Barrios, Arantza et al. (2009) International Worm Meeting "Regulation of C. elegans male mate-searching behavior by hermaphrodite cuticular cues."

Barrios, Arantza, Emmons, Scott

[International Worm Meeting, 2009]

Male mate-searching behavior in C. elegans provides a model to understand how animals integrate internal physiological needs and drives with external environmental stimuli to produce adaptive behavior. Unlike the self-fertile C. elegans hermaphrodite, the C. elegans male needs to mate in order to reproduce. The male has therefore evolved strategies to locate and remain within a source of both food and hermaphrodite mating partners. C. elegans males explore their environment in search of mates and will leave food if hermaphrodite mating partners are absent [1]. However, when mates and food coincide, male exploratory behavior is suppressed and males are retained on the food source [1]. What is the neural circuit that regulates male mate-searching? We have identified the male-specific ray neurons in the tail as part of the circuit that stimulates mate-searching behavior. In the absence of mates, ray neurons (mainly RnB with contribution from RnA) stimulate exploration away from food by promoting exits from the food lawn and inhibiting high angle turns upon exiting the lawn. In the presence of mates, periodic contact with a hermaphrodite, detected through the rays, changes the male''s behavior during periods of no contact, preventing the male from leaving the lawn. The hermaphrodite signal is conveyed by the male-specific EF interneurons, which are post-synaptic to the rays and send processes to the nerve ring in the head [2]. What is the nature of the hermaphrodite signal? Male retention by hermaphrodites is independent of copulation since neither the hermaphrodite vulva nor the male spicules are required for suppression of mate-searching behavior. Furthermore, males are fully retained by dead PFA-fixed hermaphrodites but not PFA-fixed males and retention is lost if the hermaphrodites are washed with an organic solvent such as hexane. These results suggest that sex-specific lipids on the hermaphrodite cuticle are important for the suppression of male exploratory behavior. Indeed, males are retained by males with feminized hypodermis (by expression of dpy-7::tra-2 IC -construct provided by W. Mowrey and D. Portman) indicating that the C. elegans cuticle is composed of sexualized chemicals that produce an effect in the behavior of other males. 1. Lipton, J. et al. (2004). J Neurosci 24, 7427-34. 2. Barrios, A. et al. (2008). Curr Biol 18, 1865-71.

Arantza Barrios et al. (2008) European Worm Meeting "Sensory regulation of C. elegans male mate-searching behaviour"

Scott W Emmons, Arantza Barrios, Stephen Nurrish

[European Worm Meeting, 2008]

C. elegans male mate searching behaviour provides a paradigm to study,. from a genetic standpoint, how neuronal networks encode decision making. C.. elegans males need to mate in order to reproduce and therefore they have. evolved strategies to locate and remain within a source of mating partners.. In the absence of hermaphrodite mating partners, sexually mature males. display mate-searching behaviour: they leave a spot of food and engage in. an exploratory activity around their environment. The presence of. hermaphrodites in the spot of food prevents the male from leaving and. engaging in this exploratory activity.. As a first step towards the identification of the neural circuit that. regulates mate-searching behaviour, we have identified, through analysis of. mutants and by laser and genetic ablation experiments, the male sensory. circuit that modulates retention by hermaphrodites.. We have found that hermaphrodites inhibit male mate searching. behaviour through contact-dependent cues since fixed hermaphrodites fully. retain males. Males are also fully retained by vulvaless (lin-39 n1760 ). hermaphrodites indicating that hermaphrodite retention cues are novel cues. different from those required for copulation. Hermaphrodite contact-cues. are sensed by male-specific neurons in the tail within the ray and spicule. sensilla. Each ray sensillum has two neurons, type A (RnA) and B (RnB).. Function of either the RnA neurons alone or the RnB neurons alone is. sufficient to retain males with hermaphrodites. Lack of RnB but not RnA. neurons results in decreased mate-searching activity, indicating that RnB. neurons also function to promote mate searching behavior in the absence of. hermaphrodites. This dual role of RnB neurons is dependent on the activity. of two different Ca2+ TRP channels: the polycystins PKD-2/LOV-1 are. required for mate-searching and OSM-9 is required for retention. Therefore,. one neuron class modulates the switch between the two behaviours (mate-. searching and retention) through the activity of two different molecular. pathways. Lack of spicules also results in males with defects in both. retention by hermaphrodites and mate searching behaviour. We are currently. assessing the individual contribution to these behaviours of each neuron. within the spicule sensillum.

Arantza Barrios et al. (2005) International Worm Meeting "Identifying molecular and cellular components involved in the male response to hermaphrodite cues"

Stephen Nurrish, Arantza Barrios, Scott Emmons

[International Worm Meeting, 2005]

Mate searching, a male specific motivated behaviour associated with the males need to mate, provides a paradigm to investigate neuronal integration in a goal-directed behaviour. C.elegans mate searching is regulated by internal physiological signals from the male and by external environmental cues from the hermaphrodite. To establish the neural pathways that lead from the male detection of hermaphrodites to the modulation of mate searching we have performed a mutant and RNAi screen of candidate genes to identify genes required for hermaphrodite detection. We have used two assays that test the ability of a male to sense and respond to hermaphrodite cues: the holding assay and the accumulation assay. In the holding assay, a tester male is placed in a spot of food in the presence of 2 hermaphrodites. In this assay wild type males remain in the spot of food indefinitely to stay within the vicinity of the hermaphrodites whereas mutant males unable to sense hermaphrodites leave. In the accumulation assay (modified from Simon and Sternberg) when a population of males is given the choice between a spot of medium conditioned with hermaphrodites and a control spot, males accumulate at the hermaphrodite conditioned spot. Four male specific sensory cells in the head, the CEMs, have been postulated to be involved in the detection of mating partners. However, we have found that mutant males whose CEMs undergo apoptosis (strain provided by Hillel Schwartz) respond normally to hermaphrodite cues in both assays, indicating that these cells are not required for the detection of hermaphrodites. In contrast, lin-32 (e1926) mutants, which lack most rays, are defective in the response to hermaphrodites in the holding assay but respond normally in the accumulation assay where only hermaphrodite secreted cues are present. As well as having abnormal rays, lin-32 mutants are also defective for general touch in the tail. However lack of general mechanosensation alone cannot account for their defect in response in the holding assay since mec-4 mutants respond normally. These results suggest that hermaphrodites present multiple cues, some detected through male specific rays (ray specific mec-4 independent mechanosensation or chemosensation of cuticule-bound molecules) and some detected through general chemosensory organs possibly the amphids. In support of amphid neurons being involved in hermaphrodite detection two mutations affecting genes expressed in gustatory neurons, daf-11 and gpa-6, also render males unresponsive to hermaphrodite cues in the accumulation assay.

Arantza Barrios et al. (2008) C.elegans Neuronal Development Meeting "Sensory Regulation of C. elegans Male Mate-searching Behavior"

Arantza Barrios, Stephen Nurrish, Scott Emmons

[C.elegans Neuronal Development Meeting, 2008]

C. elegans male mate-searching behavior provides a paradigm to study, from a genetic standpoint, how neuronal networks encode behavioral decisions. C. elegans males need to mate in order to reproduce and therefore they have evolved strategies to locate and remain within a source of mating partners. In the absence of hermaphrodite mating partners, sexually mature males display mate-searching behavior: they leave a spot of food and engage in an exploratory activity around their environment. The presence of hermaphrodites in the spot of food prevents the male from leaving and engaging in this exploratory activity. As a first step towards the identification of the neural circuit that regulates mate-searching behavior, we have identified, through analysis of mutants and by laser and genetic ablation experiments, the male sensory circuit that modulates retention by hermaphrodites. We have found that hermaphrodites inhibit male mate searching behavior through contact-dependent cues since fixed hermaphrodites fully retain males. Retention is independent of copulation (i.e. the consumatory step of mating behavior) since neither the male spicule neurons nor the hermaphrodite vulva are necessary for retention. Hermaphrodite contact-cues are sensed by male-specific ray neurons. Each ray sensillum has two neurons, type A (RnA) and B (RnB). We have killed each neuronal type individually by targeted expression of human caspases. Function of either the RnA neurons alone or the RnB neurons alone is sufficient to retain males with hermaphrodites. Lack of RnB alone but not RnA neurons alone results in decreased mate-searching activity, indicating that RnB neurons also function to promote mate searching behavior in the absence of hermaphrodites. This dual role of RnB neurons is dependent on the activity of two different Ca2+ TRP channels: the polycystins PKD-2/LOV-1 are required for mate-searching and OSM-9 is required for retention. Therefore, one neuron class can modulate the switch between the two behaviors (mate-searching and retention) through the activity of two different molecular pathways.

Arantza Barrios et al. (2007) International Worm Meeting "Sensory regulation of C. elegans male mate searching behavior by hermaphrodites."

Scott W. Emmons, Stephen Nurrish, Arantza Barrios

[International Worm Meeting, 2007]

Locating mating partners is essential for the C. elegans male to pass on its genes. In the absence of hermaphrodite mating partners, sexually mature males display mate searching behaviour: they leave a spot of food and engage in an exploratory activity around their environment. The presence of hermaphrodites in the spot of food prevents the male from leaving and engaging in this exploratory activity. We have found that male retention by hermaphrodites is independent of other known male/hermaphrodite interactions such as copulation and male attraction to hermaphrodite secreted cues. In order to begin to identify the neural circuit for mate searching behavior we have identified the male sensory neurons involved in hermaphrodite detection. We examined the role of individual neurons and classes of neurons by analysis of mutants, by laser ablation experiments and by genetic ablation through the expression of ICE caspase under specific promoters. Males that lack all ray neurons remain in the patch of food in the absence of hermaphrodites. Thus male mate searching behavior is stimulated by male specific neurons in the tail. Rays are also required for retention by hermaphrodites since residual mate searching activity in males that lack most ray neurons is not blocked by the presence of hermaphrodites. In each ray sensillum there are two neurons, RnA and RnB. Males lacking the B-type neurons reveal that the A-type neurons both stimulate mate searching and sense hermaphrodite cues. RnB neurons also stimulate mate searching behavior through a pathway dependent on the polycystin genes pkd-2 and lov-1. Their role in hermaphrodite detection has not yet been tested. Surprisingly, another set of male specific neurons, the CEMs, are not required for retention by hermaphrodites since males where the CEMs undergo apoptosis either during embryogenesis (ceh-30 n4289) or after the L4 stage (pkd-2:ICE) are fully retained by hermaphrodites. Gustatory amphids also contribute to retention by hermaphrodites. osm-3 (p802) males, in which the cilia of the gustatory amphids, RnB and CEMs are disrupted, leave the food source even in the presence of hermaphrodites. This phenotype of osm-3 males can be attributed to their defect in the amphid neurons since males with no CEMs and RnB neurons are fully retained by hermaphrodites. Mutants in the TRP channel osm-9 are also partially defective in retention by hermaphrodites. Cell specific rescue experiments suggest that osm-9 is required in the amphids for this function.

Arantza Barrios et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Regulation of C.elegans male mate searching behavior by hermaphrodite cues"

Arantza Barrios, Scott Emmons, Stephen Nurrish

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

C.elegans males display mate searching activity, an appetitive behaviour associated with the male"s need to mate. This exploratory male behaviour ceases upon encountering hermaphrodites. In order to unravel the neural mechanisms underlying the inhibition of male mate searching behaviour by the presence of hermaphrodites we have first identified the male sensory neurons involved in sensing hermaphrodites. We have used two assays that test the ability of a male to sense and respond to hermaphrodite cues: the holding assay and the accumulation assay. In the holding assay, a tester male is placed in a spot of food in the presence of 2 hermaphrodites. In this assay wild type males remain in the spot of food indefinitely to stay within the vicinity of the hermaphrodites whereas mutant males unable to sense hermaphrodites leave to explore the rest of the plate. In the accumulation assay (modified from Simon and Sternberg) when a population of males is given the choice between a spot of medium conditioned with hermaphrodites and a control spot, males accumulate at the hermaphrodite conditioned spot. We found that CEMs (male specific cephalic neurons) and touch receptor neurons are not required for male retention by hermaphrodites since ceh-30 (n4289) males whose CEMs undergo apoptosis and mec-4 (e1611) males whose touch receptor cells undergo necrosis respond normally in both assays. In contrast osm-3 (p802) males are defective in both assays implicating the gustatory amphids in hermaphrodite detection. Rays are also required for retention by hermaphrodites since mab-3 and lin-32 males (which lack most pairs of rays) as well as ray laser ablated males are defective in the holding assay but not in the accumulation assay. Importantly, rays are also involved in positively driving mate searching behaviour since mutants that lack subsets of rays as well as ray laser ablated males display much lower mate searching rates. In each ray sensillum there are two neurons, RnA and RnB. Genetic ablation of RnB neurons by expressing ICE caspase under the pkd-2 promoter does not prevent males from being retained by hermaphrodites, however RnB neurons appear to be required to promote mate searching behaviour.

Emmons, Scott W. et al. (2013) International Worm Meeting "Molecular regulators of male sex-drive."

Emmons, Scott W., Barrios, Arantza

[International Worm Meeting, 2013]

Well-fed, mate-deprived adult males make the risky choice to leave a plentiful source of food to explore their environment in search of mates. In contrast, recent experience with a mate while exposed to food produces a durable behavioral switch that restricts exploration within the limits of the food source. From a forward genetic screen for males that do not leave food (leaving assay defective -las), we isolated several mutants. We have recently shown that las-1(bx142) encodes a secretin-like G protein-coupled receptor for the neuropeptide pigment dispersing factor (pdf-1). Male exploratory behavior results from the balance of two physiological needs -feeding and reproduction- that compete for the control of a distributed network for navigation. The phenotype of pdfr-1 males reflects an imbalance in the relative contribution of the circuits that control exploration. pdfr-1 is required in gender-shared sensory neurons PHA, PQR and URY to generate the state of arousal to leave food in search of mates. Thus, pdfr-1 modulates a circuit that senses the internal environment of the animal and antagonizes the food-sensing circuit (Barrios et al., 2012, DOI 10.1038/nn.3253). We are currently identifying the molecular lesion responsible for the phenotype of las-2(bx143). We have mapped bx143 to the distal left arm of chromosome I and through whole genome sequencing we have identified missense mutations in two candidate genes. Rescue experiments and complementation tests with these two candidates are under way. bx143 males display normal locomotion on food but are strongly Las and mate response defective with no apparent morphological defects. These phenotypes suggest a role for bx143 in the regulation of the neural circuits that convey male sex drive.

Goss-Sampson, Evie et al. (2021) International Worm Meeting "Visualising neuropeptide spatial range of action within the nervous system."

Goss-Sampson, Evie, Barrios, Arantza, Jimeno-Martin, Angela

[International Worm Meeting, 2021]

Past experiences, moods and emotions change our behaviours, partly due to the neuromodulation of underlying circuits from neuropeptides. Neuropeptides are secreted, diffusible proteins that act on G-protein coupled receptors (GPCRs), providing long-lasting modulation to hard-wired neuronal circuits. Most neuropeptide receptor expressing neurons are not synaptically connected to the source, thus, the expression patterns of the ligand and its receptors represent a wireless chemical connectivity map that work in conjunction with traditional synaptic communication. Neuropeptides regulate many global states as well as more discrete modulatory functions such as learning and memory. However, it is unknown how neuropeptides encode such diverse information. For global behavioural state changes, it is thought neuropeptides may activate any target they reach in their diffusion pathway, a process termed volume transmission. For functions that require specific target modulation, such as associative learning, emerging evidence suggests diffusion could be more regulated and controlled (van den Pol 2012), however mechanisms underlying this regulation are unknown. Many affective disorders rely on exogenous application of neuromodulators, therefore improving our understanding of neuropeptide diffusion regulation, could reduce adverse side effects many patients suffer due to off-target activity. We are employing C. elegans to establish an in vivo system to visualise neuropeptide based neuronal communication to use as a tool to identify molecular and cellular regulators of neuropeptide diffusion. We are focusing on Pigment Dispersing Factor-1 (PDF-1) and its receptor PDFR-1, a highly conserved neuropeptide involved in a broad array of behaviours. By harnessing GPCR signalling cascades, we will introduce a synthetic pathway that converts the interaction between PDF-1 and PDFR-1 into a fluorescent signal, without interfering with endogenous signalling. We will control PDF-1 expression and secretion in a ligand-null mutant by co-expressing PDF-1 and channelrhodopsin in one neuronal source. This expression pattern will be used as the basis for a forward genetic screen. This project aims to create a system to visualise neuropeptide modulation in vivo which will set the foundation for an in-depth investigation to understand neuropeptide spatial range of action.

Molina-Garcia, Laura et al. (2021) International Worm Meeting "PDF-1 modulation of aversion and reward during associative learning"

Barrios, Arantza, Lin, Lucy, Colinas-Fischer, Susana, Molina-Garcia, Laura

[International Worm Meeting, 2021]

A central goal in neuroscience is to understand how neural circuits integrate conflicting (rewarding and aversive) experiences that need to be behaviourally resolved during learning. To shed light into the molecular and cellular mechanisms underlying this process we are dissecting the neuronal circuit that regulates sexual conditioning in C. elegans. Previously, the Iino lab (Sakai et al., 2013) and us (Sammut et al., 2015) have shown that C. elegans males undergo sexual conditioning, a form of associative learning by which a rewarding experience with mates overrides the behavioural consequences of an aversive association with starvation. Thus, sexual conditioning leads to a switch in behavioural responses to an environmental stimulus from avoidance to attraction. Our lab also identified the MCM interneurons and PDF-1 neuromodulation as the cellular and molecular regulators of the sexually conditioned behavioural switch (Sammut et al., 2015). These studies showed conditioned responses to salt. Here we show that other stimuli such as benzaldehyde can also be sexually conditioned and this is also regulated by the MCMs and PDF-1. We find a dual role for PDF-1 in the regulation of aversive learning and sexual conditioning in C. elegans. By using a Cre-Lox intersectional strategy we found that PDF signaling encodes both aversion and sexual conditioning by modulating two partially distinct circuits. Within these circuits, we have identified the interneurons RIA, AIY and RIM as target cells receiving PDF-1 neuromodulation to drive sexual conditioning. Expression of the PDF-1 receptor (pdfr-1) only in these neurons is sufficient to rescue attraction during sexual conditioning, but not repulsion during aversive learning. Furthermore, by removing PDF-1 in specific cells, while keeping the endogenous levels of expression in the rest of the circuit, we have identified the MCMs and AVB neurons, as a source of PDF-1 during sexual conditioning. Importantly, PDF-1 release from the MCMs and AVBs is not required during aversive learning. Thus, highlighting the importance of source specificity versus overall neuropeptide levels during fine tune modulation of specific behaviors. Currently, we are measuring neuronal activity within the chemotaxis circuit to further understand how sensory information is represented after conditioning and PDF neuromodulation to switch odour preferences.

Colinas Fischer, Susana et al. (2021) International Worm Meeting "Circuit and Molecular Mechanisms of an Associative Learning Task"

Molina-Garcia, Laura, Lin, Lucy, Barrios, Arantza, Colinas Fischer, Susana

[International Worm Meeting, 2021]

The ability of neural circuits to be changed by experience, optimising an organism's behaviour, is key to survival. We are dissecting the circuit underlying aversive olfactory learning to benzaldehyde (BZ), a bacterial metabolite innately attractive to C. elegans. When this odourant is paired with starvation, an aversive experience, the behavioural response to BZ switches from attraction to repulsion (Lee 2010, Lin 2010). Furthermore, this switch in behaviour can be overridden in males if a rewarding experience (mates) is present during aversive conditioning with starvation. This process is termed sexual conditioning and it was first shown to drive flexible learned responses to salt (Sakai et al, 2013). Our lab has previously shown that the neuropeptide PDF-1 is necessary for sexual conditioning. Here we show that both PDF-1 and PDF-2 redundantly mediate aversive learning, and seek to identify the PDF circuit for aversive learning. BZ is sensed primarily by AWC, which synapses onto AIB and AIY to regulate naive chemotaxis (Bargmann 1993, Chalasani 2007). The switch from attraction to repulsion during aversive learning has been proposed to be driven by changes at the AWC-AIB synapse (Cho 2016). We find that AWC-ablated animals have a dampened response to BZ, but we still observe a switch in response from attraction to repulsion after aversive conditioning, indicating that learning can occur elsewhere in the circuit. We are investigating neurons that are involved in PDF signalling to find the circuit that drives this experience-dependent change in behaviour. We are using an intersectional Cre-Lox strategy and a floxed transgene of PDF-1 cDNA integrated in single copy to return physiological levels of PDF-1 to specific neurons, and thus determine which combinations of neurons are the source of PDF-1 in aversive learning. We will also use an approach that exploits the strength of the Cre-Lox system to restore PDFR-1 to certain neurons selectively, to determine which neurons are the target of PDF during aversive learning. Then we will image the activity of source and target neurons to determine what changes occur in the circuit during conditioning that lead to changes in behaviour. Given that associative learning is a highly conserved behaviour, understanding how this simple circuit is capable of supporting aversive learning will provide us with principles that can be applied to further understand how learning occurs in more complex nervous systems.