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EphB receptors and ephrin-B ligands regulate spinal sensory connectivity and modulate pain processing

Nature Neuroscience volume 6, pages 339–340 (2003)Cite this article

Abstract

Eph receptor tyrosine kinases and their ephrin ligands are involved in crucial aspects of nervous system circuit assembly during development1,2, but their functional roles in the mature nervous system are poorly understood. We investigated their role in pain processing, using a combination of immunohistochemical, behavioral, biochemical and primary cell culture techniques. Here we report an in vivo role for EphB–ephrinB interactions as modulators of synaptic efficacy in the spinal cord, contributing to sensory abnormalities in persistent pain states.

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Figure 1: EphrinB2–Fc treatment induces thermal hyperalgesia via NMDA receptors and Src phosphorylation.
Figure 2: EphB1–Fc delivered i.t. prevents the development of thermal hyperalgesia, mechanical allodynia and c-fos upregulation normally induced by intraplantar carrageenin injection.

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Acknowledgements

We thank T. Cowen, M. Geymonat, M. Malcangio, C.Thravivoulou, S. Pezet and J.N. Wood for their help. Supported by Wellcome Trust PG 060455.

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Authors and Affiliations

  1. Centre for Neuroscience Research, King's College London, Guy's Campus, London, SE1 1UL, UK

    Anna Aurora Battaglia, Karina Sehayek, John Grist, Stephen B. McMahon & Isabella Gavazzi

Authors
  1. Anna Aurora Battaglia
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  2. Karina Sehayek
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  3. John Grist
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  4. Stephen B. McMahon
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  5. Isabella Gavazzi
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Correspondence to Isabella Gavazzi.

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Supplementary information

Supplementary Fig. 1.

Ephrin-B2 is expressed presynaptically (on sensory afferents) and EphB1 postsynaptically in spinal cord DH. Ephrin-B2 was the most widely expressed ephrin in lumbar DRG, present in ~44.8% (± 1.12 s.e.m) of neurons, virtually all of small diameter (<35 μm). Ephrin-B2 positive neurons were negative for RT97 (a marker for large and intermediate neurons). All VR1-expressing neurons and 79 ± 5.6% of P2X3 immunoreactive neurons were positive for ephrinB2. P2X3 and VR1 are markers for nociceptive neurons. Some small neurons showed staining for ephrin-B1, but none for ephrin-B3 (not shown). Terminals that had ephrin-B2, but not ephrin-B1, were visible in DH laminae I and II. Many DH neurons in laminae I-III showed EphB1 receptor immunoreactivity. Inset, motor neurons. Scale bars, 100 μm. (PDF 940 kb)

Supplementary Fig. 2.

EphrinB2-Fc i.t. treatment does not affect withdrawal latencies following mechanical non-noxious stimulation of the hindpaw. Repeated measurements from rats treated i.t. with human Fc fragment (control) or ephrinB2-Fc (at 2 or 10 ÎĽg/rat). (PDF 649 kb)

Supplementary Fig. 3.

Intrathecally delivered EphB1-Fc significantly reduces pain-related behavior in Phase II of the formalin response. Charts represent time course for pain behaviors induced by 1.5% formalin after i.t injection of human Fc (control) or EphB1-Fc. (a) Time course for lifting. EphB1-Fc i.t. injection significantly (P < 0.001) reduced the amount of time the animals spent with the formalin injected paw lifted off the floor. (b) Pain scores (calculated on the basis of the pain related behaviors of favouring, lifting and licking/biting). EphB1 i.t. injection significantly (P < 0.001) reduces pain scores in Phase II of the formalin response. (PDF 999 kb)

Supplementary Fig. 4.

ATP stimulation alters the intensity and pattern of ephrin-B2 staining in DRG neurons in culture. Confocal images of cultured rat DRG neurons, untreated or stimulated with 100 µM ATP and labelled with ephrin-B2 antibodies. For illustrative purposes images are pseudocolored to illustrate intensity of staining (lighter colour indicates higher intensity). Ephrin-B2 immunoreactivity was observed in neurons on soma, along neurites and at the level of growth cones. Note aggregates of staining in the ATP-treated neuron on the growth cone presented in the boxed area. Scale bar, 50 μm. ATP-treated neurons were significantly brighter and had a larger number of separate peaks of high intensity staining, presumably indicative of clustering, as compared to controls. Densitometric analysis was performed with LaserPict software on single optical sections. Grey value measurements for ATP-treated neurons were expressed as percentages of control. Clustering of ephrin-B2 staining was measured by counting the number of separate peaks above average grey value along randomly selected segments of neurite (500 pixels/field). A total of 3904 μm of neurites were sampled. Cells which were P2X3-immunopositive (and therefore ATP-responsive) showed higher levels of ephrin-B2 immunoreactivity in image analysis as compared to P2X3-immunopositive cells in untreated cultures (143 ±11% vs control; P < 0.05, unpaired t-test). The number of discrete peaks of ephrin-B2 immunofluorescence/unit length was significantly higher in ATP-treated neurons (119 ± 3.5 % vs. control, P < 0.01, unpaired t-test), indicating the presence of a higher number of ephrin-B2 clusters. (PDF 42 kb)

Supplementary Methods (PDF 75 kb)

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Battaglia, A., Sehayek, K., Grist, J. et al. EphB receptors and ephrin-B ligands regulate spinal sensory connectivity and modulate pain processing. Nat Neurosci 6, 339–340 (2003). https://doi.org/10.1038/nn1034

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