Available online at www.sciencedirect.com Neuroscience Letters 428 (2007) 88–92 The tight junction component protein, claudin-4, is expressed by enteric neurons in the rat distal colon Shin-ichiro Karaki, Izumi Kaji, Yasuko Otomo, Hideaki Tazoe, Atsukazu Kuwahara ∗ Laboratory of Physiology, Institute for Environmental Sciences, and Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan Received 25 July 2007; received in revised form 11 September 2007; accepted 16 September 2007 Abstract The expression of a tight junction (TJ) component protein, claudin-4, in the enteric neurons was investigated in the rat distal colon by immuno- histochemistry and RT-PCR. Claudin-4 immunoreactivity was detected in almost all neurofilament-positive enteric neurons both of the submucosal and the myenteric plexuses, and both of the cell bodies and the neurofibers. The immunoreactivity of enteric neurons for claudin-4 was divided into two types: strongly and weakly positive neurons. Especially in the myenteric plexus, the stained neurons were classified by Dogiel’s mor- phological classification of enteric neurons. The strongly stained claudin-4 positive neurons show Dogiel type II morphology, while the weakly stained claudin-4 positive neurons show Dogiel type I morphology. These immunohistochemical data were supported by mRNA expression in the muscle plus submucosa preparation containing the submucosal and myenteric plexuses, as well as mucosa preparation. The physiological function of claudin-4 expressed on enteric neurons is unclear up to now. It is however suggested that claudin-4 expressed on enteric neurons might play roles for the neural activity, for example as insulation between neurofibers. In conclusion, the present study clearly shows that claudin-4 is expressed by enteric neurons. This is the first evidence that the neuron itself expresses the TJ component protein, claudin-4, in the nervous system. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Tight junction; Claudin; Enteric nervous system; Neurofilament; Immunohistochemistry; RT-PCR Tight junction (TJ) is one of the intercellular junctional struc- tures forming the reticular-paired strands to separate the partition of the body fluid compartments especially in the epithelial tis- sues (e.g. skin, renal tubules, and gastrointestinal (GI) epithelia) and the endothelial tissues (e.g. blood vessels). The most pri- mary functions of TJs are considered to prevent from passing fluid, ions and some molecules paracellularly as a barrier. How- ever, TJs not only prevent from passing but also TJs expressed in diverse tissues are known to function for selective pores (or channels) to pass diverse ions and molecules, etc. [9] Therefore, the investigation of TJs is currently considered to be impor- tant for both basic and clinical aspects [2,10,12], for example, drug delivery system [4,8]. In the nervous system, the TJ-like structures have previously been observed both in central nervous system (CNS) and peripheral nervous system (PNS). In the CNS, TJs of the brain endothelial cells is reported to function as the blood–brain barrier [13,14]. ∗ Corresponding author. Tel.: +81 54 264 5707; fax: +81 54 264 5707. E-mail address: kuwahara@sea.u-shizuoka-ken.ac.jp (A. Kuwahara). URL: http://physiology.u-shizuoka-ken.ac.jp/ (A. Kuwahara). The TJ strands are composed of the assemblies of cell–cell interlinked transmembrane proteins including claudins and occludin, and cytoskeleton-anchoring intracellular proteins including ZO-1–ZO-3, etc. (for review as [3]). Claudins are four-transmembrane ∼23 kDa proteins, and to date, 24 isoforms (claudin-1–claudin-24) are identified [2]. The distinct claudins are expressed by different cells, and are considered to have different properties. For example, the claudin-4 expression is reported to decrease paracellular conductance through a selec- tive decrease in sodium (Na + ) permeability [11], while claudin-2 expression is reported to increase cation permeability [1]. The claudin-11 and claudin-19 have been reported to be expressed by myelinating oligodendrocytes and Schwann cells in CNS [6] and PNS [5], respectively. However, there is no report for the expression of any claudins in the neuron itself both in CNS and PNS. At first, we have designed to investigate the expression and the distribution of TJ proteins including claudin-1–claudin-4, occludin, and ZO-1 in the epithelia of the rat distal colon during the course of physiological study. At that time, we found that the TJ proteins were expressed by epithelium in specific distri- bution (data not shown) similar to the previous report [7]. In 0304-3940/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2007.09.059