Muscle-directed mechanosensory feedback activates egg-laying circuit activity and behavior in C. elegans

Abstract

Mechanosensory feedback of internal state regulates numerous bodily processes including feeding, defecation, and reproduction. However, how mechanosensory feedback signals to modulate neural circuits and coordinate these behaviors is incompletely understood. Here, we use the egg-laying circuit of C. elegans to test our hypothesis that mechanosensory feedback of egg accumulation promotes the active reproductive behavior state. Using an acute gonad microinjection technique to mimic changes in pressure and stretch resulting from germline activity and egg accumulation, we find that injection rapidly stimulates Ca²⁺ activity in both neurons and muscles of the egg-laying circuit. Injection-induced vulval muscle Ca²⁺ activity requires L-type Ca²⁺ channels but is independent of presynaptic input. Direct mechanical prodding activates the vulval muscles, suggesting they are the proximal targets of the stretch-dependent stimulus. Our results show that egg-laying behavior in C. elegans is regulated by a stretch-dependent homeostat that scales postsynaptic muscle responses with egg accumulation in the uterus.

INTRODUCTION

The ability to sense and convert physical stimuli into chemical signals that are then integrated and used to perform behavioral decisions is a conserved process that allows animals to survive and reproduce (Goodman and Sengupta, 2019). Mechanosensory feedback of internal reproductive state is crucial in shaping decisions about when to reproduce and where to rear offspring. For instance, the reduction in sexual receptivity of Drosophila following copulation is mediated by mechanosensory feedback (Shao et al., 2019). Following mating, the stretch produced from accumulated eggs in the reproductive tract is sufficient to induce an attraction to acetic acid to ensure oviposition occurs in favorable environments (Gou et al., 2014). Because artificial distention of the reproductive tract is sufficient to induce this attraction to acetic acid, feedback of egg accumulation itself drives the observed remodeling of sensory signaling. Likewise, mechanical stretch of the uterus is involved in initiating parturition or driving specific maternal behaviors like bonding or infanticide in various mammalian systems (Hayes and De Vries, 2007; Kendrick et al., 1991; Wray, 1993). Thus, while mechanical feedback of internal reproductive state exerts profound short- and long-term changes in neural circuit activity, the mechanosensory feedback mechanisms and circuits that mediate these processes are incompletely understood.

The C. elegans egg-laying circuit serves as an ideal system to study how different forms of mechanosensory feedback regulate reproductive behaviors. Previous work has shown that C. elegans egg laying is a stochastic, two-state behavior consisting of ~20 minute inactive states in which animals produce and retain eggs in the uterus punctuated by ~2 minute active states in which ~4-6 eggs are laid (Waggoner et al., 1998). Egg laying is controlled by two motoneuron types, the serotonergic hermaphrodite-specific neurons (HSNs; Desai and Horvitz, 1989; Waggoner et al., 1998), and the cholinergic ventral type C (VC) neurons, both of which synapse onto the vulval muscles to promote contraction and egg laying (Bany et al., 2003; Collins et al., 2016; Shyn et al., 2003; White et al., 1986; Zhang et al., 2008). The cellular Ca²⁺ activity of several cells in the egg-laying circuit is regulated by mechanosensory feedback. For example, external vibrations or gentle touches on the tail inhibit egg laying (Sawin, 1996), possibly by activating mechanoreceptors on the PLM neurons that signal to inhibit HSN activity (Zhang et al., 2008). The VC neurons are mechanically activated by the contraction and opening of the vulval muscles, releasing acetylcholine to enhance vulval opening for egg release (Kopchock et al., 2021). The uv1 neuroendocrine cells sit between the vulval canal and uterus, are mechanically deformed and activated in response to passage of eggs through the vulval canal (Collins et al., 2016; Jose et al., 2007), and provide inhibitory feedback following egg laying through the release of tyramine and neuropeptides that inhibit HSN activity (Alkema et al., 2005; Banerjee et al., 2017; Ravi et al., 2021; Zhang et al., 2010). Whether other cells in the egg-laying circuit are mechanosensitive and how their feedback signals to modulate discrete steps in egg laying is not clear.

Our previous work demonstrated that feedback of egg accumulation regulates Ca²⁺ activity in the egg-laying circuit (Ravi et al., 2021, 2018a). Young adults or animals lacking eggs in the uterus have reduced Ca²⁺ activity in the HSNs, with the HSNs showing infrequent Ca²⁺ transients. Conversely, animals recovering from an acute blockage of egg laying show greatly increased Ca²⁺ transient activity and egg laying (Ravi et al., 2018a). This homeostatic feedback to the HSNs depends on the postsynaptic vulval muscles, as presynaptic HSN Ca²⁺ bursting activity is lost in Notch receptor mutants (Li et al., 2013) lacking postsynaptic connection to the HSNs or when the vulval muscles are electrically silenced (Ravi et al., 2018a). Together, these data suggest that feedback of egg accumulation activates a stretch-dependent homeostat that regulates circuit activity and serotonin release that sustains the egg-laying active state. Interestingly, genetic ablation or electrical silencing of the HSNs delays, but does not abolish, the onset of egg laying (Ravi et al., 2021, 2018a; Waggoner et al., 1998) with animals recovering vulval muscle Ca²⁺ transient activity upon egg accumulation (Collins et al., 2016). These results show that while feedback of egg accumulation can promote HSN Ca²⁺ activity, HSN may not be its only target. However, the cellular and molecular mechanisms underlying this putative stretch response remains unknown.

Here, we tested whether a stretch-dependent mechanism activates the C. elegans egg-laying behavior circuit. Using acute microinjections to mimic feedback of germline activity and embryo accumulation, we show that injection is sufficient to drive both egg-laying circuit activity and behavior. Surprisingly, we find that animals deficient in synaptic vesicle or peptidergic transmission still show an acute injection response. This suggests that egg laying is at least partially driven by direct sensing of internal stretch by the egg-laying vulval muscles. Furthermore, we show that the vulval muscles themselves are receptive to direct mechanical stimulation and that L-type voltage-gated calcium channels are required for Ca²⁺ influx. Together, our data reveal how mechanosensory feedback, detected by the muscle target-cells, informs the circuit whether there are eggs to lay that acts alongside synaptic input to induce egg-laying behavior.

RESULTS

Microinjections induce egg-laying behavior and circuit activity

While microinjection into the gonad of C. elegans is a common technique for generating transgenic strains (Berkowitz et al., 2008; Mello et al., 1991), we noticed this procedure often led to vulval opening and egg release. While we feel this phenomenon is widely known among C. elegans researchers, injection-induced egg laying is not well-described in the literature. To determine that microinjection does stimulates egg laying, we inserted a needle into the gonad syncytium of wild-type animals and performed a brief (3 s) injection delivering ~250 pL of standard buffer lacking DNA while monitoring egg release (Figure 1A). Microinjection drove egg laying in nearly half (~46 ± 9%) of injected animals (Figure 1B). Egg-laying behavior in C. elegans is driven by Ca²⁺ activity in the vulval muscles that drives contraction (Collins and Koelle, 2013; Shyn et al., 2003; Zhang et al., 2008). To determine if egg release was a passive consequence of injection or an active process driven by cellular activity, we performed ratiometric Ca²⁺ recordings of the vulval muscles in animals expressing GCaMP5 (Collins et al., 2016). Gonad injections triggered a strong and rapid induction of vulval muscle Ca²⁺ activity which peaked on average 5.6 s after the onset of injection (Figure 1C; Movie 1). Direct contact with the injected buffer did not seem to be required for a robust muscle response, as vulval muscle Ca²⁺ activity was induced immediately upon injection while buffer reached the vulva a few seconds later (Movie 1). Egg-laying events were coincident with peak vulval muscle Ca²⁺ activity (Figure 1C), similar to that seen previously in behaving animals (Collins et al., 2016; Collins and Koelle, 2013). On average, injection-induced vulval muscle Ca²⁺ transients decayed to 37% of their peak value in 23 s. While the kinetic features of vulval muscle Ca²⁺ transient activity were significantly longer than that seen in behaving animals (Collins et al., 2016), we presume this is a consequence of the animals being immobilized during the injection procedure as observed in previous studies (Shyn et al., 2003). Injections into unc-54(e190) muscle-specific myosin mutants, in which the vulval muscles are unable to contract, still triggered vulval muscle Ca²⁺ transients (Figure 1 – figure supplement 1A), but this activity was unable to drive egg release (Figure 1B), indicating that microinjection does not simply force eggs out through the vulva. Together, these results show that microinjection causes an active response, triggering a rapid onset of vulval muscle Ca²⁺ activity whose magnitude can drive contractility and egg release.

Figure 1 – figure supplement 1: Optimization of the injection protocol.

(A) Bar graphs comparing the mean injection-induced vulval muscle Ca²⁺ transient amplitudes following injection into wild-type or unc-54(e190) muscle myosin mutant animals (± 95% confidence intervals). Points in scatterplot show responses in individual animals. Asterisks indicate p-value = 0.0004 (unpaired t-test). (B) Scatterplot with linear regression (red line) of relationship between injection flowrate and resulting vulval muscle Ca²⁺ transient amplitude; p-value = 0.4860 (not significant; standard least squares regression for vulval muscle Ca²⁺ transient amplitude; see ‘statistical analyses’ in methods). (C) Logistic regression comparing the relationship between injection flowrate and egg release. Binomial responses with successful egg release (y) and no egg release (n); p-value = 0.1665 (nominal logistic fit for egg laid; see ‘statistical analyses’ in methods). (D) Representative Ca²⁺ trace after sequential injections into a single animal. Vertical yellow lines represent injection pulses and orange trace indicates resulting vulval muscle Ca²⁺ activity. A single egg-laying event following the first injection is indicated with an inverted triangle. (E) Bar graph with scatterplot showing mean vulval muscle Ca²⁺ transient amplitude (±95% confidence intervals) after a 3 s injection with K⁺ buffer (black, n = 19), Na⁺ buffer (green, n = 18), deionized water (blue, n = 7), or Milli-Q water (cyan, n = 8). Asterisks indicate p-value = 0.003; n.s. indicates p-value > 0.05 (Kruskal-Wallis test with Dunn’s correction for multiple comparisons). Average bar graphs with scatterplot showing vulval muscle Ca²⁺ transient amplitude (F) or percent of injection-induced egg laying (G) when injections were performed into either anterior or posterior gonad.

Figure 1: Acute microinjection triggers vulval muscle Ca²⁺ activity and egg-laying behavior.

(A) Cartoon of C. elegans and micrograph demonstrating the injection technique. A microinjection needle (arrowhead; red outline) is inserted into the gonad (yellow outline) of an immobilized C. elegans worm (top). A 3 s pulse is applied to inject a standard microinjection buffer into the gonad syncytium. Unlaid eggs (cyan asterisks) are then monitored for injection-induced egg release through the vulva (white bracket). (B) Bar graphs showing the percent of wild-type or unc-54(e190) muscle myosin mutants animals laying eggs in response to injection. Error bars represent ±95% confidence interval for proportions; asterisks indicate p-value < 0.0001 (Fisher’s exact test, n indicates number of animals injected per genotype). (C) Representative vulval muscle Ca²⁺ trace, determined from GCaMP5/mCherry fluorescence ratio, following a 3 s injection pulse (vertical cyan bar). Key features observed from a typical injection response are egg release (inverted triangle), peak amplitude (ΔR/R; open green circle), and decay time (s; filled red circle; time from peak amplitude to 37%). (D) Distribution of estimated injection flowrates obtained by measuring the change in diameter over time of injection buffer drops (yellow vertical line in inserted micrograph) into halocarbon oil immediately following needle withdrawal after worm injection. Error bars represent ±95% confidence interval for the mean proportion across 96 injections. (E) Scatterplot of time to egg-laying onset and number of eggs accumulated at egg-laying onset in wild-type (black) and egl-1(n986dm) HSN-deficient mutant animals (brown). Asterisks indicate p-value < 0.0001 (unpaired t-tests for differences in egg-laying onset and in egg accumulation). (F) Bar graphs comparing mean vulval muscle Ca²⁺ transient amplitudes (±95% confidence interval) after injections that did not (black) and those that did (red) result in egg release. Points in scatterplot show responses in individual animals. Asterisks indicate p-value < 0.0001 (unpaired t-test). (G) Scatter plot, with a bivariate linear regression comparing the relationship between the number of eggs accumulated inside the uterus at the time of injection and the peak vulval muscle Ca²⁺ response following injection (p-value = 0.0095; standard least squares regression for vulval muscle Ca²⁺ transient amplitude; see ‘statistical analyses’ in methods).

To determine if the magnitude of stretch induced by injections affected the vulval muscle Ca²⁺ response, we estimated the flowrates across multiple injections to determine total volume delivered per injection. Injection flowrates ranged between ~10 and ~366 pL/s with a median of 82 pL/s, corresponding to a total volume delivered ranging between 30 pL – 1 nL with a median of 246 pL for each 3 s injection (Figure 1D). We estimated the volume of one embryo to be ~25 pL, as such, our injections corresponded to an equivalent stretch produced by the addition of 1.2 – 40 eggs (with a median of ~10 egg-volumes per injection). To determine if this range of injection volumes reflects a physiologically relevant number of eggs required to activate the stretch-dependent homeostat, we determined the number of eggs accumulated in the uterus at the onset of egg laying. Wild-type animals begin laying eggs at ~6 hours after reaching adulthood, having accumulated ~8 eggs in the uterus (Figure 1E). We have previously shown that the onset of egg laying is delayed in egl-1(dm) animals lacking the HSN command neurons (Ravi et al., 2018a) and in animals with strongly reduced HSN activity (Ravi et al., 2021). In HSN-deficient animals, the onset of egg laying is delayed to ~20 hours after adulthood, a point when they have accumulated an average of 45 eggs in their uterus (Figure 1E). We interpret this difference in ~37 eggs required to activate egg laying in the absence of HSNs as providing a threshold estimate of how much volume increase (~925 pL) is required to induce the stretch-dependent homeostat. As such, the volumes typically injected in our experiments are quantitatively similar to the volume changes that accompany egg production and induce egg laying in vivo.

Do injections with greater volumes elicit a stronger vulval muscle Ca²⁺ response and more frequent egg laying? To answer this question, we analyzed the correlation between injection flowrate, injected at a range of 30 – 40 psi, and the resulting vulval muscle Ca²⁺ peak amplitudes and probability of egg release. Linear and logistic regressions showed no correlation between injection flowrate and vulval muscle Ca²⁺ transient amplitude, nor between flowrate and egg release, respectively (Figure 1 – figure supplement 1B – C). This lack of correlation indicates that the range of flowrates in our 3 s injection protocol does not significantly affect the observed vulval muscle Ca²⁺ transient responses. We observed that egg-laying events correlated with larger vulval muscle Ca²⁺ peaks (Figure 1F), a result consistent with our previous observations of in vivo Ca²⁺ imaging of the vulval muscles (Collins et al., 2016; Collins and Koelle, 2013). Interestingly, the number of eggs accumulated in animals prior to injection also correlated significantly with the resulting vulval muscle Ca²⁺ peak amplitude elicited by injections (Figure 1G). This result suggests that animals with greater egg accumulation at the time of injection were more likely to have stronger vulval muscle Ca²⁺ transients, and therefore more likely to lay eggs, independent of injection volume delivered (Figure 1 – figure supplement 1C). We explored additional parameters that affected the injection response (Figure 1 – figure supplement 1D – G; see Methods). Additional attempts to inject at low flowrates, with lower injection pressures (25 psi instead of 30 – 40psi), while tracking liquid delivery with a fluorescent reporter, revealed that the vulval muscle Ca²⁺ response was weaker when liquid delivery was slow, independent of total volume delivered (Figure 1 – figure supplement 2E). Injection pressures >30 psi used in all subsequent microinjection experiments were above this threshold, allowing for rapid fluid delivery of >30 pL (~2 eggs) within 3 s and a reliable Ca²⁺ response (Figure 1 – figure supplement 1B). Together, these results show that acute microinjection triggers rapid vulval muscle Ca²⁺ activity that drives egg release. These results further suggest that microinjection may activate the same homeostatic mechanism engaged by feedback of egg accumulation in the uterus which promotes the egg-laying active state (Ravi et al., 2018a).

Figure 1 – figure supplement 2:

Quantifying correlation of injection flowrate and vulval muscle Ca²⁺ responses using bromophenol blue.

(A) Cartoon of C. elegans and micrograph demonstrating injection with bromophenol blue reporter (magenta). Dashed yellow lines represent ventral and dorsal border of animal injected with bromophenol blue (magenta) and the resulted GCaMP5 (green) fluorescence in the vulval muscles (vm). (B) Representative traces of vulval muscle GCaMP5 (green; left axis) and bromophenol blue fluorescence (magenta; right axis) after injection used to estimate flowrate and volume delivered. Yellow vertical bar represents time of injection and dotted line represents the change in bromophenol blue fluorescence used to estimate volume injected per 3 s injection time (slope). (C) Scatterplot showing significant correlation between injection fluorescence intensity slope and the resulting vulval muscle Ca²⁺ transient peak amplitude across 35 injected animals (p-value = 0.0442). (D) Distribution of all injection fluorescence intensity slopes obtained. Slopes were binned as ‘slow’ (highlighted in orange) or ‘fast’ depending on whether they were below or above the median slope, respectively. (E) Bar graphs with scatterplot showing the mean vulval muscle Ca²⁺ transient peak amplitudes seen with ‘slow’ or ‘fast’ injections binned based on slope. Asterisks indicate p-value = 0.0012 (unpaired t-test, n ≥ 17 per group).

Figure 2: Vulval muscle injection response is independent of egg availability or reproductive maturation.

(A) Diagram of experimental rationale. Floxuridine (FUDR)-treated wild-type or fog-2(oz40) sperm-deficient mutant animals were injected and vulval muscle Ca²⁺ activity was followed using GCaMP5. Orange ovals represent fertilized embryos in the uterus of control animals. (B) Representative injection-induced vulval muscle Ca²⁺ responses of control wild-type (black), wild-type sterilized with FUDR (red), and fog-2(oz40) mutant animals (green). (C) Bar graphs with scatterplot showing mean vulval muscle Ca²⁺ transient amplitudes following microinjection. Asterisk indicates p-value = 0.0105, and n.s. indicates p-value > 0.05 (Kruskal-Wallis with Dunn’s correction for multiple comparisons; n ≥ 20 animals per condition). (D) Injection-induced Ca²⁺ responses for 1-day old adult (top; black trace) and late-L4 (bottom; orange trace) animals. Solid lines show the average Ca²⁺ responses, and lighter lines show responses from individual animals (adults, n = 19; L4 juveniles, n = 15). (E-F) Bar graphs with scatterplots showing mean vulval muscle Ca²⁺ amplitudes (E) and decay times (F). Asterisk indicates p-value = 0.0139; n.s. indicates p-value > 0.05 (Mann-Whitney test). Vertical cyan bars show timing of 3 s microinjection. To facilitate comparisons, one data point with vulval muscle Ca²⁺ transient amplitude value of 2.99 (ΔR/R) from an adult animal is not visible in D and E.

The vulval muscle injection response is independent of egg availability and neural circuit development

As worms mature from juveniles to adults, the pattern of activity in the egg-laying circuit also matures into clear active and inactive states. We have previously shown that mature patterns of neural activity are dependent on the temporal development of the worm as well as the presence of fertilized embryos (Ravi et al., 2018a). To test if the vulval muscle Ca²⁺ response to acute injection was similarly dependent on egg availability, we injected into adult worms lacking fertilized embryos or into L4 juvenile worms that had not yet begun egg production. As shown in Figure 2A, we eliminated fertilized egg accumulation either genetically using a sperm-deficient fog-2 mutant (Schedl and Kimble, 1988) or chemically with floxuridine (FUDR; Mitchell et al., 1979) which disrupts proper formation of oocytes. We observed a strong induction of vulval muscle Ca²⁺ activity following injections into either fog-2 mutant or FUDR-sterilized adults (Figure 2B). While fog-2 sperm-deficient mutant worms did not show any significant differences compared to similarly injected wild-type control animals, Ca²⁺ transient amplitudes in FUDR-sterilized animals were significantly increased (Figure 2C). Although these animals lacked embryos to lay, gonad injection still induced strong contraction of the vulval muscles and vulval opening (data not shown). These results show that adult animals lacking endogenous feedback of egg production and/or accumulation in the uterus are still capable of mounting a robust vulval muscle Ca²⁺ response to injection.

To determine if developmental age influenced vulval muscle activation, we compared injection responses of juvenile animals at the late 4^th larval (L4) stage to egg-laying adults (picked 1 day earlier). Injections into late-L4 or adult animals showed similar acute vulval muscle Ca²⁺ transient responses (Figure 2D) that were not significantly different in amplitude (Figure 2E), although this Ca²⁺ activity in late-L4 juveniles was unable to drive vulval opening. Late-L4 juvenile animals showed a greater variety of vulval muscle Ca²⁺ responses that led to a significant decrease in average Ca²⁺ activity decay time (Figure 2F). We saw a similar heterogeneity in spontaneous vulval muscle Ca²⁺ responses as they develop from L4 animals into adults, coincident with induction of ERG K⁺ channel expression in the vulval muscles (Ravi et al., 2018a). Together, these results show that juvenile animals, prior to the onset of egg production, or even sterile adults remain responsive to acute changes in internal pressure and/or stretch that follow gonad microinjection.

Microinjection induces sequential Ca²⁺ activity in other cells of the egg-laying behavior circuit

Egg laying in behaving animals is driven by a stereotypical pattern of rhythmic and sequential cell Ca²⁺ activity. The HSN command neurons go first, with Ca²⁺ transients peaking ~2 s before each egg-laying event when Ca²⁺ activity is seen to peak in both the VC motor neurons and vulval muscles (Collins et al., 2016). uv1 Ca²⁺ activity is last, typically peaking ~2 s after each egg-laying event (Figure 3A). To determine if injection drives a similar, sequential pattern of Ca²⁺ activity, we performed injections in animals expressing GCaMP reporters in either the HSNs, VCs, or uv1 cells. We found that all cells of the circuit showed a robust induction of Ca²⁺ activity in response to injection, but their order and kinetics of onset differed significantly from that seen in behaving animals. The vulval muscles and uv1 neuroendocrine cells which sit proximal to the vulval muscles showed the most rapid injection response (Movie 2), reaching half-maximal Ca²⁺ levels at 2.4 ± 1.2 and 3.4 ± 3.3 s (mean ± SD), respectively, after the onset of injection (Figure 3B and 3C). The VC neurons were next, showing a half-max Ca²⁺ activity at 4.0 ± 1.5 s (Figure 3D; Movie 3). Surprisingly, the HSNs showed the slowest response with half-max Ca²⁺ activity observed 6.4 ± 4.0 s (Figure 3E and 3F; Movie 4). Injection-induced Ca²⁺ responses typically lasted longer and decayed more slowly compared to those seen in freely behaving animals which may result from the absence of rhythmic phasing of Ca²⁺ activity that accompanies locomotion (Collins et al., 2016). The robust and rapid onset of vulval muscle Ca²⁺ activity upon acute microinjection, and the out-of-sequence onset of activity seen in other cells in the circuit (Figure 3F), suggests microinjection acts directly on the vulval muscles with Ca²⁺ activity in the other cells of the circuit being a consequence of vulval opening and/or egg laying.

Movie 1.

Recording of vulval muscle GCaMP5 (green) and bromophenol blue (magenta) fluorescence during microinjection overlayed over a brightfield recording of C. elegans. Wild-type animals display an increase in vulval muscle (vm) Ca²⁺ activity that correlates with the increase in injection volume (represented as traces within the video). Egg release is coincident with peak vulval muscle GCaMP5 fluorescence.

Movie 2.

GCaMP5 to mCherry fluorescence ratio in uv1 cells of wild-type animals overlaid on top of brightfield recording during injection. Blue indicates low Ca²⁺, and red indicates high Ca²⁺. Red trace denotes uv1 Ca²⁺ response, triangle denotes egg release, and white horizontal bar denotes timing of the 3 s injection pulse.

Movie 3.

GCaMP5 to mCherry fluorescence ratio in VC neurons of wild-type animals overlaid on top of brightfield recording during injection. Blue indicates low Ca²⁺, and red indicates high Ca²⁺. Red trace denotes VC Ca²⁺ response, triangle denotes egg release, and white horizontal bar denotes timing of the 3 s injection pulse.

Movie 4.

GCaMP5 to mCherry fluorescence ratio in HSN neurons of wild-type animals overlaid on top of brightfield recording during injection. Blue indicates low Ca²⁺, and red indicates high Ca²⁺. Red trace denotes HSN Ca²⁺ response, triangle denotes egg release, and white horizontal bar denotes timing of the 3 s injection pulse.

Figure 3: Acute microinjection induces sequential Ca²⁺ activity across the entire egg-laying behavior circuit.

(A) Connectivity of the C. elegans egg-laying circuit (adapted from Collins et al., 2016). (B-E) Egg laying and normalized Ca²⁺ responses of the vulval muscles (B), uv1 neuroendocrine cells (C), VC motoneurons (D), and HSN command motoneurons (E), before and after a 3 s microinjection. Vertical lines show timing of egg-laying events pooled from all injected animals. Micrographs show Ca²⁺ activity in cell bodies (outlined in white) before and after injection; rainbow scale indicates GCaMP5/mCherry ratio in responding cells; blue indicates low Ca²⁺ and red indicates elevated Ca²⁺. White scale bar indicates 10 µm. Black solid line is a trace of the average Ca²⁺ response; colored bands indicate the 95% confidence interval (n ≥ 19 animals per trace). Vertical cyan line shows 3 s injection period. (F) Bar graphs with scatter plots showing time of half-max Ca²⁺ responses. **** indicates p-value < 0.0001, ** indicates p-value = 0.0013, and n.s. indicates p-value > 0.05 (Kruskal-Wallis test with Dunn’s correction for multiple comparisons).

Figure 4: The vulval muscle Ca²⁺ and egg-laying response to microinjection does not require synaptic or peptidergic input.

(A) Diagram highlighting the synaptic connections between cells of the C. elegans egg-laying circuit. Red “x” symbol on top of the HSNs represents the genetic removal of any input from the HSNs cells through use of egl-1(n986dm) mutants which lack HSN neurons. Tetanus Toxin (TeTx) was also transgenically expressed in the VC neurons to block neurotransmitter release. (B) Representative injection-induced vulval muscle Ca²⁺ responses of wild-type (black), HSN-deficient egl-1(dm) mutants co-expressing Tetanus Toxin in the VCs (brown), unc-13(s69) mutants with defective synaptic transmission, (magenta), and unc-31(e928) mutant animals with defective peptidergic transmission (blue). Vertical cyan bar represents the 3 s gonad injection. (C-E) Bar graphs with scatter plot showing mean time to vulval muscle Ca²⁺ transient peak (C), Ca²⁺ transient peak amplitude (D), and Ca²⁺ transient decay times (E) following injections (error bars represent mean ±95% confidence intervals). n.s. indicates p-value > 0.05 (one-way ANOVA with Bonferroni’s correction for multiple comparisons). (F) Bar graphs showing percent of injections that resulted in an egg-laying event (mean ±95% confidence intervals for the proportion); n.s. indicates p-value > 0.05 (Fisher’s exact test with Bonferroni’s correction for multiple comparisons). Numbers inside bar graph indicate sample sizes; n ≥ 19 animals for all conditions.

The vulval muscle microinjection Ca²⁺ response does not require synaptic or peptidergic input

To test the hypothesis that the vulval muscles respond to mechanical stretch cell-autonomously, we removed synaptic and peptidergic input into the vulval muscles and tested their Ca²⁺ response to microinjection. Specifically, we tested egl-1(dm) mutant animals lacking HSNs and in which we blocked synaptic transmission from the VC neurons through transgenic expression of Tetanus Toxin (Figure 4A). Despite lacking most synaptic input and showing strong defects in egg laying, the vulval muscles of these animals showed strong injection-induced Ca²⁺ activity and egg laying (Figure 4B). We observed no significant differences in the vulval muscle Ca²⁺ response time, peak amplitude, decay time, or efficiency of injection-induced egg release when compared to injected wild-type worms (Figure 4C – F). These results indicate the main synaptic inputs into the vulval muscles are not required for the vulval muscle injection response, consistent with previous work showing that worms lacking HSNs and/or VCs still enter egg-laying active states (Waggoner et al., 1998) with robust vulval muscle Ca²⁺ activity (Collins et al., 2016; Kopchock et al., 2021; Shyn et al., 2003; Zhang et al., 2008).

To account for the possibility that other cells, including those outside the egg-laying circuit, respond to acute injection and signal to activate the vulval muscles, we injected into unc-13(s69) mutants lacking synaptic vesicle transmission (Richmond et al., 1999) and unc-31(e928) mutants with defective dense core vesicle release (Ann et al., 1997). Both mutants showed essentially normal vulval muscle Ca²⁺ responses after acute microinjection (Figure 4B – E). In addition, these neurotransmission mutants were just as likely to lay eggs in response to injection as wild-type animals (Figure 4F). Together, these results show that the vulval muscles can respond to acute microinjection independent of synaptic or peptidergic input. This suggests that the injection-induced activation of egg-laying circuit activity is not simply a consequence of nociceptive neuron activation but may instead represent a muscle-dependent mechanism that detects acute changes in internal pressure or stretch that accompany germline activity and egg accumulation.

The vulval muscles are receptive to direct mechanical stimulation

If the vulval muscles are direct targets of stretch being activated in response to acute microinjection, they may respond to other forms of mechanical stimulation. We used a glass capillary probe to prod either the anterior or posterior vulval muscles while recording changes in muscle Ca²⁺ activity (Figure 5A). Mechanical stimulation of the vulval muscles triggered a strong and immediate Ca²⁺ transient in the prodded vulval muscles (Figure 5B; Movie 5). To determine optimal displacement distances, we exposed animals to a train of prodding events in which each subsequent prodding event displaced the vulval muscles by an additional 10 µm (ranging from 10 – 60 µm; Figure 5C, top). From this, we found that the vulval muscles responded most efficiently to 50 µm displacements (Figure 5C, bottom). We then applied 1 s, 50 µm prodding stimulations and compared the induced Ca²⁺ responses in pairs of vulval muscles where one set of muscles on either side of the vulval slit either did or did not receive the prodding stimulus. We observed a significantly greater induction of Ca²⁺ in the vulval muscles that were prodded (Figure 5D – E). Prodded muscles showed a half-maximal Ca²⁺ response of 0.7 ± 0.4 s (mean ± SD). This response was faster than the observed injection-induced response (2.4 ± 1.2 s), likely reflecting the difference in how the mechanical stimulation is applied: direct (prodding) vs. indirect (injection). However, unlike microinjections, direct vulval muscle prodding stimulations never led to egg-laying events. This is consistent with previous work showing that successful egg-laying events require coordinated contractions from both anterior and posterior vulval muscle groups (Li et al., 2013). Together, these results demonstrate that the vulval muscles are receptive to direct mechanical stimulation.

Movie 5.

Recording of vulval muscle mCherry (magenta) and vulval muscle GCaMP5 (green) fluorescence during mechanical prodding of the posterior but not anterior vulval muscles. Cyan outline border of the prodding needle. White horizontal bar denotes timing of 1 s prodding stimulus.

Figure 5: The vulval muscles are receptive to direct mechanical input.

(A) Diagram of experimental rationale. A glass capillary probe was used to mechanically stimulate either the anterior or posterior vulval muscles while observing changes in vulval muscle Ca²⁺ activity. (B) Representative still images of vulval muscle mCherry (magenta) and GCaMP5 (green) fluorescence prior to (top, t = 0 s) or immediately after (bottom, t = 30 s) mechanical stimulation with a stimulation probe (red outline). (C) Prodding protocol (top) in which the vulval muscles were prodded for 5 s every 30 s with increasing displacement distances (10 µm steps). Dose-response curve (bottom) showing the relationship between the displacement distance placed upon the vulval muscles and the percent of prodding events that resulted in a vulval muscle Ca²⁺ response (error bars represent ±95% confidence interval for proportions). (D) Representative vulval muscle Ca²⁺ traces following a 1 s prodding pulse (vertical cyan line) in unprodded (black, bottom trace) and prodded (red, top trace) vulval muscles. (E) Pair-wise comparison of the mean vulval muscle Ca²⁺ responses 5 s in the unprodded (black dots) and their adjacent prodded muscles (red dots). Asterisks indicate p-value < 0.0001 (paired t-test, n > 20 animals).

L-type voltage-gated calcium channels are required for the vulval muscle injection response

Previous work has shown the mechanosensory neurons in C. elegans depend on Ca²⁺ entry mediated by EGL-19, the sole L-type voltage-gated Ca²⁺ channel in C. elegans (Frøkjær-Jensen et al., 2006; Suzuki et al., 2003). EGL-19 channels are expressed in the vulval muscles and are required for proper egg laying (Kwok et al., 2006; Schafer et al., 1996; Shyn et al., 2003; Trent et al., 1983). To test if the acute injection response requires L-type voltage-gated Ca²⁺ channels, we recorded the vulval muscle Ca²⁺ in animals where channel function was blocked genetically or pharmacologically (Figure 6A). Injection into egl-19(n582) loss-of-function mutants triggered vulval muscle Ca²⁺ transients with significantly reduced amplitude (Figure 6B – C). Treatment of animals with 25 µM nemadipine, a specific blocker of L-type Ca²⁺ channels (Kwok et al., 2006), caused a dramatic and significant decrease in vulval muscle Ca²⁺ transient amplitude following injection when compared to 0.1% DMSO controls (Figure 6B – E; Movie 6). Injections were also less likely to elicit egg laying in nemadipine-exposed animals when compared to DMSO-treated worms (Figure 6F). Interestingly, a small level of residual Ca²⁺ activity that localized to the vm1 muscles was still observed in nemadipine-treated animals (Movie 6). Whether this residual Ca²⁺ activity is caused by insufficient blocking by nemadipine, differential expression of EGL-19 channels in the vm1 vs. vm2 vulval muscles, or is mediated by other molecules, for example, Ca²⁺ influx mediated by mechanoreceptors, is not yet clear. Nonetheless, these results indicate that L-type voltage-gated Ca²⁺ channels are required for the normal injection-induced vulval muscle response.

Movie 6.

GCaMP5 to mCherry fluorescence ratio in vulval muscles of a DMSO-treated control animal (top) and a nemadipine-treated animal (bottom) overlaid on top of respective brightfield recording during injection. Blue indicates low Ca²⁺, and red indicates high Ca²⁺. Red traces denote vulval muscle Ca²⁺ responses and white horizontal bar denotes timing of the 3 s injection pulse. Inserts on right side of movie show a zoomed in recording of vulval muscle Ca²⁺ response for control and Nemadipine-treated animals to emphasize localization of Ca²⁺ activity. White arrowhead shows localization of residual Ca²⁺ activity from vm1 muscles in nemadipine-treated animals.

Figure 6: L-type voltage-gated Ca²⁺ channels are required for the vulval muscle injection response.

(A) Diagram (adapted from Lagoy et al., 2018) of experimental treatments. Worms were placed on NGM plates containing either DMSO (normal condition) or 25 µM nemadipine. (B) Representative injection-induced vulval muscle Ca²⁺ responses for wild-type (black) or egl-19(n582) voltage-gated Ca²⁺ channel loss-of-function mutant animals (orange), worms exposed to 0.1% DMSO (grey), and worms exposed to 25 µM nemadipine (burgundy). Vertical cyan bar represents 3 s injection pulse. (C) Bar graphs with scatterplot of mean vulval muscle Ca²⁺ transient amplitude in wild-type (black), egl-19(lf) (orange), 0.1% DMSO-treated worms (grey), and nemadipine-treated animals (burgundy) after injection. Asterisks indicates p-value < 0.0001 and n.s. indicates p-value > 0.05 (one-way ANOVA with Bonferroni’s correction for multiple comparisons; n ≥ 18 animals). (D-E) Heatmaps of vulval muscle Ca²⁺ before and after injections in animals treated for ~2 hours with either 0.1% DMSO (D) or 25 µM nemadipine (E). Each row represents Ca²⁺ responses from an individual animal (n>20 animals). Negative time values represent vulval muscle Ca²⁺ activity prior to the injection, vertical purple line represents the start of the 3 s injection, and the diagram above the heatmap indicates the 3 s injection pulse. (F) Bar graphs showing percent of injections that resulted in an egg-laying event (mean ±95% confidence intervals for the proportion). Numbers inside bar graph indicate sample sizes; n ≥ 18 animals per condition. Asterisks indicate p-value < 0.0001; n.s. p-value > 0.05 (Pairwise Fisher’s exact test).

TMC and PIEZO mechanosensitive channels are not required for the injection-induced vulval muscle Ca²⁺ response

PIEZO mechanosensitive ion channels have been previously shown to be expressed in the reproductive system of C. elegans, including the vulva (Bai et al., 2020). To test if Piezo channels mediate the injection-induced response we injected into pezo-1(av149) mutants while recording vulval muscle Ca²⁺ activity. As shown in Figure 7A, injection into pezo-1 mutants led to robust vulval muscle Ca²⁺ responses with no significant differences in peak amplitude (Figure 7B) or egg laying (Figure 7C) when compared to wild-type animals. Thus, Piezo channels are not strictly required for the injection-induced Ca²⁺ response seen in the vulval muscles.

Figure 7: PIEZO and TMC channels are not required for the injection-induced vulval muscle Ca²⁺ and egg-laying response.

(A) Representative injection-induced vulval muscle Ca²⁺ traces after injection into wild-type (black) and pezo-1(av149) mutant animals (magenta). (B) Bar graphs with scatterplot showing mean vulval muscle Ca²⁺ responses of wild-type (black, n = 17) and pezo-1 mutants (magenta, n = 20). Error bars indicate 95% confidence interval; n.s. indicates p-value > 0.05 (unpaired t-test). (C) Bar graphs showing percent of injections that resulted in an egg-laying event (mean ±95% confidence intervals for the proportion); n.s. indicates p-value > 0.05 (Fisher’s exact test) (D) Representative injection-induced vulval muscle Ca²⁺ traces after injection into wild-type (black) and tmc-1(ok1859); tmc-2(ok1302) double mutant animals (blue). (E) Bar graphs with scatterplot showing mean vulval muscle Ca²⁺ responses of wild-type (black, n = 18) and tmc-1; tmc-2 double mutants (blue, n = 21). Error bars indicate 95% confidence interval for the mean; n.s. indicates p-value > 0.05 (unpaired t-test). (F) Bar graphs showing percent of injections that resulted in an egg-laying event (mean ±95% confidence intervals for the proportion); n.s. indicates p-value > 0.05 (Fisher’s exact test). Vertical cyan bars indicate 3 s injection. Numbers inside bar graphs indicate sample sizes.

Next, we sought to determine if TMC channels, a different class of ion channels that contribute to mechanosensory responses (Pan et al., 2018), were mediating the vulval muscle injection response. Within the egg-laying circuit of C. elegans, TMC-1 has been previously shown to be expressed in both the HSNs and vulval muscles while TMC-2 expression has only been found in the vulval muscles (Yue et al., 2018). Loss of one or both channels leads to an egg-laying defective phenotype (Yue et al., 2018), while tmc-1 mutants have impaired inhibition of egg laying in response to harsh touch (Kaulich et al., 2021), making these channels promising candidates for mediating a stretch response. We injected into tmc-1; tmc-2 double mutants while recording the vulval muscle Ca²⁺ activity and egg-laying responses. Injections into tmc-1; tmc-2 mutants still led to a robust Ca²⁺ response (Figure 7D) which did not differ significantly in peak amplitude (Figure 7E) or egg laying (Figure 7F) when compared to wild type animals. Overall, these results indicate that neither Piezo nor TMC channels are required for the vulval muscle injection-induced response.

DISCUSSION

We developed a quantitative microinjection technique to test whether acute changes in mechanical stretch that might accompany egg accumulation drive the activation of egg-laying behavior in C. elegans. We found that acute microinjection induces Ca²⁺ activity in each cell of the egg-laying circuit along with efficient egg laying. Surprisingly, we found that the vulval muscles, the final synaptic targets in the egg-laying circuit, showed the strongest and most rapid Ca²⁺ responses. Injection-induced vulval muscle Ca²⁺ activity did not require input from the HSNs or VC neurons and was still observed in unc-13 and unc-31 mutant animals lacking synaptic or peptidergic transmission, respectively. Direct prodding of the vulval muscles similarly induced Ca²⁺ transient activity, suggesting the vulval muscles themselves are direct targets of a stretch-homeostat that regulates egg laying. While the mechanosensory mechanism that mediates the injection-induced response remains unclear, loss of TMC or PIEZO channels did not significantly affect injection-induced vulval muscle Ca²⁺ responses. However, we found vulval muscle Ca²⁺ transient responses were significantly diminished by genetic or pharmacological inhibition of EGL-19 L-type voltage-gated Ca²⁺ channels known to regulate egg laying in vivo (Schafer et al., 1996; Trent et al., 1983). Together, these results support a model in which feedback of egg accumulation in the uterus activates vulval muscle mechanoreceptors and L-type Ca²⁺ channels, driving Ca²⁺ transient activity, contractility, and egg laying.

While there are numerous similarities between patterns of egg-laying circuit activity seen in behaving animals and the acute injection response, the differences are interesting and may help reveal the nature of the stretch-dependent homeostat. Analysis of circuit Ca²⁺ activity in behaving animals shows frequent, rhythmic ‘twitch’ Ca²⁺ transients in the vm1 vulval muscles phased with each body bend of locomotion and stronger ‘egg-laying’ Ca²⁺ transients that contract both the vm1 and vm2 vulval muscles (Brewer et al., 2019; Collins and Koelle, 2013). Injection-induced Ca²⁺ transients are much longer (~20 s) than those seen in behaving animals (~2 s; Collins et al., 2016). Similarly long Ca²⁺ transients have been previously reported in mechanically activated neurons of immobilized animals (Suzuki et al., 2003) as well as from cells of the egg-laying circuit (Shyn et al., 2003; Zhang et al., 2008). We propose that locomotion provides additional negative feedback onto the egg-laying circuit, possibly to reset activity in the circuit to allow subsequent bouts of egg release when the vulva can open fully at a specific phase of the body bend (Collins and Koelle, 2013). In behaving animals, Ca²⁺ activity in the HSN command neurons peaks about ~2 s before each egg-laying event (Collins et al., 2016). In response to injection, we first observe Ca²⁺ activity in the vulval muscles and the uv1s, followed by the VCs, and then finally in the HSNs. We propose that serotonin and NLP-3 signaling from HSNs normally acts to increase the electrical excitability of the vulval muscles already sensitized by stretch, as optogenetic stimulation of the HSNs induces significantly weaker vulval muscle Ca²⁺ responses in sterile animals (Ravi et al., 2018a). Injection bypasses HSNs, directly activating a stretch signal on the vulval muscles that can drive contraction and egg laying. Contraction of the vulval muscles then opens the vulva, activating tyramine and neuropeptide release from the uv1 cells (Alkema et al., 2005; Banerjee et al., 2017; Collins et al., 2016) which signal to inhibit HSN Ca²⁺ activity (Ravi et al., 2021). In this model, rebound from fast-acting tyramine inhibition would allow subsequent egg-laying events within each active state, while uv1-released neuropeptides drive long-term inhibition to terminate the active state. Consistent with this idea, we see delayed onset of HSN Ca²⁺ transient activity after injection, and vulval muscle Ca²⁺ transients in subsequent injections are weaker in amplitude (Figure 1 – supplementary Figure 1D).

The robust injection response suggests the egg-laying circuit is primed to respond to acute changes in embryo accumulation in the uterus. In support of this, C. elegans typically lays 3 – 5 eggs about every 20-minutes (Waggoner et al., 1998) while producing one egg from each gonad arm every 10 minutes (McCarter et al., 1999). Thus, the egg-laying rate is matched by the rate of egg production and, as such, dramatic swings in egg accumulation are normally small and likely averaged out over the 12-15 eggs found in the uterus at steady-state. Deposition of one or more eggs into the uterus from the spermatheca (McCarter et al., 1999), or detection of some critical stretch threshold by unidentified cells, may serve as an acute stretch signal to induce the active state. This is supported by our analyses of the effects of flowrate on Ca²⁺ transient amplitude and egg-laying responses. We find a wide range of flowrates are equally as likely to induce a strong vulval muscle Ca²⁺ transient sufficient to drive egg release (Figure 1 – figure supplement 1B), indicating that stretch induced by our injections is above a critical threshold. Future studies will focus on establishing a stretch threshold for egg-laying behavior while identifying the molecular basis for this threshold.

The ability of the vulval muscles to respond to the injection stimulus independent of synaptic input suggests a bottom-up control of egg laying wherein stretch caused by egg accumulation activates the vulval muscles and egg laying. This mechanism of control is commonly found in various autonomic functions such as the myogenic reflex that detects stretch in the gut (Alvarez, 1922; Mercado-Perez and Beyder, 2022), baroreceptor reflex that maintains arterial pressure (Guyton, 1976; Kandel et al., 2012), and micturition reflex that detects bladder stretch (Guyton, 1976). Decoupling of feedback from these cells to higher control centers does not abolish the reflex but removes the ability of higher control centers to regulate and maintain homeostasis. For example in micturition, damage to the spinal cord above the sacral region leads to loss of faciliatory inputs from the brain and causes an initial suppression of the micturition reflex that is eventually restored over time (Guyton, 1976). A strikingly similar pattern occurs in the egg-laying circuit of C. elegans as removal of the HSN command neurons leads to a marked reduction in the frequency of egg-laying active states (Waggoner et al., 1998) and a dramatic delay in the onset of egg laying (Ravi et al., 2018a) which is restored only after the accumulation of 45 eggs (Figure 1E) when vulval muscle Ca²⁺ activity resumes active / inactive state behavior transitions. Similarly, injection can bypass the absence of synaptic input from the HSN command neurons, although we expect that sensory feedback onto the HSNs ensures that eggs are normally laid in favorable environmental conditions (Fenk and de Bono, 2015; Hallem and Sternberg, 2008; Ringstad and Horvitz, 2008). Thus, it is likely that feedback from a similar bottom-up mechanism informs the HSNs of egg availability to ensure a transition into and out of egg-laying active states.

What is the molecular mechanism mediating the injection-induced vulval muscle response? While L-type calcium channels mediate mechanosensory responses in mechanosensitive neurons of C. elegans, these channels have not previously been shown to be mechanosensitive in C. elegans. However, in the human GI tract, Ca_v1.2 L-type voltage-gate Ca²⁺ channels have been shown to be activated by increases in intracellular pressure and by mechanical shear stress (Alcaino et al., 2017; Farrugia et al., 1999). Mechanosensitivity was retained by the α_1c subunit of Ca_v1.2 during heterologous expression in HEK-293 or CHO cells (Lyford et al., 2002). L-type calcium channels have also been shown to be required for the mechanosensitive response of rat cardiomyocytes (Takahashi et al., 2019). Given the expression of EGL-19 L-type Ca²⁺ channels in the vulval muscles and other cells of the egg-laying circuit (Taylor et al., 2021), stretch produced by accumulation of eggs may directly modulate the opening of L-type Ca²⁺ channels to excite the vulval muscles. However, this work cannot rule out previously proposed models in which serotonin-mediated activation of EGL-19 channels, through a Gα_q-signaling pathway, indirectly promotes egg laying via an increase in the excitability of the vulval muscles (Dhakal et al., 2022; Schafer, 2006; Waggoner et al., 1998). This model would predict that lack of an injection-induced response after L-type Ca²⁺ channel block is a consequence of reduced vulval muscle excitability. As such, future work should focus on determining the role of EGL-19 L-type voltage-gated calcium channels in regulating vulval muscle excitability and their potential mechanosensitive role in C. elegans.

Gap junctions may also be important molecules driving the vulval muscle injection-induced Ca²⁺ response. The vulval muscles show extensive electrical coupling via gap junctions among themselves and with other cells (Cook et al., 2019; White et al., 1986), with the vm2 vulval muscles making gap junctions both with the uterine muscles and the vm1 muscles where we observe rhythmic input during locomotion (Collins et al., 2016). In this model, egg accumulation activates mechanoreceptors on the uterine muscles, leading to the spread of electrical excitation through gap junctions into the vm2 vulval muscle ‘hub’ to induce the active state, as seen previously for the extensively gap-junctioned RMG neurons that facilitate pheromone attraction (Macosko et al., 2009). Once eggs are laid and the stretch signal is lost, excitation is shunted out of the vulval muscles, making it harder for the circuit to remain in the active state, similar to the electrical shunting mechanism that mediates touch perception in the nose touch hub- and-spoke circuit of C. elegans (Rabinowitch et al., 2013). Egg-induced stretch signals may also feedback onto the egg-laying neurons as the HSN and VC neurons express several innexins (Altun et al., 2009; Taylor et al., 2021) and make gap junctions among themselves, between each other, and with VC3 making a gap junction with the vm2 muscles (White et al., 1986). Interestingly, UNC-7 innexins have been recently shown to function as mechanosensitive hemichannels (Walker and Schafer, 2020), suggesting a possible mechanosensory role for other innexins expressed in the egg-laying circuit.

C. elegans egg-laying behavior is regulated by a variety of external sensory cues including environmental CO₂ (Fenk and de Bono, 2015; Hallem and Sternberg, 2008), male pheromones (Aprison and Ruvinsky, 2019), mechanical stimulation (Sawin, 1996), food availability (Trent, 1982), and osmolarity (Zhang et al., 2008). The regulation of egg laying via changes in osmotic conditions is particularly interesting in the context of our results. While the molecular and cellular mechanisms by which osmolarity regulates egg laying are unclear, it is interesting that osmotic regulation of egg laying might have the same physical consequences as acute injection. Shifting C. elegans into hypoosmotic or hyperosmotic environments results in inhibition or promotion of egg laying, respectively (Zhang et al., 2010, 2008). Given that C. elegans is hydrostatically pressurized (Avery and Thomas, 1997; Gilpin et al., 2015; Harris and Crofton, 1957), and vulval opening is sufficient for egg expulsion from the uterus (Kopchock et al., 2021), changes in osmotic conditions may reflect a physical regulation of egg-laying behavior. Acute shifts of animals to hypoosmotic conditions drives a rapid influx of water into animals (Lamitina et al., 2004) that presumably increases the hydrostatic pressure inside the animal, thus promoting circuit activity and egg-laying based on a physical model. Conversely, a decrease in hydrostatic pressure would similarly be predicted to inhibit egg-laying circuit activity and behavior, as previously observed (Zhang et al., 2010, 2008). Our data shows that the initial level of egg accumulation is a primary predictor for vulval muscle Ca²⁺ transient amplitude and the probability for an egg-laying response. Future work should test whether the hydrostatic pressure gradient is used to drive physical expulsion of eggs in response to vulval opening.

Overall, our work reveals a new model by which egg laying is regulated in C. elegans. Sensory feedback converges at the HSN command neurons, regulating serotonin and neuropeptide release, informing the circuit about favorable environmental conditions, e.g., the external state. This top-down processing decides ‘when’ and ‘where’ egg laying should occur. Feedback of germline activity and egg accumulation directly stimulates the vulval muscles, communicating information to the circuit about the internal state, namely that the animal has eggs to lay. This ‘bottom-up’ processing determines ‘if’ and ‘for how long’ eggs can be laid. These distinct forms and sources of modulatory input help drive optimal patterns of circuit activity that allow animals to adjust their behavior to a changing environment.

Materials and Methods

Nematode culture and staging

Caenorhabditis elegans strains were grown at 20°C on Nematode Growth Medium (NGM) agar plates with E. coli OP50 grown in B broth as a food source (Brenner, 1974). All assays involving adult animals were performed using adult hermaphrodites staged 24 h past the late L4 larval stage. For assays involving L4 animals, late-stage L4 worms were picked 30 minutes prior to the assay. A list of strains, mutants, and transgenes used in this study can be found in Table 1.

Table 1:

List of C. elegans strains used in this study

Calcium reporter transgenes and strain construction

Vulval muscle Ca²⁺

Vulval muscle Ca²⁺ activity was recorded in adult animals using LX1918 vsIs164 [unc-103e::GCaMP5::unc-54 3’UTR + unc-103e::mCherry::unc-54 3’UTR + lin-15(+)], lite-1(ce314), lin-15(n765ts) X, as described (Collins et al., 2016). Because the unc-103e promoter does not express strongly in the vulval muscles of younger L4 animals (Ravi et al., 2018a), MIA51 keyIs12 [ceh-24::GCaMP5::unc-54 3’UTR ceh-24::mCherry::unc-54 3’UTR lin-15(+)] IV, lite-1(ce314), lin-15(n765ts) X, as described (Ravi et al., 2018a) was used in experiments comparing injection responses in late L4 animals.

To image vulval muscle Ca²⁺ activity in animals defective for muscular contractions, MIA206 [unc-54(e190) I; vsIs164 X lite-1(ce314) lin-15(n765ts) X] strain was generated. To generate this strain, LX1918 males were crossed with CB190 [unc-54(e190) I] myosin heavy chain null mutant hermaphrodites (Brenner, 1974; Dibb et al., 1985). Offspring were allowed to self and uncoordinated, mCherry(+) F2 progeny were then selected and allowed to self for multiple generations until both phenotypes were observed to be homozygous.

To record Ca²⁺ activity from the vulval muscles of animals lacking fertilized embryos, BS553 [fog-2(oz40) V)] spermatogenesis mutant (Schedl and Kimble, 1988) males were crossed with LX1918 hermaphrodites. Cross-progeny hermaphrodites were picked and were crossed back to BS553 fog-2 males to generate mCherry(+), fog-2/+ males and mCherry(+), fog-2 hermaphrodites which were identified from single mCherry(+) L4 hermaphrodites that then developed into sterile, fog-2 adults. Male and hermaphrodite progeny continued to be crossed until the fog-2, mCherry(+) phenotypes were homozygous. The resulting strain, MIA34 [fog-2(oz40) V; lite-1(ce314) lin-15(n765) vsIs164 X], was then maintained by male and female self-progeny.

To determine if injection-induced vulval muscle Ca²⁺ responses were dependent on input from the HSNs or VC neurons, a strain lacking HSNs while simultaneously expressing Tetanus Toxin in the VC neurons was generated. To do this, N2 males were crossed with the HSN-deficient LX1938 egl-1(n986dm) V; lite-1(ce314) vsIs164 lin-15(n765ts) X hermaphrodites (Collins et al., 2016). Resulting male cross-progeny were then mated with MIA144 keyIs33; lite-1(ce314) lin15(n765ts) X hermaphrodites (Kopchock et al., 2021) expressing Tetanus Toxin in the VC neurons from a minimal lin-11 enhancer (Bany et al., 2003) which imparts a mild uncoordinated reversal phenotype. Resulting cross-progeny L4s were then picked, allowed to self, and were then selected for mCherry(+), Egl, and Unc reversal phenotypes. Animals were then positively genotyped for egl-1(n986dm) using the following oligos: 5’-CTC TGT TCC AGC TCA AAT TTC C-3’ (forward) and 5’-GTA GTT GAG GAT CTC GCT TCG GC-3’ (reverse) followed by NciI enzyme digestion. Confirmed homozygous animals were kept as strain MIA289 keyIs33; egl-1(n986dm) V; vsIs164 lite-1(ce314) lin-15(n765ts) X. To eliminate neuropeptide signaling while monitoring injection-induced Ca²⁺ activity in the vulval muscles, the dense core vesicle release mutant, CB928 unc-31(e928) IV (Brenner, 1974), was crossed with LX1918 males. vsIs164 mCherry(+) hermaphrodite cross-progeny were allowed to self and were then selected for mCherry(+), Unc, and Egl phenotypes, resulting in strain MIA312 [unc-31(e928) IV; vsIs164 X; lite-1(ce314) lin-15(n765ts) X]. Similarly, to eliminate neurotransmitter release while monitoring injection-induced Ca²⁺ activity from the vulval muscles, the neurotransmitter release mutant, EG9631 [unc-13(s69) I] (Rose and Baillie, 1980), was crossed with vsIs164 mCherry(+) males. mCherry(+) hermaphrodite cross-progeny were picked as L4s, allowed to self, and mCherry(+), Unc F2 progeny were kept, resulting in strain MIA316 unc-13(s69) I; vsIs164 lite-1(ce314) lin-15(n765ts) X.

To image Ca²⁺ activity in the vulval muscles of animals lacking the EGL-19, L-type voltage-gated Ca²⁺ channel (Trent et al., 1983), LX1918 males were crossed with MT1212 egl-19(n582) IV hermaphrodites. Cross-progeny were isolated and allowed to self and resulting mCherry(+) non-Egl progeny were picked and allowed to self until homozygous for mCherry(+) and Egl phenotypes. The resulting strain MIA185 (egl-19(n582) IV; lite-1(ce314) lin-15(n765ts) vsIs164 X) was used in this study.

To monitor Ca²⁺ activity in the vulval muscles of mechanosensory mutants, vsIs164 males were crossed with pezo-1 mutants provided by Drs. Xiaofei Bai and Andy Golden (Bai et al., 2020). This resulted in the generation of the AG527 strain [pezo-1 (av149) IV; vsIs164 lite-1(ce314) lin-15(n765ts) X.

To visualize vulval muscle Ca²⁺ activity in tmc-1, tmc-2 mechanosensory mutants, N2 males were crossed with LX1919 vsIs165 [unc-103e::GCaMP5::unc-54 3’UTR + unc-103e::mCherry::unc-54 3’UTR + lin-15(+); lite-1(ce314) lin-15(n765ts) X] hermaphrodites (Collins et al., 2016; Ravi et al., 2021). mCherry(+) vsIs165/+ male cross-progeny were then crossed with RB1546 tmc-1(ok1859) X hermaphrodites. mCherry(+) cross-progeny hermaphrodites were then picked and allowed to self. mCherry(+) cross-progeny were then genotyped for tmc-1(ok1859) using a duplex PCR strategy with the following primers: 5’-CGA CTA CTG CAA TTC TCT TTA AGG-3’ (outside forward), 5’-GTT TAC AGT ATA CAA AAT TAG GAC TCT G-3’ (inside reverse), 5’-GTG CAG TAC ATT CTC CGC CAC C-3’ (outside reverse), generating MIA486 vsIs165; tmc-1(ok1859) X strain used in this study. To generate MIA487 vsIs165 tmc-2(ok1302) X, the same approach was used but the RB1237 tmc-2(ok1302) X hermaphrodites was used instead when crossed with vsIs165/+ males. mCherry(+) progeny were confirmed to be homozygous for tmc-2(ok1302) using the following primers: 5’-CAA TCG TCG TGG GAC CTT ATC ATC-3’ (outside forward), 5’-CTA CAG TGT CAT TAC TGA TGT TCT TC-3’ (inside forward), 5’-CTC TTC TGT CCG TTC CTG GAA ATG-3’ (outside reverse). During construction of MIA486 and MIA487, we noticed the loss of the lite-1(ce314) X mutation present in the original LX1919 starting strain as tmc-1 and tmc-2 genes, both of which are near lite-1, were being homozygosed. To account for any changes in vulval muscle Ca²⁺ activity that arise from blue light illumination rather than acute injection, a control strain MIA485 vsIs165 was generated by crossing N2 males with LX1919 hermaphrodites. Heterozygous cross-progeny hermaphrodites with mCherry expression were selected, selfed, and loss of lite-1(ce314) in the progeny was confirmed through genotyping.

MIA494 vsIs165; tmc-1(ok1859) tmc-2(ok1302) X was generated to image vulval muscle Ca²⁺ activity in animals lacking both TMC-1 and TMC-2. Briefly, N2 males were mated to RB1546 tmc-1(ok1959) hermaphrodites, and the resulting hemizygous (tmc-1/Ø) males were then crossed with MIA487 hermaphrodites. The resulting cross-progeny were then allowed to self until tmc-1 and tmc-2 mutations were confirmed to be homozygous through genotyping.

uv1 neuroendocrine cell Ca²⁺

Ca²⁺ activity of uv1 neuroendocrine cells was monitored in adult animals using MIA136 keyIs34 lite-1(ce314) lin-15(n765ts) X which expresses GCaMP5 and mCherry from the tdc-1 promoter (Alkema et al., 2005) which provides stronger uv1 expression compared to the ocr-2 promoter previously used (Collins et al., 2016). To generate this strain, the ocr-2 promoter was replaced with a 1.5 Kb region of the tdc-1 promoter amplified from Ptdc-1::ChR2 generously provided by Mark Alkema using oligonucleotides Ptdc-1new-fwd: GCG GCA TGC CAC CTA ACT TCG TCG GTC and Ptdc-1-new-rev: GCG CCC GGG GAT CCT TGG GCG GTC CTG, digested with SphI/XmaI, and ligated into similarly digested pKMC281 [Pocr-2::mCherry::ocr-2(3’UTR)] or pKMC284 [Pocr-2::GCaMP5::ocr-2(3’UTR)], generating pAB5 [Ptdc-1::mCherry::ocr-2(3’UTR)] and pAB6 [Ptdc-1::GCaMP5::ocr-2(3’UTR)]. pAB5 [Ptdc-1::mCherry (5 ng/µL)], pAB6 [Ptdc-1::GCaMP5 (20 ng/µL)], and pL15EK (50 ng/µL) were injected into LX1832 lite-1(ce314), lin-15(n765ts) animals, generating the extrachromosomal transgene keyEx28 which demonstrated bright and specific expression in the uv1 cells along with dim expression in the RIM neurons. This transgene was then integrated to chromosomes using UV/TMP to generate four independent integrants keyIs34–37, of which keyIs34 was used for this study.

VC Ca²⁺

Ca²⁺activity in the VC neurons was monitored in adult animals using strain LX1960 vsIs172; lite-1(ce314) lin-15(n765ts) X, as described (Collins et al., 2016). Ca²⁺ activity levels were averaged from all VC cell bodies and process (VC1-VC6). However, due to expression patterns, differences in focus, and the position of worms during recording, not all VCs were captured in all experiments, especially VC1 and VC6 which are more distant from the vulval-proximal VC4 and VC5.

HSN Ca²⁺

Ca²⁺ activity in the HSNs was monitored in adult animals using LX2004 vsIs183 lite-1(ce314) lin-15(n765ts) X, as described (Collins et al., 2016; Ravi et al., 2018b).

Egg accumulation at onset of egg laying

To determine the extent of egg accumulation at onset of egg laying, both wild-type and MIA26 egl-1(n986dm) mutant animals were staged following the L4 molt as previously described (Ravi et al., 2018a). Animals were observed for the first instance of egg laying every 30 minutes (after a 4 h period for wild type and after a 16 h period for egl-1(dm) mutants). Once an animal had laid its first egg, it was dissolved in a 20% bleach solution, and the eggs in the uterus were counted as described (Chase and Koelle, 2004). The number of eggs in the uterus plus the number of eggs laid on the plate were considered the total number of eggs accumulated at the onset of egg laying.

Microinjection protocol and flowrate estimate

To induce acute increases in pressure within C. elegans worms, we performed microinjections essentially as described (Berkowitz et al., 2008). To create microinjection needles, filamented glass capillaries (4”, 1.0 mm outer diameter; World Precision Instruments, 1B100F-4) were pulled using a Sutter P-97 Flaming/Brown type micropipette puller using a custom program with the following parameters: Pressure = 500, Heat = 632 (ramp test value), Pull = 45, Velocity = 75, Delay = 90. Needles were back-filled with injection buffer (2% polyethylene glycol, 20 mM potassium phosphate, 3 mM potassium citrate, pH 7.5) and then placed into a Narishige HI-7 injection holder. Worms were injected using a Narishige IM-300 pneumatic microinjector with injection pressure between 30 – 40 psi, unless otherwise noted. Injections were programmed for a 3 s injection pulse and initiated manually using a foot pedal. To synchronize injection onset with captured brightfield and fluorescence micrographs, the ‘sync’ voltage signals of the microinjector were controlled and/or monitored using an Arduino Uno microcontroller running the Firmata library in Bonsai (Lopes et al., 2015). Injections (3 s) were triggered either using a foot-pedal or a TTL digital pin, and the timing of injection onset was recorded using an analog pin. To assess consistency of injection needles, the average flowrate of needles was calculated from brightfield recordings by measuring the volume of a spherical drop of injection buffer injected into halocarbon oil. The volume was calculated by measuring the expanding diameter of the drop in the first 20 frames of a three second injection (10 msec exposures recorded at 20 Hz). The average flowrate of multiple needles pulled using this protocol was calculated to be 47 pL/s with a standard deviation of 15 pL/s (n = 10).

To perform microinjections, worms were placed into a small drop of halocarbon oil on a 2% agarose pad to immobilize them (Berkowitz et al., 2008). Animals were then placed onto an inverted Zeiss Axio Observer.Z1 fluorescence microscope for imaging. A Narishige coarse manipulator (model MMN-1) and a Narishige three-axis oil hydraulic micromanipulator (model MMO-203) were used to position the tip of the needle next to the gonad of the worm. Needles were then introduced along the dorsal surface into either anterior or posterior gonad of the worm before initiating the recording (see fluorescence imaging). The first 3 s injection was triggered 30 seconds after the onset of synchronous brightfield and fluorescence recording. Subsequent injections were performed in one-minute intervals. Before the end of the recording, the injection needle was withdrawn from each animal, and the flowrate of the needle was once again recorded to determine if the flowrate had changed materially during the injection. The median flowrate from a subsample of 104 animal injections was 82 pL/s and ranged between 10 and 365 pL/s. Such variation was seen to result from either needle tips clogging or being open further as a consequence of injection, but this variability did not seem to alter injection-induced responses (see optimization of microinjection protocol).

Brightfield and fluorescence recordings

To record egg-laying behavior during injections, a Grasshopper 3 camera (FLIR) was used to capture 2 x 2 binned, 1,024 x 1,024 8-bit JPEG image sequences. Vulval muscle, HSN, VC, or uv1 Ca²⁺ responses were recorded as described (Collins et al., 2016; Ravi et al., 2018b). GCaMP5 and mCherry fluorescence was excited at 470 nm and 590 nm, respectively, for 10 msec using a Colibri.2 LED illumination system. Recordings were collected at 20 fps and a 256×256 pixel resolution (4×4 binning) using a Hamamatsu Flash 4.0 V2 sCMOS camera recording at 16-bit depth through a 20x Apochromatic objective (0.8NA). GCaMP5 and mCherry channels were separated via a Gemini beam-splitter. Worms were recorded for 30 seconds prior to injection to establish baseline levels of cell activity and were completed within five minutes to reduce effects from worm desiccation after prolonged exposure to halocarbon oil. Image sequences were imported into Volocity (Version 6.5.1, Quorum Technologies Inc.) for GCaMP5/mCherry ratiometric analysis, image segmentation, Ca²⁺ quantitation of GCaMP5/mCherry ratio changes (ΔR/R), and Ca²⁺ transient peak finding, as described previously (Collins et al., 2016; Ravi et al., 2018b).

Optimization of microinjection protocol

To ensure injections into control animals and the different mutants were comparable, the correlation between flowrate and recorded calcium transient amplitudes was analyzed. From this comparison, we found that flowrate had no significant effect on the amplitude of the resulting vulval muscle Ca²⁺ transients (p-value = 0.5816; Figure 1 – figure supplement 1B) and, as a result, the injection flowrate was allowed to vary. Additionally, the correlation between flowrate and an egg-laying response was also analyzed. This relationship also showed no significant correlation after performing a logistic regression on the data (p-value = 0.0712; Figure 1 – figure supplement 1C). Individual animals were injected multiple times to determine if subsequent responses were comparable. We found that subsequent injections resulted in lower Ca²⁺ transient amplitudes and were also less likely to result in an egg-laying event (Figure 1 – figure supplement 1D). However, given the wide range of flowrates obtained among multiple injections, we limited our analyses of injection-induced Ca²⁺ responses to each animal’s first injection.

To determine if the composition of the injection buffer influenced the Ca²⁺ response, worms were injected with deionized water or Milli-Q water. Injections with both types of water did not differ in their ability to induce comparable levels of Ca²⁺ transient activity from the vulval muscles (p-value >0.05; Figure 1 – figure supplement 1E). However, we observed that when the injection buffer was made with Na⁺ salts instead of K⁺ salts, a decrease in the average vulval muscle Ca²⁺ response was observed (p-value = 0.0003; Figure 1 – figure supplement 1E) when compared to the standard K⁺ salt containing buffer. To ensure that the injection-induced response is not a consequence of a disruption of the Na⁺/K⁺ gradient, low flowrate injections were performed by decreasing injection pressure to 20 psi. In these low-flow experiments, we observed that injections were more likely to fail to elicit a Ca²⁺ response even when the ‘optimal’ K⁺ buffer was used. Injections were performed into either the anterior or posterior gonad, with no significant difference in vulval muscle Ca²⁺ transient amplitude or proportion of injection-induced egg laying observed (Figure 1 – figure supplement 1F – G). Attempts to determine if injections performed in other compartments (e.g. the uterus) also drove vulval muscle Ca²⁺ activity were unsuccessful due to injection needles frequently getting clogged (data not shown).

Injections with bromophenol blue

To visualize injections, and to determine if Ca²⁺ dynamics correlated with the rate of liquid spread at lower flowrates (injection pressure 25 psi), a solution of 450 µM bromophenol blue made in injection buffer was used as a fluorescent tracker (Figure 1 – figure supplement 2A). Bromophenol blue was excited at 590 nm alongside co-expressed mCherry and collected through the same mCherry filter set. To determine if the flowrate of injections influenced vulval muscle Ca²⁺ responses, worms were manually injected (25 psi) by depression of the foot pedal until the bromophenol fluorescence was seen by eye to reach the vulval muscles (Figure 1 – figure supplement 2A). Bromophenol fluorescence was then quantified in Volocity. The Sum of individual pixel intensities was used as a proxy of total volume delivered during the course of each injection [(pixel area) x (16-bit fluorescence intensity for each pixel above baseline) and with all such pixels summed].

To determine total volume injected, the maximum fluorescence intensity Sum of all injections within a 2-minute period after injection was determined and used as a proxy for volume injected. To ensure different injections were comparable, the lowest of the Sum values for all injections was used to estimate a ‘threshold volume injected’ value (26053363 AU). Injections in which this threshold ‘volume’ was reached within a two-minute period were then analyzed. The flowrate of each injection was calculated by measuring the slope of the injection fluorescence intensity Sum during the injection period (Figure 1 – figure supplement 2B). At the same time, GCaMP5 fluorescence intensity (ΔF/F) from the vulval muscle response was quantified. From this, a significant correlation was observed between the slope of the bromophenol blue fluorescence intensity during injection and the vulval muscle GCaMP5 fluorescence peak amplitude (Figure 1 – figure supplement 2C). Injections were then binned into two groups depending on the median of all slopes obtained. All calculated slopes lower than the median were categorized as a ‘slow’ injection while injections above the median were categorized as ‘fast’ injections. From this, ‘slow’ injections were seen to elicit lower peak Ca²⁺ amplitudes when compared to ‘fast’ injections (Figure 1 – figure supplement 2D). This indicated that the rate at which an injection was performed significantly determined the strength of the resulting vulval muscle Ca²⁺ response but only for injections performed at these lower-than-typical injection pressures (25 psi). Since flowrate did not significantly correlate with Ca²⁺ dynamics at injection pressures greater than 30 psi, to optimize Ca²⁺ responses, and to reduce instances of needles clogging, injection pressures used when testing animal responses were maintained between 30 – 40 psi.

Vulval muscle prodding protocol

To determine if the vulval muscles were receptive to mechanical stimulus, worms were immobilized as per the microinjection protocol. A prodding needle with a 2.7 µm wide tip was positioned next to either the anterior or posterior part of the vulva. To drive a mechanical displacement onto the prodded vulva, a motorized stage was moved at a range of distances (10 – 60 µm) in the direction of the needle to determine the optimum displacement distance. Subsequent experiments used a blunt prodding probe (4.6 µm wide tip), displaced 50 µm for one second. Image sequences were then segmented into paired prodded and unprodded vulval muscle recordings as per the fluorescence recording protocol. Paired Ca²⁺ responses in the 5 s period following the prodding stimulus were averaged and analyzed.

Embryo volume estimation

To estimate the volume of an embryo, brightfield images of injection-induced egg-laying events were collected, and embryos (n = 7) were measured for the lengths of the major (length) and minor (diameter) axes. Measurements were averaged and the diameter was calculated to be 27 µm while average length was 51 µm. Calculated embryo volume was ~30 pL when approximated as a cylinder and ~20 pL when approximated as an ellipsoid. Both measurements were averaged to estimate an approximate average volume per embryo of 25 pL.

Pharmacological assays

Nemadipine

Nemadipine-B (Toronto Research Chemicals Inc.) was dissolved in DMSO to 10 mM and added to melted NGM at 25 µM final concentration prior to media solidification. Control plates containing an equivalent volume of DMSO were made on the same day (0.1% DMSO (v/v) final concentration). Plates were seeded one day prior to the experiment with a 10-fold concentrated OP50 bacteria cultured in B broth. Worms were placed on nemadipine or DMSO plates for at least 1 h prior to microinjections but no longer than 2 h to prevent excess egg accumulation.

Floxuridine

Animals were sterilized by exposure to floxuridine as described (Mitchell et al., 1979; Ravi et al., 2018a). In this assay, L4 animals were staged onto NGM plates seeded with OP50, 100 µL of floxuridine (10 mg/mL) was added directly on top of the staged worms and OP50, and animals were incubated at 20 °C for 24 h. Animals were then visually inspected for lack of embryo accumulation prior to selection for microinjection.

Data analyses

Injection-induced Ca²⁺ transients were analyzed for the following characteristics: time to peak, amplitude of peak, time to decay from peak, and whether egg laying occurred. We defined ‘Time to peak’ as the time elapsed between the start of injection onset and when the Ca²⁺ trace had reached its peak GCaMP5/mCherry (ΔR/R) value. ‘Peak amplitude’ was defined as the peak value (ΔR/R). ‘Decay time’ was defined as the time when cytosolic Ca²⁺ levels declined to 37% of their peak value. We chose 37% of the peak value as our cutoff value to allow us to distinguish between different-order kinetics of decay. Individual Ca²⁺ transient peaks were detected using a custom MATLAB script, as described (Ravi et al., 2018b) and then confirmed via visual inspection of the GCaMP5/mCherry traces from fluorescence recordings. Although multiple injections per animal were often performed, quantitative analyses were performed only on the responses elicited by the first injection. Data were omitted from analyses only for cases in which liquid delivery failed (due to a clogged needle or spillage outside of animal).

Statistical analyses

Results were pooled over several days from at least 20 animals (unless otherwise noted). No explicit power analysis was performed prior to the study, but these numbers are in line with prior behavior experiments (Chase et al., 2004; Collins et al., 2016; Collins and Koelle, 2013). Statistical analyses were performed using Prism 8 (GraphPad). Isogenic control animals were always tested alongside mutants to account for possible batch effects. Animals were excluded from analyses when liquid delivery failed to spread within the worm (visually determined from brightfield recordings). To determine if there were any statistical differences between the Ca²⁺ responses elicited by injections between wild-type and mutant worms, a Student’s T-Test was performed. In cases in which multiple genotypes or conditions were compared, the data were analyzed by one-way ANOVA. The distributions of responses were tested for normality, and in cases in which data were found to be non-normal, a nonparametric equivalence test was performed (e.g., Mann-Whitney or Kruskal-Wallis). All tests were corrected for multiple comparisons (Bonferroni for ANOVA, Dunn for Kruskal-Wallis). Details of which test was performed for specific strains can be found in figure legends.

Standard least squares regression for vulval muscle Ca²⁺ transient amplitude

To determine which factor, as a consequence of injection, best predicted the vulval muscle Ca²⁺ response, a standard least squares regression analysis was performed with ‘time to peak’, ‘decay time’, ‘number of eggs in uterus’, and ‘flowrate’ as factors. From this analysis, only ‘number of eggs in uterus’ significantly correlated with vulval muscle Ca²⁺ transient amplitude (p = 0.0095).

Nominal logistic fit for egg laying

A similar analysis was performed to determine which factor best predicted egg release following injection. The predictors used were ‘vulval muscle Ca²⁺ transient amplitude’, ‘flowrate’, ‘time to peak’, ‘decay time’, and ‘number of eggs in uterus’. Only ‘vulval muscle Ca²⁺ transient amplitude was a significant predictor (p < 0.0001).

Acknowledgements

We thank Dr. James Baker and members of the Collins Lab for constructive feedback on the manuscript. We thank Dr. Ilya Ruvinsky for helpful discussions. We also thank Drs. Xiaofei Bai and Andy Golden for constructing and providing the AG527 strain and Dr. Addys Bode Hernandez for construction of the MIA136 strain. This work was funded by grants from the National Institutes of Health (NS086932) and the National Science Foundation (IOS-1844657) to KMC. EM was supported by the NSF Graduate Research Fellowship and McKnight Doctoral Fellowship. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40-OD010440).