Rex Kerr

Optical Imaging of Calcium Transients in Neurons and Pharyngeal Muscle of C. elegans

Electrophysiology and optical indicators have been used in vertebrate systems to investigate excitable cell firing and calcium transients, but both techniques have been difficult to apply in organisms with powerful reverse genetics. To overcome this limitation, we expressed cameleon proteins, genetically encoded calcium indicators, in the pharyngeal muscle of the nematode worm Caenorhabditis elegans. In intact transgenic animals expressing cameleons, fluorescence ratio changes accompanied muscular contraction, verifying detection of calcium transients. By comparing the magnitude and duration of calcium influx in wild-type and mutant animals, we were able to determine the effects of calcium channel proteins on pharyngeal calcium transients. We also successfully used cameleons to detect electrically evoked calcium transients in individual C. elegans neurons. This technique therefore should have broad applications in analyzing the regulation of excitable cell activity in genetically tractable organisms.

In Vivo Imaging of C. elegans Mechanosensory Neurons Demonstrates a Specific Role for the MEC-4 Channel in the Process of Gentle Touch Sensation

In the nematode C. elegans, genes encoding components of a putative mechanotransducing channel complex have been identified in screens for light-touch-insensitive mutants. A long-standing question, however, is whether identified MEC proteins act directly in touch transduction or contribute indirectly by maintaining basic mechanoreceptor neuron physiology. In this study, we used the genetically encoded calcium indicator cameleon to record cellular responses of mechanosensory neurons to touch stimuli in intact, behaving nematodes. We defined a gentle touch sensory modality that adapts with a time course of approximately 500 ms and primarily senses motion rather than pressure. The DEG/ENaC channel subunit MEC-4 and channel-associated stomatin MEC-2 are specifically required for neural responses to gentle mechanical stimulation, but do not affect the basic physiology of touch neurons or their in vivo responses to harsh mechanical stimulation. These results distinguish a specific role for the MEC channel proteins in the process of gentle touch mechanosensation.

In vivo imaging of C. elegans ASH neurons: cellular response and adaptation to chemical repellents

ASH sensory neurons are required in Caenorhabditis elegans for a wide range of avoidance behaviors in response to chemical repellents, high osmotic solutions and nose touch. The ASH neurons are therefore hypothesized to be polymodal nociceptive neurons. To understand the nature of polymodal sensory response and adaptation at the cellular level, we expressed the calcium indicator protein cameleon in ASH and analyzed intracellular Ca2+ responses following stimulation with chemical repellents, osmotic shock and nose touch. We found that a variety of noxious stimuli evoked strong responses in ASH including quinine, denatonium, detergents, heavy metals, both hyper‐ and hypo‐osmotic shock and nose touch. We observed that repeated chemical stimulation led to a reversible reduction in the magnitude of the sensory response, indicating that adaptation occurs within the ASH sensory neuron. A key component of ASH adaptation is GPC‐1, a G‐protein γ‐subunit expressed specifically in chemosensory neurons. We hypothesize that G‐protein γ‐subunit heterogeneity provides a mechanism for repellent‐specific adaptation, which could facilitate discrimination of a variety of repellents by these polymodal sensory neurons.

Fast Monte Carlo Simulation Methods for Biological Reaction-Diffusion Systems in Solution and on Surfaces

Many important physiological processes operate at time and space scales far beyond those accessible to atom-realistic simulations, and yet discrete stochastic rather than continuum methods may best represent finite numbers of molecules interacting in complex cellular spaces. We describe and validate new tools and algorithms developed for a new version of the MCell simulation program (MCell3), which supports generalized Monte Carlo modeling of diffusion and chemical reaction in solution, on surfaces representing membranes, and combinations thereof. A new syntax for describing the spatial directionality of surface reactions is introduced, along with optimizations and algorithms that can substantially reduce computational costs (e.g., event scheduling, variable time and space steps). Examples for simple reactions in simple spaces are validated by comparison to analytic solutions. Thus we show how spatially realistic Monte Carlo simulations of biological systems can be far more cost-effective than often is assumed, and provide a level of accuracy and insight beyond that of continuum methods.

Serotonin and Go Modulate Functional States of Neurons and Muscles Controlling C. elegans Egg-Laying Behavior

From nematodes to humans, animals employ neuromodulators like serotonin to regulate behavioral patterns and states. In the nematode C. elegans, serotonin has been shown to act in a modulatory fashion to increase the rate and alter the temporal pattern of egg laying. Though many candidate effectors and regulators of serotonin have been identified in genetic studies, their effects on specific neurons and muscles in the egg-laying circuitry have been difficult to determine. Using the genetically encoded Ca(2+) indicator cameleon, we found that serotonin acts directly on the vulval muscles to increase the frequency of Ca(2+) transients. In contrast, we found that the spontaneous activity of the egg-laying motorneurons was silenced by serotonin. Mutations in G protein alpha subunit genes altered the responses of both vulval muscles and egg-laying neurons to serotonin; specifically, mutations in the G(q)alpha homolog egl-30 blocked serotonin stimulation of vulval muscle Ca(2+) transients, while mutations in the G(o)alpha homolog goa-1 prevented the silencing of motorneuron activity by serotonin. These data indicate that serotonin stimulates egg laying by directly modulating the functional state of the vulval muscles. In addition, serotonin inhibits the activity of the motorneurons that release it, providing a feedback regulatory effect that may contribute to serotonin adaptation.

Rapid atomic density methods for molecular shape characterization.

Two methods for rapid characterization of molecular shape are presented. Both techniques are based on the density of atoms near the molecular surface. The Fast Atomic Density Evaluation (FADE) algorithm uses fast Fourier transforms to quickly estimate densities. The Pairwise Atomic Density Reverse Engineering (PADRE) method derives modified density measures from the relationship between atomic density and total potentials. While many shape-characterization techniques define shape relative to a surface, the descriptors returned by FADE and PADRE can measure local geometry from points within the three-dimensional space surrounding a molecule. The methods can be used to find crevices and protrusions near the surface of a molecule and to test shape complementarity at the interface between docking molecules.

A Self-Regulating Feed-Forward Circuit Controlling C. elegans Egg-Laying Behavior

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.

Intracellular Ca 2+ Imaging in C. elegans

Optical methods provide a noninvasive way to monitor calcium transients in Caenorhabditis elegans. Imaging techniques are particularly appealing in C. elegans because worms are optically transparent and can be imaged while fully intact. Furthermore, a variety of genetically encoded calcium indicators are available that can be targeted to cells of interest with appropriate tissue-specific promoters. Here, we describe a specific protocol, suitable for monitoring neuronal activity, for rapid calcium imaging in C. elegans using the cameleon indicator. Notes are provided to assist with adapting this protocol for use with other indicators and slower time scales.

A consistent muscle activation strategy underlies crawling and swimming in Caenorhabditis elegans

Although undulatory swimming is observed in many organisms, the neuromuscular basis for undulatory movement patterns is not well understood. To better understand the basis for the generation of these movement patterns, we studied muscle activity in the nematode Caenorhabditis elegans. Caenorhabditis elegans exhibits a range of locomotion patterns: in low viscosity fluids the undulation has a wavelength longer than the body and propagates rapidly, while in high viscosity fluids or on agar media the undulatory waves are shorter and slower. Theoretical treatment of observed behaviour has suggested a large change in force–posture relationships at different viscosities, but analysis of bend propagation suggests that short-range proprioceptive feedback is used to control and generate body bends. How muscles could be activated in a way consistent with both these results is unclear. We therefore combined automated worm tracking with calcium imaging to determine muscle activation strategy in a variety of external substrates. Remarkably, we observed that across locomotion patterns spanning a threefold change in wavelength, peak muscle activation occurs approximately 45° (1/8th of a cycle) ahead of peak midline curvature. Although the location of peak force is predicted to vary widely, the activation pattern is consistent with required force in a model incorporating putative length- and velocity-dependence of muscle strength. Furthermore, a linear combination of local curvature and velocity can match the pattern of activation. This suggests that proprioception can enable the worm to swim effectively while working within the limitations of muscle biomechanics and neural control.

Tracking Movement Behavior of Multiple Worms on Food

Neurobiological research in genetically tractable organisms relies heavily on robust assays for behavioral phenotypes. The simple body plan of the nematode Caenorhabditis elegans makes it particularly amenable to the use of automated microscopy and image analysis to describe behavioral patterns quantitatively. Forward genetic screens and screens of drug libraries require high-throughput phenotyping, a task traditionally incompatible with manual scoring of quantitatively varying behaviors. High-throughput automated analysis of C. elegans movement behavior is now possible with several different tracking software packages. The Multiworm Tracker (MWT) described here is designed for high-throughput analysis: it can record dozens of worms simultaneously at 30 frames per second for hours or days at a time. This is accomplished by performing all image analysis in real time, saving only the worm centroid, bearing, and outline data to the disk. To simplify image processing, the system focuses only on worms that have moved, and detects and discards worms that are touching rather than trying to isolate them computationally. Because the software is entirely automated, protocols can run unattended once the worms have been placed and the software has been started. The MWT does not save images for later analysis, but behavior can be validated manually with a companion analysis tool that replays recorded body postures. This protocol describes a basic basal movement assay on food using the MWT; similar protocols apply to related assays and to similar multiple animal trackers. The protocol can be extended to a variety of assays ranging from tap response to chemotaxis.