Elsevier

Brain Research

Volume 1141, 13 April 2007, Pages 92-98
Brain Research

Research Report
Primary sensory neurons containing choline acetyltransferase of the peripheral type in the rat trigeminal ganglion and their relation to neuropeptides-, calbindin- and nitric oxide synthase-containing cells

https://doi.org/10.1016/j.brainres.2007.01.025Get rights and content

Abstract

We have previously demonstrated that a variant form of choline acetyltransferase (pChAT) is expressed in rat trigeminal neurons. To assess the significance of pChAT in sensory functions, we characterized immunohistochemically pChAT-positive trigeminal neurons in the rat. pChAT-immunoreactivity was observed in a rather uniform pattern in about half of all trigeminal neurons throughout the trigeminal ganglion. The majority of pChAT-positive neurons had small to medium-sized cell bodies. Double immunofluorescent study showed that more than 90% of substance P (SP)-positive trigeminal cells and about 80% of calcitonin gene-related peptide (CGRP)-positive cells exhibited pChAT-immunoreactivity. pChAT-positive cells formed a larger population of neurons than SP-positive or CGRP-positive cells, but they were a different population from calbindin-D28k-positive neurons. In addition, pChAT-immunoreactivity was present in a subset of neurons positive for neuronal nitric oxide synthase. The present results suggest that pChAT plays roles not only in nociception, but also in other sensory functions such as mechanoreception mediating tactile sensation.

Introduction

Choline acetyltransferase (ChAT; E.C. 2.3.1.6) is the synthesizing enzyme of acetylcholine (ACh), and has been accepted as the most reliable marker for cholinergic structures. Recently, we have cloned a splice variant of ChAT cDNA, which lacks exons 6–9 in the coding region (Tooyama and Kimura, 2000). The protein product of the variant mRNA was designated ChAT of the peripheral type (pChAT), because pChAT immunohistochemistry readily reveals peripheral cholinergic structures (Nakanishi et al., 1999, Nakajima et al., 2000, Chiocchetti et al., 2003, Yasuhara et al., 2004, Yasuhara et al., in press). The conventional ChAT protein was found in both central and peripheral neurons, and therefore called ChAT of the common type (cChAT). Interestingly, the antibody against pChAT labeled additional populations of neurons that have not previously been identified as cholinergic. For example, pChAT-immunoreactivity was observed in magnocellular neurons in the tuberomammillary nucleus of the posterior hypothalamus (Kanayama et al., 2003) and in some retinal ganglion cells (Yasuhara et al., 2003). In addition, the rat dorsal root ganglia (Bellier and Kimura, in press) and trigeminal ganglia (Yasuhara et al., 2004) were shown to possess pChAT-positive neurons, suggesting the presence of cholinergic sensory neurons. The cholinergic nature of some primary sensory neurons was supported by our previous results with Western blotting, RT-PCR and ChAT enzyme assay (Yasuhara et al., 2004, Bellier and Kimura, in press).

Primary afferent neurons in the dorsal root ganglia and trigeminal ganglia have been classified into several subpopulations on the basis of their neurochemical profiles. Substance P (SP) and calcitonin gene-related peptide (CGRP) are found mainly in small to medium-sized ganglion cells, and assumed to be neurotransmitters for nociceptive sensory neurons (Tervo et al., 1981, Wisenfeld-Hallin et al., 1984, Lee et al., 1985a, Skofitsch and Jacobowitz, 1985, Matsuyama et al., 1986). In contrast, some calcium binding proteins including calbindin-D28k have been found predominantly in medium-sized to large neurons, and some of these positive neurons are implicated in proprioception (Ichikawa et al., 1994, Ichikawa et al., 1996, Ichikawa et al., 2005, Wakisaka et al., 1996). Neuronal nitric oxide synthase (nNOS) has been localized to some populations of ganglion neurons with small to large cell bodies (Alm et al., 1995, Zhang et al., 1996, Lazarov and Dandov, 1998, Cheng et al., 2001).

It is of interest to know in which sensory modality pChAT may play a role. We have previously shown that pChAT-immunoreactivity is present in small to medium-sized neurons in the rat trigeminal ganglia, indicating that pChAT play roles in nociception (Yasuhara et al., 2004). However, such positive neurons were not characterized in detail. In the present study, therefore, we further characterized pChAT-positive trigeminal neurons in the rat by analyzing cell size distribution and colocalization with other chemical markers such as SP, CGRP, nNOS, calbindin-D28k and a general neuronal marker, protein gene product 9.5 (PGP9.5).

Section snippets

Results

Fig. 1 shows immunohistochemical localization of pChAT-positive neuronal cells in the ophthalmic, maxillary and mandibular areas of the trigeminal ganglion. Locations of these nerve territories in the ganglion were based on the previous reports (Marfurt, 1981, Klein et al., 1986, Oyagi et al., 1989). As shown, pChAT-positive cells were distributed in a rather uniform pattern throughout the trigeminal ganglion. A moderate number of immunoreactive fibers ran in the ophthalmic (Fig. 1B), maxillary

Discussion

The present study describes several characteristics of pChAT-positive cells in the rat trigeminal ganglion. First, pChAT-positive cells are distributed in a rather uniform pattern throughout the trigeminal ganglion, including the ophthalmic, maxillary and mandibular areas. Second, pChAT-positive cells are neurons, because all pChAT-positive cells show immunoreactivity for PGP9.5, a general neuronal marker. It is also shown that about a half of all trigeminal neurons (49.4% of PGP9.5-positive

Animals

Three male Wistar rats (Clea Japan Inc., Tokyo, Japan), weighing 200–300 g, were used. Procedures involving animals and their care were conducted in conformity with the standards for animal experiments in our university and are in compliance with the NIH Guide for the Care and Use of Laboratory Animals, US (1996). All efforts were made to minimize both the number of animals used and any suffering that they might experience.

Under pentobarbital anesthesia (80 mg/kg), the rats were perfused on

Acknowledgment

We thank Mr. T. Yamamoto (Shiga University of Medical Science) for technical assistance.

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