Establishing C. elegans as a Model for Studying the Bioeffects of Therapeutic Ultrasound.

Ultrasound is widely used in diagnostic and therapeutic medical procedures and it is becoming an important tool in biomedical research. During exposure, as an ultrasound beam interacts with the tissues in its path, changes known as "bioeffects" can result. Animal studies have suggested that these changes can alter survival, movement, reproduction, development and learning in various species. Additional studies in animals could provide valuable information about the mechanisms of therapeutic ultrasound and may contribute to the development of additional exciting laboratory techniques. Therefore, we developed methods for exposing C. elegans nematode worms to ultrasound and observed that they exhibited exposure-dependent reductions in movement, fecundity and survival. These effects were prevented by polyvinyl alcohol, which suggested that cavitation was the main mechanism of damage. This work provides a foundation for capitalizing on the advantages of C. elegans as a model to thoroughly characterize ultrasound's bioeffects at the cellular and molecular levels.

General Genetic Strategies.

It is difficult to study the genetics and molecular mechanisms of anesthesia in humans. Fortunately, the genetic approaches in model organisms can, and have, led to profound insights as to the targets of anesthetics. In turn, the organization of these putative targets into meaningful pathways has begun to elucidate the mechanisms of action of these agents. However, it is important to first appreciate the strengths, and limitations, of genetic approaches to understand the anesthetic action. Here we compare the commonly used genetic model organisms, various anesthetic endpoints, and different modes of genetic screens. Coupled with the more specific data presented in subsequent chapters, this chapter places those results in a framework with which to analyze the discoveries across organisms and eventually extend the resulting models to humans.

Approaches to Anesthetic Mechanisms: The C. elegans Model.

Understanding the mechanisms of volatile anesthetics has been a complex problem that has intrigued investigators for decades. Through the use of relatively simple model organisms-including the nematode Caenorhabditis elegans-progress has been made. Like any model system, C. elegans has both advantages and disadvantages, which are discussed in this chapter. Methods are provided for exposing worms to volatile anesthetics in airtight glass chambers, and for measuring the concentrations of anesthetic in the chambers by gas chromatography. In addition, various behavioral assays are described for characterizing the worms' responses to anesthetics. C. elegans identified proteins that play a role in anesthetic sensitivity that are highly conserved in other organisms, including humans. With precisely characterized neural development, C. elegans has also afforded an excellent opportunity to study anesthetic-induced neurotoxicity. Continued progress in understanding anesthetic action is anticipated from the ongoing study of C. elegans and other animal models.

Early developmental exposure to volatile anesthetics causes behavioral defects in Caenorhabditis elegans.

BACKGROUND: Mounting evidence from animal studies shows that anesthetic exposure in early life leads to apoptosis in the developing nervous system. This loss of neurons has functional consequences in adulthood. Clinical retrospective reviews have suggested that multiple anesthetic exposures in early childhood are associated with learning disabilities later in life as well. Despite much concern about this phenomenon, little is known about the mechanism by which anesthetics initiate neuronal cell death. Caenorhabditis elegans, a powerful genetic animal model, with precisely characterized neural development and cell death pathways, affords an excellent opportunity to study anesthetic-induced neurotoxicity. We hypothesized that exposing the nematode to volatile anesthetics early in life would induce neuron cell death, producing a behavioral defect that would be manifested in adulthood. METHODS: After synchronization and hatching, larval worms were exposed to volatile anesthetics at their 95% effective concentration for 4 hours. On day 4 of life, exposed and control worms were tested for their ability to sense and move to an attractant (i.e., to chemotax). We determined the rate of successful chemotaxis using a standardized chemotaxis index. RESULTS: Wild-type nematodes demonstrated striking deficits in chemotaxis indices after exposure to isoflurane (ISO) or sevoflurane (SEVO) in the first larval stage (chemotaxis index: untreated, 85 ± 2; ISO, 52 ± 2; SEVO, 47 ± 2; P < 0.05 for both exposures). The mitochondrial mutant gas-1 had a heightened effect from the anesthetic exposure (chemotaxis index: untreated, 71 ± 2; ISO, 29 ± 12; SEVO, 24 ± 13; P < 0.05 for both exposures). In contrast, animals unable to undergo apoptosis because of a mutation in the pathway that mediates programmed cell death (ced-3) retained their ability to sense and move toward an attractant (chemotaxis index: untreated, 76 ± 10; ISO, 73 ± 9; SEVO, 76 ± 10). Furthermore, we discovered that the window of greatest susceptibility to anesthetic neurotoxicity in nematodes occurs in the first larval stage after hatching (L1). This coincides with a period of neurogenesis in this model. All values are means ± SD. CONCLUSION: These data indicate that anesthetics affect neurobehavior in nematodes, extending the range of phyla in which early exposure to volatile anesthetics has been shown to cause functional neurological deficits. This implies that anesthetic-induced neurotoxicity occurs via an ancient underlying mechanism. C elegans is a tractable model organism with which to survey an entire genome for molecules that mediate the toxic effects of volatile anesthetics on the developing nervous system.

Alternatives to mammalian pain models 1: use of C. elegans for the study of volatile anesthetics.

Performing genetic studies in model organisms is a powerful approach for investigating the mechanisms of volatile anesthetic action. Striking similarities between the results observed in Caenorhabditis elegans and in other organisms suggest that many of the conclusions can be generalized across disparate phyla, and that findings in these model organisms will be applicable in humans. In this chapter, we provide detailed protocols for working with C. elegans to study volatile anesthetics. First, we explain how to fabricate chambers for exposing worms to these compounds. Then, we describe how to use the chambers to perform a variety of experiments, including behavioral assays, dose-response studies, and mutant screening or selection. Finally, we discuss a convenient strategy for performing mutant rescue assays. These methods are the building blocks for designing and interpreting genetic experiments with volatile anesthetics in C. elegans. Genetic studies in this simple, easy-to-use organism will continue to contribute to a more thorough understanding of anesthetic mechanisms, and may lead to the development and safer use of anesthetic agents.

Nucleosomes can form a polar barrier to transcript elongation by RNA polymerase II.

Nucleosomes uniquely positioned on high-affinity DNA sequences present a polar barrier to transcription by human and yeast RNA polymerase II (Pol II). In one transcriptional orientation, these nucleosomes provide a strong, factor- and salt-insensitive barrier at the entry into the H3/H4 tetramer that can be recapitulated without H2A/H2B dimers. The same nucleosomes transcribed in the opposite orientation form a weaker, more diffuse barrier that is largely relieved by higher salt, TFIIS, or FACT. Barrier properties are therefore dictated by both the local nucleosome structure (influenced by the strength of the histone-DNA interactions) and the location of the high-affinity DNA region within the nucleosome. Pol II transcribes DNA sequences at the entry into the tetramer much less efficiently than the same sequences located distal to the nucleosome dyad. Thus, entry into the tetramer by Pol II facilitates further transcription, perhaps due to partial unfolding of the tetramer from DNA.