Beyond Auditory Relay: Dissecting the Inferior Colliculus’s Role in Sensory Prediction, Cognitive Decision-Making, and Reward Prediction

Reviewer #1 (Public review):

Summary:

This work made a lot of efforts to explore the multifaceted roles of the inferior colliculus (IC) in auditory processing, extending beyond traditional sensory encoding. The authors recorded neuronal activitity from the IC at single unit level when monkeys were passively exposed or actively engaged in behavioral task. They concluded that 1）IC neurons showed sustained firing patterns related to sound duration, indicating their roles in temporal perception, 2) IC neuronal firing rates increased as sound sequences progress, reflecting modulation by behavioral context rather than reward anticipation, 3) IC neurons encode reward prediction error and their capability of adjusting responses based on reward predictability, 4) IC neural activity correlates with decision-making. In summary, this study tried to provide a new perspective on IC functions by exploring its roles in sensory prediction and reward processing, which are not traditionally associated with this structure.

Strengths:

The major strength of this work is that the authors performed electrophysiological recordings from the IC of behaving monkeys. Compared with the auditory cortex and thalamus, the IC in monkeys has not been adequately explored.

Weaknesses:

(1) The authors cited several papers focusing on dopaminergic inputs in the IC to suggest the involvement of this brain region in cognitive functions. However, all those cited work were done in rodents. Whether monkey's IC shares similar inputs is not clear.
(2) The authors confused the two terms, novelty and deviation. According to their behavioral paradigm, deviation rather than novelty should be used in the paper because all the stimuli have been presented to the monkeys during training. Therefore, there is actually no novel stimuli but only deviant stimuli. This reflects that the author has misunderstood the basic concept.
(3) Most of the conclusions were made based on correlational analysis or speculation without providing causal evidences.
(4) Results are presented in a very "straightforward" manner with too many detailed descriptions of phenomena but lack of summary and information synthesis. For example, the first section of Results is very long but did not convey clear information.
(5) The logic between different sections of Results is not clear.
(6) In the Discussion, there is excessive repetition of results, and further comparison with and discussion of potentially related work are very insufficient. For example, Metzger, R.R., et al. (J Neurosc, 2006) have shown similar firing patterns of IC neurons and correlated their findings with reward.

Reviewer #2 (Public review):

Summary:

The inferior colliculus (IC) has been explored for its possible functions in behavioral tasks and has been suggested to play more important roles rather than simple sensory transmission. The authors revealed the climbing effect of neurons in IC during decision-making tasks, and tried to explore the reward effect in this condition.

Strengths:

Complex cognitive behaviors can be regarded as simple ideals of generating output based on information input, which depends on all kinds of input from sensory systems. The auditory system has hierarchic structures no less complex than those areas in charge of complex functions. Meanwhile, IC receives projections from higher areas, such as auditory cortex, which implies IC is involved in complex behaviors. Experiments in behavioral monkeys are always time-consuming works with hardship, and this will offer more approximate knowledge of how the human brain works.

Weaknesses:

These findings are more about correlation but not causality of IC function in behaviors. And I have a few major concerns.

Comparing neurons' spike activities in different tests, a 'climbing effect' was found in the oddball paradigm. The effect is clearly related to training and learning process, but it still requires more exploration to rule out a few explanations. First, repeated white noise bursts with fixed inter-stimulus-interval of 0.6 seconds was presented, so that monkeys might remember the sounds by rhymes, which is some sort of learned auditory response. It is interesting to know monkeys' responses and neurons' activities if the inter-stimuli-interval is variable. Second, the task only asked monkeys to press one button and the reward ratio (the ratio of correct response trials) was around 78% (based on the number from Line 302). so that, in the sessions with reward, monkeys had highly expected reward chances, does this expectation cause the climbing effect?

"Reward effect" on IC neurons' responses were showed in Fig. 4. Is this auditory response caused by physical reward action or not? In reward sessions, IC neurons have obvious response related to the onset of water reward. The electromagnetic valve is often used in water-rewarding system and will give out a loud click sound every time when the reward is triggered. IC neurons' responses may be simply caused by the click sound if the electromagnetic valve is used. It is important to find a way to rule out this simple possibility.

Reviewer #3 (Public review):

Summary:

The authors aimed to investigate the multifaceted roles of the Inferior Colliculus (IC) in auditory and cognitive processes in monkeys. Through extracellular recordings during a sound duration-based novelty detection task, the authors observed a "climbing effect" in neuronal firing rates, suggesting an enhanced response during sensory prediction. Observations of reward prediction errors within the IC further highlight its complex integration in both auditory and reward processing. Additionally, the study indicated IC neuronal activities could be involved in decision-making processes.

Strengths:

This study has the potential to significantly impact the field by challenging the traditional view of the IC as merely an auditory relay station and proposing a more integrative role in cognitive processing. The results provide valuable insights into the complex roles of the IC, particularly in sensory and cognitive integration, and could inspire further research into the cognitive functions of the IC.

Weaknesses:

Major Comments:

(1) Structural Clarity and Logic Flow:
The manuscript investigates three intriguing functions of IC neurons: sensory prediction, reward prediction, and cognitive decision-making, each of which is a compelling topic. However, the logical flow of the manuscript is not clearly presented and needs to be well recognized. For instance, Figure 3 should be merged into Figure 2 to present population responses to the order of sounds, thereby focusing on sensory prediction. Given the current arrangement of results and figures, the title could be more aptly phrased as "Beyond Auditory Relay: Dissecting the Inferior Colliculus's Role in Sensory Prediction, Reward Prediction, and Cognitive Decision-Making."

(2) Clarification of Data Analysis:
Key information regarding data analysis is dispersed throughout the results section, which can lead to confusion. Providing a more detailed and cohesive explanation of the experimental design would significantly enhance the interpretation of the findings. For instance, including a detailed timeline and reward information for the behavioral paradigms shown in Figures 1C and D would offer crucial context for the study. More importantly, clearly presenting the analysis temporal windows and providing comprehensive statistical analysis details would greatly improve reader comprehension.

(3) Reward Prediction Analysis:
The conclusion regarding the IC's role in reward prediction is underdeveloped. While the manuscript presents evidence that IC neurons can encode reward prediction, this is only demonstrated with two example neurons in Figure 6. A more comprehensive analysis of the relationship between IC neuronal activity and reward prediction is necessary. Providing population-level data would significantly strengthen the findings concerning the IC's complex functionalities. Additionally, the discussion of reward prediction in lines 437-445, which describes IC neuron responses in control experiments, does not sufficiently demonstrate that IC neurons can encode reward expectations. It would be valuable to include the responses of IC neurons during trials with incorrect key presses or no key presses to better illustrate this point.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

This work made a lot of efforts to explore the multifaceted roles of the inferior colliculus (IC) in auditory processing, extending beyond traditional sensory encoding. The authors recorded neuronal activitity from the IC at single unit level when monkeys were passively exposed or actively engaged in behavioral task. They concluded that 1）IC neurons showed sustained firing patterns related to sound duration, indicating their roles in temporal perception, 2) IC neuronal firing rates increased as sound sequences progress, reflecting modulation by behavioral context rather than reward anticipation, 3) IC neurons encode reward prediction error and their capability of adjusting responses based on reward predictability, 4) IC neural activity correlates with decision-making. In summary, this study tried to provide a new perspective on IC functions by exploring its roles in sensory prediction and reward processing, which are not traditionally associated with this structure.

Strengths:

The major strength of this work is that the authors performed electrophysiological recordings from the IC of behaving monkeys. Compared with the auditory cortex and thalamus, the IC in monkeys has not been adequately explored.

We appreciate the reviewer’s acknowledgment of the efforts and strengths of our study. Indeed, our goal was to provide a comprehensive exploration of the multifaceted roles of the inferior colliculus (IC) in auditory processing and beyond, particularly in sensory prediction and reward processing. The use of electrophysiological recordings in behaving monkeys was central to our approach, as we sought to uncover the underexplored aspects of IC function in these complex cognitive domains. We are pleased that the reviewer recognizes the value of investigating the IC, a structure that has not been adequately explored in primates compared to other auditory regions like the cortex and thalamus. This feedback reinforces our belief that our work contributes significantly to advancing the understanding of the IC's roles in cognitive processing.

We look forward to addressing any further points the reviewers may have and refining our manuscript accordingly. Thank you for your constructive feedback and for recognizing the strengths of our research approach.

Weaknesses:

(1) The authors cited several papers focusing on dopaminergic inputs in the IC to suggest the involvement of this brain region in cognitive functions. However, all those cited work were done in rodents. Whether monkey's IC shares similar inputs is not clear.

We appreciate the reviewer's insightful comment on the limitations of extrapolating findings from rodent models to monkeys, particularly concerning dopaminergic inputs to the Inferior Colliculus (IC). While it is true that most studies on dopaminergic inputs to the IC have been conducted in rodents, to our knowledge, no studies have been conducted specifically in primates. To address the reviewer's concern, we have added a statement in both the introduction and discussion sections of our manuscript:

- Introduction: " However, these studies were conducted in rodents, and the existence and role of dopaminergic inputs in the primate IC remain underexplored."

- Discussion: " However, the exact mechanisms and functions of dopamine modulation in the inferior colliculus are still not fully understood, particularly in primates. "

(2) The authors confused the two terms, novelty and deviation. According to their behavioral paradigm, deviation rather than novelty should be used in the paper because all the stimuli have been presented to the monkeys during training. Therefore, there is actually no novel stimuli but only deviant stimuli. This reflects that the author has misunderstood the basic concept.

We appreciate the reviewer's clarification regarding the distinction between "novelty" and "deviation" in the context of our behavioral paradigm. We agree that, given the nature of our experimental design where all stimuli were familiar to the monkeys during training, the term "deviation" more accurately describes the stimuli used in our study rather than "novelty."

To address this, we have revised the manuscript to replace the term "novelty" with "deviation" wherever applicable. This change has been made to ensure accurate terminology is used throughout the paper, thereby eliminating any potential misunderstanding of the concepts involved in our study.

We thank the reviewer for pointing out this important distinction, which has improved the clarity and precision of our manuscript.

(3) Most of the conclusions were made based on correlational analysis or speculation without providing causal evidences.

We appreciate the reviewer’s concern regarding the reliance on correlational analyses in our study. Indeed, we acknowledge that the conclusions drawn primarily reflect correlations between neuronal activity and behavioral outcomes, rather than direct causal evidence. This limitation is inherent to many electrophysiological studies, particularly those conducted in behaving primates, where direct manipulation of specific neural circuits to establish causality is often challenging.

This limitation becomes even more complex when considering the IC’s role as a key lower-level relay station in the auditory pathway. Manipulating IC activity could potentially affect auditory responses in downstream pathways, which, in turn, may influence sensory prediction and decision-making processes. Moreover, we hypothesize that the sensory prediction and reward signals observed in the IC may not have direct causal effects but may instead be driven by top-down projections from higher cognitive regions. However, it is important to emphasize that our study provides novel evidence that the IC may exhibit multiple facets of cognitive signaling, which could inspire future research into the underlying mechanisms and broader functional implications of these signals.

To address this, we have taken the following steps in our revised manuscript:

(1) Clarified the Scope of Conclusions: We have revised the language in the Results and Discussion sections to explicitly state that our findings represent correlational relationships rather than causal mechanisms. For example, we now refer to the associations observed between IC activity and behavioral outcomes as "correlational" and have refrained from making definitive causal claims without supporting experimental evidence.

(2) Proposed Future Directions: In the Discussion section, we have included suggestions for future studies to directly test the causality of the observed relationships. We acknowledge the need for further investigation to substantiate the causal links between IC activity and cognitive functions such as sensory prediction, decision-making, and reward processing.

We believe these revisions provide a more balanced interpretation of our findings while emphasizing the importance of future research to build on our results and establish causal relationships. Thank you for raising this critical point, which has led to a more rigorous and transparent presentation of our study.

(4) Results are presented in a very "straightforward" manner with too many detailed descriptions of phenomena but lack of summary and information synthesis. For example, the first section of Results is very long but did not convey clear information.

We appreciate the reviewer’s feedback regarding the presentation of our results. We understand that the detailed descriptions of phenomena may have made it difficult to discern the key findings and overarching themes in the study. We recognize the importance of balancing detailed reporting with clear summaries and synthesis to effectively communicate our findings.

To address this concern, we have made the following revisions to the manuscript:

(1) Condensed and Synthesized Key Findings: We have streamlined the presentation of the Results section by condensing overly detailed descriptions and focusing on the most critical aspects of the data. Key findings are now summarized at the end of each subsection to ensure that the main points are clearly conveyed.

(2) Enhanced Section Summaries: We have added summary statements at the end of each major results section to synthesize the findings and highlight their significance. This should help guide the reader through the narrative and emphasize the key takeaways from each part of the study.

(3) Improved Flow and Clarity: We have revised the structure and organization of the Results section to improve the flow of information. By rearranging certain paragraphs and refining the language, we aim to present the results in a more cohesive and coherent manner.

We believe these changes will make the Results section more accessible and informative, allowing readers to more easily grasp the significance of our findings. Thank you for your valuable suggestion, which has significantly improved the clarity and impact of our manuscript.

(5) The logic between different sections of Results is not clear.

We appreciate the reviewer’s observation regarding the lack of clear logical connections between different sections of the Results. We acknowledge that a coherent flow is essential for effectively communicating the progression of findings and their implications.

To address this concern, we have made the following revisions:

(1) Enhanced Transitions Between Sections: We have introduced clearer transitional statements between sections of the Results. These transitions explicitly state how each new section builds upon or relates to the previous findings, creating a more cohesive narrative.

(2) Integration of Findings: In several places within the Results, we have added brief synthesis paragraphs that integrate findings across sections. These integrative summaries help to tie together the different aspects of our study, demonstrating how they collectively contribute to our understanding of the Inferior Colliculus’s (IC) role in sensory prediction, decision-making, and reward processing.

(3) Clarified Rationale: At the beginning of each major section, we have clarified the rationale behind why certain experiments were conducted, connecting them more clearly to the overarching goals of the study. This should help the reader understand the purpose of each set of results in the context of the broader research objectives.

We believe these changes improve the overall coherence and readability of the Results section, allowing readers to better follow the logical progression of our study. We are grateful for this constructive feedback and believe it has significantly enhanced the manuscript.

(6) In the Discussion, there is excessive repetition of results, and further comparison with and discussion of potentially related work are very insufficient. For example, Metzger, R.R., et al. (J Neurosc, 2006) have shown similar firing patterns of IC neurons and correlated their findings with reward.

We appreciate the reviewer's insightful critique regarding the excessive repetition in the Discussion and the lack of sufficient comparison with related work. We acknowledge that a well-balanced Discussion should not only interpret findings but also place them in the context of existing literature to highlight the novelty and significance of the study.

To address these concerns, we have made the following revisions:

(1) Reduction of Repetition: We have carefully revised the Discussion to minimize redundant repetition of the Results. Instead of restating the findings, we now focus more on their implications, limitations, and how they advance the current understanding of the Inferior Colliculus (IC) and its broader cognitive roles.

(2) Incorporation of Related Work: We have expanded the Discussion to include a more comprehensive comparison with existing literature, specifically highlighting studies that have reported similar findings. For example, we now discuss the work by Metzger et al. (2006), which demonstrated similar firing patterns of IC neurons and correlated these with reward-related processes. This comparison helps contextualize our results and emphasizes the novel contributions our study makes to the field.

We believe these revisions have significantly improved the quality of the Discussion by reducing unnecessary repetition and providing a more thorough engagement with the relevant literature. We are grateful for the reviewer's valuable feedback, which has helped us refine and strengthen the manuscript.

Reviewer #2 (Public review):

Summary:

The inferior colliculus (IC) has been explored for its possible functions in behavioral tasks and has been suggested to play more important roles rather than simple sensory transmission. The authors revealed the climbing effect of neurons in IC during decision-making tasks, and tried to explore the reward effect in this condition.

Strengths:

Complex cognitive behaviors can be regarded as simple ideals of generating output based on information input, which depends on all kinds of input from sensory systems. The auditory system has hierarchic structures no less complex than those areas in charge of complex functions. Meanwhile, IC receives projections from higher areas, such as auditory cortex, which implies IC is involved in complex behaviors. Experiments in behavioral monkeys are always time-consuming works with hardship, and this will offer more approximate knowledge of how the human brain works.

We greatly appreciate the reviewer's positive summary of our work and recognition of the effort involved in conducting experiments on behaving monkeys. We agree with the reviewer that the inferior colliculus (IC) plays a significant role beyond mere sensory transmission, particularly in integrating sensory inputs with higher cognitive functions. Our study aims to shed light on these complex functions by revealing the climbing effect of IC neurons during decision-making tasks and exploring how reward influences this dynamic.

We are encouraged that the reviewer acknowledges the importance of investigating the IC's role within the broader framework of complex cognitive behaviors and appreciates the hierarchical nature of the auditory system. The reviewer's comments reinforce the value of our research in contributing to a more nuanced understanding of how the IC might contribute to sensory-cognitive integration.

We thank the reviewer for highlighting the significance of using behavioral monkey models to approximate human brain function. We are hopeful that our findings will serve as a stepping stone for further research exploring the multifaceted roles of the IC in cognition and behavior.

We will now proceed to address the specific concerns and suggestions provided by the reviewer in the following sections.

Weaknesses:

These findings are more about correlation but not causality of IC function in behaviors. And I have a few major concerns.

We appreciate the reviewer’s concern regarding the reliance on correlational analyses in our study. We acknowledge the importance of distinguishing between correlation and causality. As detailed in our response to Question 3 from Reviewer #1, we recognize the limitations of relying on correlational data and the challenges of establishing direct causal links in electrophysiological studies involving behaving primates.

We have taken steps to clarify this distinction throughout our manuscript. Specifically, we have revised the Results and Discussion sections to ensure that the findings are presented as correlational, not causal, and we have proposed future studies utilizing more direct manipulation techniques to assess causality. We hope these revisions adequately address your concerns.

Comparing neurons' spike activities in different tests, a 'climbing effect' was found in the oddball paradigm. The effect is clearly related to training and learning process, but it still requires more exploration to rule out a few explanations. First, repeated white noise bursts with fixed inter-stimulus-interval of 0.6 seconds was presented, so that monkeys might remember the sounds by rhymes, which is some sort of learned auditory response. It is interesting to know monkeys' responses and neurons' activities if the inter-stimuli-interval is variable. Second, the task only asked monkeys to press one button and the reward ratio (the ratio of correct response trials) was around 78% (based on the number from Line 302). so that, in the sessions with reward, monkeys had highly expected reward chances, does this expectation cause the climbing effect?

We thank the reviewer for raising these insightful points regarding the 'climbing effect' observed in the oddball paradigm and its potential relationship with training, learning processes, and reward expectation. Below, we address each of the reviewer's specific concerns:

(1) Inter-Stimulus Interval (ISI) and Rhythmic Auditory Response:

The reviewer suggests that the fixed inter-stimulus interval (ISI) of 0.6 seconds might lead to a rhythmic auditory response, where monkeys could anticipate the sounds. We appreciate this perspective. However, we believe that rhythm is unlikely to play a significant role in the 'climbing effect' for the following reason: The 'climbing effect' starts from the second sound in the block (Fig.2D and Fig.3B), before any rhythm or pattern could be fully established, as a rhythm generally requires at least three repetitions to form. Unfortunately, we did not explore variable ISIs in the current study, so we cannot directly address this concern with the data at hand.

(2) Reward Expectation and Climbing Effect:

The reviewer raises an important concern about whether the 'climbing effect' could be influenced by the monkeys' high reward expectation, especially given the high reward ratio (~78%) in the sessions. While it is plausible that reward expectation could contribute to the observed increase in neuronal firing rates, we believe the results from our reward experiment (Fig. 4) suggest otherwise. In this experiment, even though reward expectation was likely formed due to the consistent pairing of sounds with rewards (100%), we did not observe a climbing effect in the auditory response. The presence of reward prediction error (Fig. 4D) further suggests that while the monkeys may form reward expectations, these expectations do not directly drive the climbing effect.

To clarify this point, we have added sentences in the revised manuscript to explicitly discuss the relationship between reward expectation and the climbing effect, emphasizing that our findings indicate the climbing effect is not primarily due to reward expectation.

We believe these revisions provide a clearer understanding of the factors contributing to the climbing effect and address the reviewer's concerns effectively. Thank you for these valuable suggestions.

"Reward effect" on IC neurons' responses were showed in Fig. 4. Is this auditory response caused by physical reward action or not? In reward sessions, IC neurons have obvious response related to the onset of water reward. The electromagnetic valve is often used in water-rewarding system and will give out a loud click sound every time when the reward is triggered. IC neurons' responses may be simply caused by the click sound if the electromagnetic valve is used. It is important to find a way to rule out this simple possibility.

We appreciate the reviewer’s concern regarding the potential confounding factor introduced by the electromagnetic valve’s click sound during water reward delivery, which could be misinterpreted as an auditory response rather than a response to the reward itself. Anticipating this possibility, we took measures to eliminate it by placing the electromagnetic valve outside the soundproof room where the neuronal recordings were performed.

To address your concern more explicitly, we have added sentences in the Methods section of the revised manuscript detailing this setup, ensuring that readers are aware of the steps we took to eliminate this potential confound. By doing so, we believe that the observed reward-related neural activity in the IC is attributable to the reward processing itself rather than an auditory response to the valve click. We appreciate you bringing this important aspect to our attention, and we hope our clarification strengthens the interpretation of our findings.

Reviewer #3 (Public review):

Summary:

The authors aimed to investigate the multifaceted roles of the Inferior Colliculus (IC) in auditory and cognitive processes in monkeys. Through extracellular recordings during a sound duration-based novelty detection task, the authors observed a "climbing effect" in neuronal firing rates, suggesting an enhanced response during sensory prediction. Observations of reward prediction errors within the IC further highlight its complex integration in both auditory and reward processing. Additionally, the study indicated IC neuronal activities could be involved in decision-making processes.

Strengths:

This study has the potential to significantly impact the field by challenging the traditional view of the IC as merely an auditory relay station and proposing a more integrative role in cognitive processing. The results provide valuable insights into the complex roles of the IC, particularly in sensory and cognitive integration, and could inspire further research into the cognitive functions of the IC.

We appreciate the reviewer’s positive summary of our work and recognition of its potential impact on the field. We are pleased that the reviewer acknowledges the significance of our findings in challenging the traditional view of the Inferior Colliculus (IC) as merely an auditory relay station and in proposing its integrative role in cognitive processing.

Our study indeed aims to provide new insights into the multifaceted roles of the IC, particularly in the context of sensory and cognitive integration. We believe that this research could pave the way for future studies that further explore the cognitive functions of the IC and its involvement in complex behavioral processes.

We are encouraged by the reviewer’s positive assessment and are committed to continuing to refine our work in response to the constructive feedback provided. We hope that our findings will contribute to advancing the understanding of the IC’s role in the broader context of neuroscience.

We will now proceed to address the specific concerns and suggestions provided by the reviewer in the following sections.

Weaknesses:

Major Comments:

(1) Structural Clarity and Logic Flow:

The manuscript investigates three intriguing functions of IC neurons: sensory prediction, reward prediction, and cognitive decision-making, each of which is a compelling topic. However, the logical flow of the manuscript is not clearly presented and needs to be well recognized. For instance, Figure 3 should be merged into Figure 2 to present population responses to the order of sounds, thereby focusing on sensory prediction. Given the current arrangement of results and figures, the title could be more aptly phrased as "Beyond Auditory Relay: Dissecting the Inferior Colliculus's Role in Sensory Prediction, Reward Prediction, and Cognitive Decision-Making."

We appreciate the reviewer’s detailed feedback on the structural clarity and logical flow of the manuscript. We understand the importance of presenting our findings in a clear and cohesive manner, especially when addressing multiple complex topics such as sensory prediction, reward prediction, and cognitive decision-making.

To address the reviewer's concerns, we have made the following revisions:

(1) Reorganization of Figures and Results:

We agree with the suggestion to merge Figure 3 into Figure 2. By doing so, we can present the population responses to the order of sounds more effectively, thereby streamlining the focus on sensory prediction. This will allow readers to more easily follow the progression of the results related to this key function of the IC.

We have reorganized the Results section to ensure a smoother transition between the different aspects of IC function that we are investigating. The new structure will better guide the reader through the narrative, aligning with the themes of sensory prediction, reward prediction, and cognitive decision-making.

(2) Revised Title:

In line with the reviewer's suggestion, we have revised the title to "Beyond Auditory Relay: Dissecting the Inferior Colliculus's Role in Sensory Prediction, Reward Prediction, and Cognitive Decision-Making." We believe this title more accurately reflects the scope and focus of our study, as it highlights the three core functions of the IC that we are investigating.

(3) Improved Logic Flow:

We have added introductory statements at the beginning of each section within the Results to clarify the rationale behind the experiments and the logical connections between them. This should help to improve the overall flow of the manuscript and make the progression of our findings more intuitive for readers.

We believe these changes significantly enhance the clarity and logical structure of the manuscript, making it easier for readers to understand the sequence and importance of our findings. Thank you for your valuable suggestion, which has led to a more coherent and focused presentation of our work.

(2) Clarification of Data Analysis:

Key information regarding data analysis is dispersed throughout the results section, which can lead to confusion. Providing a more detailed and cohesive explanation of the experimental design would significantly enhance the interpretation of the findings. For instance, including a detailed timeline and reward information for the behavioral paradigms shown in Figures 1C and D would offer crucial context for the study. More importantly, clearly presenting the analysis temporal windows and providing comprehensive statistical analysis details would greatly improve reader comprehension.

We appreciate the reviewer’s insightful comment regarding the need for clearer and more cohesive explanations of the data analysis and experimental design. We recognize that a well-structured presentation of this information is essential for the reader to fully understand and interpret our findings. To address this, we have made the following revisions:

(1) Detailed Explanation of Experimental Design:

We have included a more detailed explanation of the experimental design, particularly for the behavioral paradigms shown in Figures 1C and 1D. This includes a comprehensive timeline of the experiments, along with explicit information about the reward structure and timing. By providing this context upfront, we aim to give readers a clearer understanding of the conditions under which the neuronal recordings were obtained.

(2) Cohesive Presentation of Data Analysis:

Key information regarding data analysis, which was previously dispersed throughout the Results section, has been consolidated and moved to a dedicated subsection within the Methods. This subsection now provides a step-by-step description of the analysis process, including the temporal windows used for examining neuronal activity, as well as the specific statistical methods employed.

We have also ensured that the temporal windows used for different analyses (e.g., onset window, late window, etc.) are clearly defined and consistently referenced throughout the manuscript. This will help readers track the use of these windows across different figures and analyses.

(3) Enhanced Statistical Analysis Details:

We have expanded the description of the statistical analyses performed in the study, including the rationale behind the choice of tests, the criteria for significance, and any corrections for multiple comparisons. These details are now presented in a clear and accessible format within the Methods section, with relevant information also highlighted in the Result section or the figure legends to facilitate understanding.

We believe these changes will significantly improve the clarity and comprehensibility of the manuscript, allowing readers to better follow the experimental design, data analysis, and the conclusions drawn from our findings. Thank you for this valuable feedback, which has helped us to enhance the rigor and transparency of our presentation.

(3) Reward Prediction Analysis:

The conclusion regarding the IC's role in reward prediction is underdeveloped. While the manuscript presents evidence that IC neurons can encode reward prediction, this is only demonstrated with two example neurons in Figure 6. A more comprehensive analysis of the relationship between IC neuronal activity and reward prediction is necessary. Providing population-level data would significantly strengthen the findings concerning the IC's complex functionalities. Additionally, the discussion of reward prediction in lines 437-445, which describes IC neuron responses in control experiments, does not sufficiently demonstrate that IC neurons can encode reward expectations. It would be valuable to include the responses of IC neurons during trials with incorrect key presses or no key presses to better illustrate this point.

We deeply appreciate the detailed feedback provided regarding the conclusions on the inferior colliculus (IC)'s role in reward prediction within our manuscript. We acknowledge the importance of a robust and comprehensive presentation of our findings, particularly when discussing complex neural functionalities.

In response to the reviewers' concerns, we have made the following revisions to strengthen our manuscript:

(1) Inclusion of Population-Level Data for IC Neurons:

In the revised manuscript, we have included population-level results for IC neurons in a supplementary figure. Initially, we focused on two example neurons that did not exhibit motor-related responses to key presses to isolate reward-related signals. However, most IC neurons exhibit motor responses during key presses (as indicated in Fig.7), which can complicate distinguishing between reward-related activity and motor responses. This complexity is why we initially presented neurons without motor responses. To clarify this point, we have added sentences in the Results section to explain the rationale behind our selection of neurons and to address the potential overlap between motor and reward responses in the IC.

(2) Addition of Data on Key Press Errors and No-Response Trials:

In response to the reviewer’s suggestion, we have demonstrated Peri-Stimulus Time Histograms (PSTHs) for two example neurons during error trials as below, including incorrect key presses and no-response trials. Given that the monkeys performed the task with high accuracy, the number of error trials is relatively small, especially for the control condition (as shown in the top row of the figure). While we remain cautious in drawing definitive conclusions from this limited trials, we observed that no clear reward signals were detected during the corresponding window (typically centered around 150 ms after the end of the sound). It is important to note that the experiment was initially designed to explore decision-making signals in the IC, rather than focusing specifically on reward processing. However, the data in Fig. 6 demonstrated intriguing signals of reward prediction error, which is why we believe it is important to present them.

When combined with the results from our reward experiment (Fig. 5), we believe these findings provide compelling evidence of reward prediction errors being processed by IC neurons. Additionally, we observed that the reward prediction error in the IC appears to be signed, meaning that IC neurons showed robust responses to unexpected rewards but not to unexpected no-reward scenarios. However, the sign of the reward prediction error should be explored in greater depth with specifically designed experiments in future studies.

Author response image 1.

(A) PSTH of the neuron from Figure 6a during a key press trial under control condition. The number in the parentheses in the legend represents the number of trials for control condition. (B) PSTHs of the neuron from Figure 6a during non-key press trials under experimental conditions. The numbers in the parentheses in the legend represent the number of trials for experimental conditions. (C-D) Equivalent PSTHs as in A-B but from the neuron in Figure 6b.

We are grateful for the reviewer's insightful suggestions, which have allowed us to improve the depth and rigor of our analysis. We believe these revisions significantly enhance our manuscript's conclusions regarding the complex functionalities of IC.