To navigate different environments, an animal must be able to adapt its locomotory gait to its physical surroundings. The nematode Caenorhabditis elegans, between swimming in water and crawling on surfaces, adapts its locomotory gait to surroundings that impose approximately 10,000-fold differences in mechanical resistance. Here we investigate this feat by studying the undulatory movements of C. elegans in Newtonian fluids spanning nearly five orders of magnitude in viscosity. In these fluids, the worm undulatory gait varies continuously with changes in external load: As load increases, both wavelength and frequency of undulation decrease. We also quantify the internal viscoelastic properties of the worm’s body and their role in locomotory dynamics. We incorporate muscle activity, internal load, and external load into a biomechanical model of locomotion and show that (i) muscle power is nearly constant across changes in locomotory gait, and (ii) the onset of gait adaptation occurs as external load becomes comparable to internal load. During the swimming gait, which is evoked by small external loads, muscle power is primarily devoted to bending the worm’s elastic body. During the crawling gait, evoked by large external loads, comparable muscle power is used to drive the external load and the elastic body. Our results suggest that C. elegans locomotory gait continuously adapts to external mechanical load in order to maintain propulsive thrust.

A mechanistic understanding of animal navigation requires quantitative assessment of the sensorimotor strategies used during navigation and quantitative assessment of how these strategies are regulated by cellular sensors. Here, we examine thermotactic behavior of the Drosophila melanogaster larva using a tracking microscope to study individual larval movements on defined temperature gradients. We discover that larval thermotaxis involves a larger repertoire of strategies than navigation in smaller organisms such as motile bacteria and Caenorhabditis elegans. Beyond regulating run length (i.e., biasing a random walk), the Drosophila melanogaster larva also regulates the size and direction of turns to achieve and maintain favorable orientations. Thus, the sharp turns in a larva's trajectory represent decision points for selecting new directions of forward movement. The larva uses the same strategies to move up temperature gradients during positive thermotaxis and to move down temperature gradients during negative thermotaxis. Disrupting positive thermotaxis by inactivating cold-sensitive neurons in the larva's terminal organ weakens all regulation of turning decisions, suggesting that information from one set of temperature sensors is used to regulate all aspects of turning decisions. The Drosophila melanogaster larva performs thermotaxis by biasing stochastic turning decisions on the basis of temporal variations in thermosensory input, thereby augmenting the likelihood of heading toward favorable temperatures at all times.

We present an inexpensive apparatus for bright field and fluorescence microscopy with video capture, suitable for introductory laboratory courses. Experiments on Brownian motion and the Boltzmann distribution of suspended particles in a gravitational field are described. The Boltzmann constant is measured in three ways, and the results fall within 15% of the accepted value.

The nematode Caenorhabditis elegans is an excellent model organism for a systems-level understanding of neural circuits and behavior. Advances in the quantitative analyses of behavior and neuronal activity, and the development of new technologies to precisely control and monitor the workings of interconnected circuits, now allow investigations into the molecular, cellular, and systems-level strategies that transform sensory inputs into precise behavioral outcomes.

Caenorhabditis elegans is a filter feeder: it draws bacteria suspended in liquid into its pharynx, traps the bacteria, and ejects the liquid. How pharyngeal pumping simultaneously transports and filters food particles has been poorly understood. Here, we use high-speed video microscopy to define the detailed workings of pharyngeal mechanics. The buccal cavity and metastomal flaps regulate the flow of dense bacterial suspensions and exclude excessively large particles from entering the pharynx. A complex sequence of contractions and relaxations transports food particles in two successive trap stages before passage into the terminal bulb and intestine. Filtering occurs at each trap as bacteria are concentrated in the central lumen while fluids are expelled radially through three apical channels. Experiments with microspheres show that the C. elegans pharynx, in combination with the buccal cavity, is tuned to specifically catch and transport particles of a size range corresponding to most soil bacteria.

Summary 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.

The molecular and cellular mechanisms that allow adult-stage neurons to regenerate following damage are poorly understood. Recently, axons of motoneurons and mechanosensory neurons in adult C. elegans were found to regrow after being snipped by femtosecond laser ablation. Here, we explore the molecular determinants of adult-stage axon regeneration using the AVM mechanosensory neurons. The first step in AVM axon development is a pioneer axonal projection from the cell body to the ventral nerve cord. We show that regeneration of the AVM axon to the ventral nerve cord lacks the deterministic precision of initial axon development, requiring competition and pruning of unwanted axon branches. Nevertheless, axons of injured AVM neurons regrow to the ventral nerve cord with over 60% reliability in adult animals. In addition, in contrast to initial development, axon guidance during regeneration becomes heavily dependent on cytoplasmic protein MIG-10/Lamellipodin but independent of UNC-129/TGF-β repellent and UNC-40/DCC receptor, and axon growth during regeneration becomes heavily dependent on UNC-34/Ena and CED-10/Rac actin regulators. Thus, C. elegans may be used as a genetic system to characterize novel cellular and molecular mechanisms underlying adult-stage nervous system regeneration.

Caenorhabditis elegans responds to chemical cues using a small number of chemosensory neurons that detect a large variety of molecules in its environment. During chemotaxis, C. elegans biases its migration in spatial chemical gradients by lengthening (/shortening) periods of forward movement when it happens to be moving toward (/away) from preferred locations. In classical assays of chemotactic behavior, a group of crawling worms is placed on an agar plate containing a point source of chemical, the group is allowed to navigate for a period of time, and aggregation of worms near the source is quantified. Here we show that swimming worms exhibit acute motile responses to temporal variations of odor in their surrounding environment, allowing our development of an automated assay of chemotactic behavior with single-animal resolution. By placing individual worms in small microdroplets and quantifying their movements as they respond to the addition and removal of odorized airstreams, we show that the sensorimotor phenotypes of swimming worms (wild-type behavior, the effects of certain mutations, and the effects of laser ablation of specific olfactory neurons) are consistent with aggregation phenotypes previously obtained in crawling assays. The microdroplet swimming assay has certain advantages over crawling assays, including flexibility and precision in defining the stimulus waveform and automated quantification of motor response during stimulus presentation. In this study, we use the microdroplet assay to quantify the temporal dynamics of the olfactory response, the sensitivity to odorant concentration, combinations, and gradients, and the contribution of specific olfactory neurons to overall behavior.

Advances in the application of microfluidics technology to biological assays using the model organism Caenorhabditis elegans help to automate otherwise time-consuming experiments.

Caenorhabditis elegans navigates thermal gradients by using a behavioral strategy that is regulated by a memory of its cultivation temperature (T _c). At temperatures above or around the T _c, animals respond to temperature changes by modulating the rate of stochastic reorientation events. The bilateral AFD neurons have been implicated as thermosensory neurons, but additional thermosensory neurons are also predicted to play a role in regulating thermotactic behaviors. Here, we show that the AWC olfactory neurons respond to temperature. Unlike AFD neurons, which respond to thermal stimuli with continuous, graded calcium signals, AWC neurons exhibit stochastic calcium events whose frequency is stimulus-correlated in a T _c-dependent manner. Animals lacking the AWC neurons or with hyperactive AWC neurons exhibit defects in the regulation of reorientation rate in thermotactic behavior. Our observations suggest that the AFD and AWC neurons encode thermal stimuli via distinct strategies to regulate C. elegans thermotactic behavior.

When navigating spatial thermal gradients, the nematode C. elegans migrates toward colder temperatures until it reaches its previous cultivation temperature, exhibiting cryophilic movement. The strategy for effecting cryophilic movement is the biased random walk: C. elegans extends (shortens) periods of forward movement that are directed down (up) spatial thermal gradients by modulating the probability of reorientation. Here, we analyze the temporal sensory processor that enables cryophilic movement by quantifying the movements of individual worms subjected to defined temperature waveforms. We show that step increases in temperature as small as 0.05°C lead to transient increases in the probability of reorientation followed by gradual adaptation to the baseline level; temperature downsteps leads to similar but inverted responses. Short-term adaptation is a general property of sensory systems, allowing organisms to maintain sensitivity to sensory variations over broad operating ranges. During cryophilic movement C. elegans also uses the temporal dynamics of its adaptive response to compute the time derivative of gradual temperature variations with exquisite sensitivity. On the basis of the time derivative, the worm determines how it is oriented in spatial thermal gradients during each period of forward movement. We show that the operating range of the cryophilic response extends to lower temperatures in ttx-3 mutants, which affects the development of the AIY interneurons. We show that the temporal sensory processor for the cryophilic response is affected by mutation in the EAT-4 glutamate vesicular transporter. Regulating the operating range of the cryophilic response and executing the cryophilic response may have separate neural mechanisms.

Our understanding of the operation of neurons and neuronal circuits has come primarily from probing their activity in dissected, anesthetized, or restrained animals. However, the behaviorally relevant operation of neurons and neuronal circuits occurs within intact animals as they freely perform behavioral tasks. The small size and transparency of the nematode Caenorhabditis elegans make it an ideal system for noninvasive, optical measurements of neuronal activity. Here, we use a high signal-to-noise version of cameleon, a fluorescent calcium-binding protein, to quantify the activity of the AFD thermosensory neuron of individual worms freely navigating spatial thermal gradients. We find that AFD activity is directly coupled to the worm's exploratory movements in spatial thermal gradients. We show that the worm is able, in principle, to evaluate and guide its own thermotactic behaviors with respect to ambient spatial thermal gradients by monitoring the activity of this single thermosensory neuron.

Animals move through their environments by selecting gaits that are adapted to the physical nature of their surroundings. The nematode Caenorhabditis elegans swims through fluids or crawls on surfaces by propagating flexural waves along its slender body and offers a unique opportunity for detailed analysis of locomotory gait at multiple levels including kinematics,biomechanics and the molecular and physiological operation of sensory and motor systems. Here, we study the swimming gait of C. elegans in viscous fluids in the range 0.05-50 Pa s. We find that the spatial form of the swimming gait does not vary across this range of viscosities and that the temporal frequency of the swimming gait only decreases by about 20% with every 10-fold increase in viscosity. Thus, C. elegans swims in low gear,such that its musculature can deliver mechanical force and power nearly 1000-fold higher than it delivers when swimming in water. We find that mutations that disrupt mechanosensation, or the laser killing of specific touch receptor neurons, increase the temporal frequency of the undulating gait, revealing a novel effect of mechanosensory input in regulating the putative central pattern generator that produces locomotion. The adaptability of locomotory gait in C. elegans may be encoded in sensory and motor systems that allow the worm to respond to its own movement in different physical surroundings.

The nematode Caenorhabditis elegans deliberately crawls toward the negative pole in an electric field. By quantifying the movements of individual worms navigating electric fields, we show that C. elegans prefers to crawl at specific angles to the direction of the electric field in persistent periods of forward movement and that the preferred angle is proportional to field strength. C. elegans reorients itself in response to time-varying electric fields by using sudden turns and reversals, standard reorientation maneuvers that C. elegans uses during other modes of motile behavior. Mutation or laser ablation that disrupts the structure and function of amphid sensory neurons also disrupts electrosensory behavior. By imaging intracellular calcium dynamics among the amphid sensory neurons of immobilized worms, we show that specific amphid sensory neurons are sensitive to the direction and strength of electric fields. We extend our analysis to the motor level by showing that specific interneurons affect the utilization of sudden turns and reversals during electrosensory steering. Thus, electrosensory behavior may be used as a model system for understanding how sensory inputs are transformed into motor outputs by the C. elegans nervous system.

Thermotactic behavior in the nematode Caenorhabditis elegansexhibits long-term plasticity. On a spatial thermal gradient, C. elegans tracks isotherms near a remembered set-point(T_S) corresponding to its previous cultivation temperature. When navigating at temperatures above its set-point(T>T_S), C. elegans crawls down spatial thermal gradients towards the T_S in what is called cryophilic movement. The T_S retains plasticity in the adult stage and is reset by ∼4 h of sustained exposure to a new temperature. Long-term plasticity in C. elegans thermotactic behavior has been proposed to represent an associative learning of specific temperatures conditioned in the presence or absence of bacterial food. Here,we use quantitative behavioral assays to define the temperature and food-dependent determinants of long-term plasticity in the different modes of thermotactic behavior. Under our experimental conditions, we find that starvation at a specific temperature neither disrupts T_Sresetting toward the starvation temperature nor induces learned avoidance of the starvation temperature. We find that prolonged starvation suppresses the cryophilic mode of thermotactic behavior. The hen-1 and tax-6 genes have been reported to affect associative learning between temperature and food-dependent cues. Under our experimental conditions,mutation in the hen-1 gene, which encodes a secreted protein with an LDL receptor motif, does not significantly affect thermotactic behavior or long-term plasticity. Mutation in the tax-6 calcineurin gene abolishes thermotactic behavior altogether. In summary, we do not find evidence that long-term plasticity requires association between temperature and the presence or absence of bacterial food.

The nematode C. elegans, a millimeter-long roundworm, is a well-established model organism for studies of neural development and behavior, however physiological methods to manipulate and monitor the activity of its neural network have lagged behind the development of powerful methods in genetics and molecular biology. The small size and transparency of C. elegans make the worm an ideal test-bed for the development of physiological methods derived from optics and microscopy. We present the development and application of a new physiological tool: femtosecond laser dissection, which allows us to selectively ablate segments of individual neural fibers within live C. elegans. Femtosecond laser dissection provides a scalpel with submicrometer resolution, and we discuss its application in studies of neural growth, regenerative growth, and the neural basis of behavior.

Background

Caenorhabditis elegans actively crawls down thermal gradients until it reaches the temperature of its prior cultivation, exhibiting what is called cryophilic movement. Implicit in the worm's performance of cryophilic movement is the ability to detect thermal gradients, and implicit in regulating the performance of cryophilic movement is the ability to compare the current temperature of its surroundings with a stored memory of its cultivation temperature. Several lines of evidence link the AFD sensory neuron to thermotactic behavior, but its precise role is unclear. A current model contends that AFD is part of a thermophilic mechanism for biasing the worm's movement up gradients that counterbalances the cryophilic mechanism for biasing its movement down gradients.

Results

We used tightly-focused femtosecond laser pulses to dissect the AFD neuronal cell bodies and the AFD sensory dendrites in C. elegans to investigate their contribution to cryophilic movement. We establish that femtosecond laser ablation can exhibit submicrometer precision, severing individual sensory dendrites without causing collateral damage. We show that severing the dendrites of sensory neurons in young adult worms permanently abolishes their sensory contribution without functional regeneration. We show that the AFD neuron regulates a mechanism for generating cryophilic bias, but we find no evidence that AFD laser surgery reduces a putative ability to generate thermophilic bias. In addition, although disruption of the AIY interneuron causes worms to exhibit cryophilic bias at all temperatures, we find no evidence that laser killing the AIZ interneuron causes thermophilic bias at any temperature.

Conclusion

We conclude that laser surgical analysis of the neural circuit for thermotaxis does not support a model in which AFD opposes cryophilic bias by generating thermophilic bias. Our data supports a model in which the AFD neuron gates a mechanism for generating cryophilic bias.

The thermotactic behaviors of Caenorhabditis elegans indicate that its thermosensory system exhibits exquisite temperature sensitivity, long-term plasticity, and the ability to transform thermosensory input into different patterns of motor output. Here, we study the physiological role of the AFD thermosensory neurons by quantifying intracellular calcium dynamics in response to defined temperature stimuli. We demonstrate that short-term adaptation allows AFD to sense temperature changes as small as 0.05°C over temperature ranges as wide as 10°C. We show that a bidirectional thermosensory response (increasing temperature raises and decreasing temperature lowers the level of intracellular calcium in AFD) allows the AFD neurons to phase-lock their calcium dynamics to oscillatory thermosensory inputs. By analyzing the thermosensory response of AFD dendrites severed from their cell bodies by femtosecond laser ablation, we show that long-term plasticity is encoded as shifts in the operating range of a putative thermoreceptor(s) in the AFD sensory endings. Finally, we demonstrate that AFD activity is directly coupled to stimulation of its postsynaptic partner AIY. These observations indicate that many functions underlying thermotactic behavior are properties of one sensory neuronal type. Encoding multiple functions in individual sensory neurons may enable C. elegans to perform complex behaviors with simple neuronal circuits.

A memory of prior thermal experience governs Caenorhabditis elegans thermotactic behavior. On a spatial thermal gradient, C. elegans tracks isotherms near a remembered temperature we call the thermotactic set-point (T_S). The T_S corresponds to the previous cultivation temperature and can be reset by sustained exposure to a new temperature. The mechanisms underlying this behavioral plasticity are unknown, partly because sensory and experience-dependent components of thermotactic behavior have been difficult to separate. Using newly developed quantitative behavioral analyses, we demonstrate that the T_S represents a weighted average of a worm's temperature history. We identify the DGK-3 diacylglycerol kinase as a thermal memory molecule that regulates the rate of T_S resetting by modulating the temperature range of synaptic output, but not temperature sensitivity, of the AFD thermosensory neurons. These results provide the first mechanistic insight into the basis of experience-dependent plasticity in this complex behavior.