Ardiel, Evan et al. (2017) International Worm Meeting "Behaviour in an eggshell."

Shroff, Hari, Ardiel, Evan

[International Worm Meeting, 2017]

With its small and well-documented connectome, C. elegans has emerged as a powerful system for the modeling of complex behaviours from sensory input to motor output. Adult locomotion has been the focus of much research, but the emergence of coordinated behaviour during embryogenesis remains almost completely unexplored. Spontaneous muscle contractions begin about 2 h after the transition from proliferation to organogenesis (~8 h post-fertilization), before the appearance of neuromuscular junctions. Over the next 6-7 h, the embryo's length approximately doubles and myogenic contractions give way to more coordinated behaviour mediated by the nervous system. The embryo's small size, entangled posture, and rapid movements have traditionally complicated the collection and interpretation of behavioural data during this developmental period. To describe postural changes over the final hours of embryogenesis, we used light-sheet fluorescence microscopy for fast and gentle 3D imaging of a strain expressing a fluorescent marker in the seam cells. We were able to capture embryonic volumes at 3 Hz for more than 5 h without delaying hatch time. We tracked the labelled cells over the 50000+ volumes and used the lateral positions of the seam cells to construct a simple, dynamic model of the embryo in the eggshell. We observed distinct behavioural states and trajectories as the embryo developed. Detailed description of movement during the three-fold stage promises to yield insights into motor circuit assembly.

Ardiel, Evan L. et al. (2011) International Worm Meeting "Investigating neural coding and interacting circuits."

Schafer, William R., Rankin, Catharine H., Rabinowitch, Ithai, Giles, Andrew C., Ardiel, Evan L.

[International Worm Meeting, 2011]

ASH mediates backward locomotion in response to osmotic pressure, nose touch, high ambient oxygen, and a variety of volatile and non-volatile compounds. We used optogenetics to simulate these stimuli to better understand their perception and how this changes with experience. Using an automated multi-worm tracker to measure behaviour, we found that a short light pulse (200-ms) of low intensity blue light elicited a reversal response of about 200-mm in 80-90% animals expressing ChR2 in ASH. A longer light pulse (2-s) elicited a larger reversal response (1-mm) that often ended in an omega turn. Previous work demonstrated that ASH-mediated responses to different stimuli were genetically dissociable (Mellem et al., 2002). We monitored the responses of eat-4, glr-1, nmr-1, and glr-1;nmr-1 mutants and found that the behavioural impairments associated with nose touch and osmo-avoidance assays were also apparent in response to the short and long light pulses, respectively. This suggests that a part of the neural code distinguishing nose touch and chemoaversion is the duration of depolarization of ASH. We next tested habituation of ASH to our "nose touch" and "chemoaversive" light pulses. We found that both responses decremented from repeated exposure, but with differential kinetics. The proportion of worms responding to the longer light pulse decreased faster and further than responses to the nose touch-like stimulus. Disrupting neuropeptide signalling blocked this deep habituation. The interneurons downstream of ASH largely overlap with those mediating reversals to body touch. We were interested in addressing how activity in one circuit affected the other. We found that the tap response was decremented by a single light, but facilitated by repeated presentation of long-light pulses. We are currently investigating the cellular mechanisms underlying this cross-circuit plasticity.

Ardiel, Evan L. et al. (2009) International Worm Meeting "A species specific chemosensory cue can modulate body size in C. elegans."

Rankin, Catharine H., Ardiel, Evan L.

[International Worm Meeting, 2009]

Previous research on C. elegans has shown that sensory perception mutants are smaller than wild-type worms (Fujiwara et al., 2002). This suggests that sensory input from the environment regulates adult body size. But what are the relevant external cues? Work in our lab suggests that interactions with conspecifics are one source. We have found that worms reared in isolation are dwarfed compared to worms reared in colonies. Furthermore, adding another worm to the plate of an isolated worm rescues colony body size. C. briggsae are also dwarfed when reared in isolation, but the relevant conspecific cue appears to be species specific, as the presence of C. briggsae cannot rescue colony body size in an isolated C. elegans. Using sensory perception mutants with cell structure (che-2, che-3, osm-3, daf-6), signal transduction (odr-1, odr-3, tax-4, osm-9), and cell specification (ceh-36, lim-4, odr-7) defects and cell-type specific rescues, we have identified a subset of chemosensory neurons with a role in the modulation of body size in response to conspecifics. Because isolated worms exposed to the stable water-soluble or volatile chemical cues of a colony are still smaller than colony worms, we propose that a species specific contact pheromone triggers growth to a larger body size. Fujiwara, M., Segupta, P., & McIntire, S.L. (2002). Regulation of body size and behavioral state of C. elegans by sensory perception and the egl-4 cGMP-dependent protein kinase. Neuron, 366 (6), 1091-1102.

Ardiel, Evan L. et al. (2013) International Worm Meeting "Response to repeated activation of ASH requires glutamate, dopamine, and neuropeptide signaling."

Giles, Andrew C., Rabinowitch, Ithai, Lindsay, Theodore, Lockery, Shawn, Ardiel, Evan L., Schafer, William, Rankin, Catharine H.

[International Worm Meeting, 2013]

The objective of this research was a comparative analysis of habituation in two neural circuits that synapse onto the same interneurons. Previous work in the lab has focused on habituation to a non-localized mechanical stimulus - a plate tap. Here we studied the response to repeated activation of the ASH sensory neurons, which detect a variety of aversive stimuli. A strain expressing ChR2 exclusively in ASH (Ezcurra et al., 2011) was used for consistent and discrete delivery of simulated aversive stimuli to worm populations being tracked by real-time computer vision software (Swierczek et al., 2011). In addition to increasing throughput, this optogenetic approach allowed us to prevent sensory adaptation and specifically activate ASH. Whole-plate blue light illumination elicited backward crawling (reversal) in the majority of animals. As with the tap-withdrawal response, the magnitude of ASH-mediated reversals decremented with repeated stimulation in a manner dependent on the stimulus intensity and frequency and recovered to baseline in minutes. Electrophysiological recordings of repeatedly activated photocurrents in ASH demonstrated that the decrement was not caused by ChR2 desensitization. Furthermore, a tap could dishabituate decremented responding. Cross-modal habituation was evident in only one-direction, ie the tap-withdrawal response was decremented by repeated activation of ASH, thereby localizing the site of plasticity to the shared circuitry. Although their magnitude decremented over the trial, ASH reversals persisted far longer than tap-induced reversals. This response maintenance was dependent on glutamate and dopamine signaling. Repeated ASH activation also led to increased speed of forward locomotion and a greater suppression of spontaneous reversals in the periods between stimuli, as compared to tap. This change was dependent on neuropeptide signaling. These distinct behavioural strategies are hypothesized to facilitate escape from a hazardous area.

Evan Ardiel et al. (2008) C.elegans Neuronal Development Meeting "Conspecific Cues Mediate Developmental Plasticity"

Evan Ardiel, Catharine Rankin

[C.elegans Neuronal Development Meeting, 2008]

Environmental cues derived from conspecific s play an important role in mediating the developmental program of C. elegans. We previously showed that worms reared in isolation have a decreased response to mechanical stimulation, a down-regulation of pre- (snb-1) and post-synaptic (glr-1) markers in the mechanosensory neural circuit, a smaller body size, and a delayed onset of egg-laying (Rose et al., 2005). Non-localized mechanical stimulation delivered anytime during development was sufficient to rescue the effect of isolation on response to mechanical stimulation and glr-1 and snb-1 expression (Rai and Rankin, 2007). This suggests that tactile interactions with conspecifics impact neurodevelopment of the mechanosensory circuit. However, neither localized (sephadex beads on the agar) nor non-localized (taps) mechanical stimulation had any influence on body size, which we hypothesize is modulated by chemosensory cues from conspecifics. Sensory structure (che-3 and osm-3), signal transduction (osm-9 and tax-4), and cell specification (ceh-36) mutants were used to identify the subset of neurons through which the cues are sensed. Loss of TGF-beta signaling (dbl-1 and sma-6) disrupts the effect of isolation on body size, suggesting this is the pathway through which conspecific cues exert their influence.

Evan L Ardiel et al. (2007) International Worm Meeting "Developmental plasticity mediated by conspecific cues."

Susan Rai, Evan L Ardiel, Catharine H Rankin

[International Worm Meeting, 2007]

Environmental cues derived from conspecifics play an important role in mediating the developmental program of C. elegans. For example, high concentrations of dauer pheromone promote entry into larval diapause. Removal of conspecific cues also has profound impacts on worm biology. We previously showed that worms reared in isolation have a decreased response to mechanical stimulation, a down-regulation of pre- (snb-1) and post-synaptic (glr-1) markers in the mechanosensory neural circuit, a smaller body size, and a delayed onset of egg-laying (Rose et al., 2005). A small amount of non-localized mechanical stimulation delivered anytime during development was sufficient to rescue the effect of isolation on mechanical stimulation response and glr-1 expression (Rose et al., 2005). However, as the larvae aged, a greater amount of stimulation was required to rescue snb-1 expression (Rai & Rankin, 2007). This suggests that tactile interactions with conspecifics impact neurodevelopment of the mechanosensory circuit. However, neither localized (sephadex beads on the agar) nor non-localized (taps) mechanical stimulation had any influence on body size. The conspecific cue mediating body size may be specific conspecific mechanosensory stimulation, chemosensory stimuli, or an integration of the two. A smaller body size and delayed onset of egg-laying are indicative of delayed development. A hypothetical ecological explanation for more rapid development in colony worms is the selective pressure to produce progeny which can utilize an ephemeral food source. Rai, S. & Rankin, C.H. (2007) Critical and sensitive periods for reversing the effects of mechanosensory deprivation on behavior, nervous system and development in Caenorhabditis elegans. Developmental Neurobiology, in press. Rose, J. K., Sangha, S., Rai, S., Norman, K., & Rankin, C. H. (2005) Decreased sensory stimulation reduces behavioral responding, retards development and alters neuronal connectivity in Caenorhabditis elegans. Journal of Neuroscience, 25 (31), 7159-7168.

McDiarmid, Troy et al. (2015) International Worm Meeting "A glycine receptor subunit homologue, AVR-14, alters short-term memory in an interstimulus interval-dependant manner in C. elegans. ."

Rankin, Catharine, McDiarmid, Troy, Ardiel, Evan

[International Worm Meeting, 2015]

Habituation is a learned decrement in responding following repeated exposure to a stimulus. Despite its importance the mechanisms underlying habituation remain largely unknown. Repeated exposure to taps (non-localized mechanosensory stimulation) leads to habituation of a reversal withdrawal response in C. elegans that is dependent on glutamate transmission and postsynaptic AMPA receptors. Here we use high throughput behavioural analysis to characterize the role of AVR-14, an inhibitory glutamate gated chloride channel homologous to vertebrate glycine subunits that is expressed on the mechosensory neurons. Avr-14 loss of function mutants display a larger initial reversal duration in response to tap and faster habituation to tap stimuli than wild-type animals at a 10s interstimulus interval (ISI). At long ISIs (60s), avr-14 mutants habituated significantly less than wild-type animals. The stark contrast in phenotypes at short and long ISIs necessitated analysis of habituation across ISIs (10-60s ISIs). This revealed that mutations in avr-14 result in faster habituation at short ISIs, wild-type habituation at intermediate ISIs, and slower habituation and longer ISIs. Together these studies suggest mutations in avr-14 alter habituation in an ISI-dependent manner. Experiments using cell-specific knockdown, rescue, and stimulation will localize the memory functions of AVR-14 to elucidate how it modulates the tap habituation circuit. These studies will determine how the inhibitory functions of glutamate mediate short-term habituation in C. elegans, furthering our understanding of the processes underlying learning and memory. .

Yu, Alex et al. (2015) International Worm Meeting "The effects of two neuropeptide receptors on general arousal level in C. elegans."

Ardiel, Evan, Rankin, Catharine, Yu, Alex

[International Worm Meeting, 2015]

Habituation is a form of non-associative learning, the deficits of which have been associated with many psychiatric disorders. Animals are constantly bombarded with various stimuli in the environment, and habituation helps them to selectively attribute their attention, a limited resource. In C. elegans, a loss-of-function mutation in the AMPA ionotropic glutamate receptor subunit, glr-1, is known to disrupt habituation of ASH-mediated reversals. glr-1 loss of function mutants are unable to maintain their responses to ASH-stimulation towards the end of habituation training, and the latency of the response to a stimulus is longer. ASH sensory neurons detect aversive stimuli in the natural environment, such as osmotic pressure and chemical repellents that can harm or kill the worm. Therefore, normal functioning of these neurons is essential for the animal's survival. Recent studies from our lab found that the defective behavioural phenotype in ASH-mediated habituation caused by loss of glr-1 can be suppressed by loss of egl-3, which encodes a proprotein convertase; a loss-of-function egl-3 mutation knocks out neuropeptide synthesis. We are interested in identifying which neuropeptide signaling components cause the suppression of glr-1 behavioural phenotype. To investigate this, we conducted a systemic RNAi suppressor screen targeting 57 candidate genes, each encoding a known or predicted neuropeptide receptor. We observed suppressing effects in the proportion of animals responding with pdfr-1 knockdown, and in the latency of the initial response with npr-26 knockdown. pdfr-1 and npr-26 encode an ortholog of Drosophila pigment dispersing factor (PDF) receptor, and an ortholog of human urotensin-II receptor, respectively. We subsequently tested the behaviour of C. elegans with loss of function alleles of these two genes of interest. We found that pdfr-1 promotes dispersal behaviour following ASH-mediated habituation training, whereas npr-26 regulates the animal's arousal level, particularly affecting locomotion in the inter-stimulus interval. Our unexpected findings have led us to scrutinize the role of neuropeptides in general arousal level.

Hofmann ER et al. (2000) Cold Spring Harb Symp Quant Biol "DNA-damage-induced checkpoint pathways in the nematode Caenorhabditis elegans."

Milstein S, Hengartner MO, Hofmann ER

[Cold Spring Harb Symp Quant Biol, 2000]

Genomic instability is believed to be an enabling characteristic of cancer (Hanahan and Weinberg 2000). Therefore, it is not surprising that sophisticated mechanisms exist to maintain the integrity of the genome. Damage to DNA triggers checkpoint controls that result in cell cycle arrest and repair of the lesion (Nurse, 1997, 2000; Weinert 1998). In multicellular organisms, when DNA damage is extensive, these potentially harmful cells are eliminated by apoptosis (Enoch and Norbury 1995; Evan and Littlewood 1998)...

Yu, A. et al. (2017) International Worm Meeting "Non-associative learning as a behavioral strategy to promote dispersal."

Rankin, C., Yu, A., Ardiel, E.

[International Worm Meeting, 2017]

Habituation is a form of non-associative learning defined as decremented likelihood and/or magnitude in response to repeatedly presented stimuli. Habituation is thought to help animal ignore recurring innocuous events and free up its neural resources to attend to other novel or salient stimuli in the environment. C. elegans is known to habituate to harmless stimuli such as non-localized tap (Rankin et al., 1990). Recently, we have reported that worms also displayed behavioral characteristics of habituation when a pair of nociceptor neurons, ASH, were repeatedly stimulated with optogenetics (Ardiel et al., 2016). Because ASH detects natural aversive stimuli that are harmful and potentially lethal to worms, it seems puzzling that animals habituate to these noxious stimuli. Our analysis used a non-response-centric approach and found that habituation of ASH-mediated reversal responses are accompanied by the simultaneous sensitization of forward locomotion (Ardiel et al., 2017, unpublished). This behavioral plasticity was found to be mediated by the Pigment Dispersal Factor (PDF) signaling pathway. These coordinated changes in different behavioral components form an optimal behavioral strategy to guide animals to spend less time responding to the recurrent noxious stimuli and more time moving away from them. In this present study, we investigated whether tap habituation was associated with changes in other behavioral components. We observed similar patterns of behavioral plasticity in tap habituation that is also mediate by PDF signaling. Our findings suggest that different forms of non-associative learning participate in a suite of coordinated behavioral changes that together promote a shift in behavioral strategy. Our study also highlights the needs for more detailed behavioral analyses.