Roundworms need a good night’s sleep to maintain the memories associated with particular smells, a study suggests.
When trained to avoid a sweet scent, Caenorhabditis elegans (C. elegans) nematodes can still remember to do so after 16 hours, as long as their post-training nap is uninterrupted. But disturbing their sleep prevents crucial changes to their nervous system that are involved in forming long-lasting memories.
The findings, published in Cell on 2 June1, pave the way for scientists to delve more into the processes that occur at the cellular and molecular level during sleep.
“This brings cellular resolution to a question that’s ancient”, namely what is the purpose of sleep and how is it linked to memory, says study co-author Noelle L’Etoile, a cell biologist at the University of California, San Francisco.
Eating in their sleep
L’Etoile and her colleagues wanted to investigate whether this also applied to C. elegans. Studying sleep in such a simple organism “might help us to really understand the underlying mechanism”, says Hanna Zwaka, a neuroscientist at Harvard University in Cambridge, Massachusetts. “We know their individual synapses, and that’s something that we don’t have in other animals.”
The research came with an unusual challenge, says L’Etoile: defining when the one-millimetre-long worms are in fact asleep. With no limbs or clear-cut sleep stages to observe, distinguishing a sleeping C. elegans from one that is awake is a tricky task.
The researchers used video-based imaging tools to observe hundreds of worms and look for telltale features of sleep such as reduced movement, stereotypical postures and seeming to ‘wake up’ when exposed to a flashing blue light.
While napping, “the little worms become straight with just a little crook where their head is”, says L’Etoile. Although they tend not to move around, the worms can’t resist sleep eating. “Their feeding is reduced, but it’s not zero,” she says.
Memories are thought to reside in synapses — the junctions between neurons. Part of the process of consolidating memories involves reducing the number of synapses during sleep, which is thought to balance connections between neurons in the brain and keep them within an appropriate range.
With only 302 neurons, C. elegans has one of the most streamlined nervous systems of any organism, says L’Etoile, and scientists have mapped its entire connectome — the web of all of the neurons and their connecting synapses. This makes the species ideal for studying the changes in synapses that happen during sleep.
To put the worms’ memory to the test, the researchers trained them to ignore the sweet scent of the compound butanone, which they normally find attractive. The team did this by associating the smell with the removal of food.
The worms detect the odour of butanone using a neuron called AWC, which connects to a pair of neurons called AIY. Using an imaging tool called GRASP to elicit green fluorescence at synapses, the authors showed that training the worms to avoid the odour caused a reduction in the number of AWC–AIY synapses in their nervous system.
The worms also tended to rest after the training. To test whether this post-training nap is required to maintain odour memory, the researchers disrupted the worms’ sleep by either gently shaking them every 15 minutes, or removing food.
The worms that enjoyed uninterrupted sleep immediately after the odour training did not react to the smell of butanone after 16 hours. But the ones deprived of sleep in the first 1–2 hours after training showed more-intense synaptic connections than did those with undisturbed sleep, and didn’t remember to stay away from butanone after 16 hours.
“When the odour becomes a solidified memory, it requires sleep,” says L’Etoile. The results suggest that the worms have a critical sleep window of 1–2 hours after training, during which they solidify their memories. When the worms’ sleep was disrupted 2–6 hours post-training, it did not have the same effect on memory retention.
For David Raizen, a neuroscientist at the University of Pennsylvania in Philadelphia, the next step will be to study the biochemical mechanisms though which sleep behaviour induces these changes in the number and strength of synapses.