Elsevier

Brain Research

Volume 947, Issue 2, 30 August 2002, Pages 252-259
Brain Research

Research report
Localization of CaMKIIα in rat primary sensory neurons: increase in inflammation

https://doi.org/10.1016/S0006-8993(02)02932-3Get rights and content

Abstract

This study investigates Ca2+/calmodulin kinase IIα (CaMKIIα) in primary sensory neurons. Immunohistochemical staining with a CaMKIIα antibody demonstrates 28% of dorsal root ganglion (DRG) cells are positively stained and have a diameter of 27±2.4 μm (mean±S.D.). Placement of tight ligatures around the sciatic nerve demonstrates a build up of immunoreaction product proximal to the ligatures indicating that CaMKIIα is transported into the peripheral processes of DRG cells. Immunostaining of lumbar dorsal roots at the electron microscopic level demonstrates reaction product in 15.4±2.1% of unmyelinated and 2.4±1.0% of myelinated axons, indicating that CaMKIIα is transported into the central processes of DRG cells. Electron microscopic analysis of normal digital nerves demonstrates CaMKIIα labeling in 3.3±0.3% of unmyelinated and 2.0±1.1% of myelinated cutaneous axons. These percentages increase significantly to 14.1±2.3% for unmyelinated and 5.1±1.4% for myelinated axons 48 h after complete Freund’s adjuvant-induced inflammation of the hindpaw. The data indicate that CaMKIIα is present in small diameter primary sensory neurons, that it is transported into the peripheral and central processes of these cells and may play a role in processing noxious input, particularly in the inflamed state.

Introduction

Ca2+/calmodulin kinase II (CaMKII) is a multifunctional enzyme that can catalyze the phosphorylation of a variety of proteins and this action contributes to long term potentiation, release of neurotransmitters, and regulation of ion channels (see Ref. [10] for review). Recently it has been shown that CaMKIIα is preferentially localized in pain-processing regions in the central nervous system such as the superficial laminae of the dorsal horn in the spinal cord and the dorsal root ganglia (DRG) [2]. Ca2+-mediated cell signaling is important in nociceptor function and the presence of CaMKIIα in DRG cells suggests that this enzyme may contribute to nociceptive signaling. Several receptors whose activation results in Ca2+ influx have been localized on nociceptors and include purinergic P2X [24], capsaicin [3], NMDA [5] and bradykinin [13] receptors. Furthermore, it has been demonstrated that P2X-mediated responses in DRG cells are enhanced by endogenous CaMKII in normal and inflamed rats [26].

Presuming that CaMKIIα has a role in the transmission of nociceptive information, the goals of the present study were to investigate the distribution of CaMKIIα in primary sensory neurons and to analyze changes that occur in the expression of CaMKIIα following inflammation. To accomplish this, numbers of DRG cells in lumbar ganglia that contain CaMKIIα were estimated using stereological methods, the mean diameter of CaMKIIα-containing DRG cells were measured, and the axonal transport of CaMKIIα into the peripheral and central processes of DRG cells were visualized. Finally, increases in CaMKIIα-containing axons in the digital nerves of rats 48 h following inflammation of the hindpaw were documented.

Section snippets

Materials and methods

All procedures were approved by the University Animal Care and Use Committee and followed guidelines for the ethical treatment of animals [28]. Male, Sprague–Dawley rats were obtained from Harlan, Indianapolis, IN, USA.

CaMKIIα-labeling in DRG

Immunostaining for CaMKIIα in the lumbar DRG resulted in diffuse, rather homogeneous staining throughout the cytoplasm of labeled cells but the nuclei were weakly stained or unstained. The density of cytoplasmic staining varied from light, medium, to dark (Fig. 1). Positively stained cells had a diameter of 27.1±5 μm (mean±S.D.) and ranged from 13.5 to 48 μm. Numerical estimates of CaMKIIα-labeled DRG cells, obtained using stereological analysis, demonstrated that 5,829±513 cells were labeled (

Discussion

These analyses demonstrate that a subpopulation of small diameter DRG cells immunostain for the enzyme CaMKIIα. Placement of a tight ligature around the sciatic nerve demonstrates that CaMKIIα is transported into the peripheral processes of DRG cells. Analysis of the lumbar dorsal roots at the electron microscopic level demonstrates that the enzyme is also transported centrally into the spinal cord. Finally, electron microscopic analysis of the digital nerves in the hindpaw demonstrates the

Acknowledgements

The author thanks Vicki Wilson for her secretarial assistance and Greg Hargett and Zhixia Ding for their expertise in immunohistochemical staining. This work was supported by NS27910, NS11255 and NS40700 to S.M.C.

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