Neural Mechanisms of a Hyperosmotic Behavioral Response in Caenorhabditis elegans

Abstract

Maintaining water balance is essential for biological systems to retain normal molecular and cellular functions. Hyperosmotic conditions cause water loss that can be detrimental to organisms’ survival. Behavior plays an important role in stabilizing an organism’s internal osmotic condition against hyperosmotic stress. For example, mammals directly detect osmotic changes in body fluids through neuronal sensors, and regulate behaviors such as water intake to maintain osmotic homeostasis. However, the molecular and neural mechanisms underlying behavioral responses to hyperosmotic stimuli are not clear. I used the nematode Caenorhabditis elegans as a model organism to study neural mechanisms of hyperosmotic behavioral responses. C. elegans studies take advantage of the worm’s relatively simple nervous system of 302 neurons, its mapped neuronal connectivity and its amenability to molecular and cellular techniques. I discovered a novel behavioral response of C. elegans animals towards moderate hyperosmotic conditions (osmotic upshifts), and characterized the underlying neural mechanisms. The present study demonstrates for the first time that the worm gradually upregulates aversive behaviors when exposed to prolonged osmotic upshifts. By altering the solutes contributing to the osmotic stress, I demonstrated that the worm responds to the osmotic stimulus instead of specific solutes. By examining worms with high internal osmolarity, I was able to reveal that the stimulus sensed by the worm was the osmotic difference between its internal and external environments, rather than the absolute osmolarity. Despite previous studies in C. elegans reporting that the TRPV channel OSM-9 plays an essential role in the aversive response to high osmotic shocks, the behavioral response to osmotic upshifts that I describe in this work does not rely on OSM-9 and employs novel sensory mechanisms. Instead, the cyclic nucleotide-gated ion (CNG) channel subunit TAX-2 is required in the response to osmotic upshifts. Although TAX-2 is reported to function together with another CNG channel subunit TAX-4 in other sensory behaviors, in this hyperosmotic behavioral response, TAX-2 functions independently of TAX-4. Using cell-specific rescue experiments in the tax-2 mutant, I identified the sensory neurons ASJ, AQR, PQR and URX as the site of action for TAX-2 in the behavioral response to osmotic upshifts. Interestingly, the sensory neurons AQR, PQR and URX are exposed to the pseudocoelomic fluid, and may monitor the internal environment of the worm. The involvement of the ASJ sensory neuron suggests diffusible signaling playing a role in the hyperosmotic response. In addition, I have mapped the neural circuit required for this hyperosmotic behavior from the aforementioned sensory neurons to interneurons and motor neurons. Using genetic manipulations, I identified the first-layer interneurons AIB and AIY to be collectively required in the behavior, and a set of downstream inter/motor neurons in the locomotory circuit to be necessary for executing the behavioral output. Such a redundancy at the level of the first-layer interneurons serves as a protective mechanism against damages, which underlies the importance for the worm to avoid hyperosmotic conditions. Further investigating the basic neural mechanisms of the behavioral response to osmotic upshifts would provide new insights on osmosensing, and further shed light on the neural and hormonal regulation of internal osmotic stability.