The odor of a nontoxic tetrodotoxin analog, 5,6,11-trideoxytetrodotoxin, is detected by specific olfactory sensory neurons of the green spotted puffers

Abstract

Toxic puffers accumulate tetrodotoxin (TTX), a well-known neurotoxin, by feeding on TTX-bearing organisms and using it to defend themselves from predators. Our previous studies have demonstrated that toxic puffers are attracted to 5,6,11-trideoxytetrodotoxin (TDT), a nontoxic TTX analog that is simultaneously accumulated with TTX in toxic puffers and their prey. In addition, activity labeling using immunohistochemistry targeting neuronal activity marker suggests that TDT activates crypt olfactory sensory neurons (OSN) of the green spotted puffer. However, it remains to be determined whether individual crypt OSNs can physiologically respond to TDT. By employing electroporation to express GCaMP6s in OSNs, we successfully identified a distinct group of oval OSNs that exhibited a specific calcium response when exposed to TDT in green spotted puffers. These oval OSNs showed no response to amino acids (AAs), which serve as food odor cues for teleosts. Furthermore, oval morphology and surface positioning of TDT-sensitive OSNs in the olfactory epithelium closely resemble that of crypt OSNs. These findings further substantiate that TDT is specifically detected by crypt OSNs in green spotted puffer. The TDT odor may act as a chemoattractant for finding conspecific toxic puffers and for feeding TTX-bearing organisms for effective toxification.

Keywords: calcium imaging; chemical communication; electroporation; feeding; toxification.

Fig. 1.

In vivo electroporation of GCaMP6s to OSNs. Schematic diagram representing the waveform used for electroporation (A). Effects of the pattern interval between the poring and the transfer pulse (B), transfer pulse frequency (C), and transfer pulse amplitude (D) on the expression rate of GCaMP6s in OSNs. Each condition was repeated for 6 trials (for pattern interval and transfer pulse frequency) or 10 trials (for transfer pulse amplitude). Dunnett’s multiple comparison test (in comparison with t. pulses only; for (B)) or Tukey’s multiple comparison test. **P < 0.01.

Fig. 2.

GCaMP6s-expressing OSNs in green spotted puffer’s OE following electroporation using our employed protocol. (A) Distribution of the GCaMP6s-expressing cells in the electroporated OE. Differential interference contrast (DIC; a), epifluorescence (b), and a merged image of the OE surface of the OE surface from the green spotted puffer (gray: DIC, green: GCaMP6s, c). The GCaMP6s-expressing cells are dispersed in the OE, enabling the morphology of individual OSNs to be visually distinguished. Arrow heads indicate GCaMP6s expressing OSNs. (B) Pseudo-cross-sectional view of another electroporated OE. DIC (a), epifluorescence (b), and a merged image of the OE (gray: DIC, green: GCaMP6s, c). In this image, a GCaMP6s-expressing OSN extend an apical dendrite from the cell body in the middle layer of the OE to the surface. Arrowheads indicate GCaMP6s-expressing OSN. (C) Confocal laser-scanning microscopy images of the GCaMP6s-expressed OSNs. Bird’s-eye view volume-rendering x–z images of the electroporated OE surface created from Z-stacks of confocal laser-scanning microscopy. Two images from different OEs are shown in (a) and (b). The dashed rectangles in the images indicate the OSNs positions shown in c–e. Volume-rendering x–z images of a single GCaMP6sexpressing OSN generated from (a) or (b) are shown in (c–e): An oval OSN without apical dendrites in the surface layer of OE (c); a spindle-shaped OSN extending a swollen apical dendrite from the cell body in the middle layer of OE (d); an elongated OSN extending a thick and short apical dendrite from the cell body in the middle layer of OE (e). Asterisks in c–e indicate the location of the nucleus. Scale bars: 10 µm (A, B, C, a and b) and 5 µm (C, c, d and e).

Fig. 3.

Response of OSNs to AAs or TDT. (A) Fluorescence changes in AAs-sensitive OSNs to the vehicle or AAs exposure. Individual AAs-sensitive OSNs (n = 14) are represented by gray dashed lines, with the bold line and envelope showing the mean ± SEM. Representative fluorescence image frames (a–e; the vehicle, f–j; AAs) indicated by vertical lines are shown above the trace. (B) The peak amplitude of AAs-sensitive OSNs after administering the vehicle (black) or AAs (brown). Paired t-test compared the vehicle vs. AAs administration. ***P < 0.001. (C) Volume-rendered x-z image generated from z-stacks of the post-imaging confocal laser-scanning microscopy of the AAs-sensitive OSN as depicted in A. Scale bar: 5 µm. (D) Fluorescence changes in TDT-sensitive OSNs to the vehicle or TDT exposure. Individual TDT-sensitive OSNs (n = 15) are represented by gray dashed lines, with the bold line and envelope showing the mean ± SEM. Representative frames (a–e; the vehicle, f–j; TDT) indicated by vertical lines are shown above the trace. (E) The peak amplitude of TDT-sensitive OSNs after administering the vehicle (black) or TDT (green). Paired t-test compared the vehicle vs. TDT administration. ***P < 0.001. (F) Volume-rendered x-z image generated from z-stacks of the post-imaging confocal laser-scanning microscopy of the TDT-sensitive OSN as depicted in D. Scale bar: 5 µm.

Fig. 4.

TDT-sensitive OSNs did not respond to AAs. (A) Fluorescence changes in TDT-sensitive OSNs to the vehicle, TDT, and AAs exposure. Individual OSNs (n = 6) are represented by gray dashed lines, with the bold line and envelope indicating the mean ± SEM. (B) Peak amplitude of TDT-sensitive OSNs after the administration of the vehicle (black), TDT (green), and AAs (brown). One-factor repeated measures ANOVA with Holm correction. ***P < 0.001. (C) Fluorescence changes in AAs-sensitive OSNs to the vehicle, AAs, and TDT. Individual OSNs (n = 6) are represented by gray dashed lines, with the bold line and envelope indicating the mean ± SEM. (D) Peak amplitude of AAs-sensitive OSNs after the administration of the vehicle (black), TDT (green), and AAs (brown). One-factor repeated measures ANOVA with Holm correction. ***P < 0.001.