The thermosensitive potassium channel TREK-1 contributes to coolness-evoked responses of Grueneberg ganglion neurons

Abstract

Neurons of the Grueneberg ganglion (GG) residing in the vestibule of the murine nose are activated by cool ambient temperatures. Activation of thermosensory neurons is usually mediated by thermosensitive ion channels of the transient receptor potential (TRP) family. However, there is no evidence for the expression of thermo-TRPs in the GG, suggesting that GG neurons utilize distinct mechanisms for their responsiveness to cool temperatures. In search for proteins that render GG neurons responsive to coolness, we have investigated whether TREK/TRAAK channels may play a role; in heterologous expression systems, these potassium channels have been previously found to close upon exposure to coolness, leading to a membrane depolarization. The results of the present study indicate that the thermosensitive potassium channel TREK-1 is expressed in those GG neurons that are responsive to cool temperatures. Studies analyzing TREK-deficient mice revealed that coolness-evoked responses of GG neurons were clearly attenuated in these animals compared with wild-type conspecifics. These data suggest that TREK-1 channels significantly contribute to the responsiveness of GG neurons to cool temperatures, further supporting the concept that TREK channels serve as thermoreceptors in sensory cells. Moreover, the present findings provide the first evidence of how thermosensory GG neurons are activated by given temperature stimuli in the absence of thermo-TRPs.

Fig. 1

Expression of TREK-1 mRNA in the GG. a TREK-1 expression in the GG was visualized by in situ-hybridization with an antisense (as) riboprobe for TREK-1 on coronal sections through the anterior nasal region of a wild-type neonatal pup. c In control experiments, on sections incubated with the corresponding sense (s) probe for TREK-1, no staining was observable in the GG. b, d Higher magnifications of the boxed areas in a, c. Scale bars a, c = 200 μm; b, d = 50 μm

Fig. 2

TREK-1 protein is expressed in the GG. a–d Immunohistochemical staining with a TREK-1-specific antibody on coronal sections through the GG of wild-type (WT) (a, c) and TREK-KO (b, d) animals. The broken white lines denote the boundary between the mesenchyme harboring GG neurons and the adjacent cartilage tissue. In pups (a–b), TREK-1 immunoreactivity was observed in the GG of wild-type mice (a), whereas no immunolabeling was detectable in the GG of age-matched TREK-KO animals (b). c–d Similarly, in adults, TREK-1 immunoreactivity was clearly observable in the GG of wild-type mice (c) but not in the GG of TREK-KO animals (d). In adult mice, some signals were also detectable in the border region between the nasal mesenchyme and the neighboring cartilage tissue. However, these signals are non-specific since they were observed in both wild-type and TREK-KO animals. e Immunohistochemical staining with the antibody for TREK-1 on a coronal GG section from a wild-type pup. f Immunolabeling was strongly diminished after pre-incubation of the TREK-1 antibody with the corresponding blocking peptide (BP). Sections were counterstained with DAPI. Scale bars 50 μm

Fig. 3

The TREK-1 protein is expressed by GG neurons and localized to their periphery. a–c Immunohistochemical staining on a coronal section through the GG of an OMP/GFP neonatal mouse with an antibody against TREK-1 (a). Intrinsic GFP fluorescence indicative of GG neurons is shown in b. The overlay (c) demonstrates expression of TREK-1 in GG neurons. d–f Confocal laser scanning microscopy of GG neurons revealed that the TREK-1 immunoreactivity (d) encircles the GFP fluorescence (e). The merged image (f) displays a peripheral localization of the TREK-1 protein in GG neurons. The section in a–c was counterstained with DAPI. Scale bars a–c = 50 μm; d–f = 5 μm

Fig. 4

TREK-1 is present in coolness-responsive (CNGA3-positive) GG neurons. a–c Double–fluorescent immunohistochemistry with antibodies against TREK-1 (a) and CNGA3 (b) demonstrates co-expression of these two proteins in GG neurons (merged image in c). d–f Confocal laser scanning microscopy of GG neurons shows that TREK-1 immunostaining (d) and the immunoreactivity for CNGA3 (e) are localized to the same cells. The section in a–c was counterstained with DAPI. Scale bars a–c = 50 μm; d–f = 5 μm

Fig. 5

Comparison of GG responsiveness to cool ambient temperatures in wild-type and TREK-KO mice. a–d In situ-hybridization experiments with an antisense probe for c-Fos (at a dilution of 1:150) on coronal sections through the GG of wild-type (WT; left panel) or TREK-KO (right panel) pups exposed to a cool ambient temperature (22 °C) for 2 h. Figures b, d show higher magnifications of the boxed areas in a, c. Upon exposure to 22 °C, strong c-Fos expression was observed in the GG of wild-type pups (a–b), whereas c-Fos expression (notably the signal intensity) was weaker in the GG of TREK^−/− individuals (c–d). The data shown in figures a–d are representative of 4 experiments. Scale bars a, c = 200 μm; b, d = 50 μm

Fig. 6

Decreased responsiveness to coolness in the GG of TREK-KO mice. a–d Expression of c-Fos in the GG of early postnatal pups was analyzed by in situ hybridization using a c-Fos-specific antisense probe (at a dilution of 1:400). a–b Following an exposure to 22 °C for 2 h, c-Fos signals were clearly observable in the GG of wild-type (WT) mice. c–d Compared with wild-type conspecifics, the number of c-Fos-positive GG cells was reduced in the GG of TREK^−/− pups. All figures depicted are representative of 7 experiments. Scale bars a, c = 200 μm; b, d = 50 μm. e Counting the c-Fos-expressing GG cells after exposure to 22 °C for 2 h revealed that the number of these cells is clearly lower in TREK-KO than in wild-type pups. The results are derived from 7 experiments. In each experiment, the number of c-Fos-positive GG cells in a TREK^−/− pup was determined relative to that in a concomitantly processed wild-type conspecific, which was set as 100 %. A mean of values, the standard deviation and the P value were calculated (P value <0.0001)

Fig. 7

TREK-1 is not required to determine the threshold temperature of coolness-induced GG responses. a–h Expression of c-Fos in the GG of wild-type (WT) and TREK-KO pups upon exposure to different ambient temperatures for 2 h was monitored by in situ-hybridization with a c-Fos-specific antisense probe (at a dilution of 1:150) on coronal sections through the anterior nasal region. Substantial expression of c-Fos was induced in the GG of both wild-type (a) and TREK^−/− (b) mice following an exposure to 22 °C. Although c-Fos expression was clearly weaker at 26 °C, it was detectable in wild-type and TREK-KO (arrows) animals (c, d). At 28 °C, no coolness-induced response was observed in both mouse lines (e, f). Similarly, no c-Fos signals were visible upon exposure to 30 °C (g, h). The data shown in figures a–h are representative of 4 experiments. Scale bars 50 μm

Fig. 8

Schematic diagram of the molecular mechanisms underlying coolness-induced activation of GG neurons. Cool temperatures activate GG neurons via TREK-1 and additionally by means of a cGMP pathway. It is unclear whether coolness-evoked inactivation of TREK-1 might impact on cGMP signaling in these cells