The Role of Acetylcholine Signaling in the C. Elegans Egg-Laying Circuit

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The Role of Acetylcholine Signaling in the C. elegans Egg-Laying Circuit

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UNIVERSITY OF MIAMI

THE ROLE OF ACETYLCHOLINE SIGNALING IN THE C. ELEGANS EGG- LAYING CIRCUIT

Richard Kopchock III

A DISSERTATION

Submitted to the Faculty of the University of Miami in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Coral Gables, Florida

August 2021

UNIVERSITY OF MIAMI

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy

THE ROLE OF ACETYLCHOLINE SIGNALING IN THE C. ELEGANS EGG- LAYING CIRCUIT

Richard Kopchock III

Approved:

______Kevin M. Collins, Ph.D. Julia E. Dallman, Ph.D. Associate Professor of Biology Associate Professor of Biology

______Mason Klein, Ph.D. Guillermo Prado, Ph.D. Assistant Professor of Physics Dean of the Graduate School

______R. Grace Zhai, Ph.D. Professor of Molecular and Cellular Pharmacology

KOPCHOCK, RICHARD, III (Ph.D., Biology) The Role of Acetylcholine Signaling in the C. elegans (August 2021) Egg-Laying Circuit

Abstract of a dissertation at the University of Miami.

Dissertation supervised by Professor Kevin M. Collins. No. of pages in text. (95)

Successful execution of animal behavior requires coordinated activity and

communication between multiple cell types. Studies using the relatively simple neural

circuits of invertebrates have helped to uncover how conserved molecular and cellular

signaling events drive such changes in animal behavior. To understand the mechanisms

underlying neural circuit activity and behavior, I have been studying a small and relatively

isolated neural circuit that drives egg-laying behavior in the nematode worm C. elegans.

C. elegans is an ideal model organism for such studies due to its completely mapped-out

nervous system (connectome), well-established molecular toolkit, and similarity of its nervous system to higher animals, including humans. Among the similarities of the C. elegans nervous system to that of higher animals is the types of neurotransmitters used to drive neural circuit activity and behavior. One such neurotransmitter, acetylcholine (ACh), is vital for animal nervous systems and is a potential therapeutic target for a wide range of human diseases. The goal of my dissertation is to determine how the neurotransmitter acetylcholine functions within the C. elegans egg-laying circuit to regulate circuit signaling events and their resultant effects on behavior.

In Chapter 1, I show that the cholinergic (i.e. acetylcholine-releasing) Ventral C (VC) motor neurons are important for vulval muscle contractility and egg laying in response to

serotonin. Ca2+ imaging experiments show the VCs are active during times of vulval muscle contraction and vulval opening, and optogenetic stimulation of the VCs promotes vulval muscle Ca2+ activity. Blocking VC neurotransmission inhibits egg laying in response to serotonin and increases the failure rate of egg-laying attempts, indicating that

VC signaling facilitates full vulval muscle contraction and opening of the vulva for efficient egg laying. I also found the VCs are mechanically activated in response to vulval opening. Optogenetic stimulation of the vulval muscles is sufficient to drive VC Ca2+

activity, and this induction of VC Ca2+ activity requires muscle contractility, showing the presynaptic VCs and the postsynaptic vulval muscles can mutually excite each other.

Together, my results demonstrate that the cholinergic VC neurons facilitate efficient

execution of egg-laying behavior by coordinating postsynaptic muscle contractility in

response to serotonin and mechanosensory feedback.

In Chapter 2, I demonstrate that postsynaptic nicotinic ACh receptors (nAChRs)

regulate vulval muscle activity and egg-laying behavior. C. elegans lay eggs in response to

nAChR-agonists such as levamisole and nicotine, and loss-of-function mutations in the nAChR subunit genes unc-29, unc-38, and unc-63 confer insensitivity to those agonists.

Ca2+ imaging of the vulval muscles in unc-29 and unc-63 mutants reveals a decrease in the frequency of Ca2+ transients, which indicates that these nAChR subunits are involved in initiating vulval muscle activity. Optogenetic stimulation of the presynaptic cholinergic

VA and VB motor neurons leads to excitation of the vulval muscles, and this excitation is partially dependent on receptors encoded by unc-29 and unc-63, which provides evidence for an excitatory cholinergic synapse that has not been previously studied. To determine if cholinergic signaling and full vulval muscle contraction is dependent on other signaling

factors, I tested nAChR subunit mutants for their ability to lay eggs in exogenous serotonin.

Serotonin is a potent stimulator of egg laying in wild type animals, but it fails to stimulate

egg laying in unc-29 and unc-63 nAChR loss-of-function mutants. In all, my results show

that the VA and VB motor neurons provide nAChR-dependent excitatory input onto the

vulval muscles, and that cholinergic signaling acts in a common pathway with serotonin to

facilitate egg-laying behavior.

In Chapter 3, I examine how voltage-gated Ca2+ channels (VGCCs) interact with

cholinergic and serotonergic signaling to regulate vulval muscle activity and egg-laying

behavior. Both the P/Q/N-type VGCC UNC-2 and L-type VGCC EGL-19 are important for normal egg-laying behavior. UNC-2 facilitates egg laying in response to exogenous serotonin, since gain-of-function unc-2 mutants respond potently to serotonin while loss- of-function unc-2 mutants instead become inhibited. Loss-of-function egl-19 mutants are completely insensitive to serotonin, indicating EGL-19 is necessary for serotonin-induced egg laying, while gain-of-function egl-19 mutants show variable responses based on the severity of the allele. Ca2+ imaging of the vulval muscle in egl-19 mutants reveals increased

and decreased frequency of Ca2+ transients in gain-of-function and loss-of-function mutants, respectively. The decreased Ca2+ transient frequency of loss-of-function egl-19

mutants cannot be rescued by serotonin, suggesting that EGL-19 is a necessary downstream

effector of serotonergic signaling. Finally, I show that nAChR agonists stimulate egg

laying through an EGL-19 dependent pathway. Loss-of-function egl-19 mutants are

insensitive to nAChR agonists, supporting a model where nAChR signaling leads to

activation of EGL-19 to drive vulval muscle contraction and egg laying. Together, these

results show that cholinergic signaling shares a common pathway with serotonergic

signaling to influence L-type VGCC activity in the C. elegans egg-laying circuit.

The connectome of the C. elegans has greatly informed neural circuit studies, but it has also revealed that connectivity alone is not sufficient to explain nervous system operation.

The functional component of neural circuits is just as, if not more, important than the connectivity itself. The C. elegans egg-laying circuit presents a tractable model for the in vivo study of how neural circuits function at every biological level, from genes to cells to behavior, due to its relatively limited connectivity and amenability to molecular and cellular analysis. My dissertation draws upon both fundamental principles of neuroscience and work specific to the C. elegans egg-laying circuit to reveal how this circuit functions to drive behavior. Through this, I found novel roles for the previously understudied VC neurons, which make up 6 of the only 302 neurons in C. elegans. In addition, I provide evidence for a convergent pathway between the classical neurotransmitters acetylcholine and serotonin in driving muscle contraction. This convergent pathway ultimately acts on the L-type voltage-gated calcium channels to drive egg-laying behavior.

ACKNOWLEDGEMENTS

I would like to thank my advisor, Kevin Collins, for his steadfast commitment to my growth and success as both a scientist and a person. His support and guidance throughout the years has been more than I could have ever hoped for upon starting my PhD. I would also like to thank my committee members Grace Zhai, Mason Klein, and Julia Dallman for their immensely helpful perspectives and feedback on my work. I am especially grateful for Julia Dallman who has been a constant source of scientific expertise and enthusiasm toward my research. I would also like to give a shoutout to James Baker for being a cool person in general. Finally, I would like to give a special thank you to my mom and three sisters for their relentless positivity and faith in me throughout the years.

This work was funded by grants from the NIH (R01-NS086932) and NSF (IOS-

1844657) to KMC. RJK 3rd was supported by a University of Miami Maytag Fellowship.

We thank David M. Miller III for sharing plasmids and Mark Alkema for sharing strains.

Some of the strains used in this study were provided by the C. elegans Genetics Center,

which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).

iii

TABLE OF CONTENTS

Page

LIST OF FIGURES ...... v

LIST OF TABLES ...... vi

CHAPTERS

1 THE SEX-SPECIFIC VC NEURONS ARE MECHANICALLY ACTIVATED MOTOR NEURONS THAT FACILITATE SEROTONIN-INDUCED EGG LAYING IN C. elegans ...... 1

2 THE ROLE OF nAChRs IN THE C. elegans EGG-LAYING CIRCUIT ...... 33

3 THE ROLE OF VGCCs IN THE C. elegans EGG-LAYING CIRCUIT ...... 49

4 MATERIALS AND METHODS ...... 66

REFERENCES…………… ...... 80

LIST OF FIGURES Page

1.1 VC neurotransmission facilitates egg laying in response to serotonin ...... 9

1.2 The VC neurons are active during both weak vulval muscle twitching and strong egg-laying contractions ...... 12

1.3 Optogenetic VC activation induces and sustains vulval muscle Ca2+ activity 14

1.4 Blocking VC neurotransmission reduces the success rate of egg laying ...... 16

1.5 Blocking VC neurotransmission decouples vulval muscle Ca2+ activity and egg laying ...... 21

1.6 Blocking VC neurotransmission increases HSN Ca2+ activity ...... 22

1.7 Optogenetic activation and contraction of the vulval muscles drives VC neuron activity ...... 25

1.8 The VC neurons function to coordinate synchronized contraction of the vulval muscles for egg laying in response to serotonin-mediated potentiation ...... 26

2.1 nAChR mutants are defective in egg laying ...... 38

2.2 nAChRs influence vulval muscle activity...... 40

2.3 The VA/VB cholinergic motor neurons induce vulval muscle activity via nAChRs ...... 42

2.4 Serotonin-induced egg laying is facilitated through nAChRs ...... 44

3.1 VGCCs mutants are defective in egg laying ...... 53

3.2 VGCCs influence vulval muscle Ca2+ activity ...... 55

3.3 Serotonin promotes egg laying through the L-type VGCC ...... 57

3.4 VGCCs act in the same pathway as nicotine acetylcholine receptors ...... 59

3.5 The role of VGCCs and nAChRs in driving egg-laying behavior ...... 59

LIST OF TABLES Page

4.1 Strain names and genotypes for all animals used in this study ...... 76

Chapter 1: The sex-specific VC neurons are mechanically activated motor neurons that facilitate serotonin-induced egg laying in C. elegans1

Introduction

A fundamental goal of neuroscience is to understand the neural basis of behavior

(Cornelia I. Bargmann & Marder, 2013). Recent work reporting the synaptic wiring diagrams, or connectomes, of nervous systems provides an unprecedented opportunity to study how the nervous system directs animal behavior (Cook et al., 2019; Lerner et al.,

2016; Meinertzhagen, 2018). However, connectomes alone are not sufficient to predict

nervous system function (Batista-García-Ramó & Fernández-Verdecia, 2018; Swanson &

Lichtman, 2016; Taylor et al., 2019). Released neurotransmitters can signal both

synaptically and extrasynaptically through distinct receptors to drive short-term and long-

term behavior changes (Chase et al., 2004; Del-Bel & De-Miguel, 2018; Donnelly et al.,

2013; Hardingham & Bading, 2010; Koelle, 2018). Neuropeptides can be co-released from

synapses and signal alongside neurotransmitters (Brewer et al., 2019; Nusbaum et al.,

2017). Understanding how an assembly of ionotropic and metabotropic signaling events

drives the complex pattern of circuit activity is facilitated by direct tests in invertebrate

animals (Cornelia I. Bargmann & Marder, 2013) such as those amenable to genetic

investigation (Sengupta & Samuel, 2009). Such studies have the potential to reveal

conserved neural circuit signaling mechanisms that underlie behavior.

______1 This chapter was published as part of the following paper: Kopchock RJ III, Ravi B, Bode A, Collins KM. (2021) The sex-specific VC neurons are mechanically activated motor neurons that facilitate serotonin- induced egg laying in C. elegans. Journal of Neuroscience, 41(16). Movies can be found online: https://doi.org/10.1523/JNEUROSCI.2150-20.2021

The egg-laying circuit of the nematode worm C. elegans provides an ideal model for such a reductionist approach (Figure 1.1A-B). The canonical egg-laying circuit is comprised of three main cell types: the serotonergic HSN command motor neurons, the vulval muscles, and the cholinergic VC motor neurons (William R. Schafer, 2006). An active egg-laying behavior state is initiated when the HSN neurons release serotonin (Desai et al., 1988; Waggoner et al., 1998), which signals through G protein coupled serotonin receptors (Fernandez et al., 2020; Hapiak et al., 2009), and NLP-3 neuropeptides (Brewer et al., 2019). Individual eggs are laid when the vm1 and vm2 vulval muscle cells contract in synchrony to open the vulva (Collins & Koelle, 2013; Li et al., 2013). The VC motor neurons are the primary cholinergic neurons of the egg-laying circuit (Duerr et al., 2001;

Pereira et al., 2015), and are active during egg-laying behavior (Collins et al., 2016; Zhang et al., 2008), but the function of their signaling is not well understood (William R. Schafer,

2006). As in mammals, most muscle contraction events in C. elegans are ultimately driven by acetylcholine (ACh; Richmond & Jorgensen, 1999; Trojanowski et al., 2016). Nicotinic

ACh receptor (nAChR) agonists promote egg laying by acting on the vulval muscles (Kim et al., 2001; Waggoner et al., 2000), consistent with the VCs and/or other motor neurons releasing ACh to excite the vulval muscles (Waggoner et al., 1998). However, ACh synthesis and packaging mutants are hyperactive for egg laying, indicating ACh can also inhibit egg laying, possibly by signaling through inhibitory muscarinic ACh receptors

(Bany et al., 2003; Fernandez et al., 2020). This hyperactive egg-laying phenotype resembles animals in which VC neuron development has been disrupted by laser ablation or mutation (Bany et al., 2003), suggesting instead that ACh released from the VCs acts, at least in part, to inhibit egg laying, perhaps in response to sensory input. The VCs extend

processes along the vulva, leading to the proposal they might also relay mechanosensory

feedback in response to vulval opening (Zhang et al., 2010). How the VC neurons become

activated and signal to regulate egg-laying behavior remains unclear. Such insight is necessary for each cell in a neural circuit to transform static information from wiring diagrams into dynamic and meaningful understanding of animal behavior.

Here we address the function of the VC neurons during egg-laying behavior. We find that the VCs function within a serotonergic pathway to drive egg release. The VCs

provide excitatory input to convert the initial stages of vulval muscle contraction into a

successful egg-laying event. The VCs achieve this through direct activation in response to

vulval muscle excitation and contraction, forming a positive feedback loop until successful

egg laying is achieved.

Results

The VCs are a group of six, hermaphrodite-specific, cholinergic motor neurons

spaced out along the ventral cord that make synapses onto the HSNs, vm2 vulval muscles,

body wall muscles, and various motor neurons involved in locomotion (Figure 1.1A; Cook

et al., 2019; White et al., 1986). VC4 and VC5 in particular are the most proximal to the vulva and make extensive synapses onto the vm2 vulval muscles (Cook et al., 2019; White et al., 1986). Egg-laying events are always accompanied by a VC Ca2+ transient, but not

all VC Ca2+ transients coincide with egg release (Collins et al., 2016). ACh released from

VCs has been suggested to act through nAChRs including UNC-29 on the vulval muscles

to drive contraction (Kim et al., 2001; William R. Schafer, 2006), as well as through the

muscarinic ACh receptor GAR-2 on the HSNs to inhibit egg laying (Bany et al., 2003;

Fernandez et al., 2020). VC signaling has also been suggested to slow animal locomotion speed around egg-laying events (Collins et al., 2016), likely through the synapses it makes onto the body wall muscles and locomotion motor neurons (Cook et al., 2019; White et al.,

1986). In all, VC signaling appears to be complex and function through multiple pathways to regulate egg-laying circuit activity and behavior.

The vulval muscles are comprised of four vm1-type and four vm2-type muscle cells which are all electrically coupled but receive distinct synaptic input (White et al.,

1986). The vm2 vulval muscles are innervated by the HSN and VC neurons (Cook et al.,

2019; White et al., 1986). The vm1s receive cholinergic input from single VA and VB motor neurons which are part the locomotion circuit (White et al., 1986; Zhen & Samuel,

2015). Egg laying occurs when all 8 vulval muscle cells are active and pull the vulva open to expel an egg (Zhang et al., 2008). Serotonergic input from the HSNs helps to excite the vm2 vulval muscles and coordinate their activity during egg laying (Brewer et al., 2019;

Li et al., 2013; Shyn et al., 2003). The vm1 vulval muscles show rhythmic activity both during and outside of egg laying, suggesting that they are either intrinsically active or excited by VA and VB synaptic inputs (Collins et al., 2016; Collins & Koelle, 2013; Shyn et al., 2003). Vulval muscle Ca2+ transients start in the vm1 muscle cells and propagate into the vm2 muscles during egg laying events (Brewer et al., 2019; Collins & Koelle, 2013),

suggesting vm1 excitation provides a trigger for full vulval muscle contraction and egg

laying.

The pair of hermaphrodite-specific HSN neurons make synapses onto both the VCs and the vm2 vulval muscles (Cook et al., 2019; White et al., 1986). Serotonin and NLP-3

neuropeptides released by the HSNs have been shown to be critical for normal egg-laying

behavior to occur (Brewer et al., 2019; Waggoner et al., 1998). Consistent with the role of

HSNs initiating egg-laying behavior, exogenous serotonin potently increases the Ca2+

activity of the vulval muscles and VC neurons (Shyn et al., 2003; Zhang et al., 2008).

Additionally, laser ablation experiments have indicated that loss of the VC neurons disrupts

the induction of egg laying in response to serotonin (Shyn et al., 2003; Waggoner et al.,

1998). Thus, the role of the VC neurons in the egg-laying circuit and behavior may be

closely associated with serotoninergic signaling from the HSNs.

The VC neurons promote egg laying in response to serotonin

To test directly how loss of synaptic transmission from the VC motor neurons affects egg-laying circuit activity and behavior (Figure 1.1B), we used a modified lin-11

promoter/enhancer (Bany et al., 2003) to drive transgenic expression of Tetanus Toxin

(TeTx; Jose et al., 2007) which blocks both neurotransmitter and neuropeptide release

(Whim et al., 1997) from the six VC neurons. Expression of TeTx in the VC neurons did

not cause any gross defects in steady-state egg accumulation compared to non-transgenic control animals, indicating that the VC neurons are not strictly required for egg laying

(compare Figures 1.1C and 1.1D, quantified in Figure 1.1G). In contrast, egl-1(n986dm) mutant animals in which the HSNs undergo apoptosis (Trent et al., 1983), showed a dramatic impairment of egg laying, accumulating significantly more embryos (Figures

1.1E and 1.1G). Previous studies indicated that laser ablation of both the HSNs and VCs caused additive defects in egg laying (Waggoner et al., 1998). However, transgenic expression of TeTx in the VCs in HSN-deficient egl-1(n986dm) mutant animals did not significantly enhance their defects in egg laying (Figure 1.1F and 1.1G). We have

previously shown that wild-type animals lay their first egg around 7 hours after the L4- adult molt, a time when the VCs show their first activity (Ravi et al., 2018a). Animals with inhibited VC neurotransmission showed no significant change in the onset of egg laying compared to non-transgenic control animals (Figure 1.1H). In contrast, the onset of egg laying in egl-1(n968dm) mutant animals lacking HSNs is significantly delayed, occurring

about 18 hours after the L4-adult molt (Figure 1.1H). Expression of TeTx in the VCs in

HSN-deficient animals did not enhance the delay in egg laying significantly (Figure 1.1H).

These results together show that transmitter-mediated signaling from the VCs is not

required for egg-laying behavior to occur under normal culturing conditions.

We next used a drug-treatment approach to explore possible functions of the VC

neurons that may not be apparent in animals under standard laboratory conditions.

Serotonin potently stimulates egg laying even in conditions where egg laying is normally

inhibited, such as in liquid M9 buffer (Trent et al., 1983). As expected, serotonin promotes

egg laying in M9 buffer in wild-type animals (Figure 1.1I). We found that transgenic

animals expressing TeTx in the VCs failed to lay eggs in response to exogenous serotonin

in M9 buffer across varying serotonin concentrations (Figure 1.1I). Going forward, we

chose to conduct experiments at a single serotonin concentration of 18.5 mM. HSN-

deficient egl-1(n986dm) mutant animals are egg-laying defective under normal conditions

but will still lay eggs in response to exogenous serotonin (William R. Schafer et al., 1996),

and this response was suppressed in animals lacking VC neurotransmission at 18.5 mM

serotonin (Figure 1.1J). This resistance to exogenous serotonin in VC neurotransmission-

inhibited animals was not unique to M9 buffer, as transgenic animals placed on serotonin-

infused agar also showed a reduced egg-laying response (Figure 1.1K). These results show

that VC neurotransmitter release serves an important role for egg laying in response to

exogenous serotonin.

Despite the VCs being important for egg laying in response to exogenous serotonin

(Figure 1.1I-K), animals with blocked VC neurotransmission are still able to lay eggs at a

normal rate (Figure 1.1G). One potential explanation for this is that NLP-3 neuropeptide

signaling is able to compensate for the loss of the VC-mediated serotonergic signaling

pathway (Brewer et al., 2019). Based on this model, blocking VC neurotransmission in an

nlp-3 mutant background might phenocopy animals lacking all neurotransmitter release from the HSNs, as seen in the egl-1(n986dm) mutant. However, we found no significant increase in egg accumulation when blocking VC neurotransmission in an nlp-3 mutant background (Figure 1.1L). This suggests that under standard C. elegans culture conditions, serotonin is able to signal through VC-independent pathway to retain a normal rate of egg laying, likely by acting on the vulval muscles directly (Hapiak et al., 2009).

It was possible that the observed defects in serotonin response caused by inhibiting

VC neurotransmission could be due to impaired circuit development and/or by compensatory changes in circuit activity. Expression from the VC-specific promoter used to drive TeTx begins in the L4 stage as the egg-laying circuit is completing development, well before the onset of egg-laying behavior (Ravi et al., 2018a). To silence the VCs acutely after circuit development is complete, we transgenically expressed Histamine-gated chloride channels (HisCl) and treated the animals with exogenous histamine (Pokala et al.,

2014; Ravi et al., 2018a). Histamine silencing of the VCs caused no gross changes in steady-state egg accumulation (Figure 1.1M), confirming the results with TeTx that VC neurotransmission is not required for egg laying. However, acute histamine silencing of

the VCs reduced egg laying in response to serotonin in both wild-type and HSN-deficient egl-1(n986dm) mutant animals (Figure 1.1N), consistent with the results observed when blocking VC neurotransmission with TeTx. Together, these results show that both VC neuron activity and synaptic transmission are dispensable for egg laying under normal growth conditions, but the VCs do facilitate egg laying in response to exogenous serotonin.

VC Ca2+ activity is coincident with vulval muscle activity and egg laying

The VC neurons show rhythmic Ca2+ activity during the egg-laying active state

(Collins et al., 2016). However, only ~1/3 of VC Ca2+ transients were temporally

coincident with vulval muscle contractions that resulted in egg release (Collins et al.,

2016), raising questions about the function of the VC Ca2+ transients that do not coincide

with egg laying. To understand the function of VC Ca2+ activity, we expressed GCaMP5

in the VC neurons to measure Ca2+ dynamics in the VC cell bodies and processes most proximal to the vulva (VC4 & VC5; Figure 1.2A and Figure 1.2B) while simultaneously observing vulval opening and egg-laying behavior in a separate brightfield channel, as described (Ravi et al., 2018b). Since C. elegans neurons generally exhibit graded potentials, measurements of VC Ca2+ near synapses can be expected to correspond closely

with the degree of neurotransmitter and neuropeptide release (P. Liu et al., 2013; Q. Liu et

al., 2009). As expected, we found that egg-laying events were always accompanied by a

VC Ca2+ transient, but not every VC Ca2+ transient resulted in an egg-laying event (Figure

1.2C; Movie 1). These remaining VC Ca2+ transients were almost always observed with weak muscle contraction and partial opening of the vulva, termed a vulval muscle “twitch,” which are primarily mediated by the vm1 vulval muscles (Figure 1.2C and 1.2F; Collins &

Koelle, 2013).

Figure 1.1. VC neurotransmission facilitates egg laying in response to serotonin.

(A) Graphical representation of the C. elegans egg-laying circuit (modified from Collins et al., 2016). (B) Simplified circuit diagram showing the synapses and the primary neurotransmitters released between the HSN neurons, VC neurons, and vulval muscles.

The cell-specific transgene expressions performed throughout this figure are indicated. (C- F) Representative images of the C. elegans uterus showing unlaid eggs (arrowheads) in wild-type, transgenic animals expressing Tetanus Toxin (TeTx) in the VCs, and HSN- ablated egl-1(986dm) mutant animals. (G) Measurement of steady-state egg accumulation in animals from Figure 1.1C-F (±95 confidence intervals for the mean); asterisks indicate p<0.0001 and n.s. indicates p>0.05 (one-way ANOVA with Bonferroni’s correction for multiple comparisons; n≥17). (H) Scatter plot showing timing of first egg laid after the larval to adult molt in animals with blocked VC neurotransmission and ablated HSNs (asterisk indicates p<0.0001, one-way ANOVA with Bonferroni’s correction for multiple comparisons; n≥17). (I) Blockage of VC synaptic transmission inhibits serotonin-induced egg laying. Animals expressing TeTx in the VCs were placed into M9 buffer or M9 containing the indicated concentrations of serotonin and scored for number of eggs laid; asterisks indicate p<0.0001 (Kruskal-Wallis test with Dunn’s correction for multiple comparisons; n≥32). (J) Blockage of VC synaptic transmission inhibits serotonin-induced egg laying in HSN-ablated animals. HSN-ablated egl-1(986dm) mutant animals expressing TeTx in the VCs were placed in M9 buffer or M9 containing 18.5 mM serotonin; asterisks indicate p≤0.0002, pound indicates p=0.0225 and n.s. indicates p>0.05 (Kruskal-Wallis test with Dunn’s correction for multiple comparisons; n≥22). (K) Animals with inhibited VC neurotransmission lay fewer eggs compared to wild-type animals in response to 18.5 mM serotonin under otherwise normal culturing conditions on solid media agar plates; asterisks indicate p<0.0001, pound indicates p=0.0026, and n.s. indicates p>0.05 (one-way ANOVA with Bonferroni’s correction for multiple comparisons; n≥10). (L) Measurement of steady-state egg accumulation in animals expressing TeTx in the VCs in an nlp- 3(tm3023) mutant background (±95 confidence intervals for the mean); asterisks indicate p<0.0001 and n.s. indicates p>0.05 (one-way ANOVA with Bonferroni’s correction for multiple comparisons; n≥35). (M) Measurement of steady-state egg accumulation in animals expressing Histamine-gated chloride channels (HisCl) in either the VC or HSN neurons grown with or without histamine; asterisk indicates p<0.0001 and n.s. indicates p>0.05 (one-way ANOVA with Bonferroni’s correction for multiple comparisons; n≥33). (N) Acute electrical silencing of the VCs blocks serotonin-induced egg laying. Animals expressing HisCl in the VCs in either a wild-type or HSN-ablated egl-1(986dm) mutant background were incubated with 0 or 4 mM histamine for four hours, placed into wells with M9 buffer with 0 or 18.5 mM serotonin, and the number of eggs laid after 1 hour were counted; asterisks indicates p<0.05 and n.s. indicates p>0.05 (Kruskal-Wallis test with Dunn’s correction for multiple comparisons; n≥12 for control M9 and n≥24 for serotonin).

As quantified in Figure 1.2F, we found that 48% of VC Ca2+ transients were associated with vulval muscle twitch contractions, and 39% of VC Ca2+ transients were

associated with strong vulval muscle contractions that support egg release, which are the

result of simultaneous vm1 and vm2 vulval muscle contraction (Collins & Koelle, 2013).

To determine if presynaptic HSN activity was sufficient to drive VC and vulval muscle

activity downstream, we optogenetically stimulated HSNs transgenically expressing

Channelrhodopsin-2 and simultaneously recorded VC Ca2+ activity and vulval muscle

contractility (Figure 1.2A). As previously reported, optogenetic HSN stimulation was

sufficient to induce an active egg-laying behavior state (Collins et al., 2016). We found

that optogenetic stimulation of HSNs drove a robust increase in VC Ca2+ transients that

were associated with egg-laying events, but we still observed VC Ca2+ transients during

the weaker vulval muscle twitching contractions (Figure 1.2D). HSN optogenetic

stimulation rapidly elevated average Ca2+ levels in the VCs, within 5 seconds after blue

light exposure (Figure 1.2E). The light-dependent increase in VC Ca2+ activity and vulval

muscle contractions were not observed in animals grown without the essential cofactor,

all-trans-retinal (ATR; Figure 1.2D and 1.2E). During optogenetic HSN activation, 60% of

VC Ca2+ transients were associated with egg-laying events while only 35% were associated

with vulval muscle twitches, a significant difference from control animals not subjected to

HSN optogenetic stimulation that we attribute to an increase in egg laying frequency

(Figure 1.2F). Because VC Ca2+ activity rises at the same time as vulval opening and prior to egg release, these results are consistent with either a model where the VCs promote vulval muscle contractility, or a model where the VCs are activated in response to downstream vulval muscle contraction.

Figure 1.2. The VC neurons are active during both weak vulval muscle twitching and strong egg-laying contractions.

(A) Cartoon of the circuit and experimental manipulations. GCaMP5 was expressed in the VC neurons to record Ca2+ activity, and Channelrhdopsin-2 was expressed in HSNs to provide optogenetic stimulation of egg laying. (B) Representative still images of VC mCherry, GCaMP5, and GCaMP5/mCherry ratio (∆R/R) during inactive and active egg- laying behavior states. Asterisk indicates vulva. Scale bar is 20 μm. (C) Representative trace of VC GCaMP5/mCherry Ca2+ ratio in freely behaving, wild-type animals during an

egg-laying active state. (D) Representative trace of VC GCaMP5/mCherry Ca2+ ratio after optogenetic stimulation of ChR2 in the HSN neurons in animals grown in the absence (- ATR, top) and presence (+ATR, bottom) during 10 s of continuous blue light exposure. (E) Average vulval muscle Ca2+ levels (mean ±95% confidence intervals; asterisk indicates p<0.0001, Student’s t test; n=10). (F) Graph showing vulval muscle contractile activity for each VC Ca2+ transient during endogenous egg-laying active states (left) or in response to HSN optogenetic stimulation (right).

The VC neurons promote vulval muscle activity and contraction

The VC motor neurons synapse onto the vulval and body wall muscles where they

are thought to release ACh to drive contraction (Duerr et al., 2008; White et al., 1986;

Zhang et al., 2008). While VC activity and neurotransmission is not required for egg laying

(Figure 1.1G and 1.1K; Laura E. Waggoner et al., 1998), the VCs may still release ACh to

regulate vulval muscle contractility. To test if the VCs can excite the vulval muscles

directly, we expressed Channelrhodopsin-2 in the VC neurons and performed ratiometric

Ca2+ imaging in the vulval muscles after exposure to blue light (Figure 1.3A; Movie 2).

Optogenetic stimulation of the VCs led to an acute induction of vulval muscle Ca2+ activity

within 5 s but was unable to drive full vulval muscle contractions and egg release (Figure

1.3B and 1.3C). However, average vulval muscle Ca2+ transient amplitude after

optogenetic stimulation was not significantly higher through the duration of the recording

(Figure 1.3D). Vulval muscle Ca2+ transient frequency was reduced, which may result from

the increased duration of each individual transient (Figures 1.3E and 1.3F). This result

shows that VC activity alone is not able to maximally excite the vulval muscles to the point

of egg laying, but that VC activity is excitatory and can sustain ongoing vulval muscle Ca2+

activity. We find these results consistent with a model where serotonin and NLP-3

neuropeptides released from the HSNs signal to enhance the excitability and contractility

of the vulval muscles for egg laying (Brewer et al., 2019), while the ACh released from the

VCs prolong the contractile phase to facilitate egg release.

Figure 1.3. Optogenetic VC activation induces and sustains vulval muscle Ca2+ activity. (A) Cartoon of circuit and experiment. Channelrhdopsin-2 was expressed in the VC neurons and GCaMP5 was expressed in the vulval muscles. (B) Representative traces of vulval muscle GCaMP5/mCherry Ca2+ ratio (∆R/R) in animals grown in the absence (- ATR, top) or presence (+ATR, bottom) in response to 5 minutes of continuous blue light exposure. Tick marks show duration at half-maximal amplitude of each measured Ca2+ transient. Black bar indicates first 5 seconds following blue light exposure. (C) Averaged vulval muscle Ca2+ transient activity (shaded region indicates ±95% confidence intervals) during the first 5 seconds of optogenetic activation of the VC neurons in -ATR (control; blue) and +ATR conditions (orange; asterisk indicates p=0.003, Student’s t test; n≥13 animals). (D) Scatterplot showing the average peak amplitude of vulval muscle Ca2+ transients (±95% confidence intervals) per animal in response to VC optogenetic stimulation during 5 minutes of continuous blue light (n.s. indicates p>0.05; Student’s t test; n≥13 animals). (E) Scatterplot showing the average time between vulval muscle Ca2+ transients (±95% confidence intervals) per animal during VC optogenetic stimulation during 5 minutes of continuous blue light (asterisk indicates p=0.0236, Student’s t test; n≥13 animals). (F) Scatterplot showing the average vulval muscle Ca2+ transient width (±95% confidence intervals) per animal during VC optogenetic stimulation during 5

minutes of continuous blue light (asterisk indicates p<0.0001, Student’s t test; n≥13 animals; error bars indicate 95% confidence intervals for the mean).

The VCs facilitate successful vulval opening during egg laying

Loss of VC activity or synaptic transmission caused a specific defect in serotonin-

induced egg laying, suggesting the VCs are required for proper vulval muscle Ca2+ activity

and/or contractility. We recorded vulval muscle Ca2+ activity in freely behaving animals

transgenically expressing TeTx in the VCs (Figure 1.4A; Movie 3). Vulval muscle Ca2+

activity in wild-type animals is characterized by low activity during the ~20 minute egg-

laying inactive state, and periods of high activity during the ~2 minute egg-laying active

state (Figure 1.4B; Collins et al., 2016; Laura E. Waggoner et al., 1998). Expression of

TeTx in the VCs did not significantly affect the overall frequency or amplitude of vulval muscle Ca2+ transients during egg-laying inactive states compared to wild-type control

animals (Figures 1.4C and 1.4D). However, inhibition of VC neurotransmission led to

larger amplitude Ca2+ transients during the active phase (Figure 1.4D). Since previous work

suggested the VCs release ACh that inhibits egg laying (Bany et al., 2003), the simplest

explanation for this phenotype would be that the VCs normally inhibit vulval muscle Ca2+

activity. However, our results show optogenetic activation of the VCs increased vulval

muscle Ca2+ transient duration with minimal effect on amplitude (Figures 1.3C, 1.3D, and

1.3F). Further inspection of vulval muscle Ca2+ traces of individual animals showed that

transgenically expressing TeTx in the VCs had large vulval muscle Ca2+ transients of

amplitude similar to egg-laying Ca2+ transients (>1.0 ∆R/R), but without an egg being successfully released (Figure 1.4B and 1.4E; Movie 3). These large amplitude transients that did not lead to egg laying were thus termed “failed egg-laying events.” Such failed

egg-laying events were infrequent in vulval muscle Ca2+ recordings from wild-type

animals, but they occurred more frequently than successful egg-laying events in transgenic

animals expressing TeTx in the VCs (Figure 1.4E). Based on these results, it appears that

VC neurotransmission does not initiate vulval muscle Ca2+ transients but is instead critical

for coordinating vulval muscle Ca2+ activity and contraction across the vulval muscle cells

to allow for successful egg release.

Figure 1.4. Blocking VC neurotransmission reduces the success rate of egg laying. (A) Cartoon of circuit and experiment. TeTx was expressed in the VC neurons to block their neurotransmitter release and GCaMP5 was expressed in the vulval muscles to record Ca2+ activity. (B) Representative traces of vulval muscle GCaMP5/mCherry Ca2+ ratio (∆R/R) in wild-type (top) and TeTx expressing transgenic animals (bottom). Arrowheads indicate successful egg-laying events, and open circles indicate strong (>1.0 ∆R/R) vulval muscle Ca2+ transients of “failed egg-laying events.” Egg-laying behavior state (inactive or active) duration is indicated below each trace. (C) Cumulative distribution plots of all vulval muscle Ca2+ transients across wild-type and transgenic animals expressing TeTx in the VCs. Transients were parsed into egg-laying active and inactive phases (n.s. indicates

p>0.05, Kruskal-Wallis test with Dunn’s correction for multiple comparisons; n≥200 transients from 11 animals). (D) Average vulval muscle Ca2+ transient peak amplitudes per animal during the inactive and active egg-laying phase (asterisk indicates p=0.0336, one- way ANOVA with Bonferroni’s correction for multiple comparisons; n=11 animals). (E) Percentage of failed egg-laying events in wild-type and transgenic animals expressing TeTx in the VCs (asterisk indicates p=0.0014, Mann-Whitney test; n=11 animals).

Egg laying occurs when strong vulval muscle Ca2+ activity drives the synchronous

contraction of all the vulval muscle cells (Brewer et al., 2019; Li et al., 2013) that allows

for the mechanical opening of the vulva in phase with locomotion for efficient egg release

(Collins et al., 2016; Collins & Koelle, 2013). Egg-laying events are characterized by coordinated Ca2+ activity between the vm1 vulval muscles that extend to the ventral tips of

the vulva and the medial vm2 vulval muscles, leading to full contraction and egg release

(Figure 1.5A). This Ca2+ activity is distinct from weak vulval muscle twitching

contractions that are confined to the vm1 muscles (Figure 1.5A; Collins & Koelle, 2013).

Failed egg-laying events exhibit Ca2+ activity more similar to egg-laying events, where

Ca2+ signal is high across both vm1 and vm2 muscles (Figure 1.5A). To understand why

egg laying was less likely to occur during strong vulval muscle Ca2+ transients in VC

neurotransmission-inhibited animals, we measured features of contractile events during egg laying. Contraction can be directly quantified by measuring the reduction in muscle area in fluorescent micrographs, and vulval opening can be quantified by measuring the changing distance between the vulval muscle cells positioned anterior and those posterior to the vulval slit (Figure 1.5A). During egg-laying events in wild-type animals, a strong cytosolic Ca2+ transient correlates with a ~50 µm2 contraction of muscle size followed by

a rebound phase after egg release (Figure 1.5B, top and middle). Simultaneously, the

anterior and posterior muscles separate by ~10 µm, facilitating egg release (Figure 1.5B,

bottom). We found differences in the kinetics and extent of vulval muscle opening between

wild-type and TeTx-expressing transgenic animals during successful egg-laying events

(compare Figures 1.5B and 1.5C). The vulval muscles opened wider and the degree of

contraction was greater during egg-laying events in animals where VC neurotransmission

was inhibited with TeTx (Figure 1.5C), possibly because the vulval muscle Ca2+ rise started

earlier in these animals. During failed egg-laying events, the vulval muscles showed only modest contraction and only separated by ~5 µm, insufficient to allow egg release, despite reaching similar levels of cytosolic Ca2+ (Figure 1.5D). Animals with blocked VC

neurotransmission exhibited even less separation of the vulval muscles during failed egg-

laying events (compare Figures 1.5D and 1.5E). In contrast to wild type animals, animals with inhibited VC neurotransmission have many more failed egg-laying events during which they exhibit vulval opening kinetics more similar to twitches (compare Figure 1.5F and 1.5G). To understand the relationship between vulval muscle Ca2+ levels and vulval

opening, we measured the distance between anterior and posterior vulval muscles during

weak twitching, failed egg-laying events, and successful egg-laying events (Figure 1.5F).

In both wild-type and TeTx-expressing animals, we noted a linear relationship of low but positive slope between vulval opening and Ca2+ levels <1.0 ∆R/R (∆opening / ∆Ca2+) during weak twitching contractions (Figure 1.5F and 1.5G). However, as Ca2+ levels rose

above 1.0 ∆R/R, the muscles reached threshold for full opening, allowing successful egg

release (Figure 1.5F). The steep, linear ∆opening / ∆Ca2+ relationship during failed egg- laying events suggests a threshold of Ca2+ drives an all-or-none transition to full contraction, vulval opening, and egg release. In VC neurotransmission-inhibited animals, the shallow ∆opening / ∆Ca2+ relationship continued as weak twitches transitioned into

failed egg-laying events, with many strong vulval muscle Ca2+ transients failing to open

the vulva sufficiently for egg release (Figure 1.5G). However, successful egg-laying events

in VC neurotransmission-inhibited animals still showed a sharp threshold between Ca2+

levels and the degree of vulval opening. This raises the possibility of two types of failed

egg-laying events: one that is shared with wild-type animals, and another that is unique to

animals with inhibited VC neurotransmission.

VC neurotransmission regulates HSN command neuron and egg-laying circuit activity

To determine whether VC synaptic transmission regulates egg laying via HSN, we recorded HSN Ca2+ activity in wild-type and transgenic animals expressing TeTx in the

VCs (Figure 1.6A). During the egg-laying active state, the HSNs drive egg laying during

periods of increased Ca2+ transient frequency in the form of burst firing (Figure 1.6B;

Collins et al., 2016; Ravi et al., 2018a). We observed a significant increase in HSN Ca2+

transient frequency when VC synaptic transmission was blocked compared to non-

transgenic control animals (Figure 1.6C). Wild-type animals spent ~11% of their time

exhibiting high frequency burst activity in the HSN neurons, while transgenic animals

expressing TeTx in the VC neurons spent ~21% of their time exhibiting HSN burst firing activity (Figure 1.6D). These results are consistent with the interpretation that VC neurotransmission is inhibitory toward the HSNs such as proposed in previous studies

(Bany et al., 2003; Zhang et al., 2008), but the steady-state egg accumulation of animals expressing VC-specific TeTx or HisCl is normal (Figure 1.1G and 1.1K).

Figure 1.5. Blocking VC neurotransmission decouples vulval muscle Ca2+ activity and egg laying.

(A) Vulval muscle sizes and distances were quantified by measuring the changes in area and centroid of the anterior and posterior muscle groups in mCherry channel micrographs during GCaMP5/mCherry ratiometric imaging. Shown are still images of the vulval muscles during representative muscle states of relaxation, twitching, egg laying, and failed egg-laying events. Brackets indicate the distance between the centroid of vulval muscle halves, arrowheads indicate high Ca2+ activity, and asterisk indicates vulva. Scale bar is 20 μm. (B-D) Mean traces (±95 confidence intervals) of vulval muscle Ca2+ (GCaMP5/mCherry ratio), vulval muscle area (µm2), and vulval muscle centroid distance (µm) during successful (B and C; n≥26 from 11 animals) and failed egg-laying events (D and E; n≥10 from 11 animals) in wild-type (B and D) and transgenic animals expressing TeTx in the VC neurons (C and E). (F-G) Scatter plot showing peak vulval muscle Ca2+ amplitude in relation to the corresponding vulval muscle opening distance in wild-type (F) and transgenic animals expressing TeTx in the VC neurons (G). Lines through points represent simple linear regression for each labeled grouping.

We have previously shown that HSN burst firing is regulated by egg accumulation and

feedback of vulval muscle Ca2+ activity (Ravi et al., 2018a), which could be enhanced by

the high rate of failed egg-laying events observed in VC TeTx transgenic animals (Figure

1.5E). We propose that the increased HSN burst firing seen in VC TeTx transgenic animals

does not solely result from loss of inhibitory VC input, but also reflects the circuit

prolonging the active state to compensate for more failed egg-laying transients in the

absence of excitatory VC input.

Figure 1.6. Blocking VC neurotransmission increases HSN Ca2+ activity.

(A) Cartoon of circuit and the experiment. TeTx was expressed in the VC neurons to block their neurotransmitter release and HSN Ca2+ activity was recorded through GCaMP5 imaging. (B) Representative traces of HSN neuron GCaMP5/mCherry Ca2+ ratio (∆R/R) in a wild-type background (top, green) and animals expressing TeTx in the VCs (bottom, blue). (C) Cumulative distribution plots of the instantaneous frequency of HSN Ca2+ transients in wild-type and TeTx-expressing animals (asterisk indicates p=0.0006, Kolmogorov-Smirnov test; n≥154 from ≥10 animals). (D) Scatter plot for the percentage of time each animal spent with HSN Ca2+ inter-transient intervals that were less than 30 seconds, an indicator of hyperactivity (asterisk indicates p=0.00272, Student’s test; n≥10; error bars indicate 95% confidence intervals for the mean).

The VC motor neurons are responsive to vulval muscle activity and contraction

VC Ca2+ activity is coincident with strong vulval muscle twitching and egg-laying contractions (Figure 1.2; Collins et al., 2016). In addition to making synapses onto the vm2 muscles whose contraction drives egg laying, the VCs extend neurites along the vulval hypodermis devoid of synapses (White et al., 1986), suggesting the VCs may respond to

vulval opening. To test this model, we sought to induce vulval opening independent of

endogenous circuit activity and presynaptic input from the HSNs. We transgenically

expressed Channelrhodopsin-2 specifically in the vulval muscles using the ceh-24

promoter to stimulate the vulval muscles and used GCaMP5 to record blue-light induced

changes in vulval muscle Ca2+ activity (Figure 1.7A). Optogenetic stimulation of the vulval

muscles triggered an immediate rise in vulval muscle cytosolic Ca2+, tonic contraction of the vulval muscles, vulval opening, and egg release (Figures 1.7B and 1.7C). Even though optogenetic stimulation resulted in sustained vulval muscle Ca2+ activity and contraction,

vulval opening and egg release remained rhythmic and phased with locomotion, as

previously observed in wild-type animals (Collins et al., 2016; Collins & Koelle, 2013).

Simultaneous bright field recordings showed the vulva only opened for egg release when

the adjacent ventral body wall muscles were in a relaxed phase (Movie 4). We have

previously shown that eggs are preferentially released when the vulva is at a particular

phase of the body bend, typically as the ventral body wall muscles anterior to the vulva go

into a more relaxed state (Collins et al., 2016; Collins & Koelle, 2013). We now interpret

this phasing of egg release with locomotion as evidence that vulval muscle Ca2+ activity

drives contraction, but the vulva only opens for successful egg release when contraction is

initiated during relaxation of the adjacent body wall muscles. Together, these results show

that optogenetic stimulation of the vulval muscles is sufficient to induce vulval muscle

Ca2+ activity for egg-release in a locomotion phase-dependent manner.

To test the hypothesis that the VCs respond to vulval muscle activation, we

recorded changes in VC Ca2+ upon optogenetic stimulation of the vulval muscles (Figure

1.7D). We observed a robust induction of VC Ca2+ activity upon blue light illumination

(Figures 1.7E and 1.7F). The rise in VC Ca2+ occurred before the first egg-laying event, suggesting that this process is dependent on muscle activity and not necessarily the passage of an egg (Figure 1.7G; Movie 5). This result demonstrates that the VCs can become excited in response to activity of the postsynaptic vulval muscles.

How do the vulval muscles activate the VCs? The VCs make both chemical and electrical synapses onto the vm2 vulval muscles (Cook et al., 2019; White et al., 1986).

Depolarization of the vm2 vulval muscles might be expected to electrically propagate to the VC and trigger an increase in VC Ca2+ activity. Another possibility is that the VCs are

mechanically activated in response to vulval muscle contraction and/or vulval opening. To

test these alternate models, we optogenetically stimulated the vulval muscles of unc-54

muscle myosin mutants, which are unable to contract their muscles, and recorded VC Ca2+

activity (Figure 1.7D). We found optogenetic activation of the vulval muscles failed to

induce VC Ca2+ activity in unc-54 mutants compared to the wild-type background (Figures

1.7G and 1.7H). While unc-54 mutants appear to show some increase in VC Ca2+ activity

following blue light stimulation of the vulval muscles, this increase was not statistically

significant, suggesting indirect excitation of the VCs through gap junctions is insufficient

on its own to induce robust VC Ca2+ activity. Together, these results support a model where the VC neurons are mechanically activated in response to vulval muscle contraction.

Mechanical activation appears to drive VC activity and is mediated through the VC4 and

VC5 neurites which are most proximal to the vulval canal through which eggs are laid.

Figure 1.7. Optogenetic activation and contraction of the vulval muscles drives VC neuron activity.

(A) Cartoon of experiment. Channelrhodopsin-2 and GCaMP5 were expressed in the vulval muscles to monitor cytosolic Ca2+ after optogenetic stimulation. (B) Representative still images of vulval muscle mCherry, GCaMP5, and GCaMP5/mCherry ratio during optogenetic activation of the vulval muscles. Arrowheads indicate each vulval muscle half, and asterisk indicates vulva. Scale bar is 20 μm. (C) Representative traces of vulval muscle GCaMP5/mCherry Ca2+ ratio (∆R/R) in animals expressing ChR2 in the vulval muscles in the absence (-ATR , top) or presence (+ATR, bottom) of all-trans retinal during 3 minutes of continuous blue light exposure. (D) Cartoon of circuit and experiment. Channelrhdopsin-2 was expressed in the vulval muscles and GCaMP5 was expressed in the VC neurons to record Ca2+ activity in wild-type or unc-54 myosin null mutant animals. (E) Representative still images of VC neuron mCherry, GCaMP5, and GCaMP5/mCherry ratio during optogenetic activation of the vulval muscles. Arrowheads indicate VC neuron cell bodies, and asterisk indicates vulva. Scale bar is 20 μm. (F) Representative traces of VC neuron GCaMP5/mCherry Ca2+ ratio (∆R/R) in animals expressing ChR2 in the vulval muscles in the absence (-ATR , top) or presence (+ATR, bottom) of all-trans retinal during 3 minutes of continuous blue light exposure. (G) Averaged VC Ca2+ responses during the first 5 s. Error bands represent ±95 confidence intervals for the mean; n≥10 animals. (H) Scatter plot showing the average VC Ca2+ response during the first 5 seconds of optogenetic stimulation of the vulval muscles. Asterisk indicates p<0.0001; n.s. indicates p>0.05 (one- way ANOVA with Bonferroni’s correction for multiple comparisons; n≥10 animals).

Figure 1.8. The VC neurons function to coordinate synchronized contraction of the vulval muscles for egg laying in response to serotonin-mediated potentiation.

Working model of the functional connectivity within the C. elegans egg-laying circuit (Cook et al., 2019; White et al., 1986). Plus and minus signs indicate excitatory and inhibitory input, respectively. HSN command neurons release serotonin and NLP-3 to potentiate the VCs and vm2 vulval muscles (Figure 1.1 and Figure 1.2; Collins et al., 2016; Shyn et al., 2003; Waggoner et al., 1998; Zhang et al., 2008). VC neurons release acetylcholine that excites the vm2 vulval muscles, body wall muscles, and the VA/VB/VD locomotion motor neurons (Figure 1.3; Collins et al., 2016; Kim et al., 2001), as well as acetylcholine to inhibit the HSNs (Figure 1.6; Bany et al., 2003; Zhang et al., 2008). The VA/VB/VD neurons release acetylcholine and GABA onto the body wall muscles to regulate locomotion (Richmond & Jorgensen, 1999; Zhen & Samuel, 2015), and onto the vm1 vulval muscles to initiate contractions for egg laying (Collins et al., 2016; Collins & Koelle, 2013). Contraction of the vm1 vulval muscles electrically excites the vm2 vulval muscles and mechanically activates the VCs (Figure 1.7), forming a positive feedback loop until all vulval muscle cells are contracted and egg laying occurs.

Discussion

The connectome of C. elegans has greatly informed neural circuit studies and contributed to studies revealing that connectivity alone is not sufficient to explain nervous system operations (Cornelia I. Bargmann, 2012; Bentley et al., 2016). In the present study, we examined the neural circuit driving egg-laying behavior in C. elegans at a cellular

resolution to reveal functional pathways and elements of the behavior that had not been

discernable through connectome or prior genetic studies (Figure 1.8). We show that the

cholinergic VC motor neurons contribute to egg laying in a serotonergic pathway. Our data

show that HSN activity acts to excite the vulval muscles and VCs, and that VC Ca2+ activity is closely associated with visible vulval muscle contraction. In the absence of HSN- mediated potentiation, the VCs are able to excite the vulval muscles, but not to the threshold required for egg laying. This sub-threshold interaction is consistent with a model where serotonin-mediated potentiation of the VCs and vulval muscles is required before the VCs can facilitate egg laying through timely excitatory input. In the absence of VC

neurotransmission, the HSNs and the vulval muscles show excess Ca2+ activity, but this

excess activity does not correspond to increased egg laying. Instead, we find that the vulval

muscles are less efficient at opening the vulva, indicating the VCs have a role in facilitating

vulval muscle contraction. Surprisingly, optogenetic activation and contraction of the postsynaptic vulval muscles is sufficient to drive presynaptic VC Ca2+ activity. We propose

that serotonin released from the HSNs signals to promote both vulval muscle contractility

and VC sensitivity to that contraction. Following this potentiation, vulval muscle twitch contractions are able to mechanically activate the VCs to release excitatory ACh as part of a positive-feedback loop until the vulval muscles are fully contracted and egg laying occurs.

Many behaviors require downstream feedback to help fine-tune movements and make adjustments based on the changing internal and external environment, such as with wing-beat patterns in Drosophila or head-eye coordination in humans (Bartussek &

Lehmann, 2016; Fang et al., 2015). We find that the downstream target of the egg-laying neural circuit, the vulval muscles, signals upstream to facilitate proper completion of the behavior. We postulate that such feedback signaling is critical for two reasons. First, it can act to feedback inhibit the circuit to signal when the behavior has been executed. We find that vulval muscle contraction activates the VCs (Figure 1.7), which could contribute to inhibition of the egg-laying circuit through release of ACh acting on metabotropic receptors such as GAR-2 on the HSNs and vulval muscles (Bany et al., 2003; Fernandez et al., 2020; Zhang et al., 2008, 2010). GAR-2 has previously been shown to act in parallel with ionotropic receptors to differentially modulate locomotion behavior, providing both short- and longer-term effects in response to cholinergic signaling (Dittman & Kaplan,

2008; Zhen & Samuel, 2015). Second, feedback signaling could create a positive feedback

loop to facilitate full execution of a behavior. In this circuit model, the VCs are mechanically activated by vulval muscle twitches which in turn leads to the VCs further exciting the vulval muscles until ubiquitous and coordinated contraction is achieved. A similar type of feedback activity has been demonstrated in the C. elegans SMDD neurons

where TRPC channels, TRP-1 and TRP-2, are mechanically activated by neck muscle

contractions to influence neck steering behavior (Yeon et al., 2018), as well as the crab

cardiac ganglion where mechanosensitive dendrites receive feedback from the postsynaptic

muscles to modify the ganglion’s firing activity (García-Crescioni et al., 2010). Which

receptors mediate VC mechanosensation is not known, but the VCs do express innexin gap

junction proteins (Altun et al., 2009) that have recently been shown to act as

mechanosensitive hemichannels (Walker & Schafer, 2020).

Serotonergic modulation of neural circuits and behavior is a well-characterized phenomena across both invertebrate and vertebrate animals (Bacqué-Cazenave et al., 2020;

Weiger, 1997). In the mouse nociceptive circuit, serotonergic modulation has been shown to confer hyperalgesia (Bardoni, 2019; Lin et al., 2011). In this circuit, serotonin acts on dorsal root ganglion neurons to increase their sensitivity to mechanical stimuli through the

5-HT2B receptor (Su et al., 2016), and to maintain this sensitivity through 5-HT4/6/7

receptors (Godínez-Chaparro et al., 2011). In the C. elegans egg-laying circuit, serotonin

released by the HSN command neurons signals through several G-protein coupled

receptors including the 5-HT2 ortholog SER-1 and the 5-HT7 ortholog SER-7 expressed on

the vulval muscles (Hapiak et al., 2009; Hobson et al., 2006; Xiao et al., 2006). These

serotonin receptors are thought to act through Gaq and Gas signaling pathways,

respectively, which activate EGL-19 L-type voltage-gated Ca2+ channels in the vulval

muscles to enhance their response to other excitatory input (William R. Schafer, 2006;

Waggoner et al., 1998; Zhang et al., 2008). SER-7 is also expressed in the VC neurons

(Fernandez et al., 2020), and serotonin acting through SER-7 has been shown to initiate motor neurons activity in other behaviors, such as feeding (Song et al., 2013). Like animals with blocked VC neurotransmission (Figure 1.1), ser-7 mutants also fail to respond to exogenous serotonin (Hobson et al., 2006). Downstream targets of serotonergic signaling onto the VCs may include the N/P/Q-type Ca2+ channel UNC-2 to promote

neurotransmitter release (William R. Schafer et al., 1996). Following serotonergic

potentiation, the VCs may be close enough to threshold to become mechanically excited in

response to vm1-mediated vulval muscle twitches, leading to excitatory VC

neurotransmitter release onto the vm2 vulval muscles to drive complete vulval muscle contraction for egg release.

Synaptic wiring diagrams show multiple sites of cross-innervation between the canonical egg-laying circuit and locomotion circuit (Cook et al., 2019; White et al., 1986).

Consistent with this shared connectivity, egg-laying behavior is correlated with changes in locomotion behavior (Collins et al., 2016; Hardaker et al., 2001), suggesting that these two circuits actively communicate and coordinate their respective behaviors. Coordination of distinct neural circuits and behaviors has been demonstrated in the stomatogastric ganglion of the lobster, where two overlapping circuits are phase-coordinated with one another to regulate gastric peristalsis and subsequent digestion (Bartos et al., 1999; Clemens et al.,

1998). The VCs have been shown to regulate locomotion, potentially through the

GABAergic motor neurons or via direct release of ACh that drives excitation and

contraction of the body wall muscles to slow locomotion (Figure 1.8; Collins et al., 2016).

Slowing of locomotion during egg laying may provide time for the vulval muscles to fully

contract (Collins et al., 2016; Collins & Koelle, 2013). This slowing could function to hold

the animal in a body posture that is favorable for vulval opening and egg release, which

would be consistent with our finding that VC activity causes elongated vulval muscle Ca2+

twitch transients (Figure 1.3). In this role, the VCs would be signaling to the rest of the

egg-laying circuit and locomotion circuit that the vulva is open so that body posture can be

held in a favorable phase for egg release. The six VCs also make synapses onto each

another (Cook et al., 2019). The mechanical activation of the vulva-proximal VCs during

vulval opening may excite the distal VC neurons to slow locomotion and control body

posture for efficient egg release. Failed egg-laying events could occur because of disrupted

phasing of the locomotion pattern with activity in the egg-laying circuit. Since twitches and egg-laying events occur at a specific phase of the locomotion pattern (Collins & Koelle,

2013), the opening of the vulva and egg release may require not only coordinated muscle

contraction, but also the relaxation of immediately proximal body wall muscles so that they

cannot physically resist the full opening of the vulva. Thus, the VCs may act to coordinate

the excitation of the vm2 muscles with excitatory input from the VA/VB locomotion motor

neurons onto the vm1 muscles to facilitate full contraction and egg laying in phase with

locomotion.

In all, the VC neurons and the egg-laying circuit present a tractable model system for

investigating how different forms of chemical signaling and mechanosensory feedback

work together to drive a robust behavior. The elucidation of the cellular and molecular

mechanisms underlying these distinct forms of feedback could help the understanding of

32 human neurological diseases where muscle coordination and proprioception are dysregulated, such as in Parkinson’s and Huntington disease (Cornelia I. Bargmann, 2012;

Lukos et al., 2013).

Chapter 2: The role of nAChRs in the C. elegans egg-laying circuit

Introduction

Acetylcholine (ACh) is a classical neurotransmitter that is commonly found throughout the animal kingdom (Jiao et al., 2019). ACh binding to the nicotinic acetylcholine receptors (nAChRs), a ligand-gated ion channel, provides fast signaling between presynaptic neurons and their postsynaptic targets (Brown, 2019). nAChR are composed of up to four subunit-types which form a hetero- or homopentamer transmembrane channel that opens in response to ACh binding (Albuquerque et al., 2009;

Dang et al., 2016). Dysregulation of nAChR signaling has been implicated in a multitude of human diseases, including Alzheimer’s, schizophrenia, and motor neuron disease (P. T.

Francis, 2005; Higley & Picciotto, 2014; Ochoa & Lasalde-Dominicci, 2007; Palma et al.,

2016).

ACh regulates vital behaviors in C. elegans including locomotion, feeding, and egg laying (Avery & Horvitz, 1990; Dallière et al., 2016; Waggoner et al., 1998; Zhen &

Samuel, 2015). The C. elegans genome encodes at least 32 nAChR subunits (Holden-Dye

et al., 2013) which are expressed throughout both neurons and muscle cells (Cohen et al.,

2014; Kim et al., 2001; Polli et al., 2015; Saur et al., 2013). For example, UNC-29 and

UNC-63, in combination with LEV-1, LEV-8, and UNC-38, form a levamisole-sensitive

heteropentamer expressed on the body wall muscles (Boulin et al., 2008; Fleming et al.,

1997). The body wall muscles also express a levamisole-insensitive nAChR in the form of

an ACR-16 α7-like homopentamer (Touroutine et al., 2005). The body wall muscles are

innervated by cholinergic motor neurons which release ACh to act on these muscle-

expressed nAChRs to stimulate muscle contraction and drive locomotor behaviors

(Richmond & Jorgensen, 1999; Touroutine et al., 2005; Zhen & Samuel, 2015).

The vulval muscles express some of the same acetylcholine receptor subunits as the body wall muscles, such as UNC-29, UNC-38, and UNC-63 (Culetto et al., 2004; Sloan et al., 2015; Waggoner et al., 2000), but it is not known if they form a similar heteropentamer channel with LEV-1 and LEV-8 as they do in the body wall muscles (Holden-Dye et al.,

2013). Additional putative nAChRs expressed in the vulval muscles have not yet been described, but pharmacological experiments suggest the presence of a levamisole- insensitive nAChR (Waggoner et al., 2000). The vulval muscles are innervated by multiple cholinergic neuron types, including the Ventral A, B, and C neurons which also innervate the body wall muscles (Cook et al., 2019; White et al., 1986), suggesting that cholinergic signaling drives vulval muscle contraction as it does in the body wall muscles.

Egg laying occurs when the vulval muscles contract, which opens the vulva and allows egg release from the uterus into the environment (Collins et al., 2016; Zhang et al.,

2008). There are 8 total vulval muscles of two different types: vm1 and vm2 (White et al.,

1986). Positionally, each vulval muscle is separated either laterally or anteroposteriorly, such that there is one of each muscle type at all four corners around the vulval opening.

Muscle contraction across all eight muscle cells isn’t always coordinated, resulting in vulval muscle “twitches,” where partial or uncoordinated vulval muscle contraction does not fully open the vulva (Collins & Koelle, 2013). The physiological reason for whether the vulval muscles twitch or fully contract may be due to the different presynaptic inputs and gap-junctioned partners they have (Collins & Koelle, 2013; White et al., 1986). The vm2s receive synaptic input from the HSN and VC neurons both anteriorly and posteriorly,

and are electrically coupled with the vm1s (White et al., 1986). In contrast, the vm1s are electrically coupled with one another and receive synaptic input from single Ventral Cord

Type A and Type B (VA and VB, respectively; White et al., 1986). Serotonergic input from the HSNs (Brewer et al., 2019; Shyn et al., 2003; Waggoner et al., 1998)and cholinergic input from the VC neurons (Borden et al., 2020; Kopchock et al., 2021) helps to excite the vm2 vulval muscles and coordinate their activity during egg laying. The vm1s show rhythmic activity both during and outside of egg laying, suggesting that they are either intrinsically active or excited by VA and VB synaptic inputs (Borden et al., 2020; Collins et al., 2016; Collins & Koelle, 2013; Shyn et al., 2003). Vulval muscle Ca2+ transients start

in the vm1 muscle cells and propagate into the vm2 muscles during egg laying events

(Brewer et al., 2019; Collins & Koelle, 2013), leading to a model where, following

serotonergic and neuropeptidergic potentiation, cholinergic release from locomotion motor

synapses excites nAChR’s expressed on the vm1 cells, to trigger full vulval muscle

contraction and egg laying (Brewer et al., 2019; Collins et al., 2016; Kopchock et al., 2021).

Results

nAChR mutants are defective in egg laying

To determine the role of nAChRs in egg laying, we screened through various

nAChR subunit loss-of-function mutants that have either been previously reported to affect

egg-laying behavior (Kim et al., 2001; Waggoner et al., 2000), or reported to be expressed in muscles cells generally (Holden-Dye et al., 2013; Polli et al., 2015). Steady-state egg accumulation was affected in all of the nAChR mutants we tested, indicating a change in the ability of these mutant animals to lay eggs at a normal rate (Figure 2.1A). Wild-type animals retained ~16 eggs on average in their uterus, while most of the nAChR mutants retained a lower number of eggs, indicating that these mutants either have an increased rate of egg laying or are less fertile than wild-type animals. The one exception was unc-

29(e403) which prematurely terminates the protein because of an early stop codon

(wormbase.org). These mutants retained a higher number of eggs in the uterus, indicating a decreased rate of egg laying. To test if this result was allele-specific, we tested unc-

29(ok2450), a deletion allele predicted to similarly be a null mutant, and found that it retained fewer eggs compared to wild type. The egg retention phenotypes of unc-

29(ok2450) more closely resembling the phenotypes of the other nAChR mutants, indicating a more complex role for unc-29 or allele-specific effects. We next assayed the nAChR mutants on their ability to lay eggs in response to the nAChR agonists nicotine and levamisole. Egg laying is inhibited when wild-type animals are placed in M9 buffer, but

M9 buffer containing 0.25% nicotine or 250 µM levamisole induces robust egg laying

(Kim et al., 2001). Consistent with previous findings, all nAChR loss-of-function mutant animals tested laid fewer eggs in response to exogenous nicotine (Figure 2.1B) and

levamisole (Figure 2.1C) compared to wild type. The unc-29(e403) and unc-63(x37)

mutants were the least responsive to nicotine, showing no difference between M9 buffer

along vs. M9 with 0.25% nicotine (Figure 2.1B). For levamisole, the unc-29(e403), unc-

29(ok2450), and unc-38(e264) mutants were all unresponsive to levamisole (Figure 2.1C).

Interestingly, the unc-63(x37) mutants displayed abnormally high egg laying in M9 buffer,

and this egg laying was reduced when animals were incubated in M9 containing levamisole

(Figure 2.1C). Together, these results corroborate previous findings on the effects of

nAChR mutants on egg laying behavior (Kim et al., 2001; Waggoner et al., 2000), while

also finding novel effects for alternative alleles and new nAChR mutants previously

unimplicated in egg-laying behavior.

nAChRs influence vulval muscle activity

nAChRs are expressed in both neurons and muscle cells throughout C. elegans

(Holden-Dye et al., 2013; Polli et al., 2015). To test how nAChRs could be acting to

influence cell activity and egg-laying behavior, we transgenically expressed the Ca2+

reporter GCaMP5 in the vulval muscles of nAChR subunit mutant animals and recorded

changes in reporter fluorescence during egg-laying behavior, as previously described

(Figure 2.2A; Collins et al., 2016; Ravi et al., 2018). As previously described, wild type

vulval muscle Ca2+ activity in freely-behaving animals is biphasic, showing periods of relatively sparse Ca2+ transients during the egg-laying inactive phase and periods of rapid,

high amplitude Ca2+ transients during the egg-laying active state (Figure 2.2B-E; Collins

et al., 2016).

Figure 2.1. nAChR mutants are defective in egg laying

(A-B) Measurement of steady-state egg accumulation in various nicotinic acetylcholine receptor (nAChR) loss-of-function mutants (line and error bars indicate mean ±95 confidence intervals); **** indicates p<0.0001, *** indicates p≤0.006, ** indicates p≤0.0037, * indicates p=0.0236, and n.s. indicates p>0.05 (one-way ANOVA with Bonferroni’s correction for multiple comparisons). (C-D) Loss of nAChR function affects egg-laying in response to the nAChR-agonists. Wild type or the indicated nAChR loss-of- function mutants were placed in M9 buffer or either M9 containing (C) 0.25% nicotine or (D) 250 µM levamisole; **** indicate p<0.0001, *** indicates p=0.0001, ** indicates p=0.0095, and n.s. indicates p>0.05 (Kruskal-Wallis test with Dunn’s correction for multiple comparisons).

The highest amplitude Ca2+ transients typically result in maximal contraction of the vulval muscles, which opens to vulva and allows egg release (Zhang et al., 2008). In nAChR

subunit loss-of-function mutant backgrounds unc-29(e403) and unc-63(x37), we found no

significant difference in the amplitude of Ca2+ transients compared to wild type, indicating

that these nAChR subunits alone are not required for maximal Ca2+ influx in freely behaving conditions (Figure 2.2C). However, we did observe a significant increase in the inter-transient intervals in both unc-29(e403) and unc-63(x37) mutant animals during the egg-laying inactive state resulting from a lower frequency of Ca2+ transients (Figure 2.2D).

This indicates that both the UNC-29 and UNC-63 subunits contribute to the onset of Ca2+

transients during the inactive state. During the egg-laying active state, unc-63(x37) showed a normal rate of Ca2+ transients compared to wild type, while unc-29(e403) animals showed significantly fewer transients (Figure 2.2D), indicating that the UNC-63 subunit is not required for the onset of Ca2+ transients during the active state. Since the Ca2+ transients

occurring during the inactive egg-laying states are typically restricted to the vm1-type

vulval muscles (Brewer et al., 2019; Collins & Koelle, 2013), these results show that both

the UNC-29 and UNC-63 subunits are important for the initiation of vm1-type vulval

muscle Ca2+ transients.

Figure 2.2. nAChRs influence vulval muscle activity

(A) GCaMP5 was expressed in the vulval muscles to record Ca2+ activity in wild-type, unc- 63(x37), or unc-29(e403) nAChR mutant animals. (B) Representative traces of vulval muscle GCaMP5/mCherry Ca2+ ratio (∆R/R) in wild type (blue) or nAChR loss-of-function mutants (purple and green). Egg laying events (arrowheads) and demarcation of active (red lines) and inactive egg-laying behavior states (dashed lines) for each animal are indicated. (C) Scatterplots showing the average peak amplitude and (D-E) inter-transient intervals of vulval muscle Ca2+ transients (mean ±95% confidence intervals) in wild-type or nAChR mutant animals during either the egg-laying (D) active or (E) inactive state. Asterisks

indicates p<0.0001; n.s. indicates p>0.05 (one-way ANOVA with Bonferroni’s correction for multiple comparisons).

Cholinergic motor neurons induce vulval muscle activity via nAChRs

The vm1-type vulval muscles receive synaptic input from the cholinergic VA and

VB motor neurons (Cook et al., 2019; Pereira et al., 2015; White et al., 1986). To test how

these synapses influence vulval muscle activity, we expressed Channelrhodopsin-2 (ChR2)

in the VA and VB neurons under the del-1 promoter (Winnier et al., 1999) and GCaMP5

in the vulval muscles to visualize Ca2+ activity (Figure 2.3A).. In the presence of the co-

factor all-trans-retinal (ATR), ChR2 is able to activate the VA and VB neurons in response

to blue light stimulation. We found that optogenetic VA and VB neuron activation resulted

in immediate excitation of the postsynaptic vulval muscles (Figure 2.3B). Notably,

activation of the VA and VB neurons was not sufficient to induce egg laying (data not

shown). To test if this synapse depends on ACh signaling acting on postsynaptic nAChRs on the vulval muscles, we test the optogenetic response in unc-29(e403) and unc-63(x37)

nAChR mutants (Figure 2.3B). Both unc-29(e403) and unc-63(x37) mutants resulted in diminished vulval muscle Ca2+ activity upon optogenetic VA and VB neuron stimulation

(Figure 2.3B-C). In all, these results show that the VA and VB motor neurons provide

excitatory input to the vulval muscles that depends in part on nAChRs. However, this

excitation alone is not sufficient to induce egg laying, suggesting additional signaling steps

are required to initiate the full, tetanic contraction of the vulval muscles that drives egg

release.

Figure 2.3. The VA/VB cholinergic motor neurons induce vulval muscle activity via nAChRs

(A) ChR2 was expressed in the VA and VB neurons from the del-1 promoter and GCaMP5 was expressed in the vulval muscles to record Ca2+ activity in either wild type, unc-63(x37), or unc-29(e403) nAChR mutants. (B) Averaged traces of vulval muscle GCaMP5/mCherry Ca2+ ratio (∆R/R) in either wild-type (blue) or nAChR loss-of-function mutants (purple and green) in response to optogenetic VA/VB neuron stimulation. (C) Scatter plot showing the average vulval muscle Ca2+ response during the first 5 seconds of optogenetic stimulation of the VA/VB neurons. Asterisk indicates p<0.0001; n.s. indicates p>0.05 (one-way ANOVA with Bonferroni’s correction for multiple comparisons).

Serotonin-induced egg laying is facilitated through nAChRs

The HSN neurons release serotonin and NLP-3 neuropeptides to initiate the egg- laying active state (Brewer et al., 2019; Collins et al., 2016; Emtage et al., 2012; Waggoner et al., 1998). As previously reported, wild type animals exhibit robust egg laying in response to exogenous serotonin (Kim et al., 2001; Trent et al., 1983). We tested nAChR subunit loss-of-function mutants for their egg-laying response to serotonin and found that unc-63(x37) was unresponsive to serotonin compared to M9 buffer alone (Figure 2.4).

Interestingly, unc-29(e403) egg laying was significantly inhibited by serotonin, while unc-

29(ok2450) did lay eggs, again indicating potential unc-29 allele-specific effects. The remaining nAChR subunit mutants were still responsive to serotonin, however, this response may be diminished compared to the wild type response (Figure 2.4). Notably, lev-

1(e311) and unc-38(e264) mutants were still responsive to exogenous serotonin (Figure

2.4). In body wall muscle, LEV-1 and UNC-38 subunits form a nAChR which includes

UNC-29 and UNC-63 (Holden-Dye et al., 2013). The variable responses to serotonin among these subunits suggest that the same nAChR is not formed in the vulval muscles, or that some of these subunits are not required for responsiveness to serotonin. In all, these results indicate that serotonin acts through a signaling pathway involving nAChRs.

Figure 2.4. Serotonin-induced egg laying is facilitated through nAChRs

(A) Loss of nAChR function affects ability of animals to lay eggs in response to the serotonin (5-HT). Wild type or the indicated nAChR loss-of-function mutants were placed in M9 buffer or M9 containing 18.5 mM serotonin **** indicate p<0.0001, *** indicates p≤0.0006, ** indicates p≤0.0057, and n.s. indicates p>0.05; Kruskal-Wallis test with Dunn’s correction for multiple comparisons).

Discussion

Cholinergic signaling through nAChRs is critical for a wide array of nervous system processes, ranging from simple muscle contractions to regulation of cortical circuits

(Arroyo et al., 2014; Picciotto et al., 2000; Rudolf & Straka, 2019). At neuromuscular junctions (NMJs), ACh released from motor neurons acts on postsynaptic nAChRs to drive muscle contraction (Kalamida et al., 2007; Nishimune & Shigemoto, 2018; Unwin, 2013). nAChRs are well established for driving muscle contraction in a variety of species, including C. elegans (Keshishian et al., 1996; Q. Liu et al., 2009; Nishimune & Shigemoto,

2018; Unwin, 2013). In C. elegans, body wall muscles are innervated by both cholinergic and GABAergic neurons which provide competing excitatory and inhibitory input to regulate muscle activity (Richmond & Jorgensen, 1999). C. elegans locomotion is achieved through alternating contraction of dorsal and ventral body wall muscles, which are

coordinated by presynaptic motor neuron input to produce directional movement

(Gjorgjieva et al., 2014; Haspel et al., 2010; Zhen & Samuel, 2015). Despite the well-

established role of cholinergic signaling in locomotion behavior and at the body wall

muscle NMJ, the role of ACh at the vulval muscles and the nAChRs it acts through is less

clear. The vulval muscles express nAChR subunits UNC-29, UNC-38, and UNC-63, but

these subunits do not appear to be strictly necessary for egg laying under normal conditions

(Culetto et al., 2004; Kim et al., 2001; Sloan et al., 2015; Waggoner et al., 2000). Animals

lacking the UNC-29 nAChR subunit are defective for egg laying in response to nAChR

agonists, and this response can be rescued by vulval muscle-specific expression of UNC-

29 (Kim et al., 2001). However, the cholinergic neurons most prominently innervating the

vulval muscles, the VCs, are not necessary for vulval muscle contraction and egg laying

(Kopchock et al., 2021; Shyn et al., 2003; Waggoner et al., 1998). The results in our present

study show that vulval muscle Ca2+ activity is reduced in frequency in loss-of-function mutants for unc-29 and unc-63 nAChR subunit receptors (Figure 2.2), suggesting that they contribute at least in-part to vulval muscle contraction. However, it is possible that the phenotypes are observed could be due to neuronal functioning of the examined nAChRs.

Consistent with neuronal activity of these nAChRs is the fact that most nAChR mutants we observed exhibited hyperactive egg-laying phenotypes, opposite of what would be expected if these nAChRs were driving vulval muscle contraction (Figure 2.1). We also found a novel egg-laying phenotype for ACR-8, which has been reported to be expressed in the vulval muscles (Figure 2.1; Touroutine et al., 2005). ACR-8 thus presents a promising candidate for further characterization of nAChR-mediated vulval muscle activity.

In the mammalian brain, nAChRs such as the homopentamer α7 receptor are

ubiquitously expressed and play a critical function in processes such as learning and

memory (Albuquerque et al., 2009; Breese et al., 1997; Dani, 2015). Dysfunctional

cholinergic signaling, especially through the α7 nAChR, has been associated with cognitive impairment and neurodegeneration observed in Alzheimer’s disease (Coughlin et al., 2020;

Guan et al., 2000; Hernandez & Dineley, 2012; Posadas et al., 2013). The C. elegans

genome encodes at least five α7-like nAChRs which are expressed in both muscles and

neurons (Holden-Dye et al., 2013). The α7-like nAChR subunit ACR-16 is able to form a

homomeric channel (Ballivet et al., 1996) and has been shown to act in the body wall

muscles, along with the UNC-29/UNC-38/UNC-63/LEV-1/LEV-8 heteromeric nAChR

channel, to confer muscle excitation in response to synaptic input (M. M. Francis et al.,

2005; Q. Liu et al., 2009; Richmond & Jorgensen, 1999; Touroutine et al., 2005). ACR-16

is not reported to be expressed in the vulval muscles (Touroutine et al., 2005), but we did

find a hyperactive egg-laying phenotype for acr-16 null mutants, possibly related to its

neuronal expression (Figure 2.1B). We also found novel egg-laying phenotypes in animals

missing two other α7-like nAChR subunit genes, which are acr-7 and acr-23 (Figure 2.1B).

A prediction would be that animals lacking both the levamisole-sensitive UNC-29 nAChR

complex and a α7 nAChR complex would show synthetic egg-laying defects a seen for the

locomotion circuit. The amenability of egg-laying behavior to genetic and molecular

analysis could provide a tractable model in the future for understanding how aberrant

nAChR signaling can lead to nervous system and behavior dysfunction.

nAChRs are vital at the NMJ where they receive synaptic input from cholinergic

motor neurons to initiate muscle contraction leading to animal movement (Slater, 2017;

Webster, 2018). As in other animals, the C. elegans NMJ relies on cholinergic signaling through nAChRs to initiate muscle contraction required for behaviors such as feeding, locomotion, and egg laying (Borden et al., 2020; Q. Liu et al., 2009; Richmond &

Jorgensen, 1999; Trojanowski et al., 2016; Zhen & Samuel, 2015). Full vulval muscle contraction and egg laying requires the coordinated activity of both vm1-type and vm2- type vulval muscles which are electrically coupled to one another, and activity is typically observed in the vm1s before the vm2s (Brewer et al., 2019; Collins & Koelle, 2013). The vm2-type vulval muscles controlling egg laying are innervated by the cholinergic VC neurons, however, VC activity is neither necessary nor sufficient to drive full vulval muscle contraction (Cook et al., 2019; Kopchock et al., 2021; White et al., 1986). The vm1s receive small synaptic inputs from the cholinergic VA and VB motor neurons (Cook et al., 2019;

White et al., 1986) which presents a candidate for the synaptic input initiating vulval muscle contractions (Collins et al., 2016). Excitatory synaptic input from the cholinergic

VA and VB neurons could act as the site of initiation for vulval muscle contraction (Figure

2.3). However, the likelihood of this excitatory input to result in full vulval muscle contraction could depend on the synchronized activity of the cholinergic VC neurons and/or potentiation from the serotonergic HSN neurons (Brewer et al., 2019; Kopchock et al., 2021). The VA and VB motor neurons are part of the locomotion neural circuit which is controlled by multiple central pattern generators to drive rhythmic undulations (Fouad et al., 2018; Zhen & Samuel, 2015). C. elegans egg-laying behavior has been shown to be coordinated with changes in locomotion behavior and individual egg-laying events occur during a particular phase of the animal’s undulation pattern (Collins et al., 2016; Hardaker et al., 2001). The excitatory synapse between the VA and VB motor neurons and the vulval

muscles thus presents a simple model for how C. elegans coordinates these two behaviors.

Excitatory input from the VA and VB motor neurons through nAChRs provides an

explanation for how the vm1-type vulval muscles are rhythmically active, even during egg-

laying inactive phases (Figure 2.3; Collins & Koelle, 2013). Egg-laying active phases may then be initiated when the vulval muscles and other neurons in the circuit are potentiated by serotonin and NLP-3 (Brewer et al., 2019; Waggoner et al., 1998). Following vulval muscle potentiation, voltage-gated calcium channels such as the L-type EGL-19 may be closer to their activation threshold (William R. Schafer, 2006), which then allows them to open in response and drive full muscle contraction in response to nAChR-mediated excitation (Figures 2.4).

Chapter 3: The role of VGCCs in the C. elegans egg-laying circuit

Introduction

Synaptic transmission and neuromodulation function to convey information between cells within a neural circuit. This information is manifested in the form of chemicals and electrical signals to alter the membrane potential of target neurons or muscles. Changes in membrane potential can lead to the activation of voltage-dependent ion channels, which propagate an electrical signal across the cell (Catterall, 1995). This

electrical signal is typically transmitted through voltage-gated sodium channels and

voltage-gated calcium channels (VGCCs; Catterall, 1995). VGCCs are found ubiquitously

throughout animal nervous systems and play an important role in critical processes such as

muscle contraction, neurotransmitter release, and signal transduction pathways (Barbado

et al., 2009; Catterall, 2011; Senatore et al., 2016; Striessnig, 2016).

VGCCs are primarily characterized by their α1 subunit which forms the

transmembrane pore to allow passage of Ca2+ ions (Catterall, 2011). The C. elegans

genome encodes only three α1 VGCC subunits: UNC-2 (P/Q/N-type), EGL-19 (L-type),

and CCA-1 (T-type; Kwok et al., 2006). C. elegans do not have the classically-studied voltage-gated sodium channel, so VGCCs may play a larger role in cell excitability than in other commonly studied nervous systems (C. I. Bargmann, 1998; P. Liu et al., 2011).

Consistent with the idea of C. elegans VGCCs playing a uniquely significant role, inward

current in response to depolarization is dependent on L-type VGCCs in body wall muscles

(Jospin et al., 2002). Additionally, action potentials in the body wall muscles require the

L-type VGCC EGL-19 (Gao & Zhen, 2011; P. Liu et al., 2011). In C. elegans neurons, the

P/Q/N-type VGCC UNC-2 is responsible for facilitating Ca2+ entry and Ca2+-dependent 49

neurotransmitter release in presynaptic termini (Mathews et al., 2003). Additionally, UNC-

2 has recently been shown to serve a secondary function of producing membrane potential

oscillations in the soma of motor neurons (Gao et al., 2018). The T-type VGCC CCA-1

functions to promote action potentials in the pharyngeal muscles by augmenting excitatory

postsynaptic potentials following cholinergic neurotransmission (Steger et al., 2005).

All three α1 VGCC subunits have been shown to be expressed in the egg-laying

circuit and are involved in egg-laying behavior (Y. S. Lee et al., 2000, p. 2; W. R. Schafer

& Kenyon, 1995; Zang et al., 2017). The HSN and VC motor neurons both express the

P/Q/N-type VGCC UNC-2 (Kim et al., 2001; W. R. Schafer & Kenyon, 1995). Loss-of-

function and null unc-2 mutants have an egg-laying constitutive phenotype, implicating

UNC-2 regulates neurotransmitter release in cells that function in a negative feedback

pathway (Kim et al., 2001; W. R. Schafer & Kenyon, 1995). The precise mechanisms underlying unc-2 mutant phenotypes and the precise localization of UNC-2 within the HSN

and VC motor neurons has not been elucidated. One possibility is that UNC-2 is acting in

the VCs to promote ACh neurotransmission, which in turn inhibits egg laying (Mathews et

al., 2003). Consistent with this model, unc-2 loss-of-function mutants are hypersensitive to serotonin, indicating that UNC-2 normally plays a role in an inhibitory pathway to counteract serotonin signaling (W. R. Schafer & Kenyon, 1995; William R. Schafer et al.,

1996). The HSN and VC motor neurons also both express the T-type VGCC CCA-1 (Zang et al., 2017). In the HSNs, CCA-1 is suggested to modulate the effects of inhibitory neuropeptide signaling (Zang et al., 2017).

The L-type VGCC EGL-19 is expressed in the vulval muscles and is involved in

promoting calcium influx and muscle contraction (Jospin et al., 2002; Lainé et al., 2014;

P. Liu et al., 2011; Shyn et al., 2003). EGL-19 appears to be the primary source of Ca2+ in

the vulval muscles to drive contraction, since egl-19 loss-of-function mutants have severely reduced Ca2+ activity while loss-of-function mutants for the ER-based ryanodine receptors have little effect on Ca2+ influx and contraction (Liu et al., 2011; Shyn et al., 2003). It is

worth noting that C. elegans muscles have their sarcomeres close to the plasma membrane,

putting them in the vicinity of the Ca2+ entry through VGCCs (Jospin et al., 2002). Animals

with egl-19 loss-of-function mutations are severely egg-laying defective, indicating that

EGL-19 is critical for egg-laying behavior to occur (Trent et al., 1983). In addition, egl-19

loss-of-function mutants are insensitive to exogenous serotonin, suggesting a link between

serotonergic signaling and activation of EGL-19 to drive vulval muscle contraction and

egg laying (Waggoner et al., 1998).

Egg-laying behavior depends on the activity of multiple neuron types and

neurotransmitters (Collins et al., 2016; William R. Schafer, 2006). Serotonin is a critical

mediator of C. elegans egg-laying circuit activity and behavior (Brewer et al., 2019;

Waggoner et al., 1998), and ACh can act to stimulate muscle contraction and individual

egg-laying events (Borden et al., 2020; Waggoner et al., 1998, 2000). Therefore, the

initiation and downstream effects of serotonergic and cholinergic signaling could be

closely linked to the activity of the VGCCs such as EGL-19 and UNC-2. This present study

aims to elucidate the function of VGCCs within the egg-laying circuit and how this

function is related to serotonergic and cholinergic signaling.

Results

Voltage-gated Ca2+ channels regulate egg laying

To investigate the role of VGCCs in egg-laying behavior, we assayed gain-of-

function and loss-of-function mutants for the L-type (EGL-19) and P/Q/N-type (UNC-2)

VGCCs. Quantification of steady-state egg accumulation in the uterus can be used as an

indicator for overall egg-laying ability and rate. Wild-type animals retained ~12 eggs on

average in their uterus, while the egl-19(n582) loss-of-function L-type VGCC mutant

retained significantly more eggs, indicating a severe inability to lay eggs at a normal rate

(Figure 3.1A), consistent with previous studies (R. Y. Lee et al., 1997). In contrast, the

EGL-19 gain-of-function mutants, egl-19(ad695) and egl-19(n2368), retained significantly

fewer eggs (Figure 3.1A; Lee et al., 1997). (R. Y. Lee et al., 1997; W. R. Schafer &

Kenyon, 1995; Shyn et al., 2003), consistent with L-type Ca2+ channels functioning to promote egg-laying behavior. Conversely, loss-of-function unc-2 mutants retained significantly fewer eggs, indicating that UNC-2 may function in cells that play an inhibitory role for egg laying (W. R. Schafer & Kenyon, 1995; William R. Schafer et al.,

1996). VGCC mutants have previously been shown to have altered sensitivity to exogenous serotonin (William R. Schafer et al., 1996). To test this further, we placed animals in M9 buffer, which inhibits egg laying, or M9 containing 18.5 mM serotonin which promotes egg laying, as previously described (Kim et al., 2001; Kopchock et al., 2021; Trent et al.,

1983). Wild-type animals exhibit robust egg laying in response to serotonin, whereas egl-

19(n582) loss-of-function L-type Ca2+ channel mutants were significantly resistant,

indicating that EGL-19 is required for serotonin-mediated egg laying, consistent with

previous studies on their serotonin sensitivity (Figure 3.1B; Shyn et al., 2003). The strong

gain-of-function egl-19(n2368) was also insensitive to serotonin (Figure 3.1B), likely because these mutants show hyperactive egg laying and exogenous serotonin is unable to

further stimulate egg laying. Surprisingly, unc-2 loss-of-function mutants also showed egg laying activity in M9 buffer, but this egg laying was significantly inhibited by exogenous serotonin (Figure 3.1B). This result is again consistent with UNC-2 LGCC channels functioning in cells that signal to inhibit egg laying, and this suggests those cells may be additionally modulated by serotonin. In all, these results show that L-type VGCCs are critical for egg-laying behavior to occur and mediate egg laying in response to serotonin.

Figure 3.1. VGCCs mutants are defective in egg laying

(A) Measurement of steady-state egg accumulation in voltage-gated calcium channel (VGCC) mutants (line and error bars indicate mean ±95 confidence intervals); asterisks indicate p<0.0001 and n.s. indicates p>0.05 (one-way ANOVA with Bonferroni’s correction for multiple comparisons). (B) VGCC mutations affect the ability of animals to lay eggs in response to serotonin (5-HT). Animals were placed in wells containing either M9 control buffer or M9 plus 18.5 mM serotonin (**** indicates p<0.0001, *** indicates and p≤0.0004, * indicates p=0.0104, and n.s. indicates p>0.05; Kruskal-Wallis test with Dunn’s correction for multiple comparisons).

VGCCs influence vulval muscle Ca2+ activity

The EGL-19 L-type VGCC are expressed in both neurons and muscle cells

throughout C. elegans (R. Y. Lee et al., 1997). To determine the role of the L-type VGCC

in regulating egg laying, we expressed GCaMP5 in the vulval muscles in egl-19(n582)

loss-of-function and egl-19(n2368) gain-of-function mutants to record muscle Ca2+ activity

during egg laying, as previously described (Figure 3.2A; Collins et al., 2016; Ravi et al.,

2018). Vulval muscle Ca2+ activity in wild-type animals alternates between periods of low activity during the egg-laying inactive state, and periods of high activity during the egg- laying active state (Figure 3.2B; Collins et al., 2016). During the active phase in wild type animals, Ca2+ transients are more frequent and stronger in amplitude and occasionally drive

egg-laying events (Figure 3.2B). In the egl-19(n582) loss-of-function mutant background,

there was no difference in the amplitude of vulval muscle Ca2+ transients during the

inactive or active state. However, there was an increase in the amplitude of egl-19(n2368)

gain-of-function mutants during the active state (Figure 3.2B-C). The increase in egl-

19(n2368) active phase Ca2+ amplitudes appeared to be caused by animals attempting to lay eggs even when there were no egg in position next to the uterus, which is consistent with the hyperactive egg-laying phenotype observed in previous experiments (Figure 3.1A) and in animals bearing gain-of-function mutants in other signaling molecules like Gq

(Dhakal et al., 2021). During both the inactive and active states of egl-19(n582) mutants,

we found a significant decrease in the frequency of vulval muscle Ca2+ transients (Figure

3.2D and 3.2E), consistent with the inhibited egg-laying phenotypes observed (Figure

3.1A). Animals bearing the gain-of-function egl-19(n2368) mutant exhibited an increase

in the frequency of vulval muscle Ca2+ transients during the inactive state (Figure 3.2D),

but no significant changes during the active state (Figure 3.2E). Together, these results

show that the L-type VGCC EGL-19 is critical for normal vulval muscle Ca2+ activity and egg laying.

Figure 3.2. VGCCs influence vulval muscle Ca2+ activity

(A) GCaMP5 was expressed in the vulval muscles to record Ca2+ activity in either wild type or L-type VGCC mutant backgrounds. (B) Representative traces of vulval muscle GCaMP5/mCherry Ca2+ ratio (∆R/R) in wild-type or VGCC mutant animals. (C) Scatterplots showing the average peak amplitude of vulval muscle Ca2+ transients during the egg-laying active and inactive states. (F-E) Inter-transient interval of vulval muscle Ca2+ transients (±95% confidence intervals) during either the egg-laying (F) active or (E) inactive state. Asterisk indicates p≤0.0225; n.s. indicates p>0.05 (one-way ANOVA with Bonferroni’s correction for multiple comparisons).

Serotonin promotes egg laying through the L-type VGCC

Serotonin is an important modulator of egg-laying behavior (Brewer et al., 2019;

Waggoner et al., 1998). Serotonin promotes egg laying in a manner that requires both

nAChRs (Chapter 2) and VGCCs (Figure 3.1B; William R. Schafer et al., 1996). To test

how serotonin modulates vulval muscle activity in freely behaving wild type and egl-

19(n582) loss-of-function mutant animals, we treated animals with 18.5 mM serotonin for

1 hour and then recorded their vulval muscle Ca2+ activity via GCaMP5 (Figure 3.4A).

Following serotonin treatment, wild-type and egl-19(n582) animals both exhibited robust vulval muscle Ca2+ activity in both amplitude and frequency (Figure 3.4B), however, this

activity was not significantly different from untreated control animals (Figure 3.4C-D).

Following Ca2+ recording, we counted the number of eggs laid on the serotonin-infused

agar plate that the animals were treated on. We found that wild-type animals treated with

serotonin laid significantly more eggs than untreated controls, while egl-19(n582) mutants laid few or no eggs in either condition (Figure 3.4E). These results demonstrate that the serotonin-insensitivity of egl-19(n582) animals is not restricted to liquid conditions, but the results are inconclusive as to how serotonin insensitivity affects vulval muscle Ca2+

activity. Since wild-type animals did lay eggs in response to serotonin but showed little

changes in vulval muscle Ca2+ activity, it is possible that the vulval muscles are desensitized to serotonin within 1 hour.

Figure 3.3. Serotonin promotes egg laying through the L-type VGCC

(A) GCaMP5 was expressed in the vulval muscles to record Ca2+ activity in either wild type or a L-type VGCC mutant background while exposed to 0 or 18.5 mM serotonin. (B) Representative traces of vulval muscle GCaMP5/mCherry Ca2+ ratio (∆R/R) in wild-type or VGCC mutant animals following 18.5 mM serotonin treatment. (C) Scatterplots showing the average peak amplitude and (D) transient rate of vulval muscle Ca2+ transients (mean ±95% confidence intervals) in wild-type or VGCC mutant animals during either the

egg-laying active or inactive state. (E) Eggs laid on 18.5 mM serotonin plates after 1 hour in wild type or L-type VGCC mutant background. Asterisk indicates p<0.0001; n.s. indicates p>0.05 (one-way ANOVA with Bonferroni’s correction for multiple comparisons).

VGCCs act in the same pathway as nicotine acetylcholine receptors

Our previous results showed that serotonin acts through nAChRs (Chapter 2) and

VGCCs (Figures 3.1 and 3.3) to promote egg laying. These findings raise the possibility

that nAChRs and VGCCs function in the same serotonergic signaling pathway. To test this,

we treated VGCC mutants with the nAChR agonists nicotine and levamisole. Turning first

to levamisole, we found that egl-19(n582) loss-of-function mutants failed to lay eggs in

250 µM levamisole, consistent with L-type VGCCs and nAChRs acting in the same pathway. Interestingly, egl-19(ad695) gain-of-function mutants were insensitive to levamisole while the stronger egl-19(n2368) gain-of-function mutant remained sensitive

(Figure 3.4A). This is in contrast to my previous results showing that the egl-19(n2368) mutant was resistant to serotonin (Figure 3.1B). The unc-2(e55) loss-of-function mutant still responded to levamisole, while both the unc-2(gk366) putative null mutant and unc-

2(z35) gain-of-function mutants did not lay eggs in response to levamisole (Figure 3.4A).

Turning to nicotine responses, when treated with 0.25% nicotine, egl-19(n582) loss-of- function and egl-19(ad695) mutants were insensitive, while egl-19(n2368) was sensitive

(Figure 3.4B). Both unc-2(e55) and unc-2(gk366) loss-of-function mutants did not lay eggs in response to exogenous nicotine, while the unc-2(z35) gain-of-function was responsive

(Figure 3.4B). Taken together, the role of UNC-2 in egg laying appears to be complex and may be influenced by allele-specific effects. Gain of EGL-19 function appears to alter egg laying based on the strength of the gain-of-function allele, with weak gain-of-functions

showing insensitivity to nAChR agonists and strong gain-of-functions showing sensitivity

(Figure 3.4C-D). In contrast, loss of EGL-19 function reliably yields insensitivity to both

nAChR agonists (Figure 3.4C-D) and serotonin (Figure 3.1B).

Figure 3.4. VGCCs act in the same pathway as nicotine acetylcholine receptors

VGCC mutations affect the ability of animals to lay eggs in response to the nAChR- agonists (A) nicotine and (B) levamisole. Animals were placed in wells containing either M9 control buffer or M9 plus the indicated drug (250 µM levamisole or 0.25% nicotine). **** indicate p<0.0001, *** indicates p=0.0005, and n.s. indicates p>0.05; Kruskal-Wallis test with Dunn’s correction for multiple comparisons.

Figure 3.5. The role of VGCCs and nAChRs in driving egg-laying behavior Serotonergic potentiation enhances the excitability of the vulval muscles, increasing the likelihood that VGCCs open and propagate depolarization in response to acute, localized activation of nAChRs by the various cholinergic motor neurons of the egg-laying circuit.

Discussion

The C. elegans egg-laying circuit provides a tractable model for studying how

cellular and neural circuit signaling events contributes to behavior. In the present study, we

examined how VGCCs contribute to egg-laying behavior at both a behavioral and

physiological level in response to cholinergic and serotonergic signaling (Figure 3.5). We

show that animals with loss-of-function or gain-of-function mutations in L-type or P/Q/N- type VGCCs significantly alters the ability of animals to lay eggs. Loss of P/Q/N-type

VGCC UNC-2 channel function egg retention in the uterus, indicating that these channels

normally function in cells to inhibit egg laying. Conversely, loss of L-type VGCC EGL-19

function results in significantly more eggs being retained, indicating that the L-type

VGCCs are critical for egg laying to occur. In agreement with this, L-type VGCC gain-of- function mutants display the opposite phenotype in the form of fewer eggs being retained, resembling UNC-2 mutants. The egg-laying phenotypes of L-type VGCC egl-19 mutants appear to be the result of changes in vulval muscle Ca2+ dynamics, where egl-19 loss-of-

function leads to reduced frequency of Ca2+ transients and egl-19 gain-of-function leads to increased frequency of Ca2+ transients. Egg laying of VGCC mutants in response to exogenous serotonin reveals a link between VGCCs and serotonergic signaling. P/Q/N- type VGCC unc-2 loss-of-function mutant egg laying is inhibited by exogenous serotonin, indicating that UNC-2 may function in multiple pathways to both inhibit and promote egg laying, depending on the context of the circuit signaling events. In agreement with this model, P/Q/N-type VGCC unc-2 gain-of-function mutants still show egg laying in response to exogenous serotonin indicating that they are not simply inhibitory. To test if the function of downstream serotonergic signaling is to modulate EGL-19 L-type VGCCs

in the vulval muscles, we performed Ca2+ imaging on the vulval muscles following

treatment with exogenous serotonin. As predicted, egl-19 loss-of-function mutants did not show a change in vulval muscle Ca2+ activity. However, these results are difficult to fully

interpret due to the lack of change in vulval muscle Ca2+ of wild-type animals as well.

Surprisingly, we find that the ability of animals to lay eggs in response to exogenous

nAChR agonists is dependent on VGCCs. P/Q/N-type VGCC UNC-2 loss-of-function results in an overall insensitivity to nAChR agonists, while UNC-2 gain-of-function mutant animals still show sensitivity to nicotine but not levamisole. These results again suggest that UNC-2 could function in multiple signaling pathways, and the different outcomes of these pathways could depend on which types of nAChRs are involved and/or where these receptors are expressed relative to UNC-2 channel expression. L-type VGCC egl-19 loss- of-function mutants are insensitive to nAChR agonists, consistent with a model where

EGL-19 functions as the ultimate downstream effector of cholinergic signaling for egg laying. L-type VGCC egl-19 gain-of-function mutants show a mixed response to nAChR agonists depending on which allele, suggesting a more nuanced relationship between nAChR signaling and egg laying when L-type VGCC activity is increased. In all, we propose that the primary function of both serotonergic and cholinergic signaling is to activate L-type VGCCs on the vulval muscles to drive full muscle contraction and egg laying.

VGCCs are a critical component of cellular activity and signaling throughout animal nervous systems (Catterall, 2011; Snutch et al., 2013). In humans, VGCCs are ubiquitously expressed and dysfunction of these channels in either muscle or neural tissue can contribute to diseases such as epilepsy, ataxia, and long QT syndrome (Bidaud et al.,

2006; Striessnig, 2016). We find that mutants for the UNC-2 P/Q/N-type VGCC display aberrant egg-laying behavior under both normal conditions and in response to treatment with exogenous serotonin and nAChR agonists. UNC-2 is an ortholog to the human

CACNA1A which is the α1 subunit of the P/Q-type VGCC (Mathews et al., 2003; W. R.

Schafer & Kenyon, 1995). Similar to CACNA1A, UNC-2 is expressed in neurons and

where it has been shown to regulate neurotransmitter release at the presynaptic terminal

(Mathews et al., 2003; Saheki & Bargmann, 2009). UNC-2 is not reported to be expressed in the vulval muscles, but is expressed in the presynaptic HSN and VC neurons (Mathews et al., 2003). Based on this expression data, the egg-laying phenotypes of unc-2 mutants

are due to changes in presynaptic neuron activity, not the postsynaptic vulval muscles.

UNC-2 has previously been implicated to function in both the HSNs and VCs to inhibit

sensitivity to serotoninergic signaling (W. R. Schafer & Kenyon, 1995; William R. Schafer

et al., 1996). Surprisingly, we find that egg laying in unc-2 loss-of-function mutants is instead inhibited by exogenous serotonin at higher concentrations than previously examined (W. R. Schafer & Kenyon, 1995; William R. Schafer et al., 1996), indicating a more complex role for UNC-2 and its function within the HSNs and VCs. Recent studies have shown that UNC-2 can have distinct functions across difference neuron types. UNC-

2 gain-of-function leads to increased neurotransmission from cholinergic motor neurons, but decreased neurotransmission from GABAergic motor neurons (Huang et al., 2019).

The mechanism(s) underlying these seemingly contradictory results is not clear. UNC-2 has been shown to regulate rhythmic Ca2+ oscillation activity in the soma of A-type motor

neurons (Gao et al., 2018). This multifaceted functionality of the P/Q/N-type UNC-2

VGCC supports the hypothesis that UNC-2 has different functions in the HSN neurons

compared to the VC neurons which results in the context-dependent unc-2 mutant egg- laying phenotypes.

As in humans, the C. elegans L-type VGCC plays a critical role in excitation- contraction coupling of muscle cells (Calderón et al., 2014; Catterall, 2011; Sanders, 2008).

EGL-19 is the sole L-type VGCC α1 subunit and is an ortholog to human CACNA1C and

CACNA1S (Lagoy et al., 2018; Lainé et al., 2014). EGL-19 is an essential protein and has been studied for its function at the neuromuscular junction between motor neurons and body wall muscle (Jospin et al., 2002). The EGL-19 channel is primarily responsible for the body wall muscle’s inward Ca2+ current and subsequent contraction to drive locomotion

(Jospin et al., 2002; Richmond & Jorgensen, 1999). EGL-19 has been shown to be critical

for vulval muscle Ca2+ activity as well (Shyn et al., 2003). Loss-of-function mutants for egl-19 resulted in less frequent Ca2+ transients in animals (Shyn et al., 2003), which were

corroborated by the results in this present study (Figure 3.2). Importantly, we also find that

the gain-of-function egl-19 mutants exhibit the opposite phenotype in the form of increased

frequency of Ca2+ transients. In addition to Ca2+ activity, EGL-19 is required for egg laying vulval muscle Ca2+ activity in response to serotonin (Figures 3.1 and 3.3; William R.

Schafer et al., 1996). Serotonin is a potent stimulator of egg-laying behavior (Brewer et al.,

2019; Waggoner et al., 1998), and its signaling pathway has been extensively studied in both the egg-laying circuit and elsewhere throughout C. elegans (Dhakal et al., 2021, 2021;

Flavell et al., 2013; Kopchock et al., 2021; Shyn et al., 2003; Song et al., 2013; Zahratka et al., 2015). Serotonin could act as a major regulator of L-type EGL-19 VGCC activity in the vulval muscles, either through electrical potentiation or molecular signaling cascades

(Burrell & Sahley, 2005; Chapple et al., 2004; Masson et al., 2012; Mlinar et al., 2015;

Sahu et al., 2018; Zahratka et al., 2015). Several serotonin receptors are expressed in the

egg-laying circuit including SER-1, SER-4, and SER-7 (Fernandez et al., 2020). The vm1- type vulval muscles are the initiation site of vulval muscle contractions and express SER-

7, a human HTR7 ortholog (Brewer et al., 2019; Fernandez et al., 2020; Hobson et al.,

2006). However, no serotonergic neurons make synapses onto the vm1s (Cook et al., 2019;

White et al., 1986), indicating that serotonin may act extrasynaptically to potentiate the vm1-type vulval muscles (De-Miguel & Trueta, 2005; Jafari et al., 2011).

L-type VGCCs are high-voltage activated calcium channels that open in response to membrane depolarization (Catterall, 2011). A sufficient membrane depolarization can be initiated through multiple mechanisms, such as opening of low-voltage activated calcium channels, voltage-gated sodium channels, or ligand-gated ion channels (Catterall,

2011; Striessnig, 2016). The C. elegans vulval muscles express nAChRs (Culetto et al.,

2004; Sloan et al., 2015; Waggoner et al., 2000) which could act as a source of local depolarization to activate L-type VGCCs. Consistent with this model, we find that the ability of nAChR agonists to stimulate egg laying is dependent on EGL-19 (Figure 3.4).

The ability of nAChRs to lead to activation of L-type VGCCs has been shown in other systems, such as in the pinealocytes of rats (Letz et al., 1997) and in the Kenyon cells of

Drosophila (Campusano et al., 2007). The vulval muscles receive innervation from 3 cholinergic neuron types, the VA, VB, and VC motor neurons (Cook et al., 2019; White et al., 1986). However, optogenetic activation of neither the VA and VBs (Chapter 2) nor the

VC neurons (Kopchock et al., 2021) is sufficient to drive vulval muscle contractions for egg release. Additionally, optogenetic activation of all cholinergic neurons (including VA,

VB, and VC) is not sufficient to induce immediate vulval muscle contraction either (data

65 not shown). These findings reinforce a model where additional modulating factors, such as serotonin or NLP-3 (Brewer et al., 2019; Waggoner et al., 1998), must first potentiate vulval muscles to facilitate opening of L-type EGL-19 VGCCs in response to cholinergic input.

Taken together, these results support a role for the L-type EGL-19 VGCC in being the primary facilitator of vulval muscle Ca2+ activity and contraction to drive egg laying.

Vulval muscle contraction and egg laying in response to serotonin and nAChR agonists is dependent on EGL-19, which suggests that it is the final downstream effector of circuit activity. The egg-laying circuit thus presents a tractable model for in vivo studies of the integration of multiple presynaptic signaling events and transmitters in order to execute a behavior.

Chapter 4: Materials and methods

Nematode culture and strains

All C. elegans strains were maintained at 20 °C on Nematode Growth Medium (NGM) agar plates seeded with OP50 E. coli as described (Brenner, 1974). All assays were conducted on age-matched adult hermaphrodites at 24-30 h past the late L4 stage, unless otherwise stated. A list of all strains generated and used in this study can be found in Table

4.1. Strains from CGC were used in behavior experiments and crosses without performing any additional backcrossing with wild-type N2 animals.

Plasmid and strain construction

Oligonucleotides were synthesized by IDT-DNA. PCR was performed using high-fidelity

Phusion DNA polymerase (New England Biolabs) except for routine genotyping which was performed using standard Taq DNA polymerase. Plasmids were prepared using a

Qiagen miniprep spin kit. DNA concentrations were determined using a Nano-Drop spectrophotometer.

Tetanus toxin (TeTx)-expressing transgenes:

VC neuron TeTx – A ~1.4 kB DNA fragment encoding TeTx was cut from pAJ49 (Jose et

al., 2007) with AgeI/XhoI, and ligated into a similarly digested pKMC145 [lin-

11::GFP::unc-54 3’ UTR] to generate pKMC282 [lin-11::TeTx::unc-54 3’ UTR].

pKMC282 (50 ng/µl) was injected along with pL15EK (50 ng/µl; (Clark et al., 1994)) into

LX1832 lite-1(ce314) lin-15(n765ts) X generating four independent transgenic lines of

which one, MIA113 keyEx32 [lin-11::TeTx::unc-54 3’UTR + lin-15(+)]; lite-1(ce314) lin-

15(n765ts) X, was used for integration. keyEx32 was integrated with UV/TMP generating three independent integrants keyIs32-33 and keyIs46 [lin-11::TeTx::unc-54 3’UTR + lin-

15(+)]. Each transgenic line was backcrossed six times to LX1832 generating strains

MIA144-146. All transgenic strains appeared phenotypically similar, and MIA144 and

MIA146 were used for experiments and further crosses. To eliminate HSNs in animals lacking VC synaptic transmission, MIA26 egl-1(n986dm) V mutant animals were crossed with MIA146 to generate MIA173 keyIs46; egl-1(n986dm) V; lite-1(ce314) lin-15(n765ts)

Histamine-gated chloride channel (HisCl)-expressing transgenes:

VC neuron HisCl – The ~1.3 kB DNA fragment encoding Drosophila histamine-gated

chloride channel HisCl1 was PCR amplified from pNP403 (Pokala et al., 2014) using the

oligonucleotides 5’-GCG CCC GGG GTA GAA AAA ATG CAA AGC CCA ACT AGC

AAA TTG G-3’ and 5’-GCG GAG CTC TTA TCA TAG GAA CGT TGT CCA ATA

GAC AAT A-3’, cut with XmaI/SacI, and ligated into AgeI/SacI-digested pKMC145 to

generate pAB2 [lin-11::HisCl::unc-54 3’UTR]. pAB2 (20 ng/µl) was injected into LX1832

along with pL15EK (50 ng/µl), generating the extrachromosomal line MIA93 keyEx24

[lin-11::HisCl::unc-54 3’UTR + lin-15(+)]; lite-1(ce314) lin-15(n765ts) X. The

extrachromosomal transgene subsequently integrated using UV/TMP to generate the

transgenes keyIs23-30 [lin-11::HisCl::unc-54 3’UTR + lin-15(+)]. Strains bearing these

transgenes were then backcrossed to the LX1832 parent strain six times, generating strains

MIA124, MIA125, MIA130, MIA131, and MIA132. All transgenic strains appeared

phenotypically similar, and MIA125 was used for experiments and further crosses. To

eliminate the HSN neurons in animals expressing HisCl in the VC neurons, MIA125

keyIs23; lite-1(ce314) lin-15(n765ts) X was crossed with MIA26 to generate MIA176

keyIs23; egl-1(n986dm); lite-1(ce314) lin-15(n765ts) X.

Channelrhodopsin-2 (ChR2)-expressing transgenes:

Vulval muscle ChR2 – To express ChR2 in the vulval muscles, the ~1 kB DNA fragment

encoding for ChR2 was PCR amplified from pRK7 [del-1::ChR2(H34R/T159C::unc-54 3’

UTR] using oligonucleotides 5’-GCG GCT AGC ATG GAT TAT GGA GGC GCC CTG-

3’ and 5’-GCG GGT ACC TCA GGT GGC CGC GGG GAC CGC GCC AGC CTC GGC

C-3’. The amplicon and recipient plasmid, pBR3 (Ravi et al., 2018a), were digested with

NheI/KpnI, generating pRK11 [ceh-24::ChR2(H34R/T159C)::unc-54 3’ UTR]. pRK11 (50

ng/µl) was injected into LX1832 along with pL15EK (50 ng/µl) generating MIA212

keyEx43 [ceh-24::ChR2(H34R/T159C)::unc-54 3’UTR + lin-15(+)] which was

subsequently integrated with UV/TMP, generating five independent integrated transgenes keyIs47-51. Strains carrying these integrated transgenes were then backcrossed to the

LX1832 parent strain six times, generating the strains MIA229-232 and MIA242. All

transgenic strains were phenotypically similar, and MIA229 was used for experiments and

further crosses.

VC ChR2 – The allele keyIs3 was used to express ChR2 in the VC neurons under a

modified lin-11 promoter, as previously described (Collins et al., 2016).

HSN ChR2 – The allele wzIs30 was used to express ChR2 in the HSN neurons under the

egl-6 promoter, as previously described (Collins et al., 2016; Emtage et al., 2012).

VA and VB neuron ChR2 – To express ChR2 in the VA and VB motor neurons, a ~2 kB

del-1 promoter fragment was PCR amplified from pJR6 (Winnier et al., 1999) using

oligonucleotides 5’-GCG GCA TGC TCA AGT CCC ACC TCA ACC-3’ and 5’-GCG

CCC GGG TAT TTT TTC ACC CCA TCT TTT-3’. The del-1 amplicon and recipient plasmid, pKMC285 [pUNC103E::ChR2-YFP(H34R/T159C::unc-54 3’ UTR], were digested with SphI/XmaI and ligated to generate pRK7 [del-1:: ChR2-YFP

(H34R/T159C::unc-54 3’ UTR]. pRK7 (50 ng/µl) and pL15EK (50 ng/µl (Clark et al.,

1994) were injected into LX1832 lite-1(ce314) lin-15(n765ts) X hermaphrodites generating

MIA120 keyEx36 [del-1:: ChR2-YFP (H34R/T159C::unc-54 3’ UTR + lin-15(+)] which was subsequently integrated with UV/TMP, generating five independent integrated transgenes, keyIs38-45. Three of the strains carrying these integrated transgenes were then backcrossed to the LX1832 parent strain six times, generating the strains MIA179-181. All

transgenic strains were phenotypically similar, and MIA179 was used for experiments and

further crosses.

Calcium reporter transgenes:

Vulval muscle GCaMP5 – Vulval muscle Ca2+ activity was visualized using the strain

LX1918 which co-expresses GCaMP5 and mCherry in the vulval muscles from the unc-

103e promoter (Collins et al., 2016). To analyze vulval muscle Ca2+ activity in animals where VC synaptic transmission was blocked with TeTx, LX1918 was crossed with

MIA144 to generate MIA183 keyIs33; vsIs164 lite-1(ce314) lin-15(n765ts) X. To analyze

vulval muscle Ca2+ activity in animals where the VC neurons could be optogenetically

activated by ChR2, LX1918 was crossed with MIA3 (Collins et al., 2016), to generate

MIA221 keyIs3; vsIs164 lite-1(ce314) lin-15(n765ts) X. To analyze vulval muscle Ca2+

activity in animals where the vulval muscles could be optogenetically activated by ChR2,

LX1918 was crossed with MIA229 to generate MIA250 keyIs49; vsIs164 lite-1(ce314) lin-

15(n765ts) X. To analyze vulval muscle Ca2+ activity in animals with mutant L-type VGCC backgrounds, LX1918 was crossed with each MT6129 egl-19(n2368) IV and MT1212 egl-

19(n582) IV to generate strains MIA184 egl-19(n2368) IV; vsIs164; lite-1(ce314) lin-

15(n765ts) X and MIA185 egl-19(n582) IV; vsIs164; lite-1(ce314) lin-15(n765ts) X, respectively. To analyze vulval muscle Ca2+ activity in animals with mutant nAChR subunit backgrounds, LX1918 was crossed with each CB403 unc-29(e403) I and ZZ37 unc-63(x37) I to generate strains MIA177 unc-29(e403) I; vsIs164 lite-1(ce314) lin-

15(n765ts) X and MIA178 unc-63(x37) I; vsIs164 lite-1(ce314) lin-15(n765ts) X, respectively. To analyze vulval muscle Ca2+ activity in animals where the VA and VB

neurons could be optogenetically activated by ChR2, LX1918 was crossed with MIA179

to generate MIA186 keyIs40; vsIs164 lite-1(ce314) lin-15(n765ts) X. MIA179 was then

crossed with MIA177 and MIA178 to generate MIA422 unc-29(e403) I; keyIs40; vsIs164

lite-1(ce314) lin-15(n765ts) X and MIA423 unc-63(x37) I; keyIs40; vsIs164 lite-1(ce314) lin-15(n765ts) X, respectively, to test the role of nAChRs in vulval muscles being excited by the VA and VB neurons.

VC neuron GCaMP5 – VC neuron Ca2+ activity was visualized using the strain LX1960

which co-expresses GCaMP5 and mCherry in the VC neurons (Collins et al., 2016). To

visualize VC activity during optogenetic stimulation of the HSNs, the strain LX1970 was

used (Collins et al., 2016). To visualize VC activity after optogenetic stimulation of the

vulval muscles, LX1960 was crossed with MIA229 to generate MIA241 vsIs172; keyIs48

lite-1(ce314) lin-15(n765ts) X. To visualize VC activity after optogenetic stimulation of

the vulval muscles when muscle contraction is impaired, CB190 unc-54(e190) I myosin

heavy chain null mutants were crossed with LX1832 to generate MIA274 unc-54(e190) I;

lite-1(ce314) lin-15(n765ts) X. MIA274 was then crossed with MIA241 to generate

MIA298 keyIs48; vsIs172; unc-54(e190) I; lite-1(ce314) lin-15(n765ts) X.

HSN neuron GCaMP5 – HSN neuron Ca2+ activity was visualized using the strain LX2004

which co-expresses GCaMP5 and mCherry in the HSN neurons (Collins et al., 2016). To visualize HSN Ca2+ after neurotransmission from the VC neurons is blocked by TeTx,

LX2004 was crossed with MIA144 to generate MIA217 keyIs33; vsIs183 lite-1(ce314) lin-

15(n765ts) X.

Ratiometric Ca2+ imaging

Ratiometric Ca2+ imaging of the vulval muscles and VC neurons in freely behaving animals

was performed as previously described methods (Ravi et al., 2018b). Late L4

hermaphrodites were staged and then imaged 24 h later by being moved to an NGM agar

chunk between two glass coverslips. Animals were recorded on a Zeiss Axio Observer.Z1

inverted compound microscope with a 20X 0.8NA Apochromat objective. Brightfield

recordings of behavior was recorded with infrared illumination using a FLIR Grasshopper

3 CMOS camera after 2x2 binning using FlyCap software. Colibri.2 470 nm and 590 nm

LEDs were used to co-excite GCaMP5 and mCherry fluorescence which was captured at

20 Hz onto a Hamamatsu ORCA Flash-4.0V2 sCMOS camera after channel separation using a Gemini image splitter. For Ca2+ imaging of the vulval muscles, a lateral focal plane

was used to capture the anterior and posterior vm1 and vm2 cells on one side of the animal.

For Ca2+ imaging of the VC neurons, a lateral focal plane was used to capture the VC4 and

VC5 cell bodies and their presynaptic termini around the vulva. Each animal was recorded

until it entered an active egg-laying phase (up to 1 h), after which a 10-minute segment

centered around the onset of egg laying was extracted from the full recoding for analysis.

In experiments where animals were treated with exogenous serotonin, animals were only

recorded for the first 10 minutes. Two-channel fluorescence (GCaMP5/mCherry) image

sequences were processed and analyzed in Fiji (Schindelin et al., 2012), Volocity

(PerkinElmer), and a custom script for MATLAB (MathWorks) as previously described

(Ravi et al., 2018b).

Ratiometric Ca2+ imaging of the HSN neurons in freely behaving animals was performed

as previously described (Ravi et al., 2018a). Late L4 hermaphrodites were staged and then

imaged 24 h later by being moved to an NGM agar chunk and a glass coverslip being

placed over. Animals were recorded on an inverted Leica TCS SP5 confocal microscope

with a 20X 0.7NA Apochromat objective. 488 nm and 561 nm laser lines were used to co-

excite GCaMP5 and mCherry fluorescence, respectively.

Electrical silencing using HisCl

Acute electrical silencing with histamine was performed as previously described (Pokala

et al., 2014; Ravi et al., 2018a). Animals were moved onto OP50-seeded NGM agar plates

that contained either 0 mM or 10 mM histamine for four hours before the experiment.

Egg-laying behavior assays

The steady-state accumulation of eggs in the uterus was determined as previously described

(Koelle & Horvitz, 1996). Briefly, late L4 hermaphrodites were staged onto OP50-seeded

plates and grown at 20 °C for 30 h after which a single adult was placed into a 7 ul drop of

20% sodium hypochlorite (bleach) solution. The eggs, which are resistant to bleach, were

then counted using a dissecting microscope. The timing of the first egg laid was assayed

by staging a young adult (within 30 minutes of L4 to adult molt) animal onto an NGM agar

plate with food and checking every following 30 minutes for the presence of an egg on the

plate (Ravi et al., 2018a).

Egg laying in liquid in response to exogenous serotonin, levamisole, and nicotine was performed as described (Banerjee et al., 2017; Trent et al., 1983). Late L4 hermaphrodites were staged onto OP50-seed plates and grown at 20 °C for 24 h. Adult animals were placed singly into either 100 ul M9 buffer only or M9 buffer containing either 18.5 mM serotonin

(creatinine sulfate salt; Sigma Aldrich), 250 µM levamisole hydrochloride (Sigma

Aldrich), or 0.25% (-) nicotine (Sigma Aldrich) in a 96-well microtiter dish. The number of eggs laid by each animal after 1 hour were then counted. Egg laying of VC::TeTx animals on plates in response to exogenous serotonin was performed on NGM agar infused

with 18.5 mM serotonin creatine sulfate salt (Sigma-Aldrich). 3 animals were placed on each NGM agar plate, and the number of eggs laid was counted after 1 hour and divided by 3 to calculate an average-animal response per plate. For egg laying of egl-19 mutants

on plates in response to exogenous serotonin, 1 animal was placed on each plate and the

eggs laid counted after 1 hour.

Optogenetics

Optogenetic experiments with Channelrhodopsin-2 (ChR2) were performed using a Zeiss

Axio Observer.Z1 inverted compound microscope as previously described (Collins et al.,

2016). ChR2 was excited in freely behaving animals using a 470 nm (blue) LED. Late L4

animals were staged onto NGM agar plates seeded with E. coli OP50 bacterial cultures

containing either 0.4 mM all-trans retinal (ATR) or no ATR 24 h prior to the start of the

experiment. Animals were then continuously illuminated with blue light for 3-5 minutes

and the locomotion behavior and number of egg-laying events were recorded. For

experiments combining optogenetics with ratiometric Ca2+ imaging, the blue light would

excite both the ChR2 and GCaMP5 fluorescence simultaneously. Blue light intensity was

chosen based on both optimal settings to observe robust ChR2-activation and GCaMP5

fluorescence. Animals were excluded from a dataset if they entered an active egg-laying state before the onset of blue light stimulation (one animal in total across all experiments).

Experimental design and statistical analyses

Sample sizes for behavioral assays and Ca2+ imaging experiments followed previous

studies (Banerjee et al., 2017; Collins et al., 2016; Ravi et al., 2018a). A minimum of 8 animals per genotype per genotype per condition were measured across all experiments, with the exception of Ca2+ imaging of egl-19(n2368) IV GoF animals. One animal was excluded from the analysis of VC Channelrhodopsin-2; vulval muscle GCaMP5 experiment because it entered an egg-laying active state before the onset of blue light stimulation. No other data or animals were excluded. No explicit power analysis was performed prior to the study. All data were analyzed using GraphPad Prism 8. Steady-state

egg accumulation and timing of first egg laid assays were compared using a one-way

ANOVA with Bonferroni’s correction for multiple comparisons. Serotonin, nicotine, and

levamisole-induced egg laying assays were compared using either a Kruskal-Wallis test

with Dunn’s correction for multiple comparisons (in buffer with individual responses) or a

one-way ANOVA with Bonferroni’s correction for multiple comparisons (on plates with

averaged responses). Inter-transient intervals of Ca2+ transients compared between active

and inactive egg-laying behavior states were pooled together across all animals and

analyzed using either a Kolmogorov-Smirnov test or Kruskal-Wallis test with Dunn’s

correction for multiple comparisons. Per animal rates of failed egg-laying events were

analyzed using a Mann-Whitney test. Ca2+ transient peak amplitude, inter-transient

interval, or transient widths were first averaged across each animal and then these averages

were compared across animals using either a Student’s t test or a one-way ANOVA with

Bonferroni’s correction for multiple comparisons. The number of animals and instances of

76 analyzed behavior events along with exact p values resulting from defined statistical tests are reported within each figure legend.

Table 4.1. Strain names and genotypes for all animals used in this study.

Strain Genotype Feature Reference N2 wild type Bristol wild-type (Brenner, 1974) strain CB190 unc-54(e190) I Myosin heavy (Brenner, 1974) chain null mutant LX1832 lite-1(ce314) X lin- Blue light-resistant (Collins & 15(n765ts) X (optogenetics and Koelle, 2013) Ca2+ imaging), multivulva (injection rescue marker) LX1918 vsIs164 lite-1(ce314) Vulval muscles (Collins et al., lin-15(n765ts) X expressing 2016) GCaMP5, mCherry LX1960 vsIs172; lite-1(ce314) VC GCaMP5, (Collins et al., lin-15(n765ts) X mCherry 2016) LX1970 wzIs30 IV; vsIs172; lite- HSN (Collins et al., 1(ce314) lin-15(n765ts) Channelrhodopsin- 2016) X 2; VC GCaMP5, mCherry LX1978 nlp-3(tm3023) X nlp-3 null (Brewer et al., 2019) LX2004 vsIs183 lite-1(ce314) HSN expressing (Collins et al., lin-15(n765ts) X GCaMP5, 2016) mCherry MIA26 egl-1(n986dm) V No HSNs (Ravi et al., 2018a) MIA116 keyIs21; lite-1(ce314) HSN HisCl (Ravi et al., lin-15(n765ts) X 2018a) MIA123 egl-1(n986dm) V lite- No HSNs (Kopchock et 1(ce314) lin-15(n765ts) al., 2021) X MIA125 keyIs23; lite-1(ce314) VC HisCl (Kopchock et lin-15(n765ts) X al., 2021) MIA144 keyIs33; lite-1(ce314) VC TeTx (Kopchock et lin-15(n765ts) X al., 2021) MIA146 keyIs46; lite-1(ce314) VC TeTx (Kopchock et lin-15(n765ts) X al., 2021)

MIA173 keyIs46; egl-1(n986dm) VC TeTx; no (Kopchock et V lite-1(ce314) lin- HSNs al., 2021) 15(n765ts) X MIA176 keyIs23; egl-1(n986dm) VC HisCl; no (Kopchock et V; lite-1(ce314) lin- HSNs al., 2021) 15(n765ts) X MIA183 keyIs33; vsIs164 lite- VC TeTx; vulval (Kopchock et 1(ce314) lin-15(n765ts) muscle GCaMP5, al., 2021) X mCherry MIA217 keyI33; vsIs183 lite- VC TeTx; HSN (Kopchock et 1(ce314) lin-15(n765ts) GCaMP5, al., 2021) X mCherry MIA221 keyIs3; vsIs164; lite- VC (Kopchock et 1(ce314) lin-15(n765ts) Channelrhodopsin- al., 2021) X 2; vulval muscle GCaMP5, mCherry MIA229 keyIs48; lite-1(ce314) Vulval muscle (Kopchock et lin-15(n765ts) X Channelrhodopsin- al., 2021) 2 MIA241 keyIs48; vsIs172; lite- Vulval muscle (Kopchock et 1(ce314) lin-15(n765ts) Channelrhdopsin- al., 2021) X 2; VC GCaMP5, mCherry MIA250 keyIs48; vsIs164 lite- Vulval muscle (Kopchock et 1(ce314) lin-15(n765ts) Channelrhdopsin- al., 2021) X 2, GCaMP5, mCherry MIA298 keyIs48; vsIs172; unc- Vulval muscle (Kopchock et 54(e190) I; lite-1(ce314) Channelrhdopsin- al., 2021) lin-15(n765ts) X 2; VC GCaMP5, mCherry; unc-54 myosin heavy chain null mutant background MIA428 nlp-3(tm3023); keyIs33 VC TeTx; nlp-3 (Kopchock et null al., 2021) DA695 egl-19(ad695) IV L-type VGCC (R. Y. Lee et gain-of-function al., 1997) MT1212 egl-19(n582) IV L-type VGCC (R. Y. Lee et loss-of-function al., 1997) MT6129 egl-19(n2368) IV L-type VGCC (R. Y. Lee et gain-of-function al., 1997) QW47 unc-2 (zf35) X P/Q/N-type VGCC (Huang et al., gain-of-function 2019)

CB55 unc-2(e55) X P/Q/N-type VGCC (Brenner, 1974) loss-of-function CB403 unc-29(e403) I nAChR subunit (Lewis et al., loss-of-function 1980) RB1132 acr-14(ok1155) II nAChR subunit (CeDMC, 2012) knockout CB904 unc-38(e264) I nAChR subunit (Lewis et al., loss-of-function 1980) RB2294 acr-6(ok3117) I nAChR subunit (CeDMC, 2012) knockout RB1263 acr-11(ok1345) I nAChR subunit (CeDMC, 2012) knockout ZZ37 unc-63(x37) I nAChR subunit (Lewis et al., loss-of-function 1980) CB211 lev-1(e211) IV nAChR subunit (Lewis et al., loss-of-function 1980) FX863 acr-7(tm863) II nAChR subunit (CeDMC, 2012) knockout RB1195 acr-8(ok1240) X nAChR subunit (CeDMC, 2012) knockout RB1226 acr-18(ok1258) V nAChR subunit (CeDMC, 2012) knockout RB1559 acr-2(ok1887) X nAChR subunit (CeDMC, 2012) knockout RB1659 acr-3(ok2049) X nAChR subunit (CeDMC, 2012) knockout RB2119 acr-23(ok2804) V nAChR subunit (CeDMC, 2012) knockout RB2262 acr-10(ok3064) X nAChR subunit (CeDMC, 2012) knockout RB918 acr-16(ok789) V nAChR subunit (CeDMC, 2012) knockout VC1041 lev-8(ok1519) X nAChR subunit (CeDMC, 2012) knockout VC1944 unc-29(ok2450) I nAChR subunit (CeDMC, 2012) knockout MIA177 unc-29(e403) I; vsIs164 unc-29 LoF mutant This paper X; lite-1(ce314) X lin- with vulval 15(n765ts) X muscles expressing GCaMP5, mCherry MIA178 unc-63(x37) I; vsIs164 unc-63 LoF mutant This paper X; lite-1(ce314) X lin- with vulval 15(n765ts) X muscles expressing GCaMP5, mCherry

MIA184 egl-19(n2368) IV; egl-19 GoF mutant This paper vsIs164; lite-1(ce314) X with vulval lin-15(n765ts) X muscles expressing GCaMP5, mCherry MIA185 egl-19(n582) IV; egl-19 LoF mutant This paper vsIs164; lite-1(ce314) X with vulval lin-15(n765ts) X muscles expressing GCaMP5, mCherry MIA179 keyIs40; lite-1(ce314) X VA/VB neurons This paper lin-15(n765ts) X expressing ChR2 MIA186 keyIs40; vsIs164 lite- VA/VB neurons This paper 1(ce314) lin-15(n765ts) expressing ChR2; X vulval muscle GCaMP5, mCherry MIA422 unc-29(e403) I; keyIs40; VA/VB neurons This paper vsIs164 lite-1(ce314) expressing ChR2; lin-15(n765ts) X vulval muscle GCaMP5, mCherry MIA423 unc-63(x37) I; keyIs40; VA/VB neurons This paper vsIs164 lite-1(ce314) expressing ChR2; lin-15(n765ts) X vulval muscle GCaMP5, mCherry MIA120 keyEx36; lite-1(ce314) VA/VB neurons This paper X lin-15(n765ts) X expressing ChR2 (extrachromosomal

transgene)

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