A quantitative portrait of habituation in Stentor coeruleus

Reviewer #1 (Public review):

This interesting paper addresses the phenomenon of potentiation in single-cell habituation in Stentor coeruleus. This is an important "hallmark" of habituation that helps to establish single-cell learning as being similar to habituation in animals. Prior studies from Wood, as well as our own results, have shown that potentiation occurs in Stentor, but I have always remained a little bit skeptical that this effect was possibly just due to incomplete recovery after the first trial. When I first read this paper and saw the habituation curves for the first and second trials, such as in Figure 5, I thought, yes, that is definitely what is happening, and so is this really potentiation?

The authors were also clearly aware of this issue and, notably, they embraced it head-on by developing an analysis that allows potentiation effects to be detected even despite failure of the cell to fully recover after the first trial. The key is their "phase portrait" that allows the learning process to be depicted as a curve capturing how learning rates and response probability evolve over time, thus allowing the curves to be compared between trials. If my interpretation was correct that so-called potentiation was just incomplete recovery, the prediction would be that the curves for two successive trials would overlap, with the first trial curve extending beyond the second one towards higher response probabilities, which would be lost in the second trial due to failure to recover fully. But the data clearly are not consistent with that idea. I think that this result is very strong and important.

Especially nice is the approach of Figure 7C, which uses a vertical shift in the phase portrait as an indicator of potentiation. I did, however, find Figure 6 a little hard to digest at first, and I have a few suggestions about that. First, I think it would be a good idea to explicitly say which curve is the first trial and which is the second. Second, I think it would help readers if the authors could start with a cartoon that explains visually what the curves mean. For example, show a habituation curve, indicate how the slope is calculated at different parts of the curve, and then show how the slope versus response are plotted to make the phase portrait. It is all spelled out in the text, but it would help a lot of readers to see it visually, I think.

One question I have about Figure 6 is that it looks like the specific case of ITI 1 hour ISI 2 min has some kind of pathological behavior in the second trial, despite not seeing any indication of any 'weirdness' in Figure 5. I gather that this is meant to be due at least in part to the incomplete recovery seen after the first trial, but then I don't see why this would not also be an issue for ITI 1 hour ISI 3 min. I would not require the authors to explain every anomaly, but this one stands out, and I feel it could be telling us something interesting.

Reviewer #2 (Public review):

Summary:

The authors address habituation and potentiation in the single-celled organism Stentor in a large data set by systematically varying stimulus frequency and recovery duration. They analyze habituation dynamics on the level of single cells within a Bayesian inference framework to map out how the response probability of individual cells decays during training. Mapping out the progression of habituation quantified by learning rate versus decaying response probability, they observe different dynamics for different stimulus frequencies and recovery durations, which they reconcile with multiple time-scales governing the memory of prior training.

Strengths:

The authors accumulate a systematic, broad data set of Stentor habituation and potentiation, which, in combination with the Bayesian framework they developed, unfolds its power to probe underlying habituation dynamics and challenge theoretical frameworks.

Weaknesses:

The interlacing of theoretical framework, existing concepts and expectation, and experimental data in their narrative may challenge readers. The Bayesian inference of habituations is very successful in concluding that their variation with stimulus frequency and recovery duration points to multiple time scales of memory are involved. However, the authors' comprehensive analysis of potentiation may need more guidance to follow the authors' conclusions.

The combination of a dynamical systems-driven hypothesis, experimental data, and statistical analysis, as put forward in this work, is immensely powerful for uncovering the mechanisms that facilitate learning, such as habituation and potentiation, in single-celled organisms.

Author response:

We thank the reviewers and editor for their thoughtful and constructive comments. Our goal was to connect theoretical work on habituation with empirical findings on intracellular habituation in Stentor coeruleus. We developed the phase-portrait analysis to provide a more formal way to examine habituation dynamics and to address the concern that apparent potentiation might simply reflect incomplete recovery. We are glad that the reviewers found this approach useful, and we hope to build on it in future work through mechanistic modeling.

We will submit a revised version with the following changes:

(1) We will include a supplementary figure that provides visual intuition for the habituation curves and phase portraits.

(2) We agree that the anomalous behavior of the 2 min ISI / 1 hr ITI condition is noteworthy. This behavior arises from a subtle difference in the fitted shape of the trial 2 habituation curve: its Hill coefficient is less than 1, so the curve has no inflection point and its initial slope has nonzero magnitude. As a result, the corresponding phase portrait begins away from the x-axis, unlike the other conditions, whose Hill coefficients are greater than 1 and whose phase portraits are U-shaped. We will discuss this explicitly in the revision.

(3) We will reconsider the layout to make the background and results easier to follow. In particular, we will consolidate the repeated material while preserving the context needed to interpret the theoretical consequences of the empirical findings.

(4) We will revise the conclusions and discussion to leave claims about the decay of potentiation more open-ended.