Aravinthan D. T. Samuel

Optogenetic manipulation of neural activity in freely moving Caenorhabditis elegans

We present an optogenetic illumination system capable of real-time light delivery with high spatial resolution to specified targets in freely moving Caenorhabditis elegans. A tracking microscope records the motion of an unrestrained worm expressing channelrhodopsin-2 or halorhodopsin in specific cell types. Image processing software analyzes the worms position in each video frame, rapidly estimates the locations of targeted cells and instructs a digital micromirror device to illuminate targeted cells with laser light of the appropriate wavelengths to stimulate or inhibit activity. Because each cell in an unrestrained worm is a rapidly moving target, our system operates at high speed (∼50 frames per second) to provide high spatial resolution (∼30 μm). To test the accuracy, flexibility and utility of our system, we performed optogenetic analyses of the worm motor circuit, egg-laying circuit and mechanosensory circuits that have not been possible with previous methods.

Identification of thermosensory and olfactory neuron-specific genes via expression profiling of single neuron types

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. dac-1 is expressed in a subset of sensory neurons including the AFD neurons and is regulated by the TTX-1 OTX homeodomain protein. 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.

Running hot and cold: behavioral strategies, neural circuits, and the molecular machinery for thermotaxis in C. elegans and Drosophila

Like other ectotherms, the roundworm Caenorhabditis elegans and the fruit fly Drosophila melanogaster rely on behavioral strategies to stabilize their body temperature. Both animals use specialized sensory neurons to detect small changes in temperature, and the activity of these thermosensors governs the neural circuits that control migration and accumulation at preferred temperatures. Despite these similarities, the underlying molecular, neuronal, and computational mechanisms responsible for thermotaxis are distinct in these organisms. Here, we discuss the role of thermosensation in the development and survival of C. elegans and Drosophila, and review the behavioral strategies, neuronal circuits, and molecular networks responsible for thermotaxis behavior.

Biomechanical analysis of gait adaptation in the nematode Caenorhabditis elegans.

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.

The role of the AFD neuron in C. elegans thermotaxis analyzed using femtosecond laser ablation

BackgroundCaenorhabditis elegans actively crawls down thermal gradients until it reaches the temperature of its prior cultivation, exhibiting what is called cryophilic movement. Implicit in the worms 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 worms movement up gradients that counterbalances the cryophilic mechanism for biasing its movement down gradients.ResultsWe 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.ConclusionWe 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.

An olfactory neuron responds stochastically to temperature and modulates Caenorhabditis elegans thermotactic behavior

Caenorhabditis elegans navigates thermal gradients by using a behavioral strategy that is regulated by a memory of its cultivation temperature (Tc). At temperatures above or around the Tc, 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 Tc-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.

Mechanosensation and mechanical load modulate the locomotory gait of swimming C. elegans

SUMMARY 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.

Distinct cellular and molecular mechanisms mediate initial axon development and adult-stage axon regeneration in C. elegans

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.

Functional Organization of a Neural Network for Aversive Olfactory Learning in Caenorhabditis elegans

Many animals use their olfactory systems to learn to avoid dangers, but how neural circuits encode naive and learned olfactory preferences, and switch between those preferences, is poorly understood. Here, we map an olfactory network, from sensory input to motor output, which regulates the learned olfactory aversion of Caenorhabditis elegans for the smell of pathogenic bacteria. Naive animals prefer smells of pathogens but animals trained with pathogens lose this attraction. We find that two different neural circuits subserve these preferences, with one required for the naive preference and the other specifically for the learned preference. Calcium imaging and behavioral analysis reveal that the naive preference reflects the direct transduction of the activity of olfactory sensory neurons into motor response, whereas the learned preference involves modulations to signal transduction to downstream neurons to alter motor response. Thus, two different neural circuits regulate a behavioral switch between naive and learned olfactory preferences.

Temporal Activity Patterns in Thermosensory Neurons of Freely Moving Caenorhabditis elegans Encode Spatial Thermal Gradients

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 worms 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.