Neurotrophin-4 mediated TrkB activation reinforces morphine-induced analgesia (original) (raw)
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Acknowledgements
We thank D. Kaplan for TrkB antibody and M. Malcangio for advice on NT-4 release assay. Supported by Biotechnology Program of the EU QLG3-1999-00602, Swedish Medical Research Council, Swedish Cancer Society and Göran Gustafssons Foundation (P.E.); fellowship from David and Astrid Hageléns Foundation (G.L.); Academy of Finland, TEKES and Sigrid Juselius Foundation (E.C.).
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Authors and Affiliations
- Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, S-17177, Sweden
Guilherme Lucas, Tibor Harkany, Karin Agerman & Patrik Ernfors - Department of Neuroscience, Karolinska Institutet, Stockholm, S-17177, Sweden
Gustavo Paratcha, Carl Holmgren & Yuri Zilberter - Department of Neurobiology, A.I. Virtanen Institute, University of Kuopio, Kuopio, 70211, Finland
Panu Hendolin, Mikko Sairanen & Eero Castren - European Molecular Biology Laboratory, via Ramarini 32, 00016 Monterotondo, Rome, Italy
Liliana Minichiello
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- Guilherme Lucas
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Correspondence toPatrik Ernfors.
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Supplementary information
Supplementary Fig. 1.
Morphine induced phosphoTrk immunoreactivity in brainstem nuclei. Confocal images of the trigeminal spinal nucleus and locus coeruleus (insets) neurons stained with anti-Trk autophosphorylation antibody 30 min after (a) morphine (10 mg/kg), (b) morphine (10 mg/kg) followed by naloxone (1 mg/kg), and (c) naloxone alone. Note increased immunoreactivity in morphine but not morphine plus naloxone or naloxone treated mice. Arrows indicate phospho-Trk positive cells in the spinal trigeminal nucleus. Scale bar, 200 μm for the trigeminal spinal nucleus and 75 μm for the locus coeruleus. Methods: C57/BL6 mice were treated with morphine (10 mg/kg, s.c.; n = 3), or with morphine (10 mg/kg, s.c.) followed by naloxone (1 mg/kg, i.p. n = 2) 10 min later, or only naloxone (1 mg/kg, i.p. n = 1). Thirty minutes after morphine treatment or 10 min after naloxone administration, mice were anesthetized by isoflurane (5% [v/v%] in 70% N2O and 30% O2) and transcardially perfused with ice-cold physiological saline (15 ml) that was followed by 80 ml of fixative composed of 4% paraformaldehyde, 0.1% glutaraldehyde and 1mM sodium orthovanadate in sodium phosphate buffer (PB, 0.1M, pH 7.4) at a flow rate of 4 - 4.5 ml/min. Whole brains were removed from the skulls and post-fixed in 4% paraformaldehyde and 1mM sodium orthovanadate in PB overnight. Brains were then cryoprotected in 30% sucrose in physiological saline for 24 hrs, cryostat sectioned at a 40-μm thickness, and collected in 0.1M PB with 0.1% Na-azide as preservative. Series of free-floating sections of all animals were processed simultaneously in a uniform manner. After extensive rinsing in PB, sections were pre-incubated in a mixture of 10% normal donkey serum (NDS, Jackson ImmunoResearch, USA), 5% normal bovine serum (BSA) and 0.1% triton X-100 in PB for 60 min. Subsequently, sections were exposed to the primary antibody rabbit anti-Tyr674,Tyr675-phosphorylated tyrosine kinase (1:25; Calbiochem, USA) in PB containing 1% NDS, 0.1% BSA and 0.1% triton X-100 for 48 hrs at 4 °C under continuous agitation of the incubation medium. Thereafter, sections were washed in repeated changes of PB and incubated with carbocyanine 3-conjugated donkey anti-rabbit secondary antibody (1:200; affinity-pure for multiple labeling without cross-reactivity with mouse serum proteins, Jackson) in PB containing 2% BSA for 2 hrs at room temperature. Next, sections were rinsed in PB, dipped in distilled water and mounted on fluorescence-free glasses. Sections were coverslipped using Entellan (in toluene). Sections were analyzed using confocal laser-scanning microscopy with appropriate excitation and emission filters (543 nm and 560-610 nm, respectively; Zeiss 510, Germany). Selected images were captured using identical pinhole, detector gain, detector offset, amplification gain and scanning control settings, where specimen of morphine-treated animals were used for calibration of scanning conditions. Subsequently, multi-panel plates of unmodified images were created employing Paint Shop Pro (v. 7.01, Jasc Inc., USA) and Adobe Photoshop (v. 6.0, Adobe Inc., USA). Scanning data: pinhole: 60 μm, (less than 1.9 micrometer optical slice), adjusted for even light distribution. Detector gain: 960; offset: -0.3; amplificator gain: 1; 100% laser excitation on 543 nm channel; speed: 7, lines: 4; zoom: (spinal trigeminal nucleus images): 1, Locus coeruleus images: 1.5. (JPG 48 kb)
Supplementary Fig. 2.
Measurements of morphine and morphine metabolites in the plasma of NT-4-/- and NT-4+/+ mice. Mice were decapitated 30 minutes after morphine administration (10 mg/kg, s.c.) and blood samples (300 μl) collected in ice-chilled tubes containing heparin (10 IU/ml) were centrifuged for separation of the plasma. Morphine (a), Morphine-6-glucuronide (b), and Morphine-3 glucuronide (c) were measured by a reverse phase HPLC column (C18, Spherisorb S3 ODS2 100 x 4 mm I.D.; 3 μm particles) and no difference was observed between wild-type (n = 5) and NT-4-/- (n = 6) mice (Student _t_-test). This shows that the difference in morphine response between mutated mice and wild-type mice is not caused by a difference in metabolisation of morphine. (JPG 34 kb)
Supplementary Table 1 (PDF 40 kb)
Supplementary Methods (PDF 14 kb)
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Lucas, G., Hendolin, P., Harkany, T. et al. Neurotrophin-4 mediated TrkB activation reinforces morphine-induced analgesia.Nat Neurosci 6, 221–222 (2003). https://doi.org/10.1038/nn1021
- Received: 15 November 2002
- Accepted: 17 December 2002
- Published: 25 February 2003
- Issue Date: 01 March 2003
- DOI: https://doi.org/10.1038/nn1021