Neurol Sci DOI 10.1007/s10072-014-1774-9
ORIGINAL ARTICLE
Connections between EM2- and SP-containing terminals and GABAergic neurons in the mouse spinal dorsal horn Dao-Shu Luo • Jing Huang • Yu-Lin Dong • Zhen-Yu Wu • Yan-Yan Wei • Ya-Cheng Lu • Ya-Yun Wang • Yuchio Yanagawa Sheng-Xi Wu • Wei Wang • Yun-Qing Li
•
Received: 16 January 2014 / Accepted: 25 March 2014 Ó Springer-Verlag Italia 2014
Abstract Endomorphin-2 (EM2) demonstrates a potent antinociceptive effect in pain modulation. To investigate the potential interactions of EM2- and substance P (SP)containing primary afferents and c-amino butyric acid (GABA)-containing interneurons in lamina II in nociceptive transmission, connections between EM2- and SPcontaining terminals and GABAergic neurons in the spinal dorsal horn were studied. Double-immunofluorescent labeling showed that approximately 62.3 % of EM2immunoreactive neurons exhibited SP-immunostaining, and 76.9 % of SP-immunoreactive neurons demonstrated EM2-immunoreactivities in the dorsal root ganglion (DRG). Dense double-labeled EM2- and SP-immunoreactivities were mainly observed in lamina II of the lumbar
dorsal horn. Furthermore, triple-immunofluorescent labeling results revealed that EM2 and SP double-labeled terminals overlapped with GABAergic neurons. Immunoelectron microscopy confirmed that the EM2- or SPimmunoreactive terminals formed synapses with GABAimmunoreactive dendrites in lamina II of the lumbar dorsal horn. During noxious information transmission induced by formalin plantar injection, GABAergic neurons expressing FOS in their nuclei were contacted with EM2- or SPimmunoreactive terminals. These results suggest that the interactions between EM2- and SP-containing terminals and GABAergic interneurons in the lamina II influence pain transmission and modulation in the spinal dorsal horn. Keywords Endomorphin 2 Substance P C-Amino butyric acid Interaction Spinal dorsal horn
D.-S. Luo and J. Huang contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s10072-014-1774-9) contains supplementary material, which is available to authorized users. D.-S. Luo W. Wang (&) Y.-Q. Li Department of Anatomy, Histology and Embryology, Basic Medical College, Fujian Medical University, Fuzhou 350004, China e-mail: [email protected] J. Huang Y.-L. Dong Z.-Y. Wu Y.-Y. Wei Y.-C. Lu Y.-Y. Wang S.-X. Wu Y.-Q. Li (&) Department of Anatomy, Histology and Embryology, K.K. Leung Brain Research Centre, The Fourth Military Medical University, No. 169 West Changle Road, Xi’an 710032, People’s Republic of China e-mail: [email protected] Y. Yanagawa Department of Genetic and Behavioral Neuroscience, Gunma University Graduate School of Medicine, Maebashi 371-8511, Japan
Introduction The superficial laminae (laminae I and II) of the spinal dorsal horn play a critical role in the transmission and modulation of nociceptive information conveyed via primary afferents [1]. Inhibitory interneurons play an important role in regulating spinal excitability by releasing camino butyric acid (GABA) [2]. In immunocytochemical studies, approximately 30 % of the lamina II neurons show GABA-immunoreactivity [3]. Primary afferent fibers either directly contact projection neurons in lamina I or indirectly contact with projection neurons via interneurons in lamina II [4]. Endomorphin-2 (EM2) is an endogenous opioid peptide, and EM2-immunoreactive primary afferent terminals are densely located in the superficial laminae of the spinal dorsal horn [5]. Substance P (SP), a neuropeptide, is involved in the transmission and modulation of nociceptive
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information in the nervous system [6]. Previous studies have shown that EM2-like immunoreactivity co-localized with SP in both the dorsal root ganglion (DRG) and spinal dorsal horn and that EM2 might modulate the release of SP [7]. However, the connections between EM2/SP co-localized terminals and GABAergic neurons in the spinal dorsal horn remain unclear. Therefore, the principal purpose of this study was to determine whether EM2/SP co-localized terminals are in contact with GABAergic neurons in the spinal dorsal horn, using light and electron microscopic techniques. To aid in the identification of GABAergic neurons and overcome the difficulty in staining GABAergic neurons with traditional immunohistochemical methods, we used the glutamic acid decarboxylase 67-green fluorescence protein (GAD67-GFP) knock-in mouse, in which GABAergic neurons are identified by the expression of GFP.
The specificity of GFP-positive GABAergic neurons has been confirmed in our previous studies [8, 9]. According to previous studies from others [10] and ours [11], the subcutaneous injection of formalin significantly enhances the spinal dorsal horn neuronal activation indicated by nuclei FOS expression, which reaches a peak at 2 h after formalin injection in both rat and mouse [10]. Recent studies further suggested that GABAergic neurons within the spinal dorsal horn are activated during inflammatory pain. Therefore, we hypothesized that EM2/SP co-localized terminals may regulate the neuronal excitability of GABAergic neurons to regulate nociceptive transmission. To offer morphological evidence for our hypothesis, we investigated the connections between EM2/SP co-localized terminals and FOS-positive GABAergic neurons in the dorsal horn after formalin plantar injection to examine their potential interactions during noxious stimulation.
Table 1 Antisera used in each group Group
Purposes
Primary antisera
Immunofluorescence
EM2/SP
Rabbit anti-EM2 (1:200, Abcam) rat anti-SP (1:500, Millipore)
Secondary antisera
Fluorophore-conjugated avidin
Biotinylated donkey anti-rabbit IgG (1:500, vector labs) Alexa 488 donkey anti-rat IgG (1:500, Molecular probes)
EM2/SP/GFP
Rabbit anti-EM2 (1:200) rat anti-SP (1:500)
Biotinylated donkey anti-rabbit IgG (1:500)
Cy3-labeled avidin D (1:1,000, molecular probes) Cy3-labeled avidin D (1:1,000)
Alexa 647 donkey anti-rat IgG (1:500, molecular probes)
EM2/FOS/GFP
SP/FOS/GFP
Guinea pig anti-GFP (1:100,synaptic systems)
Alexa 488 donkey anti-guinea pig IgG (1:500, Molecular probes)
Rabbit anti-EM2 (1:200) mouse anti-FOS (1:500, Millipore)
Biotinylated donkey anti-rabbit IgG (1:500) Alexa 647 donkey anti-mouse IgG (1:500, molecular probes)
Guinea pig anti-GFP (1:100)
Alexa 488 donkey anti-guinea pig IgG (1:500)
Rat anti-SP (1:500) mouse anti-FOS (1:500, Millipore)
Biotinylated donkey anti-rat IgG (1:500, vector labs)
Cy3-labeled avidin D (1:1,000)
Cy3-labeled avidin D (1:1,000)
Alexa 647 donkey anti-mouse IgG (1:500)
Electronmicroscopy
Guinea pig anti-GFP (1:100)
Alexa 488 donkey anti-guinea pig IgG (1:500)
GFP/SP
Mouse anti-GFP (1:500,synaptic systems) rabbit anti-SP (1:500, Millipore)
Biotinylated anti-mouse IgG (1:100, Vector labs) goat antirabbit IgG antibody conjugated to 1.4 nm gold particles (nanoprobes)
Avidin-biotinylated peroxidase complex (1:50, Vector labs)
GFP/EM2
Mouse anti-GFP (1:500) rabbit antiEM2 (1:200)
Biotinylated anti-mouse IgG (1:100)
Avidin-biotinylated peroxidase complex (1:50)
goat anti-rabbit IgG antibody conjugated to 1.4 nm gold particles (nanoprobes)
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Six adult C57BL/6 and 24 GAD67-GFP knock-in mice were used in the present study. The experimental procedures in this study were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23) revised 1996 and IASP’s guidelines for pain research in conscious animals [12] and were approved by The Committee of
Animal Use for Research and Education in The Fourth Military Medical University (Xi’an, P.R. China). All efforts were made to minimize the number of animals used and their suffering. Mice were divided into five groups. Group 1 (six C57BL/6 mice) was used for EM2 and SP double-immunofluorescent histochemical staining in the DRG and spinal dorsal horn. Group 2 (six GAD67-GFP knock-in mice) was used for EM2/SP/GFP triple-immunofluorescent histochemical staining in the dorsal horn. Group 3 (six GAD67GFP knock-in mice) was used for subcutaneous injection of
Fig. 1 Double-immunofluorescent staining showing the labeling of endomorphin-2- (EM2, a; red) and substance P- (SP, b; green) immunoreactive neurons in the dorsal root ganglion (DRG). In the
merged image (c), some EM2-immunoreactive neurons also showed SP-immunoreactivities. Arrows point to double-labeled neurons (yellow). Scale bar = 100 lm (a–c) (color figure online)
Fig. 2 Double-labeling of EM2 and SP in the spinal dorsal horn of the mouse. EM2- (a; red) and SP-immunoreactive (b; green) terminals are concentrated in the superficial laminae of the dorsal horn. The merged image reveals extensive overlap of EM2 and SPimmunoreactive terminals (c; yellow) in lamina I and outer lamina II
of the dorsal horn. d–f Enlarged view of EM2 and SP immunoreactivity in the superficial laminae of the dorsal horn. The great majority of EM2 and SP are co-located at primary afferent terminals (arrows). Scale bars = 100 lm (a–c), 5 lm (d–f) (color figure online)
Materials and methods Animals
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5 % formalin solution (25 ll) into the plantar surface of the left hind paw 2 h before the perfusion [13]. Group 4 (six GAD67-GFP knock-in mice) as a control group, that formalin injection was replaced with 25 ll of saline. Group 3 and Group 4 were processed for EM2/FOS/GFP tripleimmunofluorescent histochemical staining. Group 5 (six GAD67-GFP knock-in mice) was used for electron-microscopic double-immunohistochemical staining of EM2/GFP or SP/GFP. Immunofluorescent histochemistry The mice from Group 1 to Group 4 were anesthetized with sodium pentobarbital (45 mg/kg) and then perfused transcardially with 0.1 M phosphate buffer (PB, pH 7.4) containing 4 % (w/v) paraformaldehyde. The lumbar spinal cord (L4–L5) was cut into 30 lm thick sections and corresponding DRG was cut into 10 lm thick sections on a freezing microtome (Kryostat 1,720; Leitz, Mannheim, Germany). The immunofluorescent histochemical staining protocols used in the present study were the same as those in our previous studies [14], and all the antisera used in each group are shown in Table 1. Briefly, the sections were incubated at room temperature sequentially with primary
Fig. 3 Triple-labeling of EM2, SP, and green fluorescence protein (GFP) in the spinal dorsal horn of the GAD67-GFP knock-in mice. a-c: EM2 (a; red), SP (b; blue), and GFP (c; green) immunoreactivity in the spinal dorsal horn. d–e: EM2 (d; red) and SP-immunoreactive terminals
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antibodies for 72 h, then incubated in the mixture of species-specific biotinylated secondary antibody and fluorochrome-labeled secondary antibody for 4 h, followed by fluorophore-conjugated avidin D for 1 h. The specificities of the staining were tested on the sections in another dish by omitting the specific primary specific antibodies. No immunoreactivities were found on the sections, as expected. The sections were observed under a confocal laserscanning microscope (Fluoview 1,000, Olympus, Tokyo, Japan). Digital images were captured using Fluoview software (Olympus). Immuno-electron microscopy For electron microscope study, six deeply anesthetized adult GAD67-GFP knock-in mice were perfused transcardially with 0.1 M PB containing 4 % paraformaldehyde, 0.05 % glutaraldehyde and 0.2 % picric acid. The lumbar spinal cord (L4–L5) was cut into 50 lm thick sections on a vibratome (Microslicer DTK-100; Dosaka, Kyoto, Japan). Sections were freeze–thawed in liquid nitrogen after cryoprotection, to enhance penetration of antibody. Details of these immuno-electron microscopy procedures were described in our previous study [15, 16]. Briefly, the
(e; blue) contacted GFP-positive (green) neurons in the dorsal horn (single arrow). f: Double-labeling of EM2 and SP-immunoreactive terminals (purple) contacting GFP-positive neurons (double arrowheads). Scale bars = 100 lm (a–c), 4 lm (d–f) (color figure online)
Neurol Sci
sections were incubated with 0.05 M Tris-buffered saline (TBS; pH7.4) containing 20 % normal goat serum for 1 h to block non-specific immunoreactivity, and then collected in two dishes. The sections in each dish were incubated for 24 h at 4 °C with a mixture of primary antibodies as GFP/ SP and GFP/EM2 (Table 1), respectively. Then, a mixture of biotinylated donkey anti-mouse IgG and goat anti-rabbit IgG conjugated with 1.4 nm gold particles for SP or EM2 (Table 1) were incubated overnight at 48 °C. Subsequently the sections were processed by the following steps: (1) postfixation with glutaraldehyde, (2) silver enhancement with HQ Silver Kit (Nanoprobes), (3) incubation with ABC kit (Vector), (4) reaction with diaminobenzidine tetrahydrochloride and H2O2, (5) osmification, (6) conterstaining with uranylacetate. Ultrathin sections at 70 nm thickness were prepared from the superficial laminae of the spinal dorsal horn, mounted on single-slot grids, and examined with an electron microscope (JEM1440, Tokyo, Japan).
immunoreactive terminals overlapped with the GFP/FOS double-labeled neurons in the spinal dorsal horn (Fig. 5).
Discussion
Results
Our previous results have shown that SP released from the primary afferent fibers may activate GABAergic inhibitory neurons in lamina II [16]. The GABAergic inhibitory neurons may inhibit the activity of ascending projection neurons in lamina I of the medullary dorsal horn [17]. These results suggest that GABAergic interneurons play an important role in regulating the transmission of nociceptive information to the spinal cord. It possibly because the difficulty in GABA staining with traditional immunohistochemical methods, the co-localization of EM2 with GABA was not observed in the spinal dorsal horn of rat [18]. To take full advantage of GAD67GFP knock-in mice to identify GABAergic neurons in our study, it is the first report showing that EM2/SP co-localized fibers/terminals make asymmetric synapses with the
In the DRG, EM2- and SP-immunoreactive were localized in small (the largest diameter \20 lm) to medium (the longest diameter 20–35 lm) sized neurons (Fig. 1a, b). Colocalization of EM2 and SP was found in the DRG neurons (Fig. 1c). Quantitative analysis showed that approximately 62.3 % of EM2-immunoreactive cell bodies contained SP-immunoreactivity and approximately 76.9 % of SP-immunoreactive cell bodies were immunoreactive for EM2. On the other hand, in the spinal cord, dense EM2- and SP-immunoreactive fibers and terminals were found to be concentrated in the superficial laminae of the dorsal horn (Fig. 2a, b, d, e), with extensive overlap (Fig. 2c, f). A large number of GFP-immunoreactive neurons were observed in lamina II of the spinal dorsal horn from the GAD67-GFP knock-in mouse (Fig. 3c). Triple-immunofluorescent labeling revealed that the EM2- and SPimmunoreactive fibers/terminals located on the periphery of the GFP-immunoreactive spinal dorsal horn neurons (Fig. 3d, e). Interestingly, some EM2/SP co-localized terminals closely connected with GFP-immunoreactive neurons in the superficial laminae, especially in lamina II of the dorsal horn (Fig. 3f. Under electron microscope, SPand EM2-immunopositive immunogold particles were observed within the thin and unmyelinated axons which make asymmetric synaptic contacts with dendritic profiles of the GFP-immunoreactive GABAergic neurons (Fig. 4a, b). In the formalin plantar injection animals, approximately 30.6 % of the GFP-positive GABAergic neurons expressed FOS in the spinal dorsal horn, and the EM2- or SP-
Fig. 4 Electron micrographs showing the synaptic connections between GABAergic dendrites and SP- or EM2-immunoreactive terminals in laminae I and II of the lumbar dorsal horn. Both (a) SPand (b) EM2-immunoreactive pre-synaptic terminals contained immune-gold particles (arrows) and formed asymmetric synapses with the post-synaptic GABA-immunopositive dendritic profiles (labeled with DAB reaction products). Scale bars = 0.2 lm (a), 0.4 lm (b)
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Neurol Sci Fig. 5 Triple-labeling of GFP (green), FOS (blue), and EM2 or SP (red) in the ipsilateral spinal dorsal horn of the GAD67-GFP knock-in mice following formalin injection into the hind paw plantar. b: A magnified image of the rectangle area is indicated in (a). Note that some of the SPimmunoreactive terminals formed close contacts with GFP/FOS double-labeled neurons (arrows). (d): The magnified image of the rectangle area is indicated in (c). Note that some of the EM2immunoreactive terminals formed close contacts with GFP/FOS double-labeled neurons (arrows). Scale bars = 10 lm (a, c), 2 lm (b, d) (color figure online)
GABAergic neurons in the superficial laminae of the spinal dorsal horn. Furthermore, EM2 and SP-immunoreactive terminals form close contacts with GABAergic neurons which were activated by peripheral noxious stimuli, suggesting these connections were involved in the processing of peripheral nociceptive transmission. As a l-opioid receptor agonist, EM2 is believed to presynaptically inhibit SP release from primary sensory neurons in DRG and the primary afferent terminals in the spinal dorsal horn, dulling the sensation of pain and alleviating neuropathic pain [7, 15, 16]. Therefore, the interaction of EM2 and SP may affect the modulation of nociceptive information transmitted from the peripheral to central nervous system. GABA is believed to be involved in presynaptic and postsynaptic inhibition in the spinal dorsal horn [19]. GABAergic inhibitory neurons in the superficial laminae of the dorsal horn can be activated by subcutaneous injection of formalin into the hind paw of rats, and these GABAergic neurons can also express FOS after noxious stimulation [20]. Therefore, the result of EM2 and SP overlapped with the FOS-positive GABAergic neurons in the dorsal horn may contribute to further study the interactions of them in response to noxious stimulation.
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The present results provide a morphological evidence of direct connections between EM2 and SP with GABAergic neurons in the spinal dorsal horn. It suggests that there are potential modulatory interactions between EM2- and SPimmunoreactive primary afferent fibers/terminals on GABAergic interneurons in nociceptive information transmission and modulation in the spinal dorsal horn. Acknowledgments This work was supported by the grants from National Natural Science Foundation of China (Nos. 30971123, 31010103909, 31100861, 81200867, 81371239).
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Neurol Sci 4. Todd AJ (2010) Neuronal circuitry for pain processing in the dorsal horn. Nat Rev Neurosci 11(12):823–836 5. Martin-Schild S, Gerall AA, Kastin AJ, Zadina JE (1999) Differential distribution of endomorphin 1- and endomorphin 2-like immunoreactivities in the CNS of the rodent. J Comp Neurol 405(4):450–471 6. Zubrzycka M, Janecka A (2000) Substance P: transmitter of nociception (minireview). Endocr Regul 34(4):195–201 7. Sanderson Nydahl K, Skinner K, Julius D, Basbaum AI (2004) Co-localization of endomorphin-2 and substance P in primary afferent nociceptors and effects of injury: a light and electron microscopic study in the rat. Eur J Neurosci 19(7):1789–1799 8. Huang J, Wang Y, Wang W, Wei Y, Li Y, Wu S (2008) Preproenkephalin mRNA is expressed in a subpopulation of GABAergic neurons in the spinal dorsal horn of the GAD67-GFP knock-in mouse. Anat Rec (Hoboken) 291(10):1334–1341 9. Wang YY, Wei YY, Huang J, Wang W, Tamamaki N, Li YQ, Wu SX (2009) Expression patterns of 5-HT receptor subtypes 1A and 2A on GABAergic neurons within the spinal dorsal horn of GAD67-GFP knock-in mice. J Chem Neuroanat 38(1):75–81 10. Zhao H, Sugawara T, Miura S, Iijima T, Kashimoto S (2007) Intrathecal landiolol inhibits nociception and spinal c-Fos expression in the mouse formalin test. Can J Anaesth 54(3):201–207 11. Liu CR, Duan QZ, Wang W, Wei YY, Zhang H, Li YQ, Wu SX, Xu LX (2011) Effects of intrathecal isoflurane administration on nociception and Fos expression in the rat spinal cord. Eur J Anaesthesiol 28(2):112–119 12. Zimmermann M (1983) Ethical guidelines for investigations of experimental pain in conscious animals. Pain 16(2):109–110 13. Zhao CS, Tao YX, Tall JM, Donovan DM, Meyer RA, Raja SN (2003) Role of micro-opioid receptors in formalin-induced pain behavior in mice. Exp Neurol 184(2):839–845
14. Chen T, Hui R, Wang XL, Zhang T, Dong YX, Li YQ (2008) Origins of endomorphin-immunoreactive fibers and terminals in different columns of the periaqueductal gray in the rat. J Comp Neurol 509(1):72–87 15. Li JL, Ding YQ, Li YQ, Li JS, Nomura S, Kaneko T, Mizuno N (1998) Immunocytochemical localization of mu-opioid receptor in primary afferent neurons containing substance P or calcitonin gene-related peptide. A light and electron microscope study in the rat. Brain Res 794(2):347–352 16. Wang D, Li YQ, Li JL, Kaneko T, Nomura S, Mizuno N (2000) gamma-aminobutyric acid- and glycine-immunoreactive neurons postsynaptic to substance P-immunoreactive axon terminals in the superficial layers of the rat medullary dorsal horn. Neurosci Lett 288(3):187–190 17. Wang D, Wu JH, Dong YX, Li YQ (2001) Synaptic connections between trigemino-parabrachial projection neurons and gammaaminobutyric acid- and glycine-immunoreactive terminals in the rat. Brain Res 921(1–2):133–137 18. Greenwell TN, Martin-Schild S, Inglis FM, Zadina JE (2007) Colocalization and shared distribution of endomorphins with substance P, calcitonin gene-related peptide, gamma-aminobutyric acid, and the mu opioid receptor. J Comp Neurol 503(2):319–333 19. Heinke B, Ruscheweyh R, Forsthuber L, Wunderbaldinger G, Sandkuhler J (2004) Physiological, neurochemical and morphological properties of a subgroup of GABAergic spinal lamina II neurones identified by expression of green fluorescent protein in mice. J Physiol 560(Pt 1):249–266 20. Todd AJ, Spike RC, Brodbelt AR, Price RF, Shehab SA (1994) Some inhibitory neurons in the spinal cord develop c-fosimmunoreactivity after noxious stimulation. Neuroscience 63(3):805–816
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ORIGINAL ARTICLE
Connections between EM2- and SP-containing terminals and GABAergic neurons in the mouse spinal dorsal horn Dao-Shu Luo • Jing Huang • Yu-Lin Dong • Zhen-Yu Wu • Yan-Yan Wei • Ya-Cheng Lu • Ya-Yun Wang • Yuchio Yanagawa Sheng-Xi Wu • Wei Wang • Yun-Qing Li
•
Received: 16 January 2014 / Accepted: 25 March 2014 Ó Springer-Verlag Italia 2014
Abstract Endomorphin-2 (EM2) demonstrates a potent antinociceptive effect in pain modulation. To investigate the potential interactions of EM2- and substance P (SP)containing primary afferents and c-amino butyric acid (GABA)-containing interneurons in lamina II in nociceptive transmission, connections between EM2- and SPcontaining terminals and GABAergic neurons in the spinal dorsal horn were studied. Double-immunofluorescent labeling showed that approximately 62.3 % of EM2immunoreactive neurons exhibited SP-immunostaining, and 76.9 % of SP-immunoreactive neurons demonstrated EM2-immunoreactivities in the dorsal root ganglion (DRG). Dense double-labeled EM2- and SP-immunoreactivities were mainly observed in lamina II of the lumbar
dorsal horn. Furthermore, triple-immunofluorescent labeling results revealed that EM2 and SP double-labeled terminals overlapped with GABAergic neurons. Immunoelectron microscopy confirmed that the EM2- or SPimmunoreactive terminals formed synapses with GABAimmunoreactive dendrites in lamina II of the lumbar dorsal horn. During noxious information transmission induced by formalin plantar injection, GABAergic neurons expressing FOS in their nuclei were contacted with EM2- or SPimmunoreactive terminals. These results suggest that the interactions between EM2- and SP-containing terminals and GABAergic interneurons in the lamina II influence pain transmission and modulation in the spinal dorsal horn. Keywords Endomorphin 2 Substance P C-Amino butyric acid Interaction Spinal dorsal horn
D.-S. Luo and J. Huang contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s10072-014-1774-9) contains supplementary material, which is available to authorized users. D.-S. Luo W. Wang (&) Y.-Q. Li Department of Anatomy, Histology and Embryology, Basic Medical College, Fujian Medical University, Fuzhou 350004, China e-mail: [email protected] J. Huang Y.-L. Dong Z.-Y. Wu Y.-Y. Wei Y.-C. Lu Y.-Y. Wang S.-X. Wu Y.-Q. Li (&) Department of Anatomy, Histology and Embryology, K.K. Leung Brain Research Centre, The Fourth Military Medical University, No. 169 West Changle Road, Xi’an 710032, People’s Republic of China e-mail: [email protected] Y. Yanagawa Department of Genetic and Behavioral Neuroscience, Gunma University Graduate School of Medicine, Maebashi 371-8511, Japan
Introduction The superficial laminae (laminae I and II) of the spinal dorsal horn play a critical role in the transmission and modulation of nociceptive information conveyed via primary afferents [1]. Inhibitory interneurons play an important role in regulating spinal excitability by releasing camino butyric acid (GABA) [2]. In immunocytochemical studies, approximately 30 % of the lamina II neurons show GABA-immunoreactivity [3]. Primary afferent fibers either directly contact projection neurons in lamina I or indirectly contact with projection neurons via interneurons in lamina II [4]. Endomorphin-2 (EM2) is an endogenous opioid peptide, and EM2-immunoreactive primary afferent terminals are densely located in the superficial laminae of the spinal dorsal horn [5]. Substance P (SP), a neuropeptide, is involved in the transmission and modulation of nociceptive
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information in the nervous system [6]. Previous studies have shown that EM2-like immunoreactivity co-localized with SP in both the dorsal root ganglion (DRG) and spinal dorsal horn and that EM2 might modulate the release of SP [7]. However, the connections between EM2/SP co-localized terminals and GABAergic neurons in the spinal dorsal horn remain unclear. Therefore, the principal purpose of this study was to determine whether EM2/SP co-localized terminals are in contact with GABAergic neurons in the spinal dorsal horn, using light and electron microscopic techniques. To aid in the identification of GABAergic neurons and overcome the difficulty in staining GABAergic neurons with traditional immunohistochemical methods, we used the glutamic acid decarboxylase 67-green fluorescence protein (GAD67-GFP) knock-in mouse, in which GABAergic neurons are identified by the expression of GFP.
The specificity of GFP-positive GABAergic neurons has been confirmed in our previous studies [8, 9]. According to previous studies from others [10] and ours [11], the subcutaneous injection of formalin significantly enhances the spinal dorsal horn neuronal activation indicated by nuclei FOS expression, which reaches a peak at 2 h after formalin injection in both rat and mouse [10]. Recent studies further suggested that GABAergic neurons within the spinal dorsal horn are activated during inflammatory pain. Therefore, we hypothesized that EM2/SP co-localized terminals may regulate the neuronal excitability of GABAergic neurons to regulate nociceptive transmission. To offer morphological evidence for our hypothesis, we investigated the connections between EM2/SP co-localized terminals and FOS-positive GABAergic neurons in the dorsal horn after formalin plantar injection to examine their potential interactions during noxious stimulation.
Table 1 Antisera used in each group Group
Purposes
Primary antisera
Immunofluorescence
EM2/SP
Rabbit anti-EM2 (1:200, Abcam) rat anti-SP (1:500, Millipore)
Secondary antisera
Fluorophore-conjugated avidin
Biotinylated donkey anti-rabbit IgG (1:500, vector labs) Alexa 488 donkey anti-rat IgG (1:500, Molecular probes)
EM2/SP/GFP
Rabbit anti-EM2 (1:200) rat anti-SP (1:500)
Biotinylated donkey anti-rabbit IgG (1:500)
Cy3-labeled avidin D (1:1,000, molecular probes) Cy3-labeled avidin D (1:1,000)
Alexa 647 donkey anti-rat IgG (1:500, molecular probes)
EM2/FOS/GFP
SP/FOS/GFP
Guinea pig anti-GFP (1:100,synaptic systems)
Alexa 488 donkey anti-guinea pig IgG (1:500, Molecular probes)
Rabbit anti-EM2 (1:200) mouse anti-FOS (1:500, Millipore)
Biotinylated donkey anti-rabbit IgG (1:500) Alexa 647 donkey anti-mouse IgG (1:500, molecular probes)
Guinea pig anti-GFP (1:100)
Alexa 488 donkey anti-guinea pig IgG (1:500)
Rat anti-SP (1:500) mouse anti-FOS (1:500, Millipore)
Biotinylated donkey anti-rat IgG (1:500, vector labs)
Cy3-labeled avidin D (1:1,000)
Cy3-labeled avidin D (1:1,000)
Alexa 647 donkey anti-mouse IgG (1:500)
Electronmicroscopy
Guinea pig anti-GFP (1:100)
Alexa 488 donkey anti-guinea pig IgG (1:500)
GFP/SP
Mouse anti-GFP (1:500,synaptic systems) rabbit anti-SP (1:500, Millipore)
Biotinylated anti-mouse IgG (1:100, Vector labs) goat antirabbit IgG antibody conjugated to 1.4 nm gold particles (nanoprobes)
Avidin-biotinylated peroxidase complex (1:50, Vector labs)
GFP/EM2
Mouse anti-GFP (1:500) rabbit antiEM2 (1:200)
Biotinylated anti-mouse IgG (1:100)
Avidin-biotinylated peroxidase complex (1:50)
goat anti-rabbit IgG antibody conjugated to 1.4 nm gold particles (nanoprobes)
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Six adult C57BL/6 and 24 GAD67-GFP knock-in mice were used in the present study. The experimental procedures in this study were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23) revised 1996 and IASP’s guidelines for pain research in conscious animals [12] and were approved by The Committee of
Animal Use for Research and Education in The Fourth Military Medical University (Xi’an, P.R. China). All efforts were made to minimize the number of animals used and their suffering. Mice were divided into five groups. Group 1 (six C57BL/6 mice) was used for EM2 and SP double-immunofluorescent histochemical staining in the DRG and spinal dorsal horn. Group 2 (six GAD67-GFP knock-in mice) was used for EM2/SP/GFP triple-immunofluorescent histochemical staining in the dorsal horn. Group 3 (six GAD67GFP knock-in mice) was used for subcutaneous injection of
Fig. 1 Double-immunofluorescent staining showing the labeling of endomorphin-2- (EM2, a; red) and substance P- (SP, b; green) immunoreactive neurons in the dorsal root ganglion (DRG). In the
merged image (c), some EM2-immunoreactive neurons also showed SP-immunoreactivities. Arrows point to double-labeled neurons (yellow). Scale bar = 100 lm (a–c) (color figure online)
Fig. 2 Double-labeling of EM2 and SP in the spinal dorsal horn of the mouse. EM2- (a; red) and SP-immunoreactive (b; green) terminals are concentrated in the superficial laminae of the dorsal horn. The merged image reveals extensive overlap of EM2 and SPimmunoreactive terminals (c; yellow) in lamina I and outer lamina II
of the dorsal horn. d–f Enlarged view of EM2 and SP immunoreactivity in the superficial laminae of the dorsal horn. The great majority of EM2 and SP are co-located at primary afferent terminals (arrows). Scale bars = 100 lm (a–c), 5 lm (d–f) (color figure online)
Materials and methods Animals
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5 % formalin solution (25 ll) into the plantar surface of the left hind paw 2 h before the perfusion [13]. Group 4 (six GAD67-GFP knock-in mice) as a control group, that formalin injection was replaced with 25 ll of saline. Group 3 and Group 4 were processed for EM2/FOS/GFP tripleimmunofluorescent histochemical staining. Group 5 (six GAD67-GFP knock-in mice) was used for electron-microscopic double-immunohistochemical staining of EM2/GFP or SP/GFP. Immunofluorescent histochemistry The mice from Group 1 to Group 4 were anesthetized with sodium pentobarbital (45 mg/kg) and then perfused transcardially with 0.1 M phosphate buffer (PB, pH 7.4) containing 4 % (w/v) paraformaldehyde. The lumbar spinal cord (L4–L5) was cut into 30 lm thick sections and corresponding DRG was cut into 10 lm thick sections on a freezing microtome (Kryostat 1,720; Leitz, Mannheim, Germany). The immunofluorescent histochemical staining protocols used in the present study were the same as those in our previous studies [14], and all the antisera used in each group are shown in Table 1. Briefly, the sections were incubated at room temperature sequentially with primary
Fig. 3 Triple-labeling of EM2, SP, and green fluorescence protein (GFP) in the spinal dorsal horn of the GAD67-GFP knock-in mice. a-c: EM2 (a; red), SP (b; blue), and GFP (c; green) immunoreactivity in the spinal dorsal horn. d–e: EM2 (d; red) and SP-immunoreactive terminals
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antibodies for 72 h, then incubated in the mixture of species-specific biotinylated secondary antibody and fluorochrome-labeled secondary antibody for 4 h, followed by fluorophore-conjugated avidin D for 1 h. The specificities of the staining were tested on the sections in another dish by omitting the specific primary specific antibodies. No immunoreactivities were found on the sections, as expected. The sections were observed under a confocal laserscanning microscope (Fluoview 1,000, Olympus, Tokyo, Japan). Digital images were captured using Fluoview software (Olympus). Immuno-electron microscopy For electron microscope study, six deeply anesthetized adult GAD67-GFP knock-in mice were perfused transcardially with 0.1 M PB containing 4 % paraformaldehyde, 0.05 % glutaraldehyde and 0.2 % picric acid. The lumbar spinal cord (L4–L5) was cut into 50 lm thick sections on a vibratome (Microslicer DTK-100; Dosaka, Kyoto, Japan). Sections were freeze–thawed in liquid nitrogen after cryoprotection, to enhance penetration of antibody. Details of these immuno-electron microscopy procedures were described in our previous study [15, 16]. Briefly, the
(e; blue) contacted GFP-positive (green) neurons in the dorsal horn (single arrow). f: Double-labeling of EM2 and SP-immunoreactive terminals (purple) contacting GFP-positive neurons (double arrowheads). Scale bars = 100 lm (a–c), 4 lm (d–f) (color figure online)
Neurol Sci
sections were incubated with 0.05 M Tris-buffered saline (TBS; pH7.4) containing 20 % normal goat serum for 1 h to block non-specific immunoreactivity, and then collected in two dishes. The sections in each dish were incubated for 24 h at 4 °C with a mixture of primary antibodies as GFP/ SP and GFP/EM2 (Table 1), respectively. Then, a mixture of biotinylated donkey anti-mouse IgG and goat anti-rabbit IgG conjugated with 1.4 nm gold particles for SP or EM2 (Table 1) were incubated overnight at 48 °C. Subsequently the sections were processed by the following steps: (1) postfixation with glutaraldehyde, (2) silver enhancement with HQ Silver Kit (Nanoprobes), (3) incubation with ABC kit (Vector), (4) reaction with diaminobenzidine tetrahydrochloride and H2O2, (5) osmification, (6) conterstaining with uranylacetate. Ultrathin sections at 70 nm thickness were prepared from the superficial laminae of the spinal dorsal horn, mounted on single-slot grids, and examined with an electron microscope (JEM1440, Tokyo, Japan).
immunoreactive terminals overlapped with the GFP/FOS double-labeled neurons in the spinal dorsal horn (Fig. 5).
Discussion
Results
Our previous results have shown that SP released from the primary afferent fibers may activate GABAergic inhibitory neurons in lamina II [16]. The GABAergic inhibitory neurons may inhibit the activity of ascending projection neurons in lamina I of the medullary dorsal horn [17]. These results suggest that GABAergic interneurons play an important role in regulating the transmission of nociceptive information to the spinal cord. It possibly because the difficulty in GABA staining with traditional immunohistochemical methods, the co-localization of EM2 with GABA was not observed in the spinal dorsal horn of rat [18]. To take full advantage of GAD67GFP knock-in mice to identify GABAergic neurons in our study, it is the first report showing that EM2/SP co-localized fibers/terminals make asymmetric synapses with the
In the DRG, EM2- and SP-immunoreactive were localized in small (the largest diameter \20 lm) to medium (the longest diameter 20–35 lm) sized neurons (Fig. 1a, b). Colocalization of EM2 and SP was found in the DRG neurons (Fig. 1c). Quantitative analysis showed that approximately 62.3 % of EM2-immunoreactive cell bodies contained SP-immunoreactivity and approximately 76.9 % of SP-immunoreactive cell bodies were immunoreactive for EM2. On the other hand, in the spinal cord, dense EM2- and SP-immunoreactive fibers and terminals were found to be concentrated in the superficial laminae of the dorsal horn (Fig. 2a, b, d, e), with extensive overlap (Fig. 2c, f). A large number of GFP-immunoreactive neurons were observed in lamina II of the spinal dorsal horn from the GAD67-GFP knock-in mouse (Fig. 3c). Triple-immunofluorescent labeling revealed that the EM2- and SPimmunoreactive fibers/terminals located on the periphery of the GFP-immunoreactive spinal dorsal horn neurons (Fig. 3d, e). Interestingly, some EM2/SP co-localized terminals closely connected with GFP-immunoreactive neurons in the superficial laminae, especially in lamina II of the dorsal horn (Fig. 3f. Under electron microscope, SPand EM2-immunopositive immunogold particles were observed within the thin and unmyelinated axons which make asymmetric synaptic contacts with dendritic profiles of the GFP-immunoreactive GABAergic neurons (Fig. 4a, b). In the formalin plantar injection animals, approximately 30.6 % of the GFP-positive GABAergic neurons expressed FOS in the spinal dorsal horn, and the EM2- or SP-
Fig. 4 Electron micrographs showing the synaptic connections between GABAergic dendrites and SP- or EM2-immunoreactive terminals in laminae I and II of the lumbar dorsal horn. Both (a) SPand (b) EM2-immunoreactive pre-synaptic terminals contained immune-gold particles (arrows) and formed asymmetric synapses with the post-synaptic GABA-immunopositive dendritic profiles (labeled with DAB reaction products). Scale bars = 0.2 lm (a), 0.4 lm (b)
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Neurol Sci Fig. 5 Triple-labeling of GFP (green), FOS (blue), and EM2 or SP (red) in the ipsilateral spinal dorsal horn of the GAD67-GFP knock-in mice following formalin injection into the hind paw plantar. b: A magnified image of the rectangle area is indicated in (a). Note that some of the SPimmunoreactive terminals formed close contacts with GFP/FOS double-labeled neurons (arrows). (d): The magnified image of the rectangle area is indicated in (c). Note that some of the EM2immunoreactive terminals formed close contacts with GFP/FOS double-labeled neurons (arrows). Scale bars = 10 lm (a, c), 2 lm (b, d) (color figure online)
GABAergic neurons in the superficial laminae of the spinal dorsal horn. Furthermore, EM2 and SP-immunoreactive terminals form close contacts with GABAergic neurons which were activated by peripheral noxious stimuli, suggesting these connections were involved in the processing of peripheral nociceptive transmission. As a l-opioid receptor agonist, EM2 is believed to presynaptically inhibit SP release from primary sensory neurons in DRG and the primary afferent terminals in the spinal dorsal horn, dulling the sensation of pain and alleviating neuropathic pain [7, 15, 16]. Therefore, the interaction of EM2 and SP may affect the modulation of nociceptive information transmitted from the peripheral to central nervous system. GABA is believed to be involved in presynaptic and postsynaptic inhibition in the spinal dorsal horn [19]. GABAergic inhibitory neurons in the superficial laminae of the dorsal horn can be activated by subcutaneous injection of formalin into the hind paw of rats, and these GABAergic neurons can also express FOS after noxious stimulation [20]. Therefore, the result of EM2 and SP overlapped with the FOS-positive GABAergic neurons in the dorsal horn may contribute to further study the interactions of them in response to noxious stimulation.
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The present results provide a morphological evidence of direct connections between EM2 and SP with GABAergic neurons in the spinal dorsal horn. It suggests that there are potential modulatory interactions between EM2- and SPimmunoreactive primary afferent fibers/terminals on GABAergic interneurons in nociceptive information transmission and modulation in the spinal dorsal horn. Acknowledgments This work was supported by the grants from National Natural Science Foundation of China (Nos. 30971123, 31010103909, 31100861, 81200867, 81371239).
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