Osmolarity regulates C. elegans egg-laying behavior via parallel chemosensory and biophysical mechanisms

Animals alter their behavior in response to changes in the environment. Upon encountering hyperosmotic conditions, the nematode worm C. elegans initiates avoidance and cessation of egg-laying behavior. While the sensory pathway for osmotic avoidance is well-understood, less is known about how egg laying is inhibited. We analyzed egg-laying behavior after acute and chronic shifts to and from hyperosmotic media. Animals on 400 mM sorbitol stop laying eggs immediately but then resume ~3 hours later, after accumulating additional eggs in the uterus. Surprisingly, the hyperosmotic cessation of egg laying did not require known osmotic avoidance signaling pathways. Acute hyperosmotic shifts in hyperosmotic-resistant mutants overproducing glycerol also blocked egg laying, but these animals resumed egg laying more quickly than similarly treated wild-type animals. These results suggest that hyperosmotic conditions disrupt a 'high-inside' hydrostatic pressure gradient required for egg laying. Consistent with this hypothesis, animals adapted to hyperosmotic conditions laid more eggs after acute shifts back to normosmic conditions. Optogenetic stimulation of the HSN egg-laying command neurons in hyper-osmotic treated animals led to fewer and slower egg-laying events, an effect not seen following direct optogenetic stimulation of the postsynaptic vulval muscles. Hyperosmotic conditions also affected egg-laying circuit activity with the vulval muscles showing reduced Ca²⁺ transient amplitudes and frequency even after egg-laying resumes. Together, these results indicate that hyperosmotic conditions regulate egg-laying via two parallel mechanisms: a sensory pathway that acts to reduce HSN excitability and neurotransmitter release, and a biophysical mechanism where a hydrostatic pressure gradient reports egg accumulation in the uterus.