Leon Avery

Active Currents Regulate Sensitivity and Dynamic Range in C. elegans Neurons

Little is known about the physiology of neurons in Caenorhabditis elegans. Using new techniques for in situ patch-clamp recording in C. elegans, we analyzed the electrical properties of an identified sensory neuron (ASER) across four developmental stages and 42 unidentified neurons at one stage. We find that ASER is nearly isopotential and fails to generate classical Na+ action potentials. Rather, ASER displays a high sensitivity to input currents coupled to a depolarization-dependent reduction in sensitivity that may endow ASER with a wide dynamic range. Voltage clamp revealed depolarization-activated K+ and Ca2+ currents that contribute to high sensitivity near the zero-current potential. The depolarization-dependent reduction in sensitivity can be attributed to activation of K+ current at voltages where it dominates the net membrane current. The voltage dependence of membrane current was similar in all neurons examined, suggesting that C. elegans neurons share a common mechanism of sensitivity and dynamic range.

Pharyngeal Pumping Continues after laser Killing of the Pharyngeal Nervous System of C. elegans

Using a laser microbeam to kill specific subsets of the pharyngeal nervous system of C. elegans, we found that feeding was accomplished by two separately controlled muscle motions, isthmus peristalsis and pumping. The single neuron M4 was necessary and sufficient for isthmus peristalsis. The MC neurons were necessary for normal stimulation of pumping in response to food, but pumping continued and was functional in MC- worms. The remaining 12 neuron types were also unnecessary for functional pumping. No operation we did, including destruction of the entire pharyngeal nervous system, abolished pumping altogether. When we killed all pharyngeal neurons except M4, the worms were viable and fertile, although retarded and starved. Since feeding is one of the few known essential actions controlled by the nervous system, we suggest that most of the C. elegans nervous system is dispensable in hermaphrodites under laboratory conditions. This may explain the ease with which nervous system mutants are isolated and handled in C. elegans.

avr‐15 encodes a chloride channel subunit that mediates inhibitory glutamatergic neurotransmission and ivermectin sensitivity in Caenorhabditis elegans

Ivermectin is a widely used anthelmintic drug whose nematocidal mechanism is incompletely understood. We have used Caenorhabditis elegans as a model system to understand ivermectins effects. We found that the M3 neurons of the C.elegans pharynx form fast inhibitory glutamatergic neuromuscular synapses. avr‐15, a gene that confers ivermectin sensitivity on worms, is necessary postsynaptically for a functional M3 synapse and for the hyperpolarizing effect of glutamate on pharyngeal muscle. avr‐15 encodes two alternatively spliced channel subunits that share ligand binding and transmembrane domains and are members of the family of glutamate‐gated chloride channel subunits. An avr‐15‐encoded subunit forms a homomeric channel that is ivermectin‐sensitive and glutamate‐gated. These results indicate that: (i) an ivermectin‐sensitive chloride channel mediates fast inhibitory glutamatergic neuromuscular transmission; and (ii) a nematocidal property of ivermectin derives from its activity as an agonist of glutamate‐gated chloride channels in essential excitable cells such as those of the pharynx.

EAT-4, a Homolog of a Mammalian Sodium-Dependent Inorganic Phosphate Cotransporter, Is Necessary for Glutamatergic Neurotransmission in Caenorhabditis elegans

The Caenorhabditis elegans gene eat-4affects multiple glutamatergic neurotransmission pathways. We find thateat-4 encodes a protein similar in sequence to a mammalian brain-specific sodium-dependent inorganic phosphate cotransporter I (BNPI). Like BNPI in the rat CNS,eat-4 is expressed predominantly in a specific subset of neurons, including several proposed to be glutamatergic. Loss-of-function mutations in eat-4 cause defective glutamatergic chemical transmission but appear to have little effect on other functions of neurons. Our data suggest that phosphate ions imported into glutamatergic neurons through transporters such as EAT-4 and BNPI are required specifically for glutamatergic neurotransmission.

LASER KILLING OF CELLS IN CAENORHABDITIS ELEGANS

Publisher Summary This chapter discusses the laser killing of cells in Caenorhabditis elegans. Individual cells can be killed in Caenorhabditis elegans by damaging them with a laser microbeam focused through the objective of a microscope. The laser beam is focused in three dimensions on a single spot in the field of view of a microscope. A cell of interest is aligned with the laser beam. Damage to the cell and adjacent structures can be visualized through the microscope during and after the operation. Identifying cells unambiguously is probably the most difficult part of the laser operation. Rigorous identification of a cell type can be accomplished by following cell lineages through embryonic or postembryonic divisions. When a laser microbeam is fired at a C. elegans nucleus, three things happen: deposition of energy in the nucleus, followed by transport, followed by energy-induced damage. Deposition places the energy with remarkable precision. Transport moves the energy from where it was deposited to other places. However, damage is the dark mystery and nothing is known about how laser energy destroys a C. elegans nucleus. Energy deposited in the irradiated nucleoplasm eventually takes the form of increased temperature and pressures, either of which can denature proteins, break DNA, and so on.

C. elegans: A Model for Exploring the Genetics of Fat Storage

To gain insights into the genetic cascades that regulate fat biology, we evaluated C. elegans as an appropriate model organism. We generated worms that lack two transcription factors, SREBP and C/EBP, crucial for formation of mammalian fat. Worms deficient in either of these genes displayed a lipid-depleted phenotype-pale, skinny, larval-arrested worms that lack fat stores. On the basis of this phenotype, we used a reverse genetic screen to identify several additional genes that play a role in worm lipid storage. Two of the genes encode components of the mitochondrial respiratory chain (MRC). When the MRC was inhibited chemically in worms or in a mammalian adipocyte model, fat accumulation was markedly reduced. A third encodes lpd-3, whose homolog is also required for fat storage in a mammalian model. These data suggest that C. elegans is a genetically tractable model to study the mechanisms that underlie the biology of fat-storing tissues.

A Cell That Dies during Wild-Type C. elegans Development Can Function as a Neuron in a ced-3 Mutant

Mutations in the C. elegans gene ced-3 prevent almost all programmed cell deaths, so that in a ced-3 mutant there are many extra cells. We show that the pharyngeal neuron M4 is essential for feeding in wild-type worms, but in a ced-3 mutant, one of the extra cells, probably MSpaaaaap (the sister of M4), can sometimes take over M4s function. The function of MSpaaaaap, unlike that of M4, is variable and subnormal. One possible explanation is that its fate, being hidden by death and not subject to selection, has drifted randomly during evolution. We suggest that such cells may play roles in the evolution of cell lineage analogous to those played by pseudogenes in the evolution of genomes.

Ordering gene function: the interpretation of epistasis in regulatory hierarchies

The order of action of genes in a regulatory hierarchy that is governed by a signal can often be determined by the method of epistasis analysis, in which the phenotype of a double mutant is compared with that of single mutants. The epistatic mutation may be in either the upstream or the downstream gene, depending on the nature of the two mutations and the type of regulation. Nevertheless, when the regulatory hierarchy satisfies certain conditions, simple rules allow the position of the epistatic locus in the pathway to be determined without detailed knowledge of the nature of the mutations, the pathway, or the molecular mechanism of regulation.

Social feeding in Caenorhabditis elegans is induced by neurons that detect aversive stimuli

Natural Caenorhabditis elegans isolates exhibit either social or solitary feeding on bacteria. We show here that social feeding is induced by nociceptive neurons that detect adverse or stressful conditions. Ablation of the nociceptive neurons ASH and ADL transforms social animals into solitary feeders. Social feeding is probably due to the sensation of noxious chemicals by ASH and ADL neurons; it requires the genes ocr-2 and osm-9, which encode TRP-related transduction channels, and odr-4 and odr-8, which are required to localize sensory chemoreceptors to cilia. Other sensory neurons may suppress social feeding, as social feeding in ocr-2 and odr-4 mutants is restored by mutations in osm-3, a gene required for the development of 26 ciliated sensory neurons. Our data suggest a model for regulation of social feeding by opposing sensory inputs: aversive inputs to nociceptive neurons promote social feeding, whereas antagonistic inputs from neurons that express osm-3 inhibit aggregation.

Dietary choice behavior in Caenorhabditis elegans

SUMMARY Animals have evolved diverse behaviors that serve the purpose of finding food in the environment. We investigated the food seeking strategy of the soil bacteria-eating nematode Caenorhabditis elegans. C. elegans bacterial food varies in quality: some species are easy to eat and support worm growth well, while others do not. We show that worms exhibit dietary choice: they hunt for high quality food and leave hard-to-eat bacteria. This food seeking behavior is enhanced in animals that have already experienced good food. When hunting for good food, worms alternate between two modes of locomotion, known as dwelling: movement with frequent stops and reversals; and roaming: straight rapid movement. On good food, roaming is very rare, while on bad food it is common. Using laser ablations and mutant analysis, we show that the AIY neurons serve to extend roaming periods, and are essential for efficient food seeking.