Cell-type-specific responses to associative learning in the primary motor cortex
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Evaluation Summary:
Using advanced live brain imaging techniques, the authors studied the activities of neurons in the primary motor cortex of mice during a classical conditional task, in which a tone is paired with water reward. They found that distinct types of neurons respond differently to the auditory cue or the reward, and the responses evolve differentially as learning proceeds. This work reveals an interesting role of the motor cortex beyond its well-recognized function in motor control, and suggests distinct functions of pyramidal neurons as well as various interneurons in reinforcement learning.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)
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Abstract
The primary motor cortex (M1) is known to be a critical site for movement initiation and motor learning. Surprisingly, it has also been shown to possess reward-related activity, presumably to facilitate reward-based learning of new movements. However, whether reward-related signals are represented among different cell types in M1, and whether their response properties change after cue–reward conditioning remains unclear. Here, we performed longitudinal in vivo two-photon Ca 2+ imaging to monitor the activity of different neuronal cell types in M1 while mice engaged in a classical conditioning task. Our results demonstrate that most of the major neuronal cell types in M1 showed robust but differential responses to both the conditioned cue stimulus (CS) and reward, and their response properties undergo cell-type-specific modifications after associative learning. PV-INs’ responses became more reliable to the CS, while VIP-INs’ responses became more reliable to reward. Pyramidal neurons only showed robust responses to novel reward, and they habituated to it after associative learning. Lastly, SOM-INs’ responses emerged and became more reliable to both the CS and reward after conditioning. These observations suggest that cue- and reward-related signals are preferentially represented among different neuronal cell types in M1, and the distinct modifications they undergo during associative learning could be essential in triggering different aspects of local circuit reorganization in M1 during reward-based motor skill learning.
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Evaluation Summary:
Using advanced live brain imaging techniques, the authors studied the activities of neurons in the primary motor cortex of mice during a classical conditional task, in which a tone is paired with water reward. They found that distinct types of neurons respond differently to the auditory cue or the reward, and the responses evolve differentially as learning proceeds. This work reveals an interesting role of the motor cortex beyond its well-recognized function in motor control, and suggests distinct functions of pyramidal neurons as well as various interneurons in reinforcement learning.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their …
Evaluation Summary:
Using advanced live brain imaging techniques, the authors studied the activities of neurons in the primary motor cortex of mice during a classical conditional task, in which a tone is paired with water reward. They found that distinct types of neurons respond differently to the auditory cue or the reward, and the responses evolve differentially as learning proceeds. This work reveals an interesting role of the motor cortex beyond its well-recognized function in motor control, and suggests distinct functions of pyramidal neurons as well as various interneurons in reinforcement learning.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
Lee et al. report on population-level cell type changes in primary motor cortex (M1) as water-deprived, head-fixed mice undergo associative learning. Mice were exposed to a condition stimulus (auditory tone), followed by a 1.5s delay and then delivery of an unconditioned stimulus (water reward), while the group simultaneously carried out in vivo two-photon calcium imaging of pyramidal neurons (PNs), somatostatin-, parvalbumin-, and vasoactive intestinal peptide-expressing inhibitory neurons (SOM-Ins, PV-Ins, and VIP-Ins, respectively) on the first and seventh day of the task. This investigation indicates that there are cell-type dependent responses encoding the cue and reward stimulus in M1. The group asserts that all of these four major cell types show distinct modifications after associative learning and …
Reviewer #1 (Public Review):
Lee et al. report on population-level cell type changes in primary motor cortex (M1) as water-deprived, head-fixed mice undergo associative learning. Mice were exposed to a condition stimulus (auditory tone), followed by a 1.5s delay and then delivery of an unconditioned stimulus (water reward), while the group simultaneously carried out in vivo two-photon calcium imaging of pyramidal neurons (PNs), somatostatin-, parvalbumin-, and vasoactive intestinal peptide-expressing inhibitory neurons (SOM-Ins, PV-Ins, and VIP-Ins, respectively) on the first and seventh day of the task. This investigation indicates that there are cell-type dependent responses encoding the cue and reward stimulus in M1. The group asserts that all of these four major cell types show distinct modifications after associative learning and that cue- and reward- related signals are coded for by major cell types in M1. In particular, it is suggested that PV-INs modulate local microcircuit activity related to the CS association in M1 and that VIP-INs act as a context-dependent switch following the reward delivery. This study provides evidence that M1 may have a broader, more diverse range of functions than previously appreciated
Strengths:
The paper takes a broad, comprehensive look at non-motor related responses in M1 during associative learning, dissecting responses across all the major cell types. This work provides a better appreciation of the heterogeneity of responses observed across the cortex and how those changes may emerge through experience. Overall, the experiments, data analysis, and results reported are highly rigorous.
Weaknesses:
The major weakness of the manuscript is the lack of analysis of motor-related activity that presumably exists in their data set. While the authors intentionally focus on non-motor responses and select a behavior that does not necessarily involve motor learning, the significance and interpretation of their findings seems incomplete without examining the motor activity and its relationship to cue and reward activity. This could better disambiguated whether the plasticity in cue and reward activity in M1 reflect local circuit changes as the authors implies or changes occurring in upstream areas that are then inherited by specific cell types in M1.
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Reviewer #2 (Public Review):
Using advanced live brain imaging techniques, the authors studied the activities of neurons in the primary motor cortex of mice during a classical conditional task, in which a tone is paired with water reward. They found that distinct types of neurons respond differently to the auditory cue or the reward, and the responses evolve differentially as learning proceeds. This work reveals an interesting role of the motor cortex beyond its well-recognized function in motor control, and suggests distinct functions of pyramidal neurons as well as various interneurons in reinforcement learning.
The investigation of the M1's role in classical condition is intriguing and the finding is interesting. The behavioral paradigm is straightforward and the systematic examination of different neuronal types is careful.
However, …
Reviewer #2 (Public Review):
Using advanced live brain imaging techniques, the authors studied the activities of neurons in the primary motor cortex of mice during a classical conditional task, in which a tone is paired with water reward. They found that distinct types of neurons respond differently to the auditory cue or the reward, and the responses evolve differentially as learning proceeds. This work reveals an interesting role of the motor cortex beyond its well-recognized function in motor control, and suggests distinct functions of pyramidal neurons as well as various interneurons in reinforcement learning.
The investigation of the M1's role in classical condition is intriguing and the finding is interesting. The behavioral paradigm is straightforward and the systematic examination of different neuronal types is careful.
However, a few technical concerns remain to be addressed or clarified and it will also be very helpful to add in some discussion to put their findings in a framework.
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Reviewer #3 (Public Review):
This study investigated how reward-associated signals are represented in layer 2/3 neurons of the primary motor cortex. Water-restricted mice were trained to respond to a conditioned auditory stimulus in order to receive a water reward. Behavior analysis showed that mice quickly learned the association between the sound stimulus and the reward as indicated by increased anticipatory lick rate. Using this behavioral paradigm, neuronal activity was monitored throughout the training (7 days). Two-photon calcium imaging was performed separately from four different types of neurons; pyramidal neurons, PV-, VIP-, and SOM-positive interneurons. Tuning of individual neurons to the tone and reward stimuli were analyzed by using Spearman correlation between the trial-averaged fluorescence and the timing of stimulus …
Reviewer #3 (Public Review):
This study investigated how reward-associated signals are represented in layer 2/3 neurons of the primary motor cortex. Water-restricted mice were trained to respond to a conditioned auditory stimulus in order to receive a water reward. Behavior analysis showed that mice quickly learned the association between the sound stimulus and the reward as indicated by increased anticipatory lick rate. Using this behavioral paradigm, neuronal activity was monitored throughout the training (7 days). Two-photon calcium imaging was performed separately from four different types of neurons; pyramidal neurons, PV-, VIP-, and SOM-positive interneurons. Tuning of individual neurons to the tone and reward stimuli were analyzed by using Spearman correlation between the trial-averaged fluorescence and the timing of stimulus delivery. Results showed that PV-positive interneuron responses became more reliable to the cue stimulus, whereas VIP-positive interneuron responses became more reliable to the reward stimulus. Some SOM-INs that were not responsive to the tone before training became responsive at day 7 of training. Activity of SOM-INs became more reliable to the reward after learning. The main findings are quite novel and may provide a new insight into the specific roles of interneurons. More representative imaging data and control experiments will make the story even more complete and convincing.
- Imaging calcium responses from individual types of interneurons are important and challenging. Tracing activity changes from same population of neurons is especially important because it will show how learning shapes the pattern of changes in each neuron. Despite such powerful approaches, activity changes from each neuron were not shown. Calcium transients measured at day 1 were re-sorted at day 7, so it is not clear whether the same neurons responsive to cue or reward stimulus are still responsive to the same stimuli and, if so, how their onset timing is changed. Knowing whether the cue- or reward-sensitive population is the same population or not may lead to a different conclusion, so plotting calcium signals over days without resorting would be important.
In addition, representative calcium images from interneurons were not shown (like Fig. 1A). It seemed that about 80-90 cells of PV-INs, VIP-INs, SOM-INs were observed (Fig. 2D). Showing some representative images from individual cell types would be helpful for readers to better understand the results.
- Identifying active cells that are above the chance level was good to define a subset of neurons responsive to a period of cue- or reward-stimulus. Quantifying the tuning of each cell's average response during the tone and reward response periods using non-parametric Spearman correlation was also a powerful way to display a subset of neurons with high or low trial-by-trial reliability. Results suggest that there are changes of less reliable neurons to more reliable ones in the case of PV-INs and VIP-INs (Fig. 4 and 5). However, whether these changes are specifically associated with learning is not clear. Running control experiments without water restriction or with random reward presentation independent of cues would be a good comparison. These experiments will help to rule out the possibility of naturally happening learning-independent changes from day 1 to day 7.
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