Luo, L., Gabel, C., Ha, H., Zhang, Y. & Samuel, A. Olfactory behavior of swimming C. elegans analyzed by measuring motile responses to temporal variations of odorants. Journal of Neurophysiology 99, 5, 2617-2625 (2008). Publisher's VersionAbstract
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.
Hulme, S., Shevkopylas, S. & Samuel, A. Microfluidics: streamlining discovery in worm biology. Nature Methods 5, 7, 589-590 (2008). Publisher's VersionAbstract
Advances in the application of microfluidics technology to biological assays using the model organism Caenorhabditis elegans help to automate otherwise time-consuming experiments.
Biron, D., Wasserman, S., Thomas, J., Samuel, A.D. & Sengupta, P. An olfactory neuron responds stochastically to temperature and modulates Caenorhabditis elegans thermotactic behavior. Proceedings of the National Academy of Sciences USA 105, 31, 11002-11007 (2008). Publisher's VersionAbstract
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.
Clark, D., Gabel, C., Lee, T. & Samuel, A. Short-Term Adaptation and Temporal Processing in the Cryophilic Response of Caenorhabditis elegans. Journal of Neurophysiology 97, 1903-1910 (2007). Publisher's VersionAbstract
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.
Clark, D., Gabel, C., Gabel, H. & Samuel, A.D. Temporal Activity Patterns in Thermosensory Neurons of Freely Moving Caenorhabditis elegans Encode Spatial Thermal Gradients. Journal of Neuroscience 27, 23, 6083– 6090 (2007). Publisher's VersionAbstract
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.
Korta, J., Clark, D.A., Gabel, C.V., Mahadevan, L. & Samuel, A.D. Mechanosensation and mechanical load modulate the locomotory gait of swimming C. elegans. Journal of Experimental Biology 210, 13, 2383-2389 (2007). Publisher's VersionAbstract
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.
Gabel, C., et al. Neural Circuits Mediate Electrosensory Behavior in Caenorhabditis elegans. Journal of Neuroscience 27, 28, 7586 –7596 (2007). Publisher's VersionAbstract
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.
Chi, C., et al. Temperature and food mediate long-term thermotactic behavioral plasticity by association-independent mechanisms in C. elegans. Journal of Experimental Biology 210, 22, 4043-4052 (2007). WebsiteAbstract
Thermotactic behavior in the nematode Caenorhabditis elegansexhibits long-term plasticity. On a spatial thermal gradient, C. elegans tracks isotherms near a remembered set-point(TS) corresponding to its previous cultivation temperature. When navigating at temperatures above its set-point(T>TS), C. elegans crawls down spatial thermal gradients towards the TS in what is called cryophilic movement. The TS 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 TSresetting 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.
Samuel, A.D.T., et al. Femtosecond laser dissection in C. elegans neural circuits. Commercial and Biomedical Applications of Ultrafast Lasers VI 6108, 1 – 6 (2006). Publisher's VersionAbstract
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.
Chung, S., Clark, D., Gabel, C., Mazur, E. & Samuel, A. The role of the AFD neuron in C. elegans thermotaxis analyzed using femtosecond laser ablation. BMC Neuroscience 7, 30 (2006). Publisher's VersionAbstract


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.


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.


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.

Clark, D., Biron, D., Sengupta, P. & Samuel, A. The AFD Sensory Neurons Encode Multiple Functions Underlying Thermotactic Behavior in Caenorhabditis elegans. Journal of Neuroscience 26, 28, 7444-7451 (2006). Publisher's VersionAbstract
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.
Biron, D., et al. A diacylglycerol kinase modulates long-term thermotactic behavioral plasticity in C. elegans. Nature Neuroscience 9, 12, 1499-1505 (2006). Publisher's VersionAbstract
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 (TS). The TS 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 TS 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 TS 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.
Luo, L., Clark, D., Biron, D., Mahadevan, L. & Samuel, A. Sensorimotor control during isothermal tracking in Caenorhabditis elegans. Journal of Experimental Biology 209, 23, 4652-4662 (2006). WebsiteAbstract
In order to purposefully navigate their environments, animals rely on precise coordination between their sensory and motor systems. The integrated performance of circuits for sensorimotor control may be analyzed by quantifying an animal's motile behavior in defined sensory environments. Here,we analyze the ability of the nematode C. elegans to crawl isothermally in spatial thermal gradients by quantifying the trajectories of individual worms responding to defined spatiotemporal thermal gradients. We show that sensorimotor control during isothermal tracking may be summarized as a strategy in which the worm changes the curvature of its propulsive undulations in response to temperature changes measured at its head. We show that a concise mathematical model for this strategy for sensorimotor control is consistent with the exquisite stability of the worm's isothermal alignment in spatial thermal gradients as well as its more complex trajectories in spatiotemporal thermal gradients.
Samuel, A. & Sengupta, P. Sensorimotor integration: locating locomotion in neural circuits. Current Biology 15, 9, R341-R343 (2005). Publisher's VersionAbstract
Neural components of the circuits that transform sensory cues into changes in motor activities are largely unknown. Several recent studies have now functionally mapped the sensorimotor circuits responsible for locomotion behaviors under defined environmental conditions in the nematode Caenorhabditis elegans.
Colosimo, M., et al. Identification of Thermosensory and Olfactory Neuron-Specific Genes via Expression Profiling of Single Neuron Types. Current Biology 14, 24, 2245-2251 (2004). Publisher's VersionAbstract
Most C. elegans sensory neuron types consist of a single bilateral pair of neurons, and respond to a unique set of sensory stimuli. Although genes required for the development and function of individual sensory neuron types have been identified in forward genetic screens, these approaches are unlikely to identify genes that when mutated result in subtle or pleiotropic phenotypes. Here, we describe a complementary approach to identify sensory neuron type-specific genes via microarray analysis using RNA from sorted AWB olfactory and AFD thermosensory neurons. The expression patterns of subsets of these genes were further verified in vivo. Genes identified by this analysis encode 7-transmembrane receptors, kinases, and nuclear factors including dac-1, which encodes a homolog of the highly conserved Dachshund protein [1]dac-1 is expressed in a subset of sensory neurons including the AFD neurons and is regulated by the TTX-1 OTX homeodomain protein [2]. On thermal gradients, dac-1 mutants fail to suppress a cryophilic drive but continue to track isotherms at the cultivation temperature, representing the first genetic separation of these AFD-mediated behaviors. Expression profiling of single neuron types provides a rapid, powerful, and unbiased method for identifying neuron-specific genes whose functions can then be investigated in vivo.
Samuel, A.D.T., Silva, R.A. & Murthy, V.N. Synaptic Activity of the AFD Neuron inCaenorhabditis elegans Correlates with Thermotactic Memory. Journal of Neuroscience 23, 2, 373–376 (2003). Publisher's VersionAbstract
Thermotactic behavior in Caenorhabditis elegans is sensitive to both a worm’s ambient temperature (Tamb) and its memory of the temperature of its cultivation (Tcult). The AFD neuron is part of a neural circuit that underlies thermotactic behavior. By monitoring the fluorescence of pH-sensitive green fluorescent protein localized to synaptic vesicles, we measured the rate of the synaptic release of AFD in worms cultivated at temperatures between 15 and 25°C, and subjected to fixed, ambient temperatures in the same range. We found that the rate of AFD synaptic release is high if either Tamb >Tcult orTamb <Tcult, but AFD synaptic release is low if Tamb ≅Tcult. This suggests that AFD encodes a direct comparison between Tamb andTcult.
Samuel, A.D.T., Murthy, V.N. & Hengartner, M.O. Calcium dynamics during fertilization in C. elegans. BMC Developmental Biology 1, 8 (2001). Publisher's VersionAbstract
Of the animals typically used to study fertilization-induced calcium dynamics, none is as accessible to genetics and molecular biology as the model organism Caenorhabditis elegans. Motivated by the experimental possibilities inherent in using such a well-established model organism, we have characterized fertilization-induced calcium dynamics in C. elegans.
Samuel, A.D.T., Peterson, J.D. & Reese, T.S. Envelope structure of Synechococcus sp. WH8113, a nonflagellated swimming cyanobacterium. BMC Microbiology 1, 4 (2001). Publisher's VersionAbstract
Many bacteria swim by rotating helical flagellar filaments [1]. Waterbury et al. [15] discovered an exception, strains of the cyanobacterium Synechococcus that swim without flagella or visible changes in shape. Other species of cyanobacteria glide on surfaces [2,7]. The hypothesis that Synechococcus might swim using traveling surface waves [6,13] prompted this investigation.
Turner, L., Samuel, A.D.T., Stern, A.S. & Berg, H.C. Temperature Dependence of Switching of the Bacterial Flagellar Motor by the Protein CheY13DK106YW. Biophysical Journal 77, 1, 597-603 (1999). Publisher's VersionAbstract
The behavior of the bacterium Escherichia coli is controlled by switching of the flagellar rotary motor between the two rotational states, clockwise (CW) and counterclockwise (CCW). The molecular mechanism for switching remains unknown, but binding of the response regulator CheY-P to the motor component FliM enhances CW rotation. This effect is mimicked by the unphosphorylated double mutant CheY13DK106YW (CheY**). To learn more about switching, we measured the fraction of time that a motor spends in the CW state (the CW bias) at different concentrations of CheY** and at different temperatures. From the CW bias, we computed the standard free energy change of switching. In the absence of CheY, this free energy change is a linear function of temperature (Turner et al., 1996. Biophys. J. 71:2227–2233). In the presence of CheY**, it is nonlinear. However, the data can be fit by models in which binding of each molecule of CheY** shifts the difference in free energy between CW and CCW states by a fixed amount. The shift increases linearly from ∼0.3kT per molecule at 5°C to ∼0.9kT at 25°C, where k is Boltzmann's constant and T is 289 Kelvin (= 16°C). The entropy and enthalpy contributions to this shift are about −0.031kT/°C and 0.10kT, respectively.
Samuel, A.D.T., et al. Flagellar determinants of bacterial sensitivity to χ-phage. Proceedings of the National Academy of Sciences 96, 17, 9863–9866 (1999). Publisher's VersionAbstract
Bacteriophage χ is known to infect motile strains of enteric bacteria by adsorbing randomly along the length of a flagellar filament and then injecting its DNA into the bacterial cell at the filament base. Here, we provide evidence for a “nut and bolt” model for translocation of phage along the filament: the tail fiber of χ fits the grooves formed by helical rows of flagellin monomers, and active flagellar rotation forces the phage to follow the grooves as a nut follows the threads of a bolt.