An animal uses stable neural circuits to generate flexible behavioral responses to its environment. We are studying the relationships between genes, the nervous system, and behaviors in the nematode Caenorhabditis elegans. We can define the circuits for specific behaviors in C. elegans because its nervous system consists of just 302 neurons with reproducible functions, morphologies, and synaptic connections. Using this information, we study genes that affect neuronal development and behavior and determine how these genes regulate the assembly and function of neural circuits.
A Circuit for Olfactory Behavior
We want to understand how the activity of each neuron in a circuit contributes to an animal's behavior. To achieve this goal, we ask how neurons detect stimuli, respond to them, and relay information to other neurons. A systematic study of the C. elegans nervous system has led to the identification of olfactory neurons and interneurons that control food- and odor-evoked behaviors. We used genetically encoded calcium indicators to monitor the activity of these neurons, and we altered the flow of information by eliminating or restoring neurotransmitter receptors in subsets of neurons. These experiments have provided a first view of information relay in the olfactory circuit. The primary olfactory neurons are activated by odor removal and suppressed by odor addition. The olfactory neurons release the neurotransmitter glutamate to inhibit one interneuron target and activate another interneuron target. As a result, one interneuron signals odor onset, and the other interneuron signals odor offset. At both a molecular level and a logical level, information processing by this circuit resembles information flow from vertebrate photoreceptors to their targets, the OFF-bipolar and ON-bipolar neurons, suggesting a conserved or convergent strategy for sensory information processing.
The analysis of neuronal activity in this circuit has led to several observations. The sensory neurons respond only transiently to the odor removal signal, but one of the target interneurons has a long-lasting response. The animal's behavioral response to odor removal is long-lasting, like the response of the interneuron. Persistent neuronal activity may help explain temporal properties of the behavior, such as its ability to outlast the sensory cues that triggered it.
Olfaction and Olfactory Learning
An important function of the olfactory system is to allow animals to evaluate potential food odors. C. elegans requires bacterial food for survival but is susceptible to infection by pathogenic bacteria following ingestion of a tainted food source. One of the most robust forms of learning in any animal is conditioned avoidance of tastes associated with visceral distress. We found that pathogenic bacteria elicit C. elegans learning behaviors similar to mammalian conditioned taste aversion. After exposure to pathogenic bacteria, C. elegans modifies its olfactory preferences to avoid odors from the pathogen. This modification is specific, suggesting that the animal associates the experience of infection with the odor of the noxious bacterium.
In another form of experience-dependent behavior, odors paired with food become more attractive to C. elegans, and odors paired with starvation become less attractive or even repulsive. We found that the behavioral switch from attraction to repulsion can occur within a single olfactory neuron. A particular olfactory neuron can send two opposite signals—one favoring attraction, the other favoring avoidance—and can switch rapidly between these two signaling modes. The switch between attraction and repulsion is regulated by a receptor-like guanylate cyclase in olfactory axons, a diacylglycerol kinase that regulates synaptic transmission, and protein kinase C. These results reveal an unexpected degree of functional plasticity within the fixed neuroanatomy of the sensory system.
Oxygen Sensation, an Environmentally Relevant Behavior
Levels of oxygen vary widely in natural soil and water environments where C. elegans can be found. C. elegans prefers an intermediate oxygen concentration, avoiding high and low oxygen levels. Oxygen detection requires specialized sensory neurons that express a number of soluble guanylate cyclase proteins (sGCs) that can directly bind oxygen through their heme domains (work of our collaborators, David Karow and Michael Marletta [University of California, Berkeley]). The sGCs are a new class of molecular receptor for environmental oxygen.
C. elegans is notable for its ability to survive in either high- or low-oxygen environments. Survival in low oxygen requires an oxygen-regulated transcription factor, HIF-1. This transcription factor also regulates behavioral responses to oxygen, shifting animals toward a lower preferred oxygen concentration. The HIF-1 pathway regulates both neurons and nonneuronal tissues that coordinate oxygen responses by long-range signaling. The complete neuroanatomical "wiring diagram" of the C. elegans nervous system does not include these regulatory pathways, indicating that functional experiments are needed to uncover circuits even in anatomically well-described nervous systems.
A Tool to Study Connections in Neural Circuits
The unit of communication between neurons is the synapse, a cell-cell junction where electrical and chemical signals are propagated between cells. The identification of synaptic partners is challenging in dense nerve bundles, where many processes occupy regions beneath the resolution of conventional light microscopy. To address this difficulty, we have developed a system to label membrane contacts and synapses between two cells in living animals. We call this method GRASP (GFP reconstitution across synaptic partners). Two complementary fragments of GFP are expressed on different cells, tethered to extracellular domains of transmembrane carrier proteins. When the complementary GFP fragments are fused to ubiquitous transmembrane proteins, GFP fluorescence appears uniformly along membrane contacts between the two cells. When one or both GFP fragments are fused to synaptic transmembrane proteins, GFP fluorescence is tightly localized to synapses. With our collaborator Kang Shen (Stanford University), we validated this method by showing that GRASP marks known synaptic contacts in C. elegans, correctly identifies changes in mutants with altered synaptic specificity, and can uncover new information about synaptic locations, as confirmed by electron microscopy. GRASP may prove particularly useful for defining connectivity in complex nervous systems.
