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. 2022 Jul 4;221(3):iyac084.
doi: 10.1093/genetics/iyac084.

Serotonin signals through postsynaptic Gαq, Trio RhoGEF, and diacylglycerol to promote Caenorhabditis elegans egg-laying circuit activity and behavior

Pravat Dhakal  1 , Sana I Chaudhry  1 , Rossana Signorelli  1 , Kevin M Collins  1
Affiliations

Serotonin signals through postsynaptic Gαq, Trio RhoGEF, and diacylglycerol to promote Caenorhabditis elegans egg-laying circuit activity and behavior

Pravat Dhakal et al. Genetics. .

Abstract

Activated Gαq signals through phospholipase-Cβ and Trio, a Rho GTPase exchange factor (RhoGEF), but how these distinct effector pathways promote cellular responses to neurotransmitters like serotonin remains poorly understood. We used the egg-laying behavior circuit of Caenorhabditis elegans to determine whether phospholipase-Cβ and Trio mediate serotonin and Gαq signaling through independent or related biochemical pathways. Our genetic rescue experiments suggest that phospholipase-Cβ functions in neurons while Trio Rho GTPase exchange factor functions in both neurons and the postsynaptic vulval muscles. While Gαq, phospholipase-Cβ, and Trio Rho GTPase exchange factor mutants fail to lay eggs in response to serotonin, optogenetic stimulation of the serotonin-releasing HSN neurons restores egg laying only in phospholipase-Cβ mutants. Phospholipase-Cβ mutants showed vulval muscle Ca2+ transients while strong Gαq and Trio Rho GTPase exchange factor mutants had little or no vulval muscle Ca2+ activity. Treatment with phorbol 12-myristate 13-acetate that mimics 1,2-diacylglycerol, a product of PIP2 hydrolysis, rescued egg-laying circuit activity and behavior defects of Gαq signaling mutants, suggesting both phospholipase-C and Rho signaling promote synaptic transmission and egg laying via modulation of 1,2-diacylglycerol levels. 1,2-Diacylglycerol activates effectors including UNC-13; however, we find that phorbol esters, but not serotonin, stimulate egg laying in unc-13 and phospholipase-Cβ mutants. These results support a model where serotonin signaling through Gαq, phospholipase-Cβ, and UNC-13 promotes neurotransmitter release, and that serotonin also signals through Gαq, Trio Rho GTPase exchange factor, and an unidentified, phorbol 12-myristate 13-acetate-responsive effector to promote postsynaptic muscle excitability. Thus, the same neuromodulator serotonin can signal in distinct cells and effector pathways to coordinate activation of a motor behavior circuit.

Keywords: Caenorhabditis elegans; DAG; G protein; Trio RhoGEF; calcium imaging; circuit activity; neurotransmission; optogenetics; serotonin; synapse.

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Figures

Fig. 1.
Fig. 1.
Trio RhoGEF acts in both neurons and muscles to regulate egg-laying behavior. a) Schematics of excitatory and inhibitory Gαq signaling pathway. C. elegans gene names are beneath the protein they encode. b) Cartoon of the C. elegans egg-laying circuit from a lateral view. Only the left side of the bilaterally symmetric circuit is shown. HSNL, Hermaphrodite Specific Neuron (left); VC4 and VC5 Ventral C neurons; vm1 and vm2 vulval muscles, um1 and um2 uterine muscles; uv1 uterine-vulval neuroendocrine cells. c–h) Bright field images of worms of the indicated genotypes; arrowheads indicate accumulated eggs. Mean number of accumulated eggs ±95% confidence intervals is also indicated. Position of the vulva is shown with an asterisk (*). i) Scatterplot of egg accumulation in wild-type, eat-16(tm761), egl-30(tg26), egl-30(ad805), egl-8(sa47), and unc-73(ce362) mutant animals. Line indicates mean eggs ± 95% confidence intervals. Asterisks indicate P ≤ 0.0001 [1-way ANOVA with Bonferroni’s correction; wild type (n = 49); eat-16(tm761) (n = 36); egl-30(tg26) (n = 47); egl-30(ad805) (n = 44); egl-8(sa47) (n = 65); unc-73(ce362) (n = 38)]. j) Transgenic rescue of egl-8 PLCβ egg-laying defects. Scatterplot of egg accumulation in transgenic animals expressing GFP only or EGL-8/PLCβ fused to GFP expressed from the rgs-1 promoter in egl-8(sa47) mutants (n = 50) compared to wild-type (n = 30) and egl-8(sa47) mutant animals (n = 30). Bar indicates mean eggs ±95% confidence intervals. Asterisks indicate P ≤ 0.0001 (1-way ANOVA with Bonferroni’s correction). k) Transgenic rescue of unc-73 Trio RhoGEF egg-laying defects. Scatterplot of egg accumulation in wild-type (n = 60), unc-73(ce362) mutants (n = 72), and transgenic animals expressing a fluorescent protein with or without Trio/UNC-73E in neurons from the rab-3 promoter (n ≥ 32) or in muscles from the myo-3 promoter (n ≥ 69), or in both neurons and muscles (n ≥ 41) in unc-73(ce362) mutants. Horizontal line indicates mean accumulated eggs ±95% confidence intervals. Asterisks indicate P ≤ 0.0145; n.s., not significant (P > 0.05; 1-way ANOVA with Bonferroni’s correction for multiple comparisons).
Fig. 2.
Fig. 2.
Serotonin signals through Gαq, Trio, and PLCβ to promote egg laying. a) A working model of serotonin and acetylcholine (ACh) signaling in the egg-laying circuit. b) Bar plots showing the percentage of animals laying eggs in M9 buffer alone (open boxes) or M9 +18.5 mM serotonin (filled boxes) after 1 hr. Bar indicates mean percent ±95% confidence intervals. Asterisks indicate P < 0.0007; n.s., not significant (P > 0.05, Fisher’s exact test with Bonferroni’s correction for multiple comparisons; n > 30 animals for each genotype and condition). c) Bar plot showing percent of animals laying eggs in M9 buffer or M9 +18.5 mM serotonin in wild-type or Trio RhoGEF mutant animals expressing nothing or Trio/UNC-73E in neurons, muscles, or both. Bar indicates mean percent ±95% confidence intervals. Asterisks indicate P < 0.0007; n.s., not significant (P > 0.05, Fisher’s exact test with Bonferroni’s correction for multiple comparisons; n > 30 animals).
Fig. 3.
Fig. 3.
Optogenetic stimulation of the HSNs or vulval muscles reveals distinct cellular specificity of Gαq effectors for egg laying. a) On left, cartoon of the egg-laying circuit and experiment showing blue light activation of HSN for 30 s. On right, scatterplot showing eggs laid per worm in the presence (+) or absence (−) of ATR cofactor during the blue light activation of ChR2 expressed in HSNs of wild-type, egl-30(ad805) Gαq strong loss-of-function mutants, egl-8(n488) and egl-8(sa47) PLCβ mutants, and unc-73(ce362) Trio mutant animals. Line indicates mean eggs laid ±95% confidence intervals. Asterisks indicate P < 0.0001; n.s., not significant (P > 0.05, 1-way ANOVA with Bonferroni’s correction for multiple comparisons; n > 10). b) On the left, cartoon of the egg-laying circuit and experiment showing blue light activation of vulval muscles for 30 s (left). On the right, scatter plots of eggs laid per worm in presence (+) or absence (−) of ATR during blue light activation of ChR2 expressed in the vulval muscles of wild type, egl-30(ad805) Gαq strong loss-of-function mutants, egl-8(n488) and egl-8(sa47) PLCβ mutants, and unc-73(ce362) Trio mutant animals. Line indicates mean eggs laid ±95% confidence intervals. Asterisk indicates P ≤ 0.0255; n.s., not significant, P > 0.05 (1-way ANOVA with Bonferroni’s correction for multiple comparisons; n > 10).
Fig. 4.
Fig. 4.
Gαq and Trio signaling promotes vulval muscle activity. a) Representative GCaMP5::mCherry (ΔR/R) ratio traces showing vulval muscle Ca2+ activity in freely behaving wild-type, egl-30(n686) Gαq weak loss-of-function mutant, egl-30(ad805) Gαq strong loss-of-function mutant, unc-73(ce362) Trio strong loss-of-function mutant, egl-8(n488) PLCβ null mutant, egl-8(sa47) PLCβ null mutant, eat-16(tm761) Gαq RGS protein null mutant, egl-30(tg26) strong Gαq gain-of-function mutant, and dgk-1(nu62) DAG Kinase null mutant animals during active (solid bar) and inactive (dotted line) egg-laying behavior states. Arrowheads indicate egg-laying events. Vertical and horizontal scale bars show GCaMP5/mCherry fluorescence ratio (ΔR/R) and time, respectively. b) Scatterplots of Ca2+ transient peak amplitudes for the indicated genotypes during twitch (closed square) and egg-laying transients (open circles). Asterisks indicate P < 0.0001, n.s. indicates not significant (P > 0.05, Kruskal–Wallis test with Dunn’s correction for multiple comparisons). c) Scatterplots of Ca2+ transient frequency for indicated genotypes. Line indicates mean eggs laid ±95% confidence intervals; asterisks indicate P ≤ 0.0340; n.s. indicates not significant (P > 0.05, 1-way ANOVA with Bonferroni’s correction for multiple comparisons; n > 10 animals recorded per genotype).
Fig. 5.
Fig. 5.
The DAG mimetic PMA rescues egg-laying circuit activity and behavior defects of Gαq signaling mutants. a) Diagrams showing working model of Gαq and DAG signaling pathway during egg-laying behavior. b) Bar plots showing the percentage of animals showing egg laying in M9 buffer (open bars) or M9 buffer +10 µM PMA (filled bars). Error bars indicate 95% confidence intervals for the proportion; asterisks indicate P < 0.0013 (Fisher’s exact test with Bonferroni’s correction for multiple comparisons; n ≥ 30 animals per genotype and condition). c) Left, representative GCaMP5::mCherry (ΔR/R) ratio traces showing vulval muscle Ca2+ activity in wild-type or the indicated Gαq signaling mutant animals in the absence or presence of 10 µM PMA. Arrowheads indicate egg-laying events. Vertical and horizontal scale bars show GCaMP5/mCherry fluorescence ratio (ΔR/R) and time, respectively. Right, heat map showing intensity modulated color spectrum of GCaMP5::mCherry (ΔR/R) ratio of vulval muscle Ca2+ activity ranging from low to high Ca2+. Rows indicate ratio changes in each of 10 animals. d) Scatterplots of Ca2+ transient frequency in the absence (−) and presence (+) of 10 µM PMA for the indicated genotypes. Lines indicate mean eggs laid ±95% confidence intervals; asterisk indicates P ≤ 0.0275; n.s., not significant (P > 0.05, 1-way ANOVA with Bonferroni’s correction for multiple comparisons; n ≥ 10 animals per genotype and condition).
Fig. 6.
Fig. 6.
DAG promotes egg laying independent of UNC-13 or PKC. a) Scatterplot of egg accumulation in wild-type, unc-13(e51) loss-of-function mutant, and unc-13(s69) null mutant animals. Lines indicate mean eggs laid ±95% confidence intervals. Asterisk indicates P ≤ 0.0021 (1-way ANOVA with Bonferroni’s correction for multiple comparisons; n ≥ 36 per genotype). b) Bar plots showing the percentage of wild-type, unc-13(e51), or unc-13(s69) mutant animals laying eggs in M9 buffer, 18.5 mM serotonin, or 10 µM PMA. Asterisks indicate P < 0.0006; n.s., not significant (P > 0.05, Fisher’s exact test with Bonferroni’s correction for multiple comparisons; n ≥ 36 animals per genotype and condition). c) Scatterplot of egg accumulation in wild type (n = 24) and the indicated PKC mutant animals (n ≥ 35 per genotype). Line indicates mean eggs accumulated ±95% confidence intervals. n.s., not significant (P > 0.05, 1-way ANOVA with Bonferroni’s correction for multiple comparisons). d) Bar plots showing the percentage of wild-type and PKC mutant animals showing egg laying in M9 buffer or 10 µM PMA. Bar indicates mean eggs ±95% confidence intervals for the proportion. Asterisks indicate P < 0.0007; n.s., not significant (P > 0.05, Fisher’s exact test with Bonferroni correction for multiple comparisons; n ≥ 35 animals per genotype and condition).
Fig. 7.
Fig. 7.
Working model of Gαq signaling in the egg-laying circuit. See text for details.
See this image and copyright information in PMC

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