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. 2017 Apr 5;37(14):3753-3763.
doi: 10.1523/JNEUROSCI.2628-16.2017. Epub 2017 Mar 7.

Compartmentalized cGMP Responses of Olfactory Sensory Neurons in Caenorhabditis elegans

Hisashi Shidara  1 , Kohji Hotta  1 , Kotaro Oka  2
Affiliations

Compartmentalized cGMP Responses of Olfactory Sensory Neurons in Caenorhabditis elegans

Hisashi Shidara et al. J Neurosci. .

Abstract

Cyclic guanosine monophosphate (cGMP) plays a crucial role as a second messenger in the regulation of sensory signal transduction in many organisms. In AWC olfactory sensory neurons of Caenorhabditis elegans, cGMP also has essential and distinctive functions in olfactory sensation and adaptation. According to molecular genetic studies, when nematodes are exposed to odorants, a decrease in cGMP regulates cGMP-gated channels for olfactory sensation. Conversely, for olfactory adaptation, an increase in cGMP activates protein kinase G to modulate cellular physiological functions. Although these opposing cGMP responses in single neurons may occur at the same time, it is unclear how cGMP actually behaves in AWC sensory neurons. A hypothetical explanation for opposing cGMP responses is region-specific behaviors in AWC: for odor sensation, cGMP levels in cilia could decrease, whereas odor adaptation is mediated by increased cGMP levels in soma. Therefore, we visualized intracellular cGMP in AWC with a genetically encoded cGMP indicator, cGi500, and examined spatiotemporal cGMP responses in AWC neurons. The cGMP imaging showed that, after odor exposure, cGMP levels in AWC cilia decreased transiently, whereas levels in dendrites and soma gradually increased. These region-specific responses indicated that the cGMP responses in AWC neurons are explicitly compartmentalized. In addition, we performed Ca2+ imaging to examine the relationship between cGMP and Ca2+ These results suggested that AWC sensory neurons are in fact analogous to vertebrate photoreceptor neurons.SIGNIFICANCE STATEMENT Cyclic guanosine monophosphate (cGMP) plays crucial roles in the regulation of sensory signal transduction in many animals. In AWC olfactory sensory neurons of Caenorhabditis elegans, cGMP also has essential and distinctive functions involving olfactory sensation and adaptation. Here, we visualized intracellular cGMP in AWC neurons with a genetically encoded cGMP indicator and examined how these different functions could be regulated by the same second messenger in single neurons. cGMP imaging showed that, after odor application, cGMP levels in cilia decreased transiently, whereas levels in dendrites and soma gradually increased. These region-specific responses indicated that the responses in AWC neurons are explicitly compartmentalized. In addition, by combining cGMP and Ca2+ imaging, we observed that AWC neurons are analogous to vertebrate photoreceptor neurons.

Keywords: C. elegans; cGMP imaging; compartment; sensory neuron.

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Figures

Figure 1.
Figure 1.
Compartmentalized cGMP responses to odor in AWC sensory neurons. A, Confocal images of pstr-2::cGi500. Black arrow indicates soma; white arrow, dendrite; and red arrow, cilia. Dotted lines indicate ROIs at either cilia or dendrites. Scale bar, 10 μm. B–G, Compartmentalized cGMP responses of AWC sensory neurons. B, D, F, Time courses of cGMP responses in cilia (B), dendrites (D), and soma (F) of AWC neurons. Traces are averages of the relative fluorescence ratio (CFP/YFP). The control corresponds to S-basal flow. Gray bar is the duration of odor stimulus. Shading in the graphs shows SEM. Dotted boxes indicate the time focused in bar graphs. C, E, G, Summaries of cGMP responses in cilia (C), dendrites (E), and soma (G) during each specified time. Error bars represent SEM. Datasets are as follows for control, 10−2, 10−3, 10−4, and 10−5 IAA, respectively: cilia and dendrites, n = 8, 9, 11, 9, and 11; soma, n = 10, 11, 11, 10, and 10 (C, left, p = 0.0801, p < 0.001, p < 0.001, p = 0.9686; C, right, p = 0.00287, p = 0.25269, p = 0.56314, and p = 0.10763; E, left, p < 0.001, p < 0.001, p = 0.0017, and p < 0.001; E, right, p = 0.0261, p = 0.0449, p = 0.8123, and p = 0.0355; G, left, p = 0.019, p < 0.001, p < 0.001, and p < 0.001; G, right, p = 0.841, p = 0.132, p = 0.939, and p = 1.000; 10−2, 10−3, 10−4, and 10−5 IAA, respectively; Dunnett's test). *p < 0.05; **p < 0.01; ***p < 0.001, significant difference. H, cGMP responses at cilia to odor removal. Time of the prior exposure to 10−4 diluted IAA was 5 min. n = 8. I, Subcellular cGMP responses at head regions. Heat maps show responses of cGMP. Horizontal axes of heat maps correspond to upper images. Resolution of the horizontal axis is 2.17 μm. Scale bar, 10 μm.
Figure 2.
Figure 2.
Compartmentalized cGMP responses to benzaldehyde in AWC sensory neurons. A–C, Time courses of cGMP responses in cilia (A), dendrites (B), and soma (C) of AWC. Traces are averages of the relative fluorescence ratio. Gray bar is the duration of odor stimulus. Shading in the graphs shows SEM. Dotted boxes indicate the time focused in bar graphs. D, Summary of transient reduction in cilia and dendrites. (p = 0.004111, p = 0.004191, and p = 0.003277; 10−3, 10−4, and 10−5, respectively). **p < 0.01, significant difference. E, F, Summaries of cGMP responses in dendrites (E) and soma (F) during 120–130 s. Error bars indicate SEM. n = 10 each region.
Figure 3.
Figure 3.
Ca2+ responses to odor in each compartment of AWC sensory neurons. A, C, E, Time courses of Ca2+ responses to IAA in cilia (A), dendrites (C), and soma (E) of AWC sensory neurons. Traces are averages of the relative fluorescence ratio (blue/green). The control corresponds to S-basal flow. Gray bar is the duration of odor stimulus. Shading in the graphs shows SEM. Dotted boxes indicate the specified time in bar graphs. B, D, F, Summaries of Ca2+ responses in cilia (B), dendrites (D), and soma (F) during each time. Error bar indicates SEM. n = 10 for each condition. (B, left, p < 10−5, p < 10−5, p < 10−5, and p < 10−5; B, right, p < 10−4, p = 0.866, p = 0.851, and p = 0.925; D, left, p < 10−9, p < 10−9, p < 10−9, and p < 10−9; D, right, p < 10−4, p = 0.968, p = 0.970, and p = 1.000; F, left, p < 0.001, p < 0.001, p < 0.001, and p = 0.0778; F, right, p < 0.001, p = 0.934, p = 0.812, and p = 0.448; 10−2, 10−3, 10−4, and 10−5 IAA, respectively; Dunnett's test). ***p < 0.001, significant difference.
Figure 4.
Figure 4.
Roles of GC for cGMP responses in AWC. A–C, Time courses of cGMP responses to IAA in cilia (A), dendrites (B), and soma (C) of AWC sensory neurons in odr-1 mutants. The control corresponds to S-basal flow. Gray bar is the duration of odor stimulus. Shading in the graphs shows SEM. Dotted boxes indicate the time focused in bar graphs. D, Summaries of cGMP responses in odr-1 mutants. Error bar indicates SEM. E–G, Time courses of cGMP responses in cilia (E), dendrites (F), and soma (G) of AWC sensory neurons in daf-11 mutants. H, Summaries of cGMP responses in daf-11 mutants. n = 10 for each condition. (D, left, p = 0.149, p = 0.0432, and p = 0.902; D, right, p = 0.467, p = 0.269, and p = 0.130; H, left, p = 0.00321, p = 0.0556, and p = 0.712; H, right, p = 0.334, p = 0.482, and p = 0.780; cilia, dendrites and soma, respectively; Student's t test). *p < 0.05 and **p < 0.01 significant difference.
Figure 5.
Figure 5.
cGMP responses after exposure to odor for 15 min. A, Experimental procedure. B, D, F, cGMP responses in cilia (B), dendrites (D), and soma (F) before and after exposure to 10−4 diluted IAA for 15 min. Gray bar is the duration of odor stimulus. Shading in the graphs shows SEM. C, E, G, Summaries of cGMP responses in cilia (C), dendrites (E), and soma (F). Error bar indicates SEM. Blue indicates cGMP responses in the first trial; red indicates cGMP responses in the second trial. Datasets are as follows: cilia and dendrite, n = 9; soma, n = 8. (C, p = 0.5565; E, p = 0.02493; and G, p = 0.9856; Student's t test). *p < 0.05, significant difference.
Figure 6.
Figure 6.
cGMP responses in cilia to repeated stimuli. A, Experimental procedure. Worms were exposed to odor for 20 s every 30 s two or four times. B, cGMP responses to odor in the two-stimulus (top) and four-stimulus conditions (bottom). Each trace was extracted from sequential data consisting of 10 s before and 20 s after odor application. Gray bar is the duration of odor stimulus. Shading in the graphs shows SEM. C, Summaries of cGMP responses. Each variation in cGMP response is normalized by the variation of the first responses. D, Third and tenth cGMP response to odor in the 10-stimulus condition. E, cGMP response to sequential application of odor for different time intervals. Each variation of the second or last cGMP responses for different time intervals was normalized by the variation of the first response. The data for 10 and 70 s are the same in C. Error bar indicates SEM. n = 10, 11, 11, and 10 (two-, four-, and 10-stimulus presentations plus 40 s time interval stimulus presentation, respectively). (C, p = 0.01977, p = 0.004951, and p = 0.003289; second, third, and last responses, respectively; D, p = 0.3652; Student's t test). *p < 0.05, significant difference with Bonferroni correction.
Figure 7.
Figure 7.
cGMP responses to relative changes in odor. A, C, cGMP responses to relative changes in diluted IAA in cilia (A) and dendrites (C). Blue bar indicates the duration of 10−4 diluted IAA application. Gray bar is the duration of the test stimulus. Shading in the graphs shows SEM. B, D, Summaries of cGMP responses. Error bar indicates SEM. Datasets are as follows for 10−4, 0.5 × 10−4, 0.25 × 10−4, 0.2 × 10−4, 0.14 × 10−4, and 10−5 IAA, respectively: cilia and dendrites, n = 10, 10, 9, 9, 11, and 10; soma, n = 10, 10, 9, 9, 11, and 10. (B, p = 1.00000, p = 0.14898, p = 0.00176, p = 0.13384, and p < 0.001; D, p = 0.953, p = 0.296, p = 0.249, p < 0.001, and p < 0.001; 0.5 × 10−4, 0.25 × 10−4, 0.2 × 10−4, 0.14 × 10−4, and 10−5 IAA, respectively; Dunnett's test). **p < 0.01 and ***p < 0.001, significant difference.
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