Elsevier

Neuroscience

Volume 162, Issue 2, 18 August 2009, Pages 453-461
Neuroscience

Pain Mechanism
Research Paper
Inflammation-induced increase in hyperpolarization-activated, cyclic nucleotide-gated channel protein in trigeminal ganglion neurons and the effect of buprenorphine

https://doi.org/10.1016/j.neuroscience.2009.04.063Get rights and content

Abstract

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are active at resting membrane potential and thus contribute to neuronal excitability. Their increased activity has recently been demonstrated in models of nerve injury–induced pain. The major aim of the current study was to investigate altered HCN channel protein expression in trigeminal sensory neurons following inflammation of the dura. HCN1 and HCN2 channel immunoreactivity was observed on the membranes of medium- to large-sized trigeminal ganglion neurons with 76% and 85% of HCN1 and HCN2 expressing neurons also containing the 200 kDa neurofilament protein (associated with myelinated fibers). Western immunoblots of lysates from rat trigeminal ganglia also showed bands with appropriate molecular weights for HCN1 and HCN2. Three days after application of complete Freund's adjuvant (CFA) to the dura mater, Western blot band densities were significantly increased; compared to control, to 166% for HCN1 and 284% for HCN2 channel protein. The band densities were normalized against alpha-actin. In addition, the number of retrogradely labeled neurons from the dura expressing HCN1 and HCN2 was significantly increased to 247% (HCN1) and 171% (HCN2), three days after inflammation. When the opioid receptor partial agonist, buprenorphine, was given systemically, immediately after CFA, the inflammation-induced increase in HCN protein expression in both Western blot and immunohistochemical experiments was not observed. These results suggest that HCN1 and HCN2 are involved in inflammation-induced sensory neuron hyperexcitability, and indicate that an opioid receptor agonist can reverse the protein upregulation.

Section snippets

Animals

Experiments were conducted on 40 male Sprague–Dawley rats of 60–150 g and 3–5 weeks of age from the Anatomy and Cell Biology colony at the University of Melbourne. All procedures were approved by the University of Melbourne Animal Experimentation Ethics Committee and comply with the guidelines of the Committee for Research and Ethical Issues of IASP (Zimmermann, 1983). Every effort was made to minimize the number of animals used and their suffering.

Retrograde tracing and induction of inflammation

Animals were anesthetized with a mixture of

HCN channel protein in trigeminal ganglion neurons

The presence of HCN isoforms 1 and 2 was determined in trigeminal ganglia using Western blot analysis. Olfactory bulb was used as a positive control tissue; it expressed both HCN isoforms as has been previously reported (Notomi and Shigemoto, 2004). Liver was used as negative control tissue and no HCN isoforms were detected, as previously reported (Arroyo et al., 2006). Bands immunoreactive for HCN1 and HCN2 antibodies were detected in both trigeminal ganglia and rat olfactory bulb (Fig. 1A).

Discussion

The present results show that levels of HCN protein in the trigeminal ganglion are increased and the proportion of dura projecting neurons that are immunoreactive for HCN more than doubles 3–4 days after an inflammation of the dura is induced. These increases were not observed after treatment with buprenorphine.

Conclusion

The present work shows that inflammation of the dura causes an increase in the protein levels and in the numbers of HCN1 and HCN2 expressing neurons in the trigeminal ganglion. These increases were not seen following buprenorphine treatment. These data suggest that HCN1 and HCN2 may contribute to inflammation-induced hyperexcitability and that one mechanism of opioid-mediated reduction of inflammatory hypersensitivity is to prevent upregulation of HCN protein. These data have potential

Acknowledgments

This work was supported by the National Health and Medical Research Council (Australia; grant # 454606). The authors thank Dr. Paul Strijbos (GlaxoSmithKline) for supplying the anti-HCN1 and anti-HCN2 antibodies. The authors thank Prof. Colin Anderson, Dr. Jason Ivanusic and Mr. Michael Williams for their helpful discussions.

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