Goal-directed motor actions drive acetylcholine dynamics in sensory cortex

Reviewer #1 (Public Review):

Summary:

This study aimed at gaining a better comprehension of the functional role of acetylcholine release within the sensory cortex. To this end, the authors measured the dynamics of cortical acetylcholine release using two-photon imaging of the GRAB-Ach3.0 fluorescent sensor, either in the mouse primary somatosensory cortex (S1), throughout the learning of a whisker-dependent object position discrimination task, or in the primary auditory cortex (A1) of mice engaged in a specific sound signal detection task.

The illustrated results suggest that variations in acetylcholine release tend to be associated, in the primary sensory areas, with goal-directed actions (whisking in the case of the object position discrimination task, and more strongly with licking), rather than with sensory inputs or rewards. They also indicate that the variations in cholinergic signal specifically associated with licking increase with learning.

Strengths:

The impact of cholinergic inputs on cortical function has intrigued neuroscientists for many decades due to the complexity of its mode of action on the molecular and cellular points of view.

Being able to image the dynamics of cortical cholinergic release in vivo on mice engaged in goal-directed tasks has moved this field into a really exciting phase, where it becomes possible to draw links between specific behavioral features and local variations of cholinergic release in given cortical areas.

This study is therefore particularly timely, it provides a set of precious and original data. Globally the experiments were rigorously designed, and the illustrated quantifications and analyses follow high standards. This work therefore constitutes a valuable contribution to this field of research and could be of interest to a large audience.

Weaknesses:

Although the manuscript reports very interesting links between behavior and cortical cholinergic release, the study remains correlative and is devoid of experiments allowing to link causally cholinergic cortical inputs with motor actions, and more globally to gauge their impact on learning and execution of the tasks. Since the nature of the link between goal-directed motor actions and acetylcholine dynamics is not really clarified here, the word "drive" in the title of the paper, which may have a causal connotation should be replaced (especially since acetylcholine-related signal fluctuations seems often to precede motor actions).

As high-speed videography of the C2 whisker was achieved during the object position discrimination task, it seems that the whisker curvature changes could have been quantified in addition to the whisker angle. This would allow appreciation of how acetylcholine related signals vary according to both whisker-related motor output and sensory input, hereby providing clearer support for the assertion that acetylcholine levels are "related to motor actions rather than sensory inputs".

The data set related to the auditory task is used here to support the claim that licks rather than rewards are linked to variations of fluorescence of the cholinergic sensor in sensory cortices. These data seem very interesting indeed but are shown here in a very incomplete manner (a figure illustrating the learning curves of the 6 recorded animals, and acetylcholine dynamics during the four types of trials would be very welcome). If the animals were placed on a treadmill and the locomotion measured, together with pupil size, during the task as in Gee et al., BioRxiv 2022, one could ask how these other motor activities are linked with acetylcholine dynamics in A1. By comparing the impact of goal-directed actions versus motor activities accompanying more global state transitions on acetylcholine dynamics, these data could provide a particularly valuable contribution to this study. They could in addition rule out potential confounding factors regarding the claim that cholinergic dynamics are here mainly linked to first licks.

Coming back to the whisker-dependent object localization task, if cholinergic-related signals have been recorded during the "no whisker sessions", analyzing these data would be very useful in the scope of this study. Indeed, during these sessions, the animals were not naive, since they went through the learning of the task, but could not resolve it anymore, still they most probably kept on licking upon the pole-in and/or pole-out cues. In these sessions, the licking is fully dissociated from tactile sensory inputs, and for this reason it would be particularly interesting to see how the fluorescence varies with first licks. In addition, plotting these sessions in Figure 6C would be informative. Indeed, if the increase of cholinergic signals with performance comes progressively due to changes in the internal state of the animal and/or plasticity mechanisms, first lick related cholinergic signal variations could remain high despite the decrease of performance in these sessions.

Finally, because the functional role of cortical cholinergic release is a hot topic, a few recent studies addressing this question with slightly different approaches in the visual cortex would be worth mentioning, at least in the discussion, as well as a recent study focusing on motor learning, which revealed an apparent decrease of acetylcholine dynamics associated with goal-directed motor actions upon learning.

Reviewer #2 (Public Review):

Summary:

The paper entitled "Goal-directed motor actions drive acetylcholine dynamics in sensory cortex" aims to characterize the dynamics of cholinergic signaling in sensory cortex during perceptual behavior. The authors showed that acetylcholine release in S1 was linked to goal-directed motor actions rather than sensory input or reward delivery, a pattern also observed in the auditory cortex (A1). This release was specifically associated with whisking and licking and was potentiated by training. The results contribute to a better understanding of neuromodulator actions. That said, several aspects of the manuscript could benefit from improved writing, data presentation, and statistical analysis.

Strengths:

The evidence provided is clear to link ACh response to different task-related events. Implementing two different tasks to show generality is appreciated. Important control analysis is included.

Weaknesses:

The quantification of ACh signal differences across different trial types or between expert and early-training mice is lacking. Although statistical significance is occasionally mentioned, the indication of significance in figures seems rare. For example, in Figures 5A and E, it is difficult to tell when p is < 0.05. Based on the sentence "small, but significant increase on Hits over False Alarm trials (Figure 5A, S Figure 4A)" there is indeed a time point where the difference is significant, and more details should be added (when and the p-value).

For Figure 5D, it seems like there is no significant difference between Hit and False alarm trials, however, for the trials with 1 or 2 lick there appears to be a difference. Is it due to a lack of power? Moreover, in Figure 5 H the first licks also seem to differ.

Linear regression: the coefficient of determination (R²) is absent, in Figures 4E, F, and 6B, H, making it hard to evaluate the goodness of the fitting.

Similar comments apply to Figure 7: the lack of quantitative comparisons between the coefficients of first lick and other regressors, and between early and expert training, as well as the change in goodness of fit by removing a regressor.

The writing of the introduction and discussion could be improved to enhance readability, and the manuscript could improve its discussion on orofacial movement and acetylcholine release by citing relevant studies demonstrating the association between neuronal activity and orofacial/body movements.