Elsevier

Brain Research

Volume 995, Issue 2, 9 January 2004, Pages 176-183
Brain Research

Research report
[3H]Resiniferatoxin autoradiography in the CNS of wild-type and TRPV1 null mice defines TRPV1 (VR-1) protein distribution

https://doi.org/10.1016/j.brainres.2003.10.001Get rights and content

Abstract

Knowledge of the distribution and function of the vanilloid receptor (VR-1 or TRPV1) in the CNS lacks the detailed appreciation of its role in the peripheral nervous system. The radiolabelled vanilloid agonist [3H]resiniferatoxin (RTX) has been used to indicate the presence of TRPV1 receptor protein in the brain but low specific binding has complicated interpretation of this data. Recently, support for a more widespread CNS distribution of TRPV1 mRNA and protein has been provided by RT-PCR and antibody data. We have exploited the availability of TRPV1 null mice and used [3H]RTX autoradiography in the CNS of TRPV1 wild-type and TRPV1 null mice to identify the component of [3H]RTX binding to TRPV1 receptor protein. In the brains of TRPV1+/+ mice, specific [3H]RTX binding was broadly localised with the greatest binding in the olfactory nuclei, the cerebral cortex, dentate gyrus, thalamus, hypothalamus, periaqueductal grey, superior colliculus, locus coeruleus and cerebellar cortex. Specific binding was also seen in the spinal cord and sensory (dorsal root and trigeminal) ganglia. This binding was much lower but not abolished in most regions in the TRPV1−/− mice. Nonspecific binding was low in all cases. The present study unequivocally demonstrates a widespread and discrete distribution pattern of the TRPV1 receptor protein in the rat central nervous system. The presence of TRPV1 receptors in several brain regions suggests that it may function as a cannabinoid-gated channel in the CNS.

Introduction

Capsaicin, the primary pungent principle from chili peppers, and related vanilloid compounds have been valuable tools for characterising the properties of vanilloid receptors in sensory neurons [17]. Resiniferatoxin (RTX) has been extensively used to study the pharmacology of the VR-1 receptor since it mimics the agonist actions of capsaicin and the radiolabelled forms show specific, saturable binding to rat sensory ganglion membranes with high affinity that is fully displaced by capsaicin [19]. The same tools have been used to explore the potential function of vanilloid receptors in the brain. Microinjection of capsaicin and RTX into various brain regions has evoked responses [6], [15] but clear description of effects at the cellular level has only been achieved in limited cases. Perhaps the best characterised response is that in the locus coeruleus, where capsaicin application evokes glutamate release [10], [12].

Localisation studies using RTX have also been equivocal. Autoradiographic studies using [3H]RTX demonstrated specific binding in the rat sensory ganglia and brain stem only [19].

In contrast, Acs et al. [2] have demonstrated [3H]RTX binding on membrane preparations from the rat ventral thalamus, locus coeruleus and hypothalamus but again with a high level of nonspecific binding.

The cloning of the vanilloid (VR-1 or TRPV1) receptor [3] has provided new tools with which to explore the distribution and function of this cation channel that is gated by heat, acid, and lipid ligands [9]. Initial attempts to detect TRPV1 mRNA from whole rat brain using Northern blot hybridisation failed [20]. Subsequently, use of PCR with rat VR-specific primers [13] has shown the presence of TRPV1 mRNA expressing neurons widely distributed in the central nervous system of the rat and human. This broad distribution has been supported at the protein level using immunocytochemistry. TRPV1 immunoreactivity was seen in regions that included cortical areas, striatum, hypothalamus, thalamus, locus coeruleus and cerebellum.

VR-1 or TRPV1 is now known to be the first member of a family of six vanilloid receptors named TRPV1–6, which are themselves part of a larger superfamily of calcium-permeable cation channels. As TRPV3, the most recent addition to this family was only described in 2002 [14], [16], it has not been possible to check the reagents used for TRPV1 localisation for specificity against all the members of this family. Thus, questions remain as to the interpretation of the existing protein localisation data.

The aim of this study was to use autoradiography to re-examine the distribution of [3H]RTX binding in the CNS in wild-type and TRPV1 null mice [5].The TRPV1 null mice enabled us to precisely define the level of binding to other binding sites in addition to TRPV1 and, hence, to accurately identify the distribution of TRPV1 binding sites. Our results clearly demonstrate a widespread distribution of TRPV1 specific binding sites in the mouse central nervous system.

Section snippets

Targeting of TRPV1 gene and generation of mutant mice

The generation of mutant mice has been reported previously [5]. A standard gene targeting approach was chosen to replace, in E14.1 ES cells, the DNA encoding amino acids 460–555 of mTRPV1 with the neomycin phosphotransferase gene. A 2.3-kilobase (kb) 3′ and 3.75-kb 5′ homology arm were isolated from a mouse 129SVJ BAC library, cloned either side of PGKneo, and flanked by diphtheria toxin-A gene as a negative selection marker. Homologous recombination in resistant ES cells was confirmed by

Distribution of [3H]resiniferatoxin (RTX) binding in the TRPV1 wild-type (+/+) mouse central nervous system

In the TRPV1 wild-type (+/+) mice, specific [3H]RTX binding was heterogeneously distributed with variable density throughout the cerebrum (Fig. 1A–F). Parallel processing of sections from similar anatomical regions showed that the density of [3H]RTX binding was greatly reduced in the CNS of TRPV1−/− mice (Fig. 1G–L). In both wild-type and TRPV1−/− sections, binding was largely displaced by 1 μM nonradioactive RTX, and remaining binding was defined as nonspecific. The distribution of specific [3

Discussion

In this study, we report the distribution of the TRPV1 receptor protein in the murine CNS based on a comparison of [3H]RTX autoradiography in tissues from TRPV1 wild-type and null mice. By measuring the difference in specific binding between TRPV1+/+ and −/− brain sections, we were able to define binding that we attribute to TRPV1 receptor protein, thus providing a valuable validation of previous studies with this ligand and a comparison with antibody localisation data. Our findings are in

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