Our lab studies the biological basis of behavior using an animal with a very simple nervous system, the roundworm Caenorhabditis elegans. C. elegans is one of the most popular organisms for studies in genetics, neurobiology, developmental biology, and other biology research fields. C. elegans produces a wide variety of behaviors which can be influenced by experience, i.e. the worm can learn and remember.
Our main focus currently concerns the mechanisms by which bacteria (the worm's food source) blocks olfactory adaptation in a pair of primary olfactory neurons called the AWC neurons. Adaptation is observed in all sensory modalities and involves a reduction in the response to a stimulus after prolonged exposure to the stimulus. The presence of bacteria decreases adaptation to odors, such that worms remain responsive to odors associated with their food source. This work is being carried out in collaboration with Dr. Noelle L'Etoile and her team of researchers at the University of California, San Francisco.
These studies address a critical and basic biological question about how, at a molecular and cellular level, multiple inputs into a nervous system are integrated to generate ecologically advantageous behaviors. Furthermore, this system is an example of sensory plasticity being regulated by a reward, and as such could reveal new and important insights into processes involved in drug addiction and eating disorders. Our lab currently is focusing on three projects aimed at elucidating how bacteria block olfactory adaptation.
We are studying adaptation in selected C. elegans mutants. The goal is to identify, and ultimately characterize, worm genes involved in the reduction of olfactory adaptation when bacteria are present. If genes that compose an essential part of these mechanisms are rendered nonfunctional by mutation, this should have a measurable effect on the influence of bacteria on olfactory adaptation.
Damien O'Halloran, while working as a postdoctoral fellow in Dr. L'Etoile's lab, generated about 20 different strains of mutant worms that not only carry a mutation for a gene that may be involved in food signal integration, but also have a known mediator of the olfactory adaptation pathway tagged with green fluorescent protein. The fluorescent tag allows us to visualize the location of this protein, called EGL-4. During olfactory adaptation, EGL-4 normally moves into the nucleus in the AWC neuron. In our tagged, mutant worms, we can observe both the movement of EGL-4 and the degree to which worms are attracted to an odor following exposure to both bacteria and the odor, and see if these behaviors differ from that of normal worms. If so, the mutation in the worm is likely to be in a gene that normally plays an important role in bacterial modification of olfactory adaptation. Once such a gene is identified, its role in the process may be further elucidated.
Preliminary data has revealed one promising candidate that appears to adapt even in the presence of food. Further studies are being done on this mutant and others.
The second experimental approach in progress is to generate new mutants that show impairment of a food block on olfactory adaptation. We performed a forward genetic screen, in which thousands of worms with random, chemically-induced mutations were generated and put through behavioral assays to help identify mutants that may exhibit an impaired food block on olfactory adaptation. Currently, we are screening through these mutants to look for mutants that do indeed show this phenotype. Ultimately, for mutants exhibiting a robust and specific impaired food block on olfactory adaptation, we plan to identify the relevant mutated genes using recently developed techniques involving whole genome sequencing.
The third experimental approach in progress is to use recently developed imaging techniques to observe and record the responses of neurons to odor and food exposure. Primary olfactory neurons have receptors that bind to odor molecules. Binding of odor initiates a cascade of molecular signals within the neuron that ultimately affects signals released from the neuron to other neurons. These neurons, in turn, signal to other neurons, generating information processing within the nervous system of the worm. Ultimately the output of this information processing determines behavior. By studying the signals produced within neurons we can gain insight into the nature of information coding about odors, and how those codes are affected during adaptation, and by food.
Recently developed techniques enable such recordings. Calcium imaging studies have been published in which specially engineered proteins introduced into worms allow monitoring of calcium levels over time within specific neurons. Increases and decreases in calcium represent a fundamental signaling system utilized by neurons and, indeed, many types of cells.
We will record calcium levels within specific neurons using worms expressing GCaMP, a fluorescent protein whose fluorescence increases when the protein binds calcium. These worms will be placed in microfluidic devices for imaging. These are microchip-sized devices that can be placed on a microscope stage, and in which an experimenter can immobilize worms and precisely apply fluid streams to the nose. Using these devices, we will record calcium signals from neurons while applying odor and/or food to the nose of the worm.
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