidergic and IB4 positive nociceptors [13]. To test the relationship between mGluR7 and TRPV1, we colabeled mGluR7 and TRPV1 in rat DRG neurons. We found that 71.09% ± 3.26% TRPV1 positive neurons could express mGluR7 (Fig. 1). These results indicate that mGluR7 might be involved in regulation of TRPV1 in DRG neurons. Former experiments showed that intraplantar-injected antagonists of group II/III mGluRs could increase the capsaicin-induced nociceptive behaviors [3]. To test whether mGluR7 could be transported to the peripheral site and exert its function, the sciatic nerve was ligated for 1 day to accumulate the proteins. Immunohistochemistry shows that mGluR7 was accumulated at the proximal site of DRG neurons. SP, which is anterogradely transported peptide and released at the nerve terminal, was also accumulated at the proximal site (Fig. 2). The above data suggest that mGluR7 could be delivered to the afferent terminals at the peripheral site and exert its function. To gain further insights into the mechanisms of mGluR7 regulation, we investigated the expression level of mGluR7 after sciatic nerve axotomy. RT-PCR experiment shows that mRNA level of mGluR7 is decreased 7 days after injury (Fig. 3A). Western blot was then performed and shows that the protein level is also decreased after axotomy (Fig. 3B). To study the expression profile of mGluR7 after nerve injury, immunostaining was carried out. As shown in Fig. 3C, the intensity of immunostaining is dramatically decreased 7 days after surgery. Moreover, only 55.87% ± 3.83% DRG neurons could express mGluR7, which is significantly lower than the normal DRG neurons (85.11% ± 2.19% are mGluR7 positive). Thus, mGluR7 is down-regulated after peripheral nerve injury. Downregulation of mGluR7 suggests the involvement of mGluR7 in the regulation of pathological pain after peripheral nerve injury.
4. Discussion In this study, we uncovered the distribution profile of mGluR7 in the peripheral nervous system. Our results indicated that mGluR7 is specifically expressed in peptidergic and large DRG neurons, but rarely in IB4 positive DRG neurons. It has been proved that peptidergic and IB4 positive fibers innervate different sites in the epidermis and project to the different lamina in the dorsal spinal cord. Peptidergic nerve endings exclusively terminate in the underlying stratum spinosum and mainly project to the lamina I and outer lamina II in the spinal cord, while IB4 positive fibers terminated about 10 m from the stratum corneum and mainly project to the inner lamina II of dorsal spinal cord [21,27]. Thus, mGluR7
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Fig. 1. mGluR7 is expressed in peptidergic and large DRG neurons but rarely in IB4 positive neurons. Double immunolabelings for mGluR7 and SP, CGRP, IB4, TRPV1 and NF200 are performed in adult rat DRG neurons. Scale bar: 100 m.
could specifically regulate the signal transduction pathway of the peptidergic DRG neurons. Previous study shows that intraplantar injection of group II/III mGluRs antagonists could increase capsaicin induced nociceptive behaviors and nociceptor activity, suggesting that endogenous
mGluRs could modulate the TRPV1 activities [3]. Here, we found that mGluR7, one of the group III mGluRs, is co-expressed with TRPV1 in the DRG neurons and could be transported to the peripheral site. Those above evidences indicate that endogenous mGluR7 might be able to modulate the TRPV1 activation.
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Fig. 2. Anterograde transport of mGluR7 in afferent fibers. After 1 day sciatic nerve ligation, immunolabeling shows that mGluR7 is accumulated at the proximal site to the DRG neuron cell bodies.
Fig. 3. mGluR7 is down-regulated after peripheral nerve injury. (A) RT-PCR shows that the mRNA level of mGluR7 is down-regulated after axotomy. (B) Western blot shows that the protein level of mGluR7 is down-regulated. (C) Seven days after sciatic nerve axotomy, immunolabeling shows that the expression of mGluR7 is down-regulated. Scale bar: 100 m.
Further sciatic nerve ligation shows that mGluR7 accumulated at the proximal site to the DRG cell bodies, indicating that mGluR7 could be delivered to the peripheral site. It is known that systematic administration of agonist AMN082, could significantly relief neuropathic and inflammatory pain [6,16]. However, AMN082 was found to be able to cross the brain–blood barrier [14], it might induce strong side effects after systematic administration. Our present study reveals the possibility that administration AMN082 at peripheral site to attenuate the neuropathic and inflammatory pain. Following the peripheral nerve injury, change in the expression of ion channels, receptors, neuropeptides or other molecules might contribute to the generation and maintenance of chronic pain [25]. As a receptor of glutamate, mGluR7 has been implicated in synaptic transmission block through inhibiting P/Q-type Ca2+ channels in many areas [15,18,22]. The excitability of neurons is significantly increased in mGluR7 knockout mice [19]. In this study, we found the expression of mGluR7 is significantly down-regulated after axotomy, which might contribute to the spontaneous activity of the
injured peripheral nerve and DRG neurons [7,23]. Down-regulation of mGluR7 supports the possibility that mGluR7 might be one of the key regulators involved in the development of chronic neuropathic pain.
Author contributions J.-Y.L., X.W. and N.W. conceived and designed the experiments; J.-Y.L., X.W., P.-T.J., X.-F.L., G.-H.G., X.-S.J. and G.-S.Z. performed the experiments; F.H., X.W. and J.-Y.L. analyzed the data; J.-Y.L., X.W. and N.W. wrote the paper.
Acknowledgments This study was supported by the grant from National Natural Science Foundation of China (31171024), Medical Scientific Research Foundation of Zhejiang Province (2009B153 and 2012RCB039),
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Huzhou Science and Technology R&D Fund (2009YS04, 2010GS09 and 2012YS02). References [1] P. Brumovsky, M. Watanabe, T. Hokfelt, Expression of the vesicular glutamate transporters-1 and -2 in adult mouse dorsal root ganglia and spinal cord and their regulation by nerve injury, Neuroscience 147 (2007) 469–490. [2] K.R. Byrnes, D.J. Loane, A.I. Faden, Metabotropic glutamate receptors as targets for multipotential treatment of neurological disorders, Neurotherapeutics 6 (2009) 94–107. [3] S.M. Carlton, S. Zhou, R. Govea, J. Du, Group II/III metabotropic glutamate receptors exert endogenous activity-dependent modulation of TRPV1 receptors on peripheral nociceptors, Journal of Neuroscience 31 (2011) 12727–12737. [4] D. Debanne, G. Daoudal, V. Sourdet, M. Russier, Brain plasticity and ion channels, Journal of Physiology (Paris) 97 (2003) 403–414. [5] G.K. Dhami, S.S. Ferguson, Regulation of metabotropic glutamate receptor signaling, desensitization and endocytosis, Pharmacology & Therapeutics 111 (2006) 260–271. [6] S. Dolan, M.D. Gunn, L. Biddlestone, A.M. Nolan, The selective metabotropic glutamate receptor 7 allosteric agonist AMN082 inhibits inflammatory pain-induced and incision-induced hypersensitivity in rat, Behavioural Pharmacology 20 (2009) 596–604. [7] K.C. Kajander, G.J. Bennett, Onset of a painful peripheral neuropathy in rat: a partial and differential deafferentation and spontaneous discharge in A beta and A delta primary afferent neurons, Journal of Neurophysiology 68 (1992) 734–744. [8] J.M. Kinzie, J.A. Saugstad, G.L. Westbrook, T.P. Segerson, Distribution of metabotropic glutamate receptor 7 messenger RNA in the developing and adult rat brain, Neuroscience 69 (1995) 167–176. [9] M.C. Lagerstrom, K. Rogoz, B. Abrahamsen, E. Persson, B. Reinius, K. Nordenankar, C. Olund, C. Smith, J.A. Mendez, Z.F. Chen, J.N. Wood, A. WallenMackenzie, K. Kullander, VGLUT2-dependent sensory neurons in the TRPV1 population regulate pain and itch, Neuron 68 (2010) 529–542. [10] J.L. Li, H. Ohishi, T. Kaneko, R. Shigemoto, A. Neki, S. Nakanishi, N. Mizuno, Immunohistochemical localization of a metabotropic glutamate receptor, mGluR7, in ganglion neurons of the rat; with special reference to the presence in glutamatergic ganglion neurons, Neuroscience Letters 204 (1996) 9–12. [11] X.J. Liu, J.R. Gingrich, M. Vargas-Caballero, Y.N. Dong, A. Sengar, S. Beggs, S.H. Wang, H.K. Ding, P.W. Frankland, M.W. Salter, Treatment of inflammatory and neuropathic pain by uncoupling Src from the NMDA receptor complex, Nature Medicine 14 (2008) 1325–1332. [12] Y. Liu, O. Abdel Samad, L. Zhang, B. Duan, Q. Tong, C. Lopes, R.R. Ji, B.B. Lowell, Q. Ma, VGLUT2-dependent glutamate release from nociceptors is required to sense pain and suppress itch, Neuron 68 (2010) 543–556. [13] G.J. Michael, J.V. Priestley, Differential expression of the mRNA for the vanilloid receptor subtype 1 in cells of the adult rat dorsal root and nodose ganglia and its downregulation by axotomy, Journal of Neuroscience 19 (1999) 1844–1854.
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Contents lists available at SciVerse ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Peripheral nerve injury decreases the expression of metabolic glutamate receptor 7 in dorsal root ganglion neurons Jian-You Li a,1 , Xiang Wang b,1 , Peng-Tian Ji b , Xiong-Feng Li a , Guo-Hua Guan a , Xue-Sheng Jiang a , Guo-shun Zhou a , Feng Hua b , Nan Wang c,∗ a
Orthopedics Department, Huzhou Central Hospital, 198 Hongqi Road, Huzhou 313000, People’s Republic of China Huzhou Key Laboratory of Molecular Medicine, Huzhou Central Hospital, 198 Hongqi Road, Huzhou 313000, People’s Republic of China c Medical Department, Huzhou Traditional Chinese Medicine Hospital, 82 Nanjie Road, Huzhou 313000, People’s Republic of China b
h i g h l i g h t s mGluR7 is expressed in peptidergic and large DRG neurons but not in IB4+ neurons. mGluR7 is anterogradely transported from DRG cell body to the peripheral site. After peripheral nerve injury, the expression of mGluR7 is down-regulated.
a r t i c l e
i n f o
Article history: Received 4 September 2012 Accepted 9 October 2012 Keywords: mGluR7 Dorsal root ganglion Axotomy Neuropathic pain
a b s t r a c t Group II and III metabolic glutamate receptors (mGluRs) are responsible for the glutamate-mediated postsynaptic excitation of neurons. Previous pharmacological evidences show that activation of mGluR7 could inhibit nociceptive reception. However, the distribution and expression patterns of mGluR7 after peripheral injury remain unclear. Herein we found that mGluR7 was expressed in the rat peptidergic dorsal root ganglion (DRG) neurons and large neurons, but rarely in isolectin B4 positive neurons. Sciatic nerve ligation experiment showed that mGluR7 was anterogradely transported from cell body to the peripheral site. Furthermore, after peripheral nerve injury, mGluR7 expression was down-regulated in both peptidergic and large DRG neurons. Our work suggests that mGluR7 might be involved in the regulation of pathological pain after peripheral nerve injury. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Glutamate is one of the most prominent excitatory transmitters in the nervous system. According to the mechanisms by which their activation gives rise to the postsynaptic current, glutamate receptors could be divided into two different groups, ionotropic glutamate receptors (iGluRs) and metabolic glutamate receptors (mGluRs) [17]. Different from the iGluRs, mGluRs belong to the seven transmembrane G-protein coupled receptors and have no channel activity. They could activate the downstream ion channels through a G-protein mediated signaling pathway [5]. Based on their signal transduction pathways and pharmacological profiles, mGluRs could be divided into three groups, groups I–III [2]. Both iGluRs and mGluRs have been shown to modulate the synaptic plasticity [4].
∗ Corresponding author. Tel.: +86 572 2021757; fax: +86 572 2023599. E-mail address: [email protected] (N. Wang). 1 These authors contribute equally to this work. 0304-3940/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2012.10.014
More and more evidences suggest that glutamate pathway is involved in the nociceptive perception and chronic pain. Vesicular glutamate transporter 2 (VGLUT2) has been proved to be involved in the TRPV1 thermal nociception and normal itch response [9,12]. VGLUT3, expressed in unmyelinated TRPV1-negative neurons, is critical for mechanical hypersensitivity caused by injury [20]. After sciatic nerve transection, the expression level of VGLUT1 and VGLUT2 is both down-regulated [1]. Pharmacological evidence shows that inhibition of the NMDA and AMPA, two major iGluRs, could significantly relief neuropathic pain and enhance the antinociceptive effects of the electroacupuncture [11,26]. Blocking the peripheral mGluR activity could increase the capsaicin-induced nociceptive behaviors and nociceptor activity [3]. Administration of AMN082, agonist of mGluR7, could significantly attenuate neuropathic pain and inflammatory pain, and enhance the analgesic effects of morphine [6,16]. Previous studies have shown that mGluR7 is widely expressed in central nervous system and peripheral nervous system [8,10], yet the expression profile of mGluR7 in the DRG neurons after peripheral nerve injury is still unclear. In this paper, our study shows that mGluR7 is expressed in peptidergic and large DRG neurons, but
J.-Y. Li et al. / Neuroscience Letters 531 (2012) 52–56
rarely in isolectin B4 (IB4) positive neurons. Sciatic nerve ligation experiment indicates that mGluR7 could be delivered to the peripheral terminal. The expression of mGluR7 was down-regulated after axotomy. The above evidences suggest the involvement of mGluR7 in regulation of pathological pain after peripheral nerve injury. 2. Materials and methods 2.1. Animals and surgery Animal care was performed in accordance with the policy of Society for Neuroscience (USA) for neuroscience research. The experimental protocol was approved by the Animal Care and Use Committee of Huzhou Central Hospital. Male Sprague-Dawley (SD) rats (∼200 g, Animal Center of Zhejiang Academy of Medical Sciences) were housed in the 12:12 h light–dark cycle room. To generate the neuropathic rat model, sciatic nerve was axotomized according to the literature [24]. In brief, after SD rats were anesthetized, 3–5 mm sciatic nerve from the left limb was transected. Rats were used for further experiments on the 2nd and 7th day after surgery. For the sciatic nerve ligation, sciatic nerve was exposed and ligated after SD rats were anesthetized. Rats were used for further experiments on the 1st day after surgery. 2.2. Immunohistochemistry After deep anesthesia, the L4–L5 DRGs were dissected and fixed in 4% paraformaldehyde for 4 h, following by cryoprotection in 20% sucrose in PBS. Samples were then sectioned on a cryostat at 10 m and mounted onto microscope slides (Fisher Scientific) for staining. Tissue was incubated in PBS with 0.3% Triton X-100 plus 5% bovine serum albumin (BSA, Sigma) for 1 h at room temperature. Primary antibodies diluted in PBS with 0.3% Triton X-100 and 1% BSA were added and incubated overnight at 4 ◦ C. After washed three times using PBS for 30 min, secondary antibodies were added and incubated with samples for 2 h at room temperature. After the slides were mounted, images were acquired with confocal microscope. The following primary antibodies were used: mGluR7 (1:500, Millipore-Chemicon), calcitonin gene-related peptide (CGRP; 1:500, Sigma), Substance P (SP; 1:500, Neuromics), IB4 (1:500, Sigma), neurofilament 200 (NF200; 1:1000, Sigma) and Vanilloid receptor (TrpV1; 1:100, Santa Cruz). 2.3. Western blot L4–L5 DRGs from normal and axotomized rats were dissected after rats were deep anesthetized. Total proteins were extracted using the RIPA buffer (Cell Signaling). Samples were then separated by SDS-PAGE, transferred and probed with primary antibodies overnight at 4 ◦ C. After reacted with secondary antibodies for 1 h at room temperature, samples were visualized with chemiluminescence (GE Healthcare Life Sciences). Primary antibodies against mGluR7 (1:500, Millipore-Chemicon) and actin (1:5000, Abcam) were used. 2.4. RT-PCR After deep anesthesia, L4–L5 DRGs were dissected and stored in −80 ◦ C. RNA from normal and axotomized rat DRGs was extracted with Trizol (Invitrogen) according to the protocol. Reverse transcript was then performed using M-MLV reverse transcriptase (Promega). The following primers were used for PCR to detect the mRNA expression: for mGluR7, 5 -TCAGTGGCGCTGGGAATGCT-3 and 5 -GCCAGGCTGGGTGACAGAAT-3 ; for GAPDH, 5 -ACAGCAAC AGGGTGGTGGAC-3 and 5 -TTTGAGGGTGCAGCGAACTT-3 .
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3. Results Previous studies have shown that mGluR7 is widely expressed in central nervous system and peripheral nervous system [8,10]. However, the detailed expression profile of mGluR7 in dorsal root ganglion neurons is still not quite clear. Immunohistochemistry experiment shows that 85.11% ± 2.19% DRG neurons could express mGluR7. To identify the subpopulation of mGluR7 positive DRG neurons, we analyzed co-expression of mGluR7 with SP, CGRP, IB4, TRPV1 and NF200. Double immunolabeling shows that 86.08% ± 3.35% SP positive neurons and 91.78% ± 2.96% CGRP positive neurons could express mGluR7. Almost all the NF200 positive large DRG neurons are mGluR7 positive. In contrast, only 21.77% ± 3.51% IB4 positive neurons could express mGluR7 (Fig. 1). These data suggest that mGluR7 is specifically expressed in the peptidergic neurons and large DRG neurons. Recent studies suggested that group II/III mGluRs could exert endogenous activitydependent modulation of TRPV1 receptors on peripheral nociceptors [3]. It has been shown that TRPV1 exclusively expressed in peptidergic and IB4 positive nociceptors [13]. To test the relationship between mGluR7 and TRPV1, we colabeled mGluR7 and TRPV1 in rat DRG neurons. We found that 71.09% ± 3.26% TRPV1 positive neurons could express mGluR7 (Fig. 1). These results indicate that mGluR7 might be involved in regulation of TRPV1 in DRG neurons. Former experiments showed that intraplantar-injected antagonists of group II/III mGluRs could increase the capsaicin-induced nociceptive behaviors [3]. To test whether mGluR7 could be transported to the peripheral site and exert its function, the sciatic nerve was ligated for 1 day to accumulate the proteins. Immunohistochemistry shows that mGluR7 was accumulated at the proximal site of DRG neurons. SP, which is anterogradely transported peptide and released at the nerve terminal, was also accumulated at the proximal site (Fig. 2). The above data suggest that mGluR7 could be delivered to the afferent terminals at the peripheral site and exert its function. To gain further insights into the mechanisms of mGluR7 regulation, we investigated the expression level of mGluR7 after sciatic nerve axotomy. RT-PCR experiment shows that mRNA level of mGluR7 is decreased 7 days after injury (Fig. 3A). Western blot was then performed and shows that the protein level is also decreased after axotomy (Fig. 3B). To study the expression profile of mGluR7 after nerve injury, immunostaining was carried out. As shown in Fig. 3C, the intensity of immunostaining is dramatically decreased 7 days after surgery. Moreover, only 55.87% ± 3.83% DRG neurons could express mGluR7, which is significantly lower than the normal DRG neurons (85.11% ± 2.19% are mGluR7 positive). Thus, mGluR7 is down-regulated after peripheral nerve injury. Downregulation of mGluR7 suggests the involvement of mGluR7 in the regulation of pathological pain after peripheral nerve injury.
4. Discussion In this study, we uncovered the distribution profile of mGluR7 in the peripheral nervous system. Our results indicated that mGluR7 is specifically expressed in peptidergic and large DRG neurons, but rarely in IB4 positive DRG neurons. It has been proved that peptidergic and IB4 positive fibers innervate different sites in the epidermis and project to the different lamina in the dorsal spinal cord. Peptidergic nerve endings exclusively terminate in the underlying stratum spinosum and mainly project to the lamina I and outer lamina II in the spinal cord, while IB4 positive fibers terminated about 10 m from the stratum corneum and mainly project to the inner lamina II of dorsal spinal cord [21,27]. Thus, mGluR7
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Fig. 1. mGluR7 is expressed in peptidergic and large DRG neurons but rarely in IB4 positive neurons. Double immunolabelings for mGluR7 and SP, CGRP, IB4, TRPV1 and NF200 are performed in adult rat DRG neurons. Scale bar: 100 m.
could specifically regulate the signal transduction pathway of the peptidergic DRG neurons. Previous study shows that intraplantar injection of group II/III mGluRs antagonists could increase capsaicin induced nociceptive behaviors and nociceptor activity, suggesting that endogenous
mGluRs could modulate the TRPV1 activities [3]. Here, we found that mGluR7, one of the group III mGluRs, is co-expressed with TRPV1 in the DRG neurons and could be transported to the peripheral site. Those above evidences indicate that endogenous mGluR7 might be able to modulate the TRPV1 activation.
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Fig. 2. Anterograde transport of mGluR7 in afferent fibers. After 1 day sciatic nerve ligation, immunolabeling shows that mGluR7 is accumulated at the proximal site to the DRG neuron cell bodies.
Fig. 3. mGluR7 is down-regulated after peripheral nerve injury. (A) RT-PCR shows that the mRNA level of mGluR7 is down-regulated after axotomy. (B) Western blot shows that the protein level of mGluR7 is down-regulated. (C) Seven days after sciatic nerve axotomy, immunolabeling shows that the expression of mGluR7 is down-regulated. Scale bar: 100 m.
Further sciatic nerve ligation shows that mGluR7 accumulated at the proximal site to the DRG cell bodies, indicating that mGluR7 could be delivered to the peripheral site. It is known that systematic administration of agonist AMN082, could significantly relief neuropathic and inflammatory pain [6,16]. However, AMN082 was found to be able to cross the brain–blood barrier [14], it might induce strong side effects after systematic administration. Our present study reveals the possibility that administration AMN082 at peripheral site to attenuate the neuropathic and inflammatory pain. Following the peripheral nerve injury, change in the expression of ion channels, receptors, neuropeptides or other molecules might contribute to the generation and maintenance of chronic pain [25]. As a receptor of glutamate, mGluR7 has been implicated in synaptic transmission block through inhibiting P/Q-type Ca2+ channels in many areas [15,18,22]. The excitability of neurons is significantly increased in mGluR7 knockout mice [19]. In this study, we found the expression of mGluR7 is significantly down-regulated after axotomy, which might contribute to the spontaneous activity of the
injured peripheral nerve and DRG neurons [7,23]. Down-regulation of mGluR7 supports the possibility that mGluR7 might be one of the key regulators involved in the development of chronic neuropathic pain.
Author contributions J.-Y.L., X.W. and N.W. conceived and designed the experiments; J.-Y.L., X.W., P.-T.J., X.-F.L., G.-H.G., X.-S.J. and G.-S.Z. performed the experiments; F.H., X.W. and J.-Y.L. analyzed the data; J.-Y.L., X.W. and N.W. wrote the paper.
Acknowledgments This study was supported by the grant from National Natural Science Foundation of China (31171024), Medical Scientific Research Foundation of Zhejiang Province (2009B153 and 2012RCB039),
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