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.

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.

Single-gene mutations that disrupt mitochondrial respiratory chain function in C. elegans change patterns of protein expression and metabolites. Our goal was to develop useful molecular fingerprints employing adaptable techniques to recognize mitochondrial defects in the electron transport chain. We analyzed mutations affecting complex I, complex II, or ubiquinone synthesis and discovered overarching patterns in the response of C. elegans to mitochondrial dysfunction across all of the mutations studied. These patterns are in KEGG pathways conserved from C. elegans to mammals, verifying that the nematode can serve as a model for mammalian disease. In addition, specific differences exist between mutants that may be useful in diagnosing specific mitochondrial diseases in patients.

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Inhaled anesthetics have been used for more than a century, and they are currently administered to millions of patients each year. Although well understood in an empirical sense, their basic molecular mechanisms of action are still unknown. During the past two decades, a large amount of evidence has been presented that is most consistent with the hypothesis that inhaled anesthetics act at multiple sites. For example, genetic mutations exist that distinguish between different inhaled anesthetics, i.e. the mutations alter sensitivity to some anesthetics differently than others. Since it is probable that multiple mechanisms contribute to inhaled anesthetic action, a genetic approach is a powerful method for sorting out which molecules are involved in specific anesthetic effects. This review describes recent pharmacogenetic studies performed using model organisms, including yeast, nematodes, fruit flies, and mice. At first glance, the results of these studies are notable for their lack of a common putative molecular target. In fact, the results suggest that anesthetics interact with a seemingly broad range of cellular components including ion channels, membrane receptors, lipid rafts, and the mitochondrial electron transport chain. However, a unifying theme is beginning to emerge, one that implicates the presynaptic neuron as a common functional target for inhaled anesthetics. Intriguing similarities among the results suggest that many of the findings obtained in model organisms can be generalized across disparate phyla, and that the findings will be applicable in humans. By continuing to exploit the power of genetics, such studies are likely to unravel the great mystery of how inhaled anesthetics produce their effects.

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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.

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