Rethinking Learning in the Simplest of Nervous Systems

Comparative research on learning has long focused on animals with complex nervous systems, from vertebrates to a handful of invertebrates such as honeybees. Yet including species at the other end of the spectrum is essential to understand how associative learning evolved in the first place. This new study turns to Caenorhabditis elegans, a tiny nematode of just one millimetre in length and 302 neurons, whose entire connectome has been completely mapped. Despite the simplicity of its nervous system, C. elegans is capable of several forms of learning —but the boundary between truly associative processes and simpler, non-associative ones has remained blurred.

The team asked whether this nematode could show behavioural signatures of conditioned inhibition (CI), a form of learning in which a stimulus predicts the absence of another. CI has traditionally been considered a hallmark of more complex nervous systems and, in invertebrates, robust evidence has only been reported in honeybees and a few gastropods. Testing this question in C. elegans offers a rare opportunity to probe the deep evolutionary roots of associative learning.

"Working with C. elegans allows us to ask fundamental questions about the origins of learning at the very base of the metazoan tree. What our results show is that behavioural changes that look like complex learning can actually rest on simpler, evolutionarily older mechanisms. This has real implications for how we interpret cognition across invertebrates."

— Miquel Llorente, co-author of the study and researcher at the Universitat de Girona.

Testing Conditioned Inhibition in a 302-Neuron Mind

To find out, the team designed an experiment in which one signal reliably predicted the absence of another. Twenty-four worms took part: half of them received the training, and the other half acted as a control group with no training at all.

Each worm was exposed to two very different kinds of signals: a chemical they tend to avoid (a mild detergent-like substance called SDS), and a light mechanical touch, delivered with a very thin, carefully calibrated fibre — similar in principle to the tools used to measure sensitivity in medical research. During the training, one of these signals was paired with the absence of the other, so that the worm could, in theory, learn to predict when the second signal would not happen.

To measure whether they had learned anything, the researchers compared the worms' behaviour before training, immediately after training, and again three hours later, to check both short- and medium-term memory. The design was carefully counterbalanced: they varied which signal was used, how strong it was, and whether it was applied to the head or the tail. This kind of rigorous control is essential in tiny animals like nematodes, where a simple sensory reflex can easily be mistaken for genuine learning.

The results were clear. The worms did change their behaviour after training, and the change was strongest just after the training session, weakening slightly three hours later. The statistical analysis confirmed that these changes were unlikely to be due to chance.

But there was a crucial twist. The change in behaviour did not actually depend on the specific signal the worms were supposed to have learned about. What predicted their new behaviour was simply the movement pattern the training had encouraged. In other words, the worms weren't really learning "signal A means signal B won't happen" —which would be true conditioned inhibition. Instead, the training was quietly reinforcing responses they already had built in from the start. This is a much older, simpler form of learning known as alpha conditioning: an experience does not create a new association, but strengthens one that was already there.

"When we designed the study, we wanted to be really careful about disentangling what is genuinely associative from what could be explained by simpler processes. Our results show that in C. elegans, alpha conditioning is enough to account for the behavioural changes we see —there is no need to invoke a more complex mechanism like true conditioned inhibition."

— Leonard Nigrini, first author of the study and predoctoral researcher at the Universitat de Girona.

Ancient Roots of Associative Learning

The implications go well beyond this small worm. If alpha conditioning is enough to explain apparent "complex learning" in C. elegans, then this basic form of learning is probably very ancient and shared by a huge range of animals —from the simplest invertebrates to ourselves. True conditioned inhibition, on the other hand, seems to require extra layers of brain processing that only appear in animals with much more elaborate nervous systems, like the honeybee (with around one million neurons) or vertebrates.

Beyond this specific result, the study makes a broader point about how comparative psychology works. Telling apart simple and complex forms of learning in tiny animals is genuinely difficult, and it requires careful experiments that can rule out the simplest explanation before jumping to more elaborate ones. This is exactly the kind of work the Comparative Minds Research Group has been developing across a wide variety of species, from nematodes to primates.

"Our work at the University of Girona aims to map the building blocks of learning and cognition across the animal kingdom. Studies like this one, at the simplest end of the neural complexity spectrum, are essential to complement our research on primates and other vertebrates. Only by looking at the whole tree can we start to understand how these capacities evolved."

— Miquel Llorente.

All data and R analysis code are publicly available through the Open Science Framework: osf.io/jevw4.

Reference: Nigrini, L., Amici, F., & Llorente, M. (2026). Caenorhabditis elegans: Learning Outcomes in Conditioned Inhibition Protocols are Consistent with Alpha Conditioning. International Journal of Comparative Psychology. https://doi.org/10.46867/ijcp.63132