Research ReportPrimary 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
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.
References (39)
- et al.
Presence of neuronal nitric oxide synthase in autonomic and sensory ganglion neurons innervating the lacrimal glands of the cat: an immunofluorescent and retrograde tracer double-labeling study
J. Chem. Neuroanat.
(2001) - et al.
Non-myelinated afferent fibers do not originate exclusively from the smallest dorsal root ganglion cells in the cat
Neurosci. Lett.
(1986) - et al.
VR1-immunoreactive primary sensory neurons in the rat trigeminal ganglion
Brain Res.
(2001) - et al.
Parvalbumin, calretinin and carbonic anhydrase in the trigeminal and spinal primary neurons of the rat
Brain Res.
(1994) - et al.
Calbindin-D28k-immunoreactivity in the trigeminal ganglion neurons and molar tooth pulp of the rat
Brain Res.
(1996) - et al.
Calretinin-containing neurons which co-express parvalbumin and calbindin D28k in the rat spinal and cranial sensory ganglia; triple immunofluorescence study
Brain Res.
(2005) - et al.
Expression of a splice variant of choline acetyltransferase in magnocellular neurons of the tuberomammillary nucleus of rat
Neuroscience
(2003) - et al.
Topographic organization of peripheral trigeminal ganglionic projections in newborn rats
Brain Res.
(1986) - et al.
Coexistence of calcitonin gene-related peptide and substance P-like peptide in single cell of the trigeminal ganglion of the rat: immunohistochemical analysis
Brain Res.
(1985) - et al.
Distribution of calcitonin gene-related peptide in the rat peripheral nervous system with reference to its coexistence with substance P
Neuroscience
(1985)
Two distinct calcitonin gene-related peptide-containing peripheral nervous systems: distribution and quantitative differences between the iris and cerebral artery with special reference to substance P
Brain Res.
Immunohistochemical demonstration of choline acetyltransferase of a peripheral type, pChAT, in the enteric nervous system of rats
J. Chem. Neuroanat.
Immunohistochemical localization of choline acetyltransferase of a peripheral type in rat larynx
J. Chem. Neuroanat.
Topographic study of the feline trigeminal ganglion via the horseradish peroxidase tracer method
Brain Res.
Choline acetyltransferase-like immunoreactivity in small diameter neurones of the rat dorsal root ganglion
Neurosci. Lett.
Calcitonin gene-related peptide coexists with substance P in capsaicin sensitive neurons and sensory ganglia of the rat
Peptides
A protein encoded by an alternative splice variant of choline acetyltransferase mRNA is localized preferentially in peripheral nerve cells and fibers
J. Chem. Neuroanat.
Partial coexistence of neuropeptide Y and calbindin D28 k in the trigeminal ganglion following peripheral axotomy of the inferior alveolar nerve in the rat
Brain Res.
Monosynaptic circuitry of trigeminal proprioceptive afferents coordinating jaw movement with visceral and laryngeal activities in rats
Neuroscience
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