Systematic and quantitative mRNA expression analysis of TRP channel genes at the single trigeminal and dorsal root ganglion level in mouse

Abstract Background Somatosensory nerve fibres arising from cell bodies within the trigeminal ganglia (TG) in the head and from a string of dorsal root ganglia (DRG) located lateral to the spinal cord convey endogenous and environmental stimuli to the central nervous system. Although several members of the transient receptor potential (TRP) superfamily of cation channels have been implicated in somatosensation, the expression levels of TRP channel genes in the individual sensory ganglia have never been systematically studied. Results Here, we used quantitative real-time PCR to analyse and compare mRNA expression of all TRP channels in TG and individual DRGs from 27 anatomically defined segments of the spinal cord of the mouse. At the mRNA level, 17 of the 28 TRP channel genes, TRPA1, TRPC1, TRPC3, TRPC4, TRPC5, TRPM2, TRPM3, TRPM4, TRPM5, TRPM6, TRPM7, TRPM8, TRPV1, TRPV2, TRPV4, TRPML1 and TRPP2, were detectable in every tested ganglion. Notably, four TRP channels, TRPC4, TRPM4, TRPM8 and TRPV1, showed statistically significant variation in mRNA levels between DRGs from different segments, suggesting ganglion-specific regulation of TRP channel gene expression. These ganglion-to-ganglion differences in TRP channel transcript levels may contribute to the variability in sensory responses in functional studies. Conclusions We developed, compared and refined techniques to quantitatively analyse the relative mRNA expression of all TRP channel genes at the single ganglion level. This study also provides for the first time a comparative mRNA distribution profile in TG and DRG along the entire vertebral column for the mammalian TRP channel family.</p

Transient receptor potential canonical 5 (TRPC5) protects against pain and vascular inflammation in arthritis and joint inflammation

Objective: Transient receptor potential canonical 5 (TRPC5) is functionally expressed on a range of cells including fibroblast-like synoviocytes, which play an important role in arthritis. A role for TRPC5 in inflammation has not been previously shown in vivo. We investigated the contribution of TRPC5 in arthritis. Methods: Male wild-type and TRPC5 knockout (KO) mice were used in a complete Freund’s adjuvant (CFA)-induced unilateral arthritis model, assessed over 14 days. Arthritis was determined by measurement of knee joint diameter, hindlimb weightbearing asymmetry and pain behaviour. Separate studies involved chronic pharmacological antagonism of TRPC5 channels. Synovium from human post-mortem control and inflammatory arthritis samples were investigated for TRPC5 gene expression. Results: At baseline, no differences were observed. CFA-induced arthritis resulted in increased synovitis in TRPC5 KO mice assessed by histology. Additionally, TRPC5 KO mice demonstrated reduced ipsilateral weightbearing and nociceptive thresholds (thermal and mechanical) following CFA-induced arthritis. This was associated with increased mRNA expression of inflammatory mediators in the ipsilateral synovium and increased concentration of cytokines in synovial lavage fluid. Chronic treatment with ML204, a TRPC5 antagonist, augmented weightbearing asymmetry, secondary hyperalgesia and cytokine concentrations in the synovial lavage fluid. Synovia from human inflammatory arthritis demonstrated a reduction in TRPC5 mRNA expression. Conclusions: Genetic deletion or pharmacological blockade of TRPC5 results in an enhancement in joint inflammation and hyperalgesia. Our results suggest that activation of TRPC5 may be associated with an endogenous anti-inflammatory/analgesic pathway in inflammatory joint conditions

Expression of functional TRPV1 receptor in primary culture of canine keratinocytes

The interest for the endovanilloid system and for transient receptor potential vanilloid 1 (TRPV1) is continuously increasing, due to their involvement in inflammation, nociception and pruritus. Even if TRPV1 enrolment was highlighted in both physiological and pathological conditions, some aspects remain unclear, mostly in veterinary medicine. This study aimed to verify the expression and functionality of TRPV1 in canine keratinocytes to investigate in vitro the role of TRPV1 in these cells that are involved in different cutaneous pathologies. Keratinocytes primary cultures were isolated from bioptical samples and cultivated. Binding assay (using 3 [H]-resiniferatoxin), displacement assay (in the presence of 1.2 nM 3 [H]-resiniferatoxin) and functional assays (in the presence of 1 μCi/45 Ca2+ ) with vanilloid agonists and antagonists, specifically addressed to TRPV1 receptor, were performed. Binding assay demonstrated the presence of measurable concentrations of TRPV1 (Bmax = 1,240 ± 120 fmol/mg protein; Kd = 0.01 ± 0.004 nM). Displacement assay highlighted the highest affinity for resiniferatoxin (RTX) and 5-iodo-resiniferatoxin (5-I-RTX), among agonists and antagonists, respectively. The same compounds results as the most potent in the functional assays. This study demonstrated the identification and the characterization of TRPV1 receptor in primary canine keratinocytes cultures. The results are promising for a clinical use, but further in vivo investigations are required

De rol van TRP kanalen in het voelen van pijnlijke hitte

Although several members of the transient receptor potential (TRP) superfamily of cation channels have been implicated in somatosensation, the expression levels of TRP channel genes in the individual sensory ganglia have never been systematically studied. In the first part of this thesis, we developed, compared, and refined techniques to quantitatively analyse the relative mRNA expression of all TRP channels in trigeminal and individual dorsal root ganglia from 27 anatomically defined segments of the spinal cord of the mouse. At the mRNA level, 17 of the 28 TRP channel genes, TRPA1, TRPC1, TRPC3, TRPC4, TRPC5, TRPM2, TRPM3, TRPM4, TRPM5, TRPM6, TRPM7, TRPM8, TRPV1, TRPV2, TRPV4, TRPML1, and TRPP2, were detectable in every tested ganglion. Notably, four TRP channels, TRPC4, TRPM4, TRPM8, and TRPV1, showed statistically significant variation in mRNA levels between dorsal root ganglia from different segments, suggesting ganglion-specific regulation of TRP channel gene expression. This study provides for the first time a comparative mRNA distribution profile in TG and DRG along the entire vertebral column for the mammalian TRP channel family. Furthermore, although it is well-known that acute heat sensing in mammals is performed by a subset of sensory neurons expressing the heat sensor TRPV1, genetic ablation of TRPV1 or other candidate heat sensors, such as TRPM3, only resulted in small deficits in noxious heat sensing. So, the molecular basis of noxious heat sensing remained largely unresolved. In the second part of this thesis, we demonstrate that acute noxious heat sensing is mediated by a set of three TRP ion channels. We compared the sensitivity to heat in wild type mice and mice deficient for TRPV1, TRPM3, and TRPA1 in combined knockouts. We found that all double knockout mice retain robust heat responses at the cellular and behavioural level. In contrast, Trpa1-/-/Trpm3-/-/Trpv1-/- mice lack heat responses in isolated sensory neurons, and show a striking deficit in the heat sensitivity of intact C and Aδ nerve fibres and in the nociceptive responses to painfully hot temperatures. Reintroducing any of the three TRP channels into triple knockout sensory neurons restores heat responsiveness. Together, these data indicate that TRPA1, TRPV1, and TRPM3 have critical but redundant roles in acute noxious heat sensing. This redundancy could be a fail-safe mechanism allowing heat avoidance, even when the function of one or two molecular heat sensors is compromised.status: publishe

Heat is absolute, cold is relative

In vivo imaging of the spinal cord provides insights into the coding of skin temperature. Intriguingly, while heat-responsive dorsal horn neurons encode absolute temperatures, cold-responsive neurons report relative drops.status: publishe

Reply to: Heat detection by the TRPM2 ion channel

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