Pan-neuronal optogenetic induction of cAMP disrupts SIS in C. elegans

Induction of IlaC22 or PaaC in cholinergic motor neurons increases locomotor activity in awake young adults (Ryu et al. 2014; Etzl et al. 2018). Because cAMP is a conserved wake-promoting second messenger (Crocker and Sehgal 2010), we hypothesized that activation of IlaC22 or PaaC in neurons that are active during times of wakefulness would inhibit sleep. To test this hypothesis, we codon-optimized IlaC22 for C. elegans expression (Redemann et al. 2011) (GeneScript), and subsequently created multiple extrachromosomal lines and an integrated transgenic strain that express IlaC22 in all 302 neurons, using the promoter from the gene snb-1 (Nonet et al. 1998). The snb-1 promoter was placed upstream of an artificial operon (Spieth et al. 1993), comprising the sequence of IlaC22 and the coding sequence for dsRed as the downstream gene, to verify expression (Figure 1A). As a first test to determine IlaC22 functionality in vivo, we quantified locomotion (body bends), feeding (pumps), and defecation (expulsions) in first-day adults following exposure to green or red light. As expected, snb-1p:IlaC22 animals performed more bends than wild-type animals both in the presence of green and red light, but significantly more during red light exposure (Figure S1A). A basal activity of IlaC22 in the absence of red light has been described in the past (Etzl et al. 2018). Next, we quantified feeding and defecation under red light and found that snb-1p:IlaC22 animals displayed a slightly higher but significant increase in pumping rate, but there was not a significant difference in their defecation rate (Figure S1, B and C).

To quantify the effects of IlaC22 activation during SIS, first-day adult wild-type, ceh-17(np1) animals that are SIS defective (Hill et al. 2014) because of a nonfunctional ALA neuron (Pujol et al. 2000), and snb-1p:IlaC22 animals were exposed to UV irradiation, as previously described (DeBardeleben et al. 2017). Locomotion quiescence was measured for 4-hr, under constant red or green light, using a WorMotel (Churgin et al. 2017). We found that snb-1p:IlaC22 animals exposed to red or green light displayed less SIS, although the reduction under green light did not reach the level of significance (Figure 1B). Additionally, we monitored the integrated and two extrachromosomal snb-1p:IlaC22 strains for 8-hr following UV exposure and found a significant reduction in total SIS (Figure 1C) and for the majority of the 8-hr time period (Figure 1D). Next, we expressed the red-light activated enzyme PaaC (Etzl et al. 2018) in snb-1–expressing neurons and again observed a reduction in SIS during constant red light (Figure 1E). Thus, induction of cAMP in the nervous system promotes arousal during SIS. Additionally, we measured total levels of DTS during L4 lethargus and observed a significant reduction in snb-1p:IlaC22 animals compared to wild type (Figure S1D).

When animals undergo SIS they become quiescent with respect to feeding and their defecation cycle (Nath et al. 2016). However, snb-1p:IlaC22 animals displayed normal levels of feeding quiescence and a similar number of defecation events 90 min post UV exposure (Figure 1, F and G). Thus, induction of cAMP, using IlaC22, in the nervous system disrupts locomotion but not feeding or defecation quiescence.

Inhibition of PKA is required for SIS

PKA is a likely downstream target of cAMP during DTS since kin-2(ce179) reduction of function mutants, where PKA is constitutively active (Charlie et al. 2006), display significantly less sleep (Belfer et al. 2013; Nagy et al. 2014b). Whether PKA inhibition is required for SIS had not been specifically tested, although PKA inhibition is not necessary for feeding quiescence during heat-induced SIS (Trojanowski et al. 2015). To explore the role of PKA in more detail, we measured locomotion, feeding, and defecation quiescence in kin-2(ce179) mutants during SIS. First, we monitored animals for 8-hr on WorMotels following a 30-min 37° heat shock. We found that locomotion quiescence was significantly decreased in kin-2(ce179) animals (Figure 2A). The same was observed for UV-induced SIS (Figure 2B). Next, we sought to quantify feeding and defecation quiescence. First, pumping rate prior to stress exposure was not significantly different between kin-2(ce179) and wild-type animals; however, kin-2(ce179) displayed impaired feeding quiescence 90 min after UV stress (Figure 2C). kin-2(ce179) animals displayed a reduced prestress defecation rate (Figure 2D), consistent with the finding that the duration of the defecation motor program is extended in kin-2 mutants (Nagy et al. 2015). Despite this, we observed a suppression of defecation quiescence 90 min after UV stress (Figure 2D). Thus, PKA inhibition is required for quiescence of locomotion, feeding, and defecation during SIS.

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Figure 2

PKA inactivation is required for both heat- and UV-induced SIS. (A) Locomotion quiescence during heat-induced SIS (37° for 30 min) in wild-type and kin-2(ce179) animals for 8 hr using a WorMotel (N = 32, *** P < 0.001). Statistical significance was calculated using Student’s t-test. (B) Locomotion quiescence during UV-induced SIS in wild-type and kin-2(ce179) animals for 8 hr using a WorMotel [N = 12 (wild type), N = 30 (kin-2), *** P < 0.001]. Statistical significance was calculated using Student’s t-test. (C) Pumping rate before and after UV-induced SIS in wild-type and kin-2(ce179) animals (N = 15 for each genotype; * P < 0.05). Statistical significance was calculated using one-way ANOVA followed by Tukey’s multiple comparisons test. (D) Defecation rate of wild-type and kin-2(ce179) animals pre-UV shock and 90 min post-UV shock (N = 20, *** P < 0.001, * P < 0.05). Statistical significance was calculated using one-way ANOVA followed by Tukey’s multiple comparisons test. All error bars represent mean ± SEM.

CRH-1/CREB does not function downstream of cAMP/PKA during SIS

CREB, coded by crh-1 in C. elegans (Kimura et al. 2002), is a downstream target of PKA (Carlezon et al. 2005) and promotes arousal in mammals and flies (Hendricks et al. 2001; Graves et al. 2003). In C. elegans, crh-1 null mutant animals display increased levels of DTS (Singh et al. 2014), making CRH-1 the likely target during DTS regulation. We wanted to determine if CRH-1 is functioning downstream of cAMP/PKA during SIS. To do this, we obtained two deletion alleles for crh-1, n3315 and tz2. crh-1(tz2) mutant animals displayed increased DTS (Figure 3A), similar to what was observed with crh-1(n3315) animals (Singh et al. 2014). To determine if CRH-1 is functioning downstream of cAMP/PKA during DTS, we measured locomotion quiescence in snb-1p:IlaC22 and crh-1(tz2);snb-1p:IlaC22 animals and found that activation of IlaC22 in this context resulted in DTS levels comparable to wild type but not increased to the levels of crh-1(tz2) animals alone (Figure 3B). Thus, as expected, CRH-1 is a likely downstream target of cAMP/PKA during the regulation of DTS, but PKA likely targets other yet to be identified molecules as well (Figure 3E, DTS model).

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Figure 3

CRH-1/CREB is required for stress-induced sleep. (A) Locomotion quiescence during L4 lethargus in wild-type and crh-1(tz2) animals (N = 15, *** P < 0.001). Statistical significance was calculated using Student’s t-test. (B) Locomotion quiescence during L4 lethargus in wild-type, snb-1p:IlaC22, and crh-1(tz2);snb-1:IlaC22 animals (N > 24, ** P < 0.01, *** P < 0.001). Statistical significance was calculated using one-way ANOVA followed by Tukey’s multiple comparisons test. (C) Locomotion quiescence during UV-induced SIS in wild-type, crh-1(tz2), and crh-1(n3315) animals for 8 hr using a WorMotel (N > 24, *** P < 0.001). Statistical significance was calculated using one-way ANOVA followed by Tukey’s multiple comparisons test. (D) Locomotion quiescence during UV-induced SIS in wild-type, snb-1p:IlaC22, and crh-1(tz2);snb-1:IlaC22 animals (N > 24, *** P < 0.001). Statistical significance was calculated using one-way ANOVA followed by Tukey’s multiple comparisons test. (E) Proposed models for cAMP/PKA and CREB signaling during DTS and SIS. All error bars represent mean ± SEM.

Next, we measured locomotion quiescence in both crh-1(tz2) and crh-1(n3315) animals during SIS and found that total quiescence was significantly decreased in both strains (Figure 3C). Also, crh-1(tz2);snb-1p:IlaC22 animals displayed a similar reduction in SIS compared to crh-1(tz2) animals, which was not additive (Figure 3D). These data suggest that cAMP/PKA signal through yet to be identified downstream factors to promote arousal during SIS and that CREB is activated via PKA-independent mechanisms to promote sleep (Figure 3E, SIS model 1). Alternatively, PKA could inhibit CREB during times of wakefulness (Figure 3E, SIS model 2).

Induction of cAMP in the DVA, RIFs, and CEPsh glial cells disrupts SIS

cAMP levels are regulated by activating and inhibitory GPCRs that modulate AC activity (Beebe 1994). Neuropeptides regulate sleep across phylogeny by signaling through GPCRs (Trojanowski and Raizen 2016); during SIS, the ALA neuron releases neuropeptides that act on unknown target cells to initiate sleep (Nelson et al. 2014; Nath et al. 2016), which may occur by inhibiting cAMP/PKA. We proposed that cells expressing the gene twk-16, including the DVA interneuron (Salkoff et al. 2001), were potential downstream targets of ALA signaling, based upon the expression pattern of the sleep-promoting neuropeptide receptor FRPR-4 (Nelson et al. 2015). To test this hypothesis, we generated transgenic lines expressing IlaC22 from the twk-16 promoter. In these lines, we observed intense expression in the DVA and RIF interneurons and the CEPsh glial cells, the latter of which has been implicated in DTS regulation (Katz et al. 2018) (Figure S2). We found that twk-16p:IlaC22 animals displayed significantly reduced quiescence compared to wild type (Figure 4A). To determine if these wake-promoting effects were downstream of known SIS regulating pathways, we activated IlaC22 in twk-16–expressing cells following overexpression of flp-13, a gene that codes for neuropeptides released from the ALA neuron during SIS that are capable of inducing sleep in active adults (Nelson et al. 2014; Nath et al. 2016). We found that FLP-13–induced quiescence was reduced in twk-16p:IlaC22 animals (Figure S2). Next, to test if PKA is acting in the twk-16 cells to promote arousal, we generated transgenic RNAi strains that express kin-2 double-strand RNA in the twk-16–expressing cells. We found that SIS was reduced when kin-2 is knocked down in these cells (Figure 4B). Thus, cAMP/PKA is functioning in the twk-16-expressing cells to promote arousal.

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Figure 4

Induction of cAMP in twk-16–expressing cells disrupts stress-induced sleep. (A) Locomotion quiescence during UV-induced SIS in wild-type (N = 53) and twk-16p:IlaC22 animals for 8 hr using a WorMotel (** P < 0.01, *** P < 0.001). Three extrachromosomal lines were analyzed (N > 15 for each line). (B) Locomotion quiescence during UV-induced SIS in wild-type (N = 51) and twk-16p:kin-2(RNAi) animals for 8 hr using a WorMotel (*** P < 0.001). Three extrachromosomal lines were analyzed (N > 21 for each line). (C) Locomotion quiescence during UV-induced SIS in wild-type (N = 35) and twk-16(cs1)p:IlaC22 animals for 8 hr using a WorMotel (* P < 0.05). Three extrachromosomal lines were analyzed (N ≥ 15 for each line). (D) Locomotion quiescence during UV-induced SIS in wild-type (N = 40) and hlh-17p:IlaC22 animals for 8 hr using a WorMotel (* P < 0.05, *** P < 0.001). Three extrachromosomal lines were analyzed (N ≥ 12 for each line). (E) Locomotion quiescence during UV-induced SIS in wild-type (N = 22) and pdfr-1p:IlaC22 animals for 8 hr using a WorMotel (** P < 0.01). Three extrachromosomal lines were analyzed (N ≥ 15 for each line). In each case, statistical significance was calculated using one-way ANOVA followed by Tukey’s multiple comparisons test. All error bars represent mean ± SEM.

Next, we made transgenic animals that express IlaC22 specifically in the DVA interneuron using the enhancer element of the twk-16 promoter, cs1 (Puckett Robinson et al. 2013) (Figure S2). Activation of IlaC22 in DVA alone reduced SIS in one of three transgenic lines, but not to the same levels seen when activated in all twk-16–expressing cells (Figure 4C). Next, we expressed IlaC22 from the promoter of the gene hlh-17, which is expressed in the CEPsh glia (McMiller and Johnson 2005) (Figure S2). When IlaC22 was activated in the CEPsh cells, we observed a reduction in SIS in three independent lines, which was less than when activated in all twk-16–expressing cells (Figure 4D). Lastly, we expressed IlaC22 in the RIF interneurons using the promoter from the gene pdfr-1 (Barrios et al. 2012) (Figure S2). Activation of IlaC22 in the RIFs also reduced SIS in one of three transgenic lines (Figure 4E). Thus, cAMP induction in the DVA or RIF neurons or the CEPsh glia is sufficient to promote wakefulness during SIS, but not to the same extent when cAMP is induced in all cells together.

cAMP dynamics in the RIF and DVA interneurons are regulated by the ALA neuron

To test the physiological relevance of the cAMP/PKA pathway in the twk-16–expressing cells during SIS, we adapted a cAMP biosensor epac1-camps (Nikolaev et al. 2004) for use in C. elegans. Before expressing epac1-camps in vivo we confirmed that we could use this biosensor in cell culture. We transfected HEK293 cells with epac1-camps and treated cells with 25 µM forskolin and 100 µM IBMX and observed a significant increase in cAMP (Figure S4A). To confirm that epac1-camps is functional in vivo we made a transgenic line expressing epac1-camps and IlaC22 in cholinergic motor neurons. We exposed these animals to either red light or darkness for 20 min and quantified the CFP:YFP ratio every 5 min. We imaged a single neuron of the paired AIYs and found that cAMP levels increased consistently over the 20-min period (Figure S4B). This suggests that epac1-camps responds to changes in cAMP and that IlaC22 produces measurable increases of cAMP in vivo in C. elegans.

Next, we codon-adapted epac1-camps for optimal expression in C. elegans (Redemann et al. 2011) and made an integrated transgenic line expressing epac1-camps from the twk-16 promoter (Figure S5A). We crossed the integrated twk-16p:epac1-camps array into the ceh-17(np1) mutant background; SIS was quantified to confirm that this strain displayed reduced levels of sleep during SIS (Figure S5B). As a first test, we quantified the CFP:YFP ratio in the DVA, RIF, and CEPsh in different populations of animals before SIS (T = 0) and every 60 min for 4-hr following the induction of SIS by UV irradiation. We found that the CFP:YFP ratio was unchanged following UV stress in the DVA and CEPsh glial cells in both the wild-type and ceh-17(np1) mutant background compared to prestress conditions. However, when we measured the CFP:YFP ratio in the RIF neurons we found that although cAMP levels were not different at 0 and 240 min post-UV shock, cAMP levels were significantly lower in wild-type populations compared to ceh-17(np1) animals at 60 and 180 min post-UV (Figure 5, A–C). These data suggest that cAMP levels are reduced at detectable levels in the RIFs during SIS in an ALA-dependent manner.

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Figure 5

cAMP levels are reduced in the DVA and RIF neurons compared to ceh-17 mutant animals. (A) Average CFP:YFP ratio in the DVA cell body during UV-induced SIS in wild-type and ceh-17(np1) animals (N > 27 for each time point). (B) Average CFP:YFP ratio in the RIF interneurons during UV-induced SIS in wild-type and ceh-17(np1) animals (N > 28 for each time point; * P < 0.05). (C) Average CFP:YFP ratio in the CEPshVL glial cell during UV-induced SIS in wild-type and ceh-17(np1) animals (N > 28 for each time point). For A–C, different populations of animals were imaged at each time point and statistical significance was calculated using one-way ANOVA followed by Tukey’s multiple comparisons test. (D) Average CFP:YFP ratio in the DVA from 45 to 75 min post-UV shock SIS in wild-type and ceh-17(np1) animals (N = 15 for each genotype; * P < 0.05). (E) Average CFP:YFP ratio in the RIFs from 45 to 75 min post-UV shock in wild-type and ceh-17(np1) animals (N = 20 for each time point; ** P < 0.01, *** P < 0.001). (F) Average CFP:YFP ratio in the CEPshVL glial cell from 45 to 75 min post-UV shock SIS in wild-type and ceh-17(np1) animals (N = 15 for each genotype). For D–F, the same animal was imaged at each time point and this was repeated over multiple trials. Statistical significance was calculated using two-way ANOVA followed by Sidak’s multiple comparisons test. All error bars represent mean ± SEM.

Next, we made more frequent measurements during the peak of UV-induced SIS, between 45 and 75 min post-UV shock in the same animal, and repeated this over multiple trials in both the wild-type and ceh-17(np1) background. In this context, we found that cAMP levels were significantly lower in wild-type animals at each time point compared to the sleep-defective ceh-17(np1) mutant animals in both the DVA and RIF interneurons, but not the CEPsh glial cells (Figure 5, D–F). Based on these observations, we propose that earlier and more frequent imaging at the peak of SIS revealed a difference in the DVA between these two strains (Figure 5D). Thus, we also propose that cAMP levels are reduced in the RIFs compared to prestress conditions during SIS in an ALA-dependent manner, and that cAMP levels in the DVA are kept at a basal level by the ALA, but not necessarily reduced below times of wake. These data suggest that cAMP levels are affected in unique ways by neuropeptide signaling in distinct cell types.

FLP-13, FLP-24, and PDF-1 neuropeptides are capable of reducing cAMP in the DVA and RIF interneurons

The ALA neuron releases a collection of sleep-promoting neuropeptides during SIS, including those coded by flp-13 (Nelson et al. 2014) and flp-24 (Nath et al. 2016). We predicted that overexpression of sleep-promoting neuropeptides would reduce cAMP in the DVA and/or RIF neurons. To test this, we used an inducible heat-shock promoter from the gene hsp-16.2 to ectopically overexpress either flp-13 or flp-24 in ceh-17(np1);twk-16p:epac1-camps animals. We measured the CFP:YFP ratio prior to induction of the hsp-16.2 promoter, and 2-hr after a 30-min 35° heat shock. Interestingly, we found that flp-13 overexpression significantly decreased cAMP in the DVA, but not the RIFs or CEPsh cells, whereas flp-24 overexpression reduced cAMP in the RIFs, but not the DVA or CEPsh cells (Figure 6, A–C). These data suggest that FLP-13 neuropeptides signal to the DVA and FLP-24 neuropeptides signal to the RIFs to promote locomotion quiescence during SIS.

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Figure 6

cAMP levels are altered in the RIF and DVA interneurons following neuropeptide overexpression. Average CFP:YFP ratio in the DVA (A), RIF (B), or CEPsh (C) cells pre- and 2 hr post-heat shock in ceh-17(np1) (controls), ceh-17(np1);hsp-16.2p:flp-13, ceh-17(np1);hsp-16.2p:flp-24, and ceh-17(np1);hsp-16.2p:pdf-1 animals (N > 30, ** P < 0.01, *** P < 0.001). Statistical significance was calculated using Student’s t-test. All error bars represent mean ± SEM.

Since the RIFs express the PDFR-1 receptor (Barrios et al. 2012) and pigment dispersing factor (PDF) neuropeptides promote arousal in D. melanogaster (Renn et al. 1999) and C. elegans (Choi et al. 2013), we hypothesized that overexpression of pdf-1 would increase cAMP in the RIFs. To test this, we overexpressed the pdf-1 gene from the hsp-16.2 promoter in ceh-17(np1);twk-16p:epac1-camps animals. Overexpression of pdf-1 resulted in a significant increase in cAMP in the RIFs, but not in the DVA or CEPsh cells (Figure 6, A–C), suggesting the PDF-1 and FLP-24 neuropeptides play antagonistic signaling roles to the RIFs during the regulation of quiescence during SIS. These data support a model where neuropeptides released by the ALA reduce cAMP in select cells to induce different aspects of sleep, and may reflect a broader role for neuropeptide signaling during sleep regulation in other animals.