Microbial light-activatable proton pumps as neuronal inhibitors to functionally dissect neuronal networks in C. elegans

Essentially any behavior in simple and complex animals depends on neuronal network function. Currently, the best-defined system to study neuronal circuits is the nematode Caenorhabditis elegans, as the connectivity of its 302 neurons is exactly known. Individual neurons can be activated by photostimulation of Channelrhodopsin-2 (ChR2) using blue light, allowing to directly probe the importance of a particular neuron for the respective behavioral output of the network under study. In analogy, other excitable cells can be inhibited by expressing Halorhodopsin from Natronomonas pharaonis (NpHR) and subsequent illumination with yellow light. However, inhibiting C. elegans neurons using NpHR is difficult. Recently, proton pumps from various sources were established as valuable alternative hyperpolarizers. Here we show that archaerhodopsin-3 (Arch) from Halorubrum sodomense and a proton pump from the fungus Leptosphaeria maculans (Mac) can be utilized to effectively inhibit excitable cells in C. elegans. Arch is the most powerful hyperpolarizer when illuminated with yellow or green light while the action spectrum of Mac is more blue-shifted, as analyzed by light-evoked behaviors and electrophysiology. This allows these tools to be combined in various ways with ChR2 to analyze different subsets of neurons within a circuit. We exemplify this by means of the polymodal aversive sensory ASH neurons, and the downstream command interneurons to which ASH neurons signal to trigger a reversal followed by a directional turn. Photostimulating ASH and subsequently inhibiting command interneurons using two-color illumination of different body segments, allows investigating temporal aspects of signaling downstream of ASH.     

A Blue-shifted Light-driven Proton Pump for Neural Silencing

Ion-transporting rhodopsins are widely utilized as optogenetic tools both for light-induced neural activation and silencing. The most studied representative is Bacteriorhodopsin (BR), which absorbs green/red light (∼570 nm) and functions as a proton pump. Upon photoexcitation, BR induces a hyperpolarization across the membrane, which, if incorporated into a nerve cell, results in its neural silencing. In this study, we show that several residues around the retinal chromophore, which are completely conserved among BR homologs from the archaea, are involved in the spectral tuning in a BR homolog (HwBR) and that the combination mutation causes a large spectral blue shift (λ_max = 498 nm) while preserving the robust pumping activity. Quantum mechanics/molecular mechanics calculations revealed that, compared with the wild type, the β-ionone ring of the chromophore in the mutant is rotated ∼130° because of the lack of steric hindrance between the methyl groups of the retinal and the mutated residues, resulting in the breakage of the π conjugation system on the polyene chain of the retinal. By the same mutations, similar spectral blue shifts are also observed in another BR homolog, archearhodopsin-3 (also called Arch). The color variant of archearhodopsin-3 could be successfully expressed in the neural cells of Caenorhabditis elegans, and illumination with blue light (500 nm) led to the effective locomotory paralysis of the worms. Thus, we successfully produced a blue-shifted proton pump for neural silencing.     

The neural circuits and synaptic mechanisms underlying motor initiation in C. elegans

C. elegans is widely used to dissect how neural circuits and genes generate behavior. During locomotion, worms initiate backward movement to change locomotion direction spontaneously or in response to sensory cues; however, the underlying neural circuits are not well defined. We applied a multidisciplinary approach to map neural circuits in freely behaving worms by integrating functional imaging, optogenetic interrogation, genetic manipulation, laser ablation, and electrophysiology. We found that a disinhibitory circuit and a stimulatory circuit together promote initiation of backward movement and that circuitry dynamics is differentially regulated by sensory cues. Both circuits require glutamatergic transmission but depend on distinct glutamate receptors. This dual mode of motor initiation control is found in mammals, suggesting that distantly related organisms with anatomically distinct nervous systems may adopt similar strategies for motor control. Additionally, our studies illustrate how a multidisciplinary approach facilitates dissection of circuit and synaptic mechanisms underlying behavior in a genetic model organism.     

The Caenorhabditis elegans AMP-activated protein kinase AAK-2 is phosphorylated by LKB1 and is required for resistance to oxidative stress and for normal motility and foraging behavior

AAK-2 is one of two alpha isoforms of the AMP-activated protein kinase in Caenorhabditis elegans and is involved in life span maintenance, stress responses, and germ cell cycle arrest upon dauer entry. We found that AAK-2 was phosphorylated at threonine 243 in response to paraquat treatment and that this phosphorylation depends on PAR-4, the C. elegans LKB1 homologue. Both aak-2 mutation and par-4 knockdown increased the sensitivity of C. elegans worms to paraquat, and the double deficiency did not further increase sensitivity, indicating that aak-2 and par-4 act in a linear pathway. Both mutations also slowed body bending during locomotion and failed to reduce head oscillation in response to anterior touch. Consistent with this abnormal motility and behavioral response, expression of the AAK-2::green fluorescent protein fusion protein was observed in the ventral cord, some neurons, body wall muscle, pharynx, vulva, somatic gonad, and excretory cell. Our study suggests that AMPK can influence the behavior of C. elegans worms in addition to its well known function in metabolic control.     

Heterologous expression of functional G-protein-coupled receptors in Caenorhabditis elegans

New strategies for expression, purification, functional characterization, and structural determination of membrane-spanning G-protein-coupled receptors (GPCRs) are constantly being developed because of their importance to human health. Here, we report a Caenorhabditis elegans heterologous expression system able to produce milligram amounts of functional native and engineered GPCRs. Both bovine opsin [(b)opsin] and human adenosine A(2A) subtype receptor [(h)A(2A)R] expressed in neurons or muscles of C. elegans were localized to cell membranes. Worms expressing these GPCRs manifested changes in motor behavior in response to light and ligands, respectively. With a newly devised protocol, 0.6-1 mg of purified homogenous 9-cis-retinal-bound bovine isorhodopsin [(b)isoRho] and ligand-bound (h)A(2A)R were obtained from C. elegans from one 10-L fermentation at low cost. Purified recombinant (b)isoRho exhibited its signature absorbance spectrum and activated its cognate G-protein transducin in vitro at a rate similar to native rhodopsin (Rho) obtained from bovine retina. Generally high expression levels of 11 native and mutant GPCRs demonstrated the potential of this C. elegans system to produce milligram quantities of high-quality GPCRs and possibly other membrane proteins suitable for detailed characterization.     

Optogenetic Control of Targeted Peripheral Axons in Freely Moving Animals

Optogenetic control of the peripheral nervous system (PNS) would enable novel studies of motor control, somatosensory transduction, and pain processing. Such control requires the development of methods to deliver opsins and light to targeted sub-populations of neurons within peripheral nerves. We report here methods to deliver opsins and light to targeted peripheral neurons and robust optogenetic modulation of motor neuron activity in freely moving, non-transgenic mammals. We show that intramuscular injection of adeno-associated virus serotype 6 enables expression of channelrhodopsin (ChR2) in motor neurons innervating the injected muscle. Illumination of nerves containing mixed populations of axons from these targeted neurons and from neurons innervating other muscles produces ChR2-mediated optogenetic activation restricted to the injected muscle. We demonstrate that an implanted optical nerve cuff is well-tolerated, delivers light to the sciatic nerve, and optically stimulates muscle in freely moving rats. These methods can be broadly applied to study PNS disorders and lay the groundwork for future therapeutic application of optogenetics.     

Paradigms for Pharmacological Characterization of C. elegans Synaptic Transmission Mutants

The nematode, Caenorhabditis elegans, has become an expedient model for studying neurotransmission. C. elegans is unique among animal models, as the anatomy and connectivity of its nervous system has been determined from electron micrographs and refined by pharmacological assays. In this video, we describe how two complementary neural stimulants, an acetylcholinesterase inhibitor, called aldicarb, and a gamma-aminobutyric acid (GABA) receptor antagonist, called pentylenetetrazole (PTZ), may be employed to specifically characterize signaling at C. elegans neuromuscular junctions (NMJs) and facilitate our understanding of antagonistic neural circuits.Of 302 C. elegans neurons, nineteen GABAergic D-type motor neurons innervate body wall muscles (BWMs), while four GABAergic neurons, called RMEs, innervate head muscles. Conversely, thirty-nine motor neurons express the excitatory neurotransmitter, acetylcholine (ACh), and antagonize GABA transmission at BWMs to coordinate locomotion. The antagonistic nature of GABAergic and cholinergic motor neurons at body wall NMJs was initially determined by laser ablation and later buttressed by aldicarb exposure. Acute aldicarb exposure results in a time-course or dose-responsive paralysis in wild-type worms. Yet, loss of excitatory ACh transmission confers resistance to aldicarb, as less ACh accumulates at worm NMJs, leading to less stimulation of BWMs. Resistance to aldicarb may be observed with ACh-specific or general synaptic function mutants. Consistent with antagonistic GABA and ACh transmission, loss of GABA transmission, or a failure to negatively regulate ACh release, confers hypersensitivity to aldicarb. Although aldicarb exposure has led to the isolation of numerous worm homologs of neurotransmission genes, aldicarb exposure alone cannot efficiently determine prevailing roles for genes and pathways in specific C. elegans motor neurons. For this purpose, we have introduced a complementary experimental approach, which uses PTZ.Neurotransmission mutants display clear phenotypes, distinct from aldicarb-induced paralysis, in response to PTZ. Wild-type worms, as well as mutants with specific inabilities to release or receive ACh, do not show apparent sensitivity to PTZ. However, GABA mutants, as well as general synaptic function mutants, display anterior convulsions in a time-course or dose-responsive manner. Mutants that cannot negatively regulate general neurotransmitter release and, thus, secrete excessive amounts of ACh onto BWMs, become paralyzed on PTZ. The PTZ-induced phenotypes of discrete mutant classes indicate that a complementary approach with aldicarb and PTZ exposure paradigms in C. elegans may accelerate our understanding of neurotransmission. Moreover, videos demonstrating how we perform pharmacological assays should establish consistent methods for C. elegans research.Download video file.^{(189M, mp4)}     

Running worms: C. elegans self-sorting by electrotaxis

The nematode C. elegans displays complex dynamical behaviors that are commonly used to identify relevant phenotypes. Although its maintenance is straightforward, sorting large populations of worms when looking for a behavioral phenotype is difficult, time consuming and hardly quantitative when done manually. Interestingly, when submitted to a moderate electric field, worms move steadily along straight trajectories. Here, we report an inexpensive method to measure worms crawling velocities and sort them within a few minutes by taking advantage of their electrotactic skills. This method allows to quantitatively measure the effect of mutations and aging on worm's crawling velocity. We also show that worms with different locomotory phenotypes can be spatially sorted, fast worms traveling away from slow ones. Group of nematodes with comparable locomotory fitness could then be isolated for further analysis. C. elegans is a growing model for neurodegenerative diseases and using electrotaxis for self-sorting can improve the high-throughput search of therapeutic bio-molecules.     

Effects of the novel anthelmintic emodepside on the locomotion, egg-laying behaviour and development of Caenorhabditis elegans

Emodepside, a cyclooctadepsipeptide, is a broad-spectrum anthelmintic previously shown to paralyse body wall muscle and pharyngeal muscle in the model nematode Caenorhabditis elegans. We demonstrate that wild-type C. elegans L4 are less sensitive than adults to emodepside in two independent assays of locomotor behaviour: body bend generation on agar (adult IC(50) 3.7 nM, L4 IC(50) 13.4 nM) and thrashing behaviour in liquid (thrashing behaviour as a % of controls after 1h in 10 microM emodepside: adults 16%, L4 worms 48%). We also show that continuous exposure of wild-type C. elegans to emodepside throughout the life-cycle from egg onwards, slows worm development, an effect that is emodepside concentration-dependent. The rate of worm-hatching from eggs on agar plates containing emodepside was not significantly different from controls, suggesting that it is development post-hatching rather than hatching itself that is affected by the drug. Emodepside also inhibits wild-type C. elegans egg-laying, with acute exposure to the drug at 500 nM resulting in an almost total inhibition within the first hour. However, the rate of egg production was not inhibited and therefore emodepside-treated worms became bloated with eggs, eventually rupturing. This suggests that the effect of emodepside on reproduction is not due to an inhibition of egg production but rather a paralytic effect on the egg-laying muscles. These results, when coupled with previous research, suggest that emodepside interferes with signalling at the neuromuscular junction on the body-wall muscles (Willson et al., 2003), pharynx (Willson et al., 2004) and egg-laying muscles and thus inhibits three important physiological functions: locomotion, feeding and reproduction.     

Optogenetic manipulation of neural activity in C. elegans: From synapse to circuits and behaviour

The emerging field of optogenetics allows for optical activation or inhibition of excitable cells. In 2005, optogenetic proteins were expressed in the nematode Caenorhabditis elegans for the first time. Since then, C. elegans has served as a powerful platform upon which to conduct optogenetic investigations of synaptic function, circuit dynamics and the neuronal basis of behaviour. The C. elegans nervous system, consisting of 302 neurons, whose connectivity and morphology has been mapped completely, drives a rich repertoire of behaviours that are quantifiable by video microscopy. This model organism's compact nervous system, quantifiable behaviour, genetic tractability and optical accessibility make it especially amenable to optogenetic interrogation. Channelrhodopsin‐2 (ChR2), halorhodopsin (NpHR/Halo) and other common optogenetic proteins have all been expressed in C. elegans. Moreover, recent advances leveraging molecular genetics and patterned light illumination have now made it possible to target photoactivation and inhibition to single cells and to do so in worms as they behave freely. Here, we describe techniques and methods for optogenetic manipulation in C. elegans. We review recent work using optogenetics and C. elegans for neuroscience investigations at the level of synapses, circuits and behaviour.     

A self-regulating feed-forward circuit controlling C. elegans egg-laying behavior

BACKGROUND: Egg laying in Caenorhabditis elegans has been well studied at the genetic and behavioral levels. However, the neural basis of egg-laying behavior is still not well understood; in particular, the roles of specific neurons and the functional nature of the synaptic connections in the egg-laying circuit remain uncharacterized. RESULTS: We have used in vivo neuroimaging and laser surgery to address these questions in intact, behaving animals. We have found that the HSN neurons play a central role in driving egg-laying behavior through direct excitation of the vulval muscles and VC motor neurons. The VC neurons play a dual role in the egg-laying circuit, exciting the vulval muscles while feedback-inhibiting the HSNs. Interestingly, the HSNs are active in the absence of synaptic input, suggesting that egg laying may be controlled through modulation of autonomous HSN activity. Indeed, body touch appears to inhibit egg laying, in part by interfering with HSN calcium oscillations. CONCLUSIONS: The egg-laying motor circuit comprises a simple three-component system combining feed-forward excitation and feedback inhibition. This microcircuit motif is common in the C. elegans nervous system, as well as in the mammalian cortex; thus, understanding its functional properties in C. elegans may provide insight into its computational role in more complex brains.     

Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses

For studying the function of specific neurons in their native circuitry, it is desired to precisely control their activity. This often requires dissection to allow accurate electrical stimulation or neurotransmitter application , and it is thus inherently difficult in live animals, especially in small model organisms. Here, we employed channelrhodopsin-2 (ChR2), a directly light-gated cation channel from the green alga Chlamydomonas reinhardtii, in excitable cells of the nematode Caenorhabditis elegans, to trigger specific behaviors, simply by illumination. Channelrhodopsins are 7-transmembrane-helix proteins that resemble the light-driven proton pump bacteriorhodopsin , and they also utilize the chromophore all-trans retinal, but to open an intrinsic cation pore. In muscle cells, light-activated ChR2 evoked strong, simultaneous contractions, which were reduced in the background of mutated L-type, voltage-gated Ca2+-channels (VGCCs) and ryanodine receptors (RyRs). Electrophysiological analysis demonstrated rapid inward currents that persisted as long as the illumination. When ChR2 was expressed in mechanosensory neurons, light evoked withdrawal behaviors that are normally elicited by mechanical stimulation. Furthermore, ChR2 enabled activity of these neurons in mutants lacking the MEC-4/MEC-10 mechanosensory ion channel . Thus, specific neurons or muscles expressing ChR2 can be quickly and reversibly activated by light in live and behaving, as well as dissected, animals.     

A high-throughput method to deliver targeted optogenetic stimulation to moving C. elegans populations

We present a high-throughput optogenetic illumination system capable of simultaneous closed-loop light delivery to specified targets in populations of moving Caenorhabditis elegans. The instrument addresses three technical challenges: It delivers targeted illumination to specified regions of the animal’s body such as its head or tail; it automatically delivers stimuli triggered upon the animal’s behavior; and it achieves high throughput by targeting many animals simultaneously. The instrument was used to optogenetically probe the animal’s behavioral response to competing mechanosensory stimuli in the the anterior and posterior gentle touch receptor neurons. Responses to more than 43,418 stimulus events from a range of anterior–posterior intensity combinations were measured. The animal’s probability of sprinting forward in response to a mechanosensory stimulus depended on both the anterior and posterior stimulation intensity, while the probability of reversing depended primarily on the anterior stimulation intensity. We also probed the animal’s response to mechanosensory stimulation during the onset of turning, a relatively rare behavioral event, by delivering stimuli automatically when the animal began to turn. Using this closed-loop approach, over 9,700 stimulus events were delivered during turning onset at a rate of 9.2 events per worm hour, a greater than 25-fold increase in throughput compared to previous investigations. These measurements validate with greater statistical power previous findings that turning acts to gate mechanosensory evoked reversals. Compared to previous approaches, the current system offers targeted optogenetic stimulation to specific body regions or behaviors with many fold increases in throughput to better constrain quantitative models of sensorimotor processing.

This study resents a new targeted illumination method for the nematode Caenorhabditis elegans, allowing delivery of optogenetic stimulation to specific body parts of many animals at once, automatically triggered by the animals’ behavior.     

A multispectral optical illumination system with precise spatiotemporal control for the manipulation of optogenetic reagents

Optogenetics is an excellent tool for noninvasive activation and silencing of neurons and muscles. Although they have been widely adopted, illumination techniques for optogenetic tools remain limited and relatively nonstandardized. We present a protocol for constructing an illumination system capable of dynamic multispectral optical targeting of micrometer-sized structures in both stationary and moving objects. The initial steps of the protocol describe how to modify an off-the-shelf video projector by insertion of optical filters and modification of projector optics. Subsequent steps involve altering the microscope's epifluorescence optical train as well as alignment and characterization of the system. When fully assembled, the illumination system is capable of dynamically projecting multispectral patterns with a resolution better than 10 μm at medium magnifications. Compared with other custom-assembled systems and commercially available products, this protocol allows a researcher to assemble the illumination system for a fraction of the cost and can be completed within a few days.     

A Na+/Cl- -coupled GABA transporter, GAT-1, from Caenorhabditis elegans: structural and functional features, specific expression in GABA-ergic neurons, and involvement in muscle function

GABA functions as an inhibitory neurotransmitter in body muscles and as an excitatory neurotransmitter in enteric muscles in Caenorhabditis elegans. Whereas many of the components of the GABA-ergic neurotransmission in this organism have been identified at the molecular and functional levels, no transporter specific for this neurotransmitter has been identified to date. Here we report on the cloning and functional characterization of a GABA transporter from C. elegans (ceGAT-1) and on the functional relevance of the transporter to the biology of body muscles and enteric muscles. ceGAT-1 is coded by snf-11 gene, a member of the sodium-dependent neurotransmitter symporter gene family in C. elegans. The cloned ceGAT-1 functions as a Na(+)/Cl(-)-coupled high-affinity transporter selective for GABA with a K(t) of approximately 15 microm. The Na(+):Cl(-):GABA stoichiometry for ceGAT-1-mediated transport process is 2:1:1. The transport process is electrogenic as evidenced from GABA-induced inward currents in Xenopus laevis oocytes that express ceGAT-1 heterologously. The transporter is expressed exclusively in GABA-ergic neurons and in two other additional neurons. We also investigated the functional relevance of ceGAT-1 to the biology of body muscles and enteric muscles by ceGAT-1-specific RNA interference (RNAi) in rrf-3 mutant, a strain of C. elegans in which neurons are not refractory to RNAi as in the wild type strain. The down-regulation of ceGAT-1 by RNAi leads to an interesting phenotype associated with altered function of body muscles (as evident from changes in thrashing frequency) and enteric muscles (as evident from the rates of defecation failure) and also with altered sensitivity to aldicarb-induced paralysis. These findings provide unequivocal evidence for a modulatory role of GABA and ceGAT-1 in the biology of cholinergic neurons and in the function of body muscles and enteric muscles in this organism.     

Optogenetic manipulation of neural activity in freely moving Caenorhabditis elegans

We present an optogenetic illumination system capable of real-time light delivery with high spatial resolution to specified targets in freely moving Caenorhabditis elegans. A tracking microscope records the motion of an unrestrained worm expressing channelrhodopsin-2 or halorhodopsin in specific cell types. Image processing software analyzes the worm's position in each video frame, rapidly estimates the locations of targeted cells and instructs a digital micromirror device to illuminate targeted cells with laser light of the appropriate wavelengths to stimulate or inhibit activity. Because each cell in an unrestrained worm is a rapidly moving target, our system operates at high speed (～50 frames per second) to provide high spatial resolution (～30 μm). To test the accuracy, flexibility and utility of our system, we performed optogenetic analyses of the worm motor circuit, egg-laying circuit and mechanosensory circuits that have not been possible with previous methods.     

Specific Expression of Channelrhodopsin-2 in Single Neurons of Caenorhabditis elegans

Optogenetic approaches using light-activated proteins like Channelrhodopsin-2 (ChR2) enable investigating the function of populations of neurons in live Caenorhabditis elegans (and other) animals, as ChR2 expression can be targeted to these cells using specific promoters. Sub-populations of these neurons, or even single cells, can be further addressed by restricting the illumination to the cell of interest. However, this is technically demanding, particularly in free moving animals. Thus, it would be helpful if expression of ChR2 could be restricted to single neurons or neuron pairs, as even wide-field illumination would photostimulate only this particular cell. To this end we adopted the use of Cre or FLP recombinases and conditional ChR2 expression at the intersection of two promoter expression domains, i.e. in the cell of interest only. Success of this method depends on precise knowledge of the individual promoters' expression patterns and on relative expression levels of recombinase and ChR2. A bicistronic expression cassette with GFP helps to identify the correct expression pattern. Here we show specific expression in the AVA reverse command neurons and the aversive polymodal sensory ASH neurons. This approach shall enable to generate strains for optogenetic manipulation of each of the 302 C. elegans neurons. This may eventually allow to model the C. elegans nervous system in its entirety, based on functional data for each neuron.     

Identification of Optogenetically Activated Striatal Medium Spiny Neurons by Npas4 Expression

Optogenetics is a powerful neuromodulatory tool with many unique advantages to explore functions of neuronal circuits in physiology and diseases. Yet, interpretation of cellular and behavioral responses following in vivo optogenetic manipulation of brain activities in experimental animals often necessitates identification of photoactivated neurons with high spatial resolution. Although tracing expression of immediate early genes (IEGs) provides a convenient approach, neuronal activation is not always followed by specific induction of widely used neuronal activity markers like c-fos, Egr1 and Arc. In this study we performed unilateral optogenetic stimulation of the striatum in freely moving transgenic mice that expressed a channelrhodopsin-2 (ChR2) variant ChR2(C128S) in striatal medium spiny neurons (MSNs). We found that in vivo blue light stimulation significantly altered electrophysiological activity of striatal neurons and animal behaviors. To identify photoactivated neurons we then analyzed IEG expression patterns using in situ hybridization. Upon light illumination an induction of c-fos was not apparent whereas another neuronal IEG Npas4 was robustly induced in MSNs ipsilaterally. Our results demonstrate that tracing Npas4 mRNA expression following in vivo optogenetic modulation can be an effective tool for reliable and sensitive identification of activated MSNs in the mouse striatum.     

Serotonin modulates locomotory behavior and coordinates egg-laying and movement in Caenorhabditis elegans.

Biogenic amines have been implicated in the modulation of neural circuits involved in diverse behaviors in a wide variety of organisms. In the nematode C. elegans, serotonin has been shown to modulate the temporal pattern of egg-laying behavior. Here we show that serotonergic neurotransmission is also required for modulation of the timing of behavioral events associated with locomotion and for coordinating locomotive behavior with egg-laying. Using an automated tracking system to record locomotory behavior over long time periods, we determined that both the direction and velocity of movement fluctuate in a stochastic pattern in wild-type worms. During periods of active egg-laying, the patterns of reversals and velocity were altered: velocity increased transiently before egg-laying events, while reversals increased in frequency following egg-laying events. The temporal coordination between egg-laying and locomotion was dependent on the serotonergic HSN egg-laying motorneurons as well as the decision-making AVF interneurons, which receive synaptic input from the HSNs. Serotonin-deficient mutants also failed to coordinate egg-laying and locomotion and exhibited an abnormally low overall reversal frequency. Thus, serotonin appears to function specifically to facilitate increased locomotion during periods of active egg-laying, and to function generally to modulate decision-making neurons that promote forward movement.