William R. Schafer
e-mail: wschafer@ucsd.edu
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The analysis of simple, genetically tractable organisms has provided critical in-sights into the nature of many complex biological processes, including gene regulation and multicellular development. We are using a similar approach to understand the molecular and cellular basis of behavior in the nematode Caenorhabditis elegans. C. elegans has a small and extremely well characterized nervous system, a completely cloned and sequenced genome, and an unparalleled accessability to genetic analysis. Using a variety of diverse experimental approaches, we ultimately hope to gain a reductionist under-standing of how specific neurons and gene products influence a whole animal's behavior. Conversely, the ability to functionally map the influence of a particular gene on a specific behavior makes it possible to use behavioral mutants to genetically dissect the signal transduction pathways mediating poorly understood processes such as addiction.
Genetic analysis of nicotine response and adatptation: One major emphasis of my lab's research has been to investigate the molecular mechanisms underlying long-term responses to nicotine. The activity and abundance of nicotinic acetylcholine receptors in vertebrates are subject to long-term downregulation by nicotine; however, despite the likely importance of these processes to nicotine addiction, little is known about their molecular mechanism. We have demonstrated using behavioral assays that C. elegans also acquires nicotine tolerance as well as dependence following long-term exposure to nicotine, effects that occur at least in part through downregulation of neuronal and neuromuscular nicotinic receptors. We have also identified, through genetic (Waggoner et al., 2000) and biochemical (Gottschalk et al., submitted) screens, a number of novel molecules whose activity is important for the regulation and/or functional activity of C. elegans nicotinic receptors. Since these molecules have close human homologues, they represent good candidates for molecules that may function in nicotine addiction.
In vivo analysis of sensory perception: To fully understand behavior at the molecular and cellular level, it is necessary to determine the effects of specific gene products on the activity of individual neurons as well as muscle cells, and to correlate neural activity with behavior. However, the impermeant cuticle, hydrostatic skeleton and small neurons of C. elegans have made it extremely difficult to monitor neural activity in intact behaving animals. We therefore became interested in surmounting this key obstacle by using the genetically encoded optical indicator cameleon as an in vivo neural sensor. We recently demonstrated for the first time that cameleon could be used to image excitable cell function in vivo, first in pharyngeal muscle and then in electrically-excited neurons (Kerr et al., 2000). We have subsequently used cameleon-based calcium imaging to address fundamental mechanisms of sensory transduction in C. elegans mechanosensory neurons. By generating transgenic animals expressing cameleon in touch receptor neurons, we were able to record the activity of single mechanosensory neurons in intact, behaving nematodes. The DEG/ENaC channel subunit MEC-4 and the channel-associated stomatin MEC-2 were specifically required for touch-evoked neural activity in response to gentle mechanical stimulation, but were not required for the response to harsh mechanical stimulation or for potassium-induced depolarisation of touch neurons. Our results demonstrated for the first time that DEG/ENaC channels, which are conserved from nematodes to mammals are not necessary for the general function of the touch receptor neurons, but rather play a direct role in the process of mechanosensation (Suzuki et al., 2003). We have also applied similar methods to analyze neuronal signal transduction mechanisms in nociceptors (Hilliard et al., submitted) and serotonergic motorneurons (Shyn et al., 2003).
Quantitative analysis of behavioral phenotypes: Along with our interest in developing methods for functional imaging of worm neurons, we have also been interested in applying imaging methodologies to the analysis of worm behavior. Although C. elegans has powerful genetics and a small, well characterized nervous system, many genes with critical roles in neuronal function have effects on behavior that are subtle or difficult to describe precisely. Therefore, to fully realize the potential of C. elegans for the genetic analysis of nervous system function, it will be necessary to develop sophisticated methods for the rapid and consistent quantitation of behavioral phenotypes. We have developed traching and imaging systems that have facilitated the quantification of nematode egg-laying (Waggoner et al., 1998) and locomotion. More recently, we have developed machine vision tools for comprehensively defining behavioral and morphological abnormalities in C. elegans mutants (Baek et al, 2002). We have begun to use this system to quantify the similarities of C. elegans mutant phenotypes and to investigate the natural clustering of mutant behavioral patterns (Geng et al., 2003). Ultimately, we hope to use these methods to generate a large-scale phenotypic database for C. elegans. With the accumulation of data on an increasing number of worm types, it should be possible to identify groups of mutants and pharmacological treatments that have similar effects on behavior or development, and therefore infer involvement in a common biological function.
Suzuki, H., Kerr, R., Bianchi, L., Frøkjær-Jensen, C., Slone, D., Xue, J., Gerstbrein, B., Driscoll, M., Schafer, W.R. (2003) "In vivo functional analysis of C. elegans mechanosensory neurons reveals a specific role for MEC-4 channels in the process of gentle touch transduction" Neuron 39: 1005-1017.
Shyn, S.I., Kerr, R., Schafer, W.R. (2003) "Serotonin and Go modulate functional states of neurons and muscles involved in C. elegans egg-laying behavior" Curr. Biol. 13: 1910-1915.
Geng, W., Cosman, P., Baek, J.-H., Berry, C.C., W.R. Schafer. (2003) "Quantitative classification and natural clustering of C. elegans behavioral phenotypes" Genetics 165: 1117-1123.
Kindt, K. S., T. Tam, S. Whiteman, W. R. Schafer. (2002) "Serotonin promotes Go/Gq-mediated neuronal migration in Caenorhabditis elegans" Curr. Biol. 12: 1738-1747.
Hardaker, L.A., E.
Singer, R. Kerr, G. T. Zhou, W. R. Schafer. (2001) "Serotonin
modulates locomotory behavior and coordinates egg-laying and
movement in Caenorhabditis elegans" J. Neurobiol. 49: 303-313.
Kim, J., D. S. Poole, L. E. Waggoner,
A. Kempf, D. S. Ramirez, P. A. Treschow, W. R. Schafer (2001) "Genes
affecting affecting the activity of nicotinic receptors involved
in Caenorhabditis elegans egg-laying behavior" Genetics 157: 1599-1610.
Waggoner, L. E., K. A. Dickinson, D. S. Poole, Y. Tabuse, J. Miwa, W. R. Schafer. (2000). Long-term nicotine adaptation in Caenorhabditis elegans involves PKC-dependent changes in nicotinic receptor abundance. J. Neurosci: 20: 8802-8811
Kerr, R., V. Lev-Ram, G. Baird, P. Vincent, R. Y. Tsien,, W. R. Schafer. (2000). Optical imaging of calcium transients in C. elegans neurons and pharyngeal muscle. Neuron 26: 583-594.
Tam, T., E. Mathews, T. Snutch., W. R. Schafer (2000). Voltage-gated calcium channels direct neuronal migration in Caenorhabditis elegans. Dev. Biol. 226: 104-117.
Waggoner, L.E. , L. A. Hardaker, S. Golik, W. R. Schafer. (2000). Effect of a neuropeptide gene on behavioral states in Caenorhabditis elegans egg-laying. Genetics 134: 1181-1192.
Waggoner, L. E., G. T. Zhou, R. W. Schafer, W. R. Schafer. (1998). Control of alternative behavioral states by serotonin in Caenorhabditis elegans. Neuron 21: 203-214.
Bill Schafer received his Ph.D. in biochemistry from UC Berkeley and did postdoctoral work at UCSF. He has been the recipient of the Presidential Early Career Award for Scientists and Engineers, a NARSAD Young Investigator Award, a Beckman Young Investigator Award, a Sloan Fellowship, and a Klingenstein Fellowship.
