David M. Raizen

Lethargus is a Caenorhabditis elegans sleep-like state

There are fundamental similarities between sleep in mammals and quiescence in the arthropod Drosophila melanogaster, suggesting that sleep-like states are evolutionarily ancient. The nematode Caenorhabditis elegans also has a quiescent behavioural state during a period called lethargus, which occurs before each of the four moults. Like sleep, lethargus maintains a constant temporal relationship with the expression of the C. elegans Period homologue LIN-42 (ref. 5). Here we show that quiescence associated with lethargus has the additional sleep-like properties of reversibility, reduced responsiveness and homeostasis. We identify the cGMP-dependent protein kinase (PKG) gene egl-4 as a regulator of sleep-like behaviour, and show that egl-4 functions in sensory neurons to promote the C. elegans sleep-like state. Conserved effects on sleep-like behaviour of homologous genes in C. elegans and Drosophila suggest a common genetic regulation of sleep-like states in arthropods and nematodes. Our results indicate that C. elegans is a suitable model system for the study of sleep regulation. The association of this C. elegans sleep-like state with developmental changes that occur with larval moults suggests that sleep may have evolved to allow for developmental changes.

Electrical activity and behavior in the pharynx of caenorhabditis elegans

The pharynx of C. elegans, a model system for neural networks and for membrane excitability, has been chiefly studied by observing its behavior in normal worms, in mutant worms, and in worms lacking pharyngeal neurons. To complement this behavioral approach, we devised a method for recording currents produced by changes in pharyngeal muscle membrane potential. The electrical records, called electropharyngeograms, contain transients caused by pharyngeal muscle action potentials and by inhibitory synaptic transmission between pharyngeal neuron M3 and the muscle. Using the electropharyngeograms, we show that gamma-aminobutyric acid is not likely to be the M3 neurotransmitter, that synaptic transmission is present but abnormal in mutants lacking synaptotagmin, and that worms mutant in the eat-4 gene are defective for M3 function or transmission.

Insulin, cGMP, and TGF-β Signals Regulate Food Intake and Quiescence in C. elegans: A Model for Satiety

Despite the prevalence of obesity and its related diseases, the signaling pathways for appetite control and satiety are not clearly understood. Here we report C. elegans quiescence behavior, a cessation of food intake and movement that is possibly a result of satiety. C. elegans quiescence shares several characteristics of satiety in mammals. It is induced by high-quality food, it requires nutritional signals from the intestine, and it depends on prior feeding history: fasting enhances quiescence after refeeding. During refeeding after fasting, quiescence is evoked, causing gradual inhibition of food intake and movement, mimicking the behavioral sequence of satiety in mammals. Based on these similarities, we propose that quiescence results from satiety. This hypothesized satiety-induced quiescence is regulated by peptide signals such as insulin and TGF-beta. The EGL-4 cGMP-dependent protein kinase functions downstream of insulin and TGF-beta in sensory neurons including ASI to control quiescence in response to food intake.

eat-2 and eat-18 Are Required for Nicotinic Neurotransmission in the Caenorhabditis elegans Pharynx

Mutations in eat-2 and eat-18 cause the same defect in C. elegans feeding behavior: the pharynx is unable to pump rapidly in the presence of food. EAT-2 is a nicotinic acetylcholine receptor subunit that functions in the pharyngeal muscle. It is localized to the synapse between pharyngeal muscle and the main pharyngeal excitatory motor neuron MC, and it is required for MC stimulation of pharyngeal muscle. eat-18 encodes a small protein that has no homology to previously characterized proteins. It has a single transmembrane domain and a short extracellular region. Allele-specific genetic interactions between eat-2 and eat-18 suggest that EAT-18 interacts physically with the EAT-2 receptor. While eat-2 appears to be required specifically for MC neurotransmission, eat-18 also appears to be required for the function of other nicotinic receptors in the pharynx. In eat-18 mutants, the gross localization of EAT-2 at the MC synapse is normal, suggesting that it is not required for trafficking. These data indicate that eat-18 could be a novel component of the pharyngeal nicotinic receptor.

Conservation of sleep: insights from non-mammalian model systems

The past 10 years have seen new approaches to elucidating genetic pathways regulating sleep. The emerging theme is that sleep-like states are conserved in evolution, with similar signaling pathways playing a role in animals as distantly related as flies and humans. We review the evidence for the presence of sleep states in non-mammalian species including zebrafish (Danio rerio), fruitflies (Drosophila melanogaster) and roundworms (Caenorhabditis elegans). We describe conserved sleep-regulatory molecular pathways with a focus on cAMP and epidermal growth factor signaling; neurotransmitters with conserved effects on sleep and wake regulation, including dopamine and GABA; and a conserved molecular response to sleep deprivation involving the chaperone protein BiP/GRP78.

Cellular Stress Induces a Protective Sleep-like State in C. elegans

Sleep is recognized to be ancient in origin, with vertebrates and invertebrates experiencing behaviorally quiescent states that are regulated by conserved genetic mechanisms. Despite its conservation throughout phylogeny, the function of sleep remains debated. Hypotheses for the purpose of sleep include nervous-system-specific functions such as modulation of synaptic strength and clearance of metabolites from the brain, as well as more generalized cellular functions such as energy conservation and macromolecule biosynthesis. These models are supported by the identification of synaptic and metabolic processes that are perturbed during prolonged wakefulness. It remains to be seen whether perturbations of cellular homeostasis in turn drive sleep. Here we show that under conditions of cellular stress, including noxious heat, cold, hypertonicity, and tissue damage, the nematode Caenorhabditis elegans engages a behavioral quiescence program. The stress-induced quiescent state displays properties of sleep and is dependent on the ALA neuron, which mediates the conserved soporific effect of epidermal growth factor (EGF) ligand overexpression. We characterize heat-induced quiescence in detail and show that it is indeed dependent on components of EGF signaling, providing physiological relevance to the behavioral effects of EGF family ligands. We find that after noxious heat exposure, quiescence-defective animals show elevated expression of cellular stress reporter genes and are impaired for survival, demonstrating the benefit of stress-induced behavioral quiescence. These data provide evidence that cellular stress can induce a protective sleep-like state in C. elegans and suggest that a deeply conserved function of sleep is to mitigate disruptions of cellular homeostasis.

A novel gain-of-function mutant of the cyclic GMP-dependent protein kinase egl-4 affects multiple physiological processes in Caenorhabditis elegans.

cGMP-dependent protein kinases are key intracellular transducers of cell signaling. We identified a novel dominant mutation in the C. elegans egl-4 cGMP-dependent protein kinase (PKG) and show that this mutation causes increased normal gene activity although it is associated with a reduced EGL-4 protein level. Prior phenotypic analyses of this gain-of-function mutant demonstrated a reduced longevity and a reduced feeding behavior when the animals were left unperturbed. We characterize several additional phenotypes caused by increased gene activity of egl-4. These phenotypes include a small body size, reduced locomotion in the presence of food, a pale intestine, increased intestinal fat storage, and a decreased propensity to form dauer larvae. The multiple phenotypes of egl-4 dominant mutants are consistent with an instructive signaling role of PKG to control many aspects of animal physiology. This is among the first reported gain-of-function mutations in this enzyme of central physiological importance. In a genetic screen we have identified extragenic suppressors of this gain-of-function mutant. Thus, this mutant promises to be a useful tool for identifying downstream targets of PKG.

FMRFamide-like FLP-13 Neuropeptides Promote Quiescence following Heat Stress in Caenorhabditis elegans

Among the most important decisions an animal makes is whether to engage in active movement and feeding behavior or to become quiescent. The molecular signaling mechanisms underlying this decision remain largely unknown. The nematode Caenorhabditis elegans displays sleep-like quiescence following exposures that result in cellular stress. The neurosecretory ALA neuron is required for this stress-induced recovery quiescence, but the mechanisms by which ALA induces quiescence have been unknown. We report here that quiescence induced by heat stress requires ALA depolarization and release of FMRFamide-like neuropeptides encoded by the flp-13 gene. Optogenetic activation of ALA reduces feeding and locomotion in a FLP-13-dependent manner. Overexpression of flp-13 is sufficient to induce quiescent behavior during normally active periods. We have here identified a major biological role for FMRFamide-like neuropeptides in nematodes, and we suggest that they may function in a similar capacity in other organisms.

Engineering adenylate cyclases regulated by near-infrared window light

Significance Microbial photoreceptors, bacteriophytochromes, absorb near-infrared light, which penetrates deep into animal tissues and is harmless. Bacteriophytochromes delivered as genes can be used to control biological activities in live animals via external light sources. However, the lack of understanding of light-induced conformational changes has hindered development of bacteriophytochrome-based optogenetic tools. Here, we offer a proof of principle that homodimeric bacteriophytochromes can be engineered to activate heterologous output domains that require homodimerization. We constructed an adenylate cyclase, which can control cAMP-dependent processes in live animals in response to light in the near-infrared spectral window. When expressed in cholinergic neurons of a roundworm Caenorhabditis elegans, near-infrared light-activated adenylate cyclase affected worm behavior in a light-dependent manner. Bacteriophytochromes sense light in the near-infrared window, the spectral region where absorption by mammalian tissues is minimal, and their chromophore, biliverdin IXα, is naturally present in animal cells. These properties make bacteriophytochromes particularly attractive for optogenetic applications. However, the lack of understanding of how light-induced conformational changes control output activities has hindered engineering of bacteriophytochrome-based optogenetic tools. Many bacteriophytochromes function as homodimeric enzymes, in which light-induced conformational changes are transferred via α-helical linkers to the rigid output domains. We hypothesized that heterologous output domains requiring homodimerization can be fused to the photosensory modules of bacteriophytochromes to generate light-activated fusions. Here, we tested this hypothesis by engineering adenylate cyclases regulated by light in the near-infrared spectral window using the photosensory module of the Rhodobacter sphaeroides bacteriophytochrome BphG1 and the adenylate cyclase domain from Nostoc sp. CyaB1. We engineered several light-activated fusion proteins that differed from each other by approximately one or two α-helical turns, suggesting that positioning of the output domains in the same phase of the helix is important for light-dependent activity. Extensive mutagenesis of one of these fusions resulted in an adenylate cyclase with a sixfold photodynamic range. Additional mutagenesis produced an enzyme with a more stable photoactivated state. When expressed in cholinergic neurons in Caenorhabditis elegans, the engineered adenylate cyclase affected worm behavior in a light-dependent manner. The insights derived from this study can be applied to the engineering of other homodimeric bacteriophytochromes, which will further expand the optogenetic toolset.

Chapter 11 Electrophysiological Methods

Publisher Summary This chapter describes the Electropharyngeogram (EPG), and how it is applied to Caenorhabditis elegans. Most C. elegans electrophysiology so far has been done on the pharynx. When pharyngeal muscle is excited, a pulse of current must flow out of the mouth to charge the apical membranes and hypodermis. The total charge that comes out of the mouth during a current pulse is proportional to the change in voltage across the basal membrane. The current, at any point in time, is proportional to the rate of change of basal membrane potential. The total charge that comes out of the mouth during a current pulse is proportional to the capacitance of that part of the apical membrane whose voltage changes. As capacitance is proportional to membrane area, bigger worms give bigger signals. Two strategies are generally used to device a saline solution for electrophysiological recording. The first determines the chemical composition of the extracellular fluid and to makes a solution with the appropriate concentrations of the major inorganic ions. The second strategy is trial and error, starting with salines from other species. It is possible to see the variation of the concentration of individual ions in dissected preparations, using an easily observable physiological response to determine the effect.