The cyclic nucleotide-gated ion channel CNGA3 contributes to coolness-induced responses of Grueneberg ganglion neurons

Abstract

Localized to the vestibule of the nasal cavity, neurons of the Grueneberg ganglion (GG) respond to cool ambient temperatures. The molecular mechanisms underlying this thermal response are still elusive. Recently, it has been suggested that cool temperatures may activate a cyclic guanosine monophosphate (cGMP) pathway in the GG, which would be reminiscent of thermosensory neurons in Caenorhabditis elegans. In search for other elements of such a cascade, we have found that the cyclic nucleotide-gated ion channel CNGA3 was strongly expressed in the GG and that expression of CNGA3 was confined to those cells that are responsive to coolness. Further experiments revealed that the response of GG neurons to cool temperatures was significantly reduced in CNGA3-deficient mice compared to wild-type conspecifics. The observation that a cGMP-activated non-selective cation channel significantly contributes to the coolness-evoked response in GG neurons strongly suggests that a cGMP cascade is part of the transduction process.

Fig. 1

Identification of CNG channel subunits expressed in the GG by PCR experiments. In PCR approaches with specific primer pairs matching to the coding sequence of CNG channel subtypes CNGA1, CNGA2, CNGA3, CNGA4, CNGB1 and CNGB3, no amplicons of the expected molecular size were obtained from GG cDNA of several-day-old postnatal animals for CNGA1, CNGB1 and CNGB3. In control experiments, using the same primers, such PCR products were easily amplified from genomic DNA (gD) or cDNA of the MOE or retina (Re), respectively (PCR reactions without template are indicated by “W”). With primers for CNGA2, CNGA3 or CNGA4 (arrowhead), PCR products of the predicted size were obtained from GG cDNA

Fig. 2

Expression of CNGA3 in numerous GG cells. a–f In situ hybridization experiments with antisense probes specific for CNGA4 (a, b), CNGA2 (c, d) or CNGA3 (e, f) on coronal sections through the GG of pups. b, d and f Show higher magnifications of the boxed areas in a, c and e. These experiments revealed that CNGA4 is absent from the GG, whereas CNGA2 is weakly expressed by a subset of cells in the GG (arrow in d). CNGA3, however, is strongly expressed in numerous GG cells. Scale bars a, c, e = 200 μm; b, d, f = 50 μm

Fig. 3

CNGA3 is expressed in a large subpopulation of OMP-positive GG neurons. a–c Two-color in situ hybridization on a coronal section through the GG from an early postnatal stage using antisense RNA probes for CNGA3 (a, red) and OMP (b, green). The overlay (c) demonstrates expression of CNGA3 in the overwhelming majority of OMP-positive GG neurons. The section was counterstained with DAPI (blue). Scale bar = 50 μm

Fig. 4

Expression of CNGA3 in a receptor-specific subset of GG neurons. a–c Double-labeling in situ hybridization on a coronal section through the GG of an early postnatal mouse with antisense riboprobes for CNGA3 (a, green) and V2r83 (b, red). The merged image (c) demonstrates expression of CNGA3 by V2r83-positive GG neurons. d–f In situ hybridization with probes for CNGA3 (d, green), TAAR6 and TAAR7 (e, red) revealed absence of CNGA3 from TAAR-positive GG neurons (f). Sections were counterstained with DAPI (blue). Scale bars = 50 μm

Fig. 5

Co-expression of CNGA3 and GC-G in the GG of pups. a–c Double fluorescent in situ hybridization with antisense probes for CNGA3 (a, red) and GC-G (b, green) reveals co-expression of these two signaling elements in GG neurons (merged image in c). The section was counterstained with DAPI (blue). Scale bar = 50 μm

Fig. 6

The CNGA3 protein is localized to the soma and the axon of GG neurons. a Immunohistochemical staining with a CNGA3-specific antibody on a coronal section through the GG of a CNGA3^+/+ pup. b High magnification image of the boxed area in a. The soma of GG neurons is clearly stained. c–d Using the anti-CNGA3 antibody for immunostaining on sagittal sections [caudal (C) is to the right and dorsal (D) to the top] through the GG of wild-type animals, in addition to the soma, the CNGA3 protein was also found in axonal processes of GG neurons (arrows in d). The boxed area in c is shown at a higher magnification in d. Sections were counterstained with propidium iodide (red, a–c). Scale bars a = 200 μm; b, d = 50 μm; c = 100 μm

Fig. 7

Attenuation of coolness-induced responses in the GG of CNGA3-deficient mice. a–h In situ hybridization experiments with an antisense probe for c-Fos on coronal sections through the GG of wild-type (left panel) or CNGA3-deficient (right panel) pups exposed to a warm (30°C; a–d) or a cool (22°C; e–h) ambient temperature for 2 h. b, d, f and h Show higher magnifications of the boxed areas in a, c, e and g. At 30°C (a–d), no c-Fos expression was detectable in the GG of both wild-type (a, b) and CNGA3^−/− (c, d) individuals. Upon exposure to 22°C, strong c-Fos expression was observed in the GG of wild-type pups (e, f), whereas c-Fos expression was hardly detectable in the GG of CNGA3-deficient individuals (g, h). The data shown in a–h are representative of four independent experiments. For each of these four independent experiments, a “novel” litter was used. From each of these litters, one to three animals were used for each temperature tested. Scale bars a, c, e, g = 200 μm; b, d, f, h = 50 μm