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.

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.