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Comparative Study
. 2011 Mar 30;31(13):5067-77.
doi: 10.1523/JNEUROSCI.6451-10.2011.

Trpv1 reporter mice reveal highly restricted brain distribution and functional expression in arteriolar smooth muscle cells

Daniel J Cavanaugh  1 , Alexander T Chesler, Alexander C Jackson, Yaron M Sigal, Hiroki Yamanaka, Rebecca Grant, Dajan O'Donnell, Roger A Nicoll, Nirao M Shah, David Julius, Allan I Basbaum
Affiliations
Free PMC article
Comparative Study

Trpv1 reporter mice reveal highly restricted brain distribution and functional expression in arteriolar smooth muscle cells

Daniel J Cavanaugh et al. J Neurosci. .
Free PMC article

Abstract

The heat and capsaicin receptor, TRPV1, is required for the detection of painful heat by primary afferent pain fibers (nociceptors), but the extent to which functional TRPV1 channels are expressed in the CNS is debated. Because previous evidence is based primarily on indirect physiological responses to capsaicin, here we genetically modified the Trpv1 locus to reveal, with excellent sensitivity and specificity, the distribution of TRPV1 in all neuronal and non-neuronal tissues. In contrast to reports of widespread and robust expression in the CNS, we find that neuronal TRPV1 is primarily restricted to nociceptors in primary sensory ganglia, with minimal expression in a few discrete brain regions, most notably in a contiguous band of cells within and adjacent to the caudal hypothalamus. We confirm hypothalamic expression in the mouse using several complementary approaches, including in situ hybridization, calcium imaging, and electrophysiological recordings. Additional in situ hybridization experiments in rat, monkey, and human brain demonstrate that the restricted expression of TRPV1 in the CNS is conserved across species. Outside of the CNS, we find TRPV1 expression in a subset of arteriolar smooth muscle cells within thermoregulatory tissues. Here, capsaicin increases calcium uptake and induces vasoconstriction, an effect that likely counteracts the vasodilation produced by activation of neuronal TRPV1.

Figures

Figure 1.
Figure 1.
Primary afferent expression of reporter molecules in TRPV1PLAP-nlacZ mice. A, Schematic showing the Trpv1 locus of TRPV1PLAP-nlacZ mice. B, nlacZ is present in nuclei of DRG neurons. C, PLAP is present both in cell bodies (arrowheads) and axonal processes (arrows) of DRG neurons. D, PLAP staining in primary afferent terminals in the SC dorsal horn. E, nlacZ staining (magenta) in the DRG section shows near-complete overlap with TRPV1 immunoreactivity (green). F–H, Cultured adult DRG neurons were imaged with Fura-2-AM dye at baseline (F) and following stimulation with 1 μm CAP (G) and HiK (H). I, nlacZ staining in these cells. J, The 340/380 ratios of cells as numbered in I. Scale bars: B, C, E, I, 100 μm; D, 200 μm.
Figure 2.
Figure 2.
nlacZ staining in coronal sections of TRPV1PLAP-nlacZ mice. A–H, We observed sparse nlacZ expression in the IF (A), entorhinal cortex (B), RLi (C), SuM (D), periaqueductal gray (PAG; E), Cajal-Retzius cells of the hippocampus (HI; F), DMH (G), and AOB (H). Insets in A, C, D, E, and G are magnified images of dashed regions. Insets in F and H show sections processed for nlacZ (magenta) and stained for reelin immunoreactivity (green). I, Outlines of coronal brains illustrating the nlacZ+ areas of the rostral midbrain and caudal hypothalamus. Scale bar, 200 μm.
Figure 3.
Figure 3.
Radioactive in situ for TRPV1 in DRGs and brain of mouse, rat, and human. A, Bright-field image of radioactive in situ in mouse DRG, counterstained with H&E. B, Double labeling with TRPV1 antibody (brown) and radioactive in situ probe (black) shows complete colocalization between the two in mouse DRG. C, Bright-field image of radioactive in situ in rat DRG, counterstained with H&E. S, Small-diameter neurons; M, medium-diameter neurons; L, large-diameter neurons. Asterisks indicate negative cells. D, Dark-field image of radioactive in situ in rat DRG. E–H, Coronal sections of rat brain processed for radioactive TRPV1 in situ hybridization showing positive signals in PH (E), SuM (F), IF (G), and DMV (10th motor nucleus; H). I–P, Close-up images of indicated regions are shown for rat (I–L) and mouse (M–P). Q, Dark-field image of radioactive in situ signal in human DRG. R, S, Representative images of radioactive in situ in human brain sections, demonstrating complete lack of staining. ac, Anterior commissure; CA1, CA1 region of hippocampus; cc, corpus callosum; Cd, caudate; CI, claustrum; DG, dentate gyrus; ec, external capsule; GP, globus pallidus; ins gyrus, insular gyrus; Pir Cx, piriform cortex; Put, putamen. Scale bars: A, B, Q, 100 μm; C, D, I–L, 50 μm; E–H, 2 mm; M–P, 25 μm; R, S, 5 mm.
Figure 4.
Figure 4.
Brain lacZ staining in TRPV1Cre/R26R-lacZ mice. A, Schematic showing the Trpv1 locus of TRPV1Cre mice. B, C, lacZ staining in DRG (B) and SC (C) of TRPV1Cre/R26R-lacZ mice. D–K, We observed lacZ+ cells in the IF (D), entorhinal cortex (E), RLi (F), SuM (G), periaqueductal gray (PAG; H), Cajal-Retzius cells of the hippocampus (HI; I), DMH (J), and AOB (K). Insets are magnified images of dashed regions. Scale bars: B, 100 μm; C–K, 200 μm.
Figure 5.
Figure 5.
Developmental expression of TRPV1 in the brain. Left, lacZ staining in 80 μm sections of TRPV1Cre/R26R-lacZ mice. Right, Lack of nlacZ staining in the same areas in TRPV1PLAP-nlacZ mice, likely attributable to developmental expression that is extinguished in the adult. A–I, Regions include the 10th and 12th motor nuclei of the medulla (A) (arrowheads indicate cell body staining in 10th and 12th motor nuclei; arrows indicate axonal lacZ label in the area postrema and nucleus of the solitary tract), nucleus interpolaris (B), perivolivary region (C), raphe magnus (D), central gray region (E), inferior colliculus (F), parabrachial nucleus (PB; arrowheads) and nucleus of the lateral lemniscus (arrows) (G), lateral hypothalamic nucleus (H, arrowheads), and paraventricular hypothalamic nucleus (PVH; I). J, RT-PCR analysis of brain regions and peripheral tissues to detect presence of TRPV1 mRNA. Note the faint band in caudal hypothalamus lane (arrow). OB, Olfactory bulb; Co, cortex; Hi, hippocampus; AH, anterior hypothalamus; CH, caudal hypothalamus; PG, periaqueductal gray; Ce, cerebellum; Li, liver; Bl, bladder; Cr, cremaster; neg, no cDNA. Scale bar, 500 μm.
Figure 6.
Figure 6.
Functional TRPV1 expression in brain regions implicated in the Trpv1 knock-in mice. A–H, Calcium imaging of SuM neurons in slices (A–D) and acutely dissociated cultures (E–H) from TRPV1Cre/R26R-EYFP mice during application of 10 μm CAP (B, F) and high K+ ACSF (C, G). D, H, Endogenous EYFP fluorescence. I, J, Representative 340/380 ratios of the EYFP-positive cells from slices (normalized to baseline; I) and acutely dissociated neurons (J). Representative 340/380 ratios of cells from another well (J, bottom) are shown, demonstrating a block of CAP-induced calcium increases by RR. Arrows in D and H point to CAP-responsive, EYPF+ cells; the arrowhead points to EYFP+ cell that did not respond to CAP. K, Whole-cell recordings of EYFP+ SuM cells in brain slices of TRPV1Cre/R26R-EYFP mice during application of 10 μm CAP. The red trace shows the average response of all CAP-responsive cells. L–N, Calcium imaging of dentate gyrus neurons in slices during application of 10 μm CAP (M) and high K+ ACSF (N). Insets are magnified images of the dashed region in L. O, Representative 340/380 ratios, normalized to baseline, of 30 dentate gyrus neurons during calcium imaging.
Figure 7.
Figure 7.
TRPV1 expression in a subset of arteriolar SMCs. A, PLAP staining in cremaster muscle, showing PLAP+ (arrows) and PLAP− (arrowheads) vessels. B, nlacZ staining in cremaster muscle. C, PLAP staining in dura, showing PLAP+ (arrows) and PLAP− (arrowheads) vessels. D, nlacZ in tongue (magenta) colocalizes with staining for SMA (green). E, F, Inset, Magnification of boxed area. PLAP+ SMCs (arrows) were also observed in trachea (E) and skin (F). Arrowheads point to the PLAP+ axonal label, most likely of primary afferent origin. G, H, RTX eliminates nlacZ staining in the ear. I, J, A similar pattern of lacZ staining was seen in TRPV1Cre/R26R-lacZ mice. Shown are images from tongue (I) and ear (J). In J, the arrow points to lacZ+ SMCs, and the arrowhead points to a lacZ+ nerve. Scale bars: A–C, E–H, J, 500 μm; D, I, 200 μm.
Figure 8.
Figure 8.
Calcium imaging of TRPV1+ arterioles. A, C, Cultured arteriole explants from TRPV1Cre/R26R-EYFP mice were imaged with Fura-2-AM dye at baseline (BL) (A) and after stimulation with 10 μm CAP (C). E, EYFP expression in arteriolar SMCs of this mouse. B, D, F, Explants from Trpv1 knock-out mice lack CAP responses (D) but maintain responses to HiK (F). G, The 340/380 ratios of boxed regions in A. H, The 340/380 ratios of boxed regions in B. I, Average responses of arterioles from TRPV1Cre and Trpv1 knock-out mice after application of CAP, RR, and HiK (n = 4). p̂ < 0.05; ^p̂ < 0.01, compared with BL; **p < 0.01, compared with TRPV1Cre; two-way ANOVA with Bonferroni post hoc test. Scale bar, 100 μm.
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Comment in

  • The expression pattern of TRPV1 in brain.
    Menigoz A, Boudes M. Menigoz A, et al. J Neurosci. 2011 Sep 14;31(37):13025-7. doi: 10.1523/JNEUROSCI.2589-11.2011. J Neurosci. 2011. PMID: 21917785 Free PMC article. No abstract available.

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