Short communicationExpression of the vanilloid receptor TRPV1 in rat dorsal root ganglion neurons supports different roles of the receptor in visceral and cutaneous afferents
Introduction
The vanilloid receptor TRPV1 (VR1) [7] is a non-selective cation channel of the transient receptor potential family of receptors [13] gated by noxious heat and capsaicin and also activated by mediators of inflammation and ischemia [19]. TRPV1, expressed by both peptidergic and non-peptidergic dorsal root ganglion (DRG) neurons at the origin of C and Aδ primary afferent fibers [14], [23], is considered an important marker for nociceptors [6]. It is transported peripherally to the skin and viscera [31] and centrally to the spinal cord and the sensory trigeminal nuclei [14]. Functionally, TRPV1 is important for thermal nociception and inflammatory hyperalgesia and allodynia [11] and has also been implicated in neuropathic pain [17].
The majority of TRPV1-positive DRG neurons express the peptidergic markers substance P (SP) and calcitonin gene-related peptide (CGRP) [23], [14], consistent with reports that stimulation causes capsaicin-sensitive afferents to release neuropeptides from their central terminals [16] and/or their peripheral terminals [24]. We found few fibers and boutons double-stained for TRPV1 and SP in the dorsal horn at spinal level L4 [20], [32] but substantial colocalization at spinal level L6/S1, particularly in areas of termination of visceral afferents [18]. This difference suggests that colocalization of TRPV1 with peptides is negligible in cutaneous afferents (which predominate at L4) but may be more pronounced in visceral afferents (which predominate at L6/S1), more specifically from the urinary bladder [2] and/or the rectum [9]. Intriguingly, very little or no colocalization of TRPV1 with SP has been observed in peripheral afferent fibers in the skin [15]. Furthermore, while the degree of colocalization of TRPV1 with CGRP in peripheral afferent axons was reported to vary with the particular viscus (in the gastrointestinal tract) [35], colocalization was less pronounced than expected on the basis of experiments with capsaicin treatment of neonatal rats [28].
We here employ a combination of neuronal tracing and multiple color immunofluorescence to identify differences in expression of TRPV1 by bladder and skin afferents as a way to explore the different role this receptor may play in visceral and cutaneous afferents. For this, we compare visceral TRPV1-positive afferents (from the urinary bladder) with cutaneous afferents to the same DRG (L6) and examine their expression of the peptidergic markers SP and CGRP and their ability to bind the non-peptidergic marker Isolectin B4 (IB4).
Section snippets
Materials and methods
Care and treatment of animals were according to University of North Carolina and NIH guidelines. Male Sprague–Dawley rats (250–350 g; Charles River) were anesthetized with a mixture of ketamine (50 mg/kg) and xylazine (8 mg/kg) and divided in two groups: 5 rats received 5 cutaneous injections of 1 μl of the tracer Fast Blue (FB, 1% aqueous solution containing 5% DMSO; EMS-Chemie) within the L6 dermatome on each side [30], and 5 rats received 5–7 injections of 0.5 μl FB via a 35 G needle
Results
Similar number of DRG neurons were labeled with FB injected into skin or bladder (Fig. 1). The cross-sectional area of DRG neurons labeled with FB injected in the skin ranged from 200 to 2200 μm2, but most (84%) were small and medium-sized (<1000 μm2, Fig. 2A). Thirty three percent of FB-positive cells with area smaller than 1000 μm2 were also TRPV1-positive. The area of FB-positive cells traced from the bladder ranged from 200 to 1600 μm2, and 90% were smaller than 1000 μm2. Of the latter, 62%
Discussion
Our results indicate that (i) TRPV1 is expressed by a higher fraction of visceral than cutaneous afferents to L6, (ii) the same fraction of visceral and cutaneous TRPV1-positive afferents co-express neuropeptides, (iii) a much higher fraction of cutaneous than visceral TRPV1-positive afferents stain for IB4, (iv) some visceral TRPV1-positive afferents do not stain for either neuropeptides or IB4, and (v) a considerable fraction of cutaneous TRPV1-positive afferents stain for both SP and IB4.
Acknowledgments
We thank Dr. R. Weinberg for careful reading of the manuscript. This work was supported by NIH grant NS40885.
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