Distinct catecholaminergic pathways projecting to hippocampal CA1 transmit contrasting signals during navigation in familiar and novel environments

  1. Chad M Heer
  2. Mark E J Sheffield

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    eLife assessment

    This study presents important findings on the differential activity of noradrenergic and dopaminergic input to dorsal hippocampus CA1 in head-fixed mice traversing a runway in a virtual environment that is familiar or novel. The data are rigorously analysed, and the observed divergence in the dynamics of activity in the dopaminergic and noradrenergic axons is solid. Future studies, using specific manipulations of the two distinct midbrain inputs combined with behavioral testing, are required to strengthen the claim that distinct signals to the hippocampus cause distinct behavioral effects.

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Abstract

Neuromodulatory inputs to the hippocampus play pivotal roles in modulating synaptic plasticity, shaping neuronal activity, and influencing learning and memory. Recently it has been shown that the main sources of catecholamines to the hippocampus, ventral tegmental area (VTA) and locus coeruleus (LC), may have overlapping release of neurotransmitters and effects on the hippocampus. Therefore, to dissect the impacts of both VTA and LC circuits on hippocampal function, a thorough examination of how these pathways might differentially operate during behavior and learning is necessary. We therefore utilized 2-photon microscopy to functionally image the activity of VTA and LC axons within the CA1 region of the dorsal hippocampus in head-fixed male mice navigating linear paths within virtual reality (VR) environments. We found that within familiar environments some VTA axons and the vast majority of LC axons showed a correlation with the animals’ running speed. However, as mice approached previously learned rewarded locations, a large majority of VTA axons exhibited a gradual ramping-up of activity, peaking at the reward location. In contrast, LC axons displayed a pre-movement signal predictive of the animal’s transition from immobility to movement. Interestingly, a marked divergence emerged following a switch from the familiar to novel VR environments. Many LC axons showed large increases in activity that remained elevated for over a minute, while the previously observed VTA axon ramping-to-reward dynamics disappeared during the same period. In conclusion, these findings highlight distinct roles of VTA and LC catecholaminergic inputs in the dorsal CA1 hippocampal region. These inputs encode unique information, with reward information in VTA inputs and novelty and kinematic information in LC inputs, likely contributing to differential modulation of hippocampal activity during behavior and learning.

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  1. Version published to 10.7554/elife.95213.3 on eLife
  2. Version published to 10.7554/elife.95213 on eLife
  3. eLife

    eLife assessment

    This study presents important findings on the differential activity of noradrenergic and dopaminergic input to dorsal hippocampus CA1 in head-fixed mice traversing a runway in a virtual environment that is familiar or novel. The data are rigorously analysed, and the observed divergence in the dynamics of activity in the dopaminergic and noradrenergic axons is solid. Future studies, using specific manipulations of the two distinct midbrain inputs combined with behavioral testing, are required to strengthen the claim that distinct signals to the hippocampus cause distinct behavioral effects.

  4. eLife

    Reviewer #1 (Public Review):

    Summary:

    Heer and Sheffield used 2 photon imaging to dissect the functional contributions of convergent dopamine and noradrenaline inputs to the dorsal hippocampus CA1 in head restrained mice running down a virtual linear path. Mice were trained to collect water reward at the end of the track and on test days, calcium activity was recorded from dopamine (DA) axons originating in ventral tegmental area (VTA, n=7) and noradrenaline axons from the locus coeruleus (LC, n=87) under several conditions. When mice ran laps in a familiar environment, VTA DA axons exhibited ramping activity along the track that correlated with distance to reward and velocity to some extent, while LC input activity remained constant across the track, but correlated invariantly with velocity and time to motion onset. A subset of recordings taken when the reward was removed showed diminished ramping activity in VTA DA axons, but no changes in the LC axons, confirming that DA axon activity is locked to reward availability. When mice were subsequently introduced to a new environment, the ramping to reward activity in the DA axons disappeared, while LC axons showed a dramatic increase in activity lasting 90s (6 laps) following the environment switch. In the final analysis, the authors sought to disentangle LC axon activity induced by novelty vs. behavioral changes induced by novelty by removing periods in which animals were immobile, and established that the activity observed in the first 2 laps reflected novelty-induced signal in LC axons.

    The revised manuscript included additional evidence of increased (but transient) signal in LC axons after a transition to a novel environment during periods of immobility, and also that a change from dark to familiar environment induces a peak in LC axon activity, showing that LC input to dCA1 may not solely signal novelty.

    Strengths:

    The results presented in this manuscript provide insights into the specific contributions of catecholaminergic input to the dorsal hippocampus CA1 during spatial navigation in a rewarded virtual environment, offering a detailed analysis at the resolution of single axons. The data analysis is thorough and possible confounding variables and data interpretation are carefully considered.

    Weaknesses:

    Aspects of the methodology, data analysis, and interpretation diminish the overall significance of the findings, as detailed below.

    The LC axonal recordings are well powered, but the DA axonal recordings are severely underpowered, with recordings taken from a mere 7 axons (compare to 87 LC axons). Additionally, 2 different calcium indicators with differential kinetics and sensitivity to calcium changes (GCaMP6S and GCaMP7b) were used (n=3, n=4 respectively) and the data pooled. This makes it very challenging to draw any valid conclusions from the data, particularly in the novelty experiment. The surprising lack of novelty-induced DA axon activity may be a false negative. Indeed, at least 1 axon (axon 2) appears to be showing novelty-induced rise in activity in Figure 3C. Changes in activity in 4/7 axons are also referred to as a 'majority' occurrence in the manuscript, which again is not an accurate representation of the observed data

    The authors conducted analysis on recording data exclusively from periods of running in the novelty experiment to isolate the effects of novelty from novelty-induced changes in behavior. However, if the goal is to distinguish between changes in locus coeruleus (LC) axon activity induced by novelty and those induced by motion, analyzing LC axon activity during periods of immobility would enhance the robustness of the results.

    The authors attribute the ramping activity of the DA axons to the encoding of the animals' position relative to reward. However, given the extensive data implicating the dorsal CA1 in timing, and the remarkable periodicity of the behavior, the fact that DA axons could be signalling temporal information should be considered.

    The authors should explain and justify the use of a longer linear track (3m, as opposed to 2m in the DAT-cre mice) in the LC axon recording experiments.

    AFTER REVISIONS:

    The authors have addressed my concerns in a thorough manner. The reviewer also appreciates the increased transparency of reporting in the revised manuscript.

    Listed below are some remaining comments.
    The increase in LC activity with any change in environment (from familiar to novel or from dark to familiar) suggests that LC input acts not solely as a novelty signal, but as a general arousal or salience signal in response to environmental changes. Based on this, I have a couple of questions:

    • Is the overall claim that LC input to the dHC signals novelty still valid based on observed findings - as claimed throughout the manuscript?
    • Would the omission of a reward be considered a salient change in the environment that activates LC signals, or is the LC not involved with processing reward-related information? Has the activity of LC and VTA axons been analysed in the seconds following reward presentation and/or omission?

  5. eLife

    Reviewer #2 (Public Review):

    Summary:

    The authors used 2-photon Ca2+-imaging to study the activity of ventral tegmental area (VTA) and locus coeruleus (LC) axons in the CA1 region of the dorsal hippocampus in head-fixed male mice moving on linear paths in virtual reality (VR) environments.

    The main findings were as follows:
    - In a familiar environment, activity of both VTA axons and LC axons increased with the mice's running speed on the Styrofoam wheel, with which they could move along a linear track through a VR environment.
    - VTA, but not LC, axons showed marked reward position-related activity, showing a ramping-up of activity when mice approached a learned reward position.
    - In contrast, activity of LC axons ramped up before initiation of movement on the Styrofoam wheel.
    - In addition, exposure to a novel VR environment increased LC axon activity, but not VTA axon activity.

    Overall, the study shows that the activity of catecholaminergic axons from VTA and LC to dorsal hippocampal CA1 can partly reflect distinct environmental, behavioral and cognitive factors. Whereas both VTA and LC activity reflected running speed, VTA, but not LC axon activity reflected approach of a learned reward and LC, but not VTA, axon activity reflected initiation of running and novelty of the VR environment.

    I have no specific expertise with respect to 2-photon imaging, so cannot evaluate the validity of the specific methods used to collect and analyse 2-photon calcium imaging data of axonal activity.

    Strengths:

    (1) Using a state-of-the-art approach to record separately the activity of VTA and LC axons with high temporal resolution in awake mice moving through virtual environments, the authors provide convincing evidence that activity of VTA and LC axons projecting to dorsal CA1 reflect partly distinct environmental, behavioral and cognitive factors.

    (2) The study will help a) to interpret previous findings on how hippocampal dopamine and norepinephrine or selective manipulations of hippocampal LC or VTA inputs modulate behavior and b) to generate specific hypotheses on the impact of selective manipulations of hippocampal LC or VTA inputs on behavior.

    Comments on revised version:

    I thank the authors for including a sample size justification.

    The justification is based on previous studies using similar sample sizes to characterize behavioral correlates of LC and VTA activity and on practical reasons. I note that to improve reproducibility, it would be preferable to have predefined target sample sizes based on predefined plans for statistical analysis.

  6. eLife

    Reviewer #3 (Public Review):

    Summary:

    Heer and Sheffield provide a well-written manuscript that clearly articulates the theoretical motivation to investigate specific catecholaminergic projections to dorsal CA1 of the hippocampus during a reward-based behavior. Using 2-photon calcium imaging in two groups of cre transgenic mice, the authors examine activity of VTA-CA1 dopamine and LC-CA1 noradrenergic axons during reward seeking in a linear track virtual reality (VR) task. The authors provide a descriptive account of VTA and LC activities during walking, approach to reward, and environment change. Their results demonstrate LC-CA1 axons are activated by walking onset, modulated by walking velocity, and heighten their activity during environment change. In contrast, VTA-CA1 axons were most activated during approach to reward locations. Together the authors provide a functional dissociation between these catecholamine projections to CA1. A major strength to their approach is the methodological rigor of 2-photon recording, data processing, and analysis approaches to accommodate their unequal LC-CA1 and VTA-CA1 sample sizes. These important systems neuroscience studies provide solid evidence that will contribute to the broader field of navigation and memory.

    Weaknesses:

    The conclusions of this manuscript are mostly well supported by the data. However, increasing the sample size of the VTA-CA1 group and using experimental methods that are identical among LC-CA1 and VTA-CA1 groups would help to fully support the author's conclusions.

  7. eLife

    Author response:

    The following is the authors’ response to the previous reviews.

    Recommendations for the authors:

    Reviewer #1 (Recommendations For The Authors):

    Please reorder the supplementary figures in the order they are referred to in the Results section for ease of reading. Supp Fig 5 b - should read 'Mean normalized fluorescence of LC ROIs (n = 87) during immobile periods aligned to the switch from familiar to novel environment.’

    We thank the reviewer for highlighting these issues and have reordered the supplementary figures and edited the figure legends appropriately.

    Reviewer #2 (Recommendations For The Authors):

    The authors should include sample size justifications (e.g. based on previous studies, considerations of statistical power, practical considerations, or a combination of these factors).

    In response to this concern, we have added a statement to the “Imaging Sessions” section of the methods. Here we highlight sample sizes were largely based on previous studies and/or limited by the difficulty of recordings and the limited number of visible axons per imaging session.

    Reviewer #3 (Recommendations For The Authors):

    The addition of Supp. Fig 5 partially addresses my previous point 3. However, the claim of dissociation between VTA-CA1 and LC-CA1 would be strengthened by showing that VTA-CA1 axons do not respond to the darkness -> familiar environment in Supp Fig 5. This is particularly important given that (1) the additional 2 VTA-CA1 axons in the revision were not recorded during transitions to novel environments and (2) the overall concern of the reviewers that the low n and heterogeneity of the VTA-CA1 dataset may lead to a false negative. Providing VTA-CA1 data for the darkness -> familiar environment would provide a within-manuscript replication that these axons are not responding to environment changes; a major claim of this manuscript.

    While we agree that data of VTA-CA1 axons during the switch from darkness to the familiar environment would provide additional evidence that these axons are not responding to environment changes, unfortunately, VTA axons were not recorded during the switch from familiar to novel.

  8. Version published to 10.7554/elife.95213.2 on eLife
  9. eLife

    eLife assessment

    This manuscript provides important results that assessed the contribution of two catecholaminergic projections to the hippocampus during environment-guided reward behavior. The authors use 2-photon imaging in the hippocampus of behaving mice to provide solid evidence that there are dissociable roles of dopamine and norepinephrine in this structure. Although of great interest to the field of learning and memory, the results would be strengthened by additional data collected from dopaminergic projections to the hippocampus.

  10. eLife

    Reviewer #1 (Public Review):

    Summary:

    Heer and Sheffield used 2 photon imaging to dissect the functional contributions of convergent dopamine and noradrenaline inputs to the dorsal hippocampus CA1 in head restrained mice running down a virtual linear path. Mice were trained to collect water reward at the end of the track and on test days, calcium activity was recorded from dopamine (DA) axons originating in ventral tegmental area (VTA, n=7) and noradrenaline axons from the locus coeruleus (LC, n=87) under several conditions. When mice ran laps in a familiar environment, VTA DA axons exhibited ramping activity along the track that correlated with distance to reward and velocity to some extent, while LC input activity remained constant across the track, but correlated invariantly with velocity and time to motion onset. A subset of recordings taken when the reward was removed showed diminished ramping activity in VTA DA axons, but no changes in the LC axons, confirming that DA axon activity is locked to reward availability. When mice were subsequently introduced to a new environment, the ramping to reward activity in the DA axons disappeared, while LC axons showed a dramatic increase in activity lasting 90s (6 laps) following the environment switch. In the final analysis, the authors sought to disentangle LC axon activity induced by novelty vs. behavioral changes induced by novelty by removing periods in which animals were immobile and established that the activity observed in the first 2 laps reflected novelty-induced signal in LC axons.

    The revised manuscript included additional evidence of increased (but transient) signal in LC axons after a transition to a novel environment during periods of immobility, and also that a change from dark to familiar environment induces a peak in LC axon activity, showing that LC input to dCA1 may not solely signal novelty.

    Strengths:

    The results presented in this manuscript provide insights into the specific contributions of catecholaminergic input to the dorsal hippocampus CA1 during spatial navigation in a rewarded virtual environment, offering a detailed analysis at the resolution of single axons. The data analysis is thorough and possible confounding variables and data interpretation are carefully considered.

    The authors have addressed my concerns in a thorough manner. The reviewer also appreciates the increased transparency of reporting in the revised manuscript.

    Weaknesses:

    Listed below are some remaining comments.
    The increase in LC activity with any change in environment (from familiar to novel or from dark to familiar) suggests that LC input acts not solely as a novelty signal, but as a general arousal or salience signal in response to environmental changes. Based on this, I have a couple of questions:

    • Is the overall claim that LC input to the dHC signals novelty still valid based on observed findings - as claimed throughout the manuscript?
    • Would the omission of a reward be considered a salient change in the environment that activates LC signals, or is the LC not involved with processing reward-related information? Has the activity of LC and VTA axons been analysed in the seconds following reward presentation and/or omission?

  11. eLife

    Reviewer #2 (Public Review):

    Summary:

    The authors used 2-photon Ca2+-imaging to study the activity of ventral tegmental area (VTA) and locus coeruleus (LC) axons in the CA1 region of the dorsal hippocampus in head-fixed male mice moving on linear paths in virtual reality (VR) environments.

    The main findings were as follows:
    - In a familiar environment, activity of both VTA axons and LC axons increased with the mice's running speed on the Styrofoam wheel, with which they could move along a linear track through a VR environment.
    - VTA, but not LC, axons showed marked reward position-related activity, showing a ramping-up of activity when mice approached a learned reward position.
    - In contrast, activity of LC axons ramped up before initiation of movement on the Styrofoam wheel.
    - In addition, exposure to a novel VR environment increased LC axon activity, but not VTA axon activity.

    Overall, the study shows that the activity of catecholaminergic axons from VTA and LC to dorsal hippocampal CA1 can partly reflect distinct environmental, behavioral and cognitive factors. Whereas both VTA and LC activity reflected running speed, VTA, but not LC axon activity reflected the approach of a learned reward and LC, but not VTA, axon activity reflected initiation of running and novelty of the VR environment.

    I have no specific expertise with respect to 2-photon imaging, so cannot evaluate the validity of the specific methods used to collect and analyse 2-photon calcium imaging data of axonal activity.

    Strengths:

    (1) Using a state-of-the-art approach to record separately the activity of VTA and LC axons with high temporal resolution in awake mice moving through virtual environments, the authors provide convincing evidence that activity of VTA and LC axons projecting to dorsal CA1 reflect partly distinct environmental, behavioral and cognitive factors.

    (2) The study will help a) to interpret previous findings on how hippocampal dopamine and norepinephrine or selective manipulations of hippocampal LC or VTA inputs modulate behavior and b) to generate specific hypotheses on the impact of selective manipulations of hippocampal LC or VTA inputs on behavior.

    Weaknesses:

    (1) The findings are correlational and do not allow strong conclusions on how VTA or LC inputs to dorsal CA1 affect cognition and behavior. However, as indicated above under Strengths, the findings will aid the interpretation of previous findings and help to generate new hypotheses as to how VTA or LC inputs to dorsal CA1 affect distinct cognitive and behavioral functions.

    (2) Some aspects of the methodology would benefit from clarification.
    First, to help others to better scrutinize, evaluate and potentially to reproduce the research, the authors may wish to check if their reporting follows the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines for the full and transparent reporting of research involving animals (https://arriveguidelines.org/). For example, I think it would be important to include a sample size justification (e.g., based on previous studies, considerations of statistical power, practical considerations or a combination of these factors). The authors should also include the provenance of the mice. Moreover, although I am not an expert in 2-photon imaging, I think it would be useful to provide a clearer description of exclusion criteria for imaging data (see below, Recommendations for the authors).
    Second, why were different linear tracks used for studies of VTA and LC axon activity (from line 362)? Could this potentially contribute to the partly distinct activity correlates that were found for VTA and LC axons?
    Third, the authors seem to have used two different criteria for defining immobility. Immobility was defined as moving at <5 cm/s for the behavioral analysis in Fig. 3a, but as <0.2 cm/s for the imaging data analysis in Fig. 4 (see legends to these figures and also see Methods, from line 447, line 469, line 498)? I do not understand why, and it would be good if the authors explained this.

    (3) In the Results section (from line 182) the authors convincingly addressed the possibility that less time spent immobile in the novel environment may have contributed to the novelty-induced increase of LC axon activity in dorsal CA1 (Fig. 4). In addition, initially (for the first 2-4 laps), the mice also ran more slowly in the novel environment (Fig. 3aIII, top panel). Given that LC and VTA axon activity were both increasing with velocity (Fig. 1F), reduced velocity in the novel environment may have reduced LC and VTA axon activity, but this possibility was not addressed. Reduced LC axon activity in the novel environment could have blunted the novelty-induced increase. More importantly, any potential novelty-induced increase in VTA axon activity could have been masked by decreases in VTA axon activity due to reduced velocity. The latter may help to explain the discrepancy between the present study and previous findings that VTA neuron firing was increased by novelty (see Discussion, from line 243). It may be useful for the authors to address these possibilities based on their data in the Results section, or to consider them in their Discussion.

    (4) Sensory properties of the water reward, which the mice may be able to detect, could account for reward-related activity of VTA axons (instead of an expectation of reward). Do the authors have evidence that this is not the case? Occasional probe trials, intermixed with rewarded trials, could be used to test for this possibility.

    REVIEW OF THE REVISED MANUSCRIPT
    I thank the authors for their responses addressing some of the weaknesses I raised in my original comments.

    Regarding their clarification of some methodological issues [Point 2) above], I have a few additional comments:
    - I appreciate that the authors clearly state the sample sizes contributing to the data. However, sample size justifications (e.g. based on previous studies, considerations of statistical power, practical considerations or a combination of these factors) are still lacking.
    - It is good that the authors have now clearly indicated how many mice they excluded due to lack of GCaMP expression or due to failure to reach the behavioral criteria. They also indicated that they discarded some of the collected datasets, based on the visual assessment of imaging sessions and the registration metrics output by suite2p. I appreciate that this may be common practice (although I am not using 2-photon imaging myself). However, I note that to minimize the risk of experimenter bias and improve reproducibility, it would be preferable to have more clearly defined quantitative criteria for such exclusions.
    - The authors clarified in their response why they used two different linear tracks for their studies of VTA and LC axon activity. I would encourage them to include this clarification in the manuscript. From the authors' response, I understand that they chose the different track lengths to facilitate comparison to previous studies involving LC and VTA axon recordings. However, given that the present paper aimed to compare LC and VTA axon recordings, the use of different track lengths for LC and VTA axon recordings remains a limitation of the present paper.

  12. eLife

    Reviewer #3 (Public Review):

    Summary:

    Heer and Sheffield provide a well-written manuscript that clearly articulates the theoretical motivation to investigate specific catecholaminergic projections to dorsal CA1 of the hippocampus during a reward-based behavior. Using 2-photon calcium imaging in two groups of cre transgenic mice, the authors examine activity of VTA-CA1 dopamine and LC-CA1 noradrenergic axons during reward seeking in a linear track virtual reality (VR) task. The authors provide a descriptive account of VTA and LC activities during walking, approach to reward, and environment change. Their results demonstrate LC-CA1 axons are activated by walking onset, modulated by walking velocity, and heighten their activity during environment change. In contrast, VTA-CA1 axons were most activated during approach to reward locations. Together the authors provide a functional dissociation between these catecholamine projections to CA1. A major strength to their approach is the methodological rigor of 2-photon recording, data processing, and analysis approaches to accommodate their unequal LC-CA1 and VTA-CA1 sample sizes. These important systems neuroscience studies provide solid evidence that will contribute to the broader field of navigation and memory.

    Weaknesses:

    The conclusions of this manuscript are mostly well supported by the data. However, increasing the sample size of the VTA-CA1 group and using experimental methods that are identical among LC-CA1 and VTA-CA1 groups would help to fully support the author's conclusions.

  13. eLife

    Author response:

    The following is the authors’ response to the original reviews.

    Public Reviews:

    Reviewer #1 (Public Review):

    Summary:

    Heer and Sheffield used 2 photon imaging to dissect the functional contributions of convergent dopamine and noradrenaline inputs to the dorsal hippocampus CA1 in head-restrained mice running down a virtual linear path. Mice were trained to collect water rewards at the end of the track and on test days, calcium activity was recorded from dopamine (DA) axons originating in the ventral tegmental area (VTA, n=7) and noradrenaline axons from the locus coeruleus (LC, n=87) under several conditions. When mice ran laps in a familiar environment, VTA DA axons exhibited ramping activity along the track that correlated with distance to reward and velocity to some extent, while LC input activity remained constant across the track, but correlated invariantly with velocity and time to motion onset. A subset of recordings taken when the reward was removed showed diminished ramping activity in VTA DA axons, but no changes in the LC axons, confirming that DA axon activity is locked to reward availability. When mice were subsequently introduced to a new environment, the ramping to reward activity in the DA axons disappeared, while LC axons showed a dramatic increase in activity lasting 90 s (6 laps) following the environment switch. In the final analysis, the authors sought to disentangle LC axon activity induced by novelty vs. behavioral changes induced by novelty by removing periods in which animals were immobile and established that the activity observed in the first 2 laps reflected novelty-induced signal in LC axons.

    Strengths:

    The results presented in this manuscript provide insights into the specific contributions of catecholaminergic input to the dorsal hippocampus CA1 during spatial navigation in a rewarded virtual environment, offering a detailed analysis of the resolution of single axons. The data analysis is thorough and possible confounding variables and data interpretation are carefully considered.

    Weaknesses:

    Aspects of the methodology, data analysis, and interpretation diminish the overall significance of the findings, as detailed below.

    The LC axonal recordings are well-powered, but the DA axonal recordings are severely underpowered, with recordings taken from a mere 7 axons (compared to 87 LC axons).

    Additionally, 2 different calcium indicators with differential kinetics and sensitivity to calcium changes (GCaMP6S and GCaMP7b) were used (n=3, n=4 respectively) and the data pooled. This makes it very challenging to draw any valid conclusions from the data, particularly in the novelty experiment. The surprising lack of novelty-induced DA axon activity may be a false negative. Indeed, at least 1 axon (axon 2) appears to be showing a novelty-induced rise in activity in Figure 3C. Changes in activity in 4/7 axons are also referred to as a 'majority' occurrence in the manuscript, which again is not an accurate representation of the observed data.

    We appreciate the reviewer's detailed feedback regarding the analysis of VTA axons in our dataset. The relatively low sample size for VTA axons is due to their sparsity in the dCA1 region of the hippocampus and the inherent difficulty in recording from these axons. VTA axons are challenging to capture due to their low baseline fluorescence and long-range axon segments, resulting in a typical yield of only a single axon per field of view (FOV) per animal. In contrast, LC axons are more abundant in dCA1.

    To address the disparity in sample sizes between LC and VTA axons, we down-sampled the LC axons to match the number of VTA axons, repeating this process 1000 times to create a distribution. However, we acknowledge the reviewer's concern that the relatively low sample size for VTA axons might result in insufficient sampling of this population. Increasing the baseline expression of GCaMP to record from VTA axons requires several months, limiting our ability to quickly expand the sample size.

    In response to the reviewer's comments, we have added recordings from 2 additional VTA axons, increasing the sample size from 7 to 9. We re-analyzed all data from the familiar environment with n=9 VTA axons, comparing them to down-sampled LC axons as previously described. However, the additional axons were not recorded in the novel environment. We agree with the reviewer that the lack of novelty-induced DA axon activity may be a false negative. To address this, we have revised the description of our results to include the following sentence:

    “However, 1 VTA ROI showed an increase in activity immediately following exposure to novelty, indicating heterogeneity across VTA axons in CA1, and the lack of a novelty signal on average may be due to a small sample size.”

    Regarding the use of two different GCaMP constructs, we understand the reviewer's concern. We used GCaMP6s and GCaMP7b variants to determine if one would improve the success rate of recording from VTA axons. Given the long duration of these experiments and the low yield, we pooled the data from both GCaMP variants to increase statistical power. However, we recognize the importance of verifying that there are no differences in the signals recorded with these variants.

    With the addition of 2 VTA DA axons expressing GCaMP6s, we now have n=5 GCaMP6s and n=4 GCaMP7b VTA DA axons. This allowed us to compare the activity of the two sensors in the familiar environment. As shown in new Supplementary Figure 2, both sets of axons responded similarly to the variables measured: position in VR, time to motion onset, and animal velocity (although the GCaMP6s expressing axons showed stronger correlations). Since all LC axons recorded expressed GCaMP6s, we also specifically compared VTA GCaMP6s axons to LC GCaMP6s axons (Supp Fig. 3). Our conclusions remained consistent when comparing this subset of VTA axons to LC axons.

    Overall, our paper now includes comparisons of combined VTA axons (n=9) and separately the GCaMP6s-expressing VTA axons (n=5) with LC axons. Both datasets support our initial conclusions that VTA axons signal proximity to reward, while LC axons encode velocity and motion initiation in familiar environments.

    The authors conducted analysis on recording data exclusively from periods of running in the novelty experiment to isolate the effects of novelty from novelty-induced changes in behavior. However, if the goal is to distinguish between changes in locus coeruleus (LC) axon activity induced by novelty and those induced by motion, analyzing LC axon activity during periods of immobility would enhance the robustness of the results.

    We appreciate the reviewer's insightful suggestion to analyze LC axon activity during periods of immobility to distinguish between changes induced by novelty and those induced by motion. This additional analysis would indeed strengthen our conclusions regarding the LC novelty signal.

    In response to this suggestion, we performed the same analysis as before, but focused on periods of immobility. Our findings indicate that following exposure to novelty, there was a significant increase in LC activity specifically during immobility. This supports the idea that LC axons produce a novelty signal that is independent of novelty-induced behavioral changes. The results of this analysis are now presented in new Supplementary Figure 5b

    The authors attribute the ramping activity of the DA axons to the encoding of the animals' position relative to reward. However, given the extensive data implicating the dorsal CA1 in timing, and the remarkable periodicity of the behavior, the fact that DA axons could be signalling temporal information should be considered.

    This is an insightful comment regarding the potential role of VTA DA axons in signaling temporal information. We agree that VTA DA axons could indeed be encoding temporal information, as previous work from our lab has shown that these axons exhibit ramping activity when averaged by time to reward (Krishnan et al., 2022).

    To address this, we have now examined DA axon activity relative to time to reward, as shown in new Supplementary Figure 4. Our analysis confirms that these axons ramp up in activity relative to time to reward. Given the periodicity of our mice's behavior in these experiments, as the reviewer correctly points out, we are unable to distinguish between spatial proximity to reward and time to reward. We have added a sentence to our paper highlighting this limitation and stating that further experiments are necessary to differentiate these two variables.

    Krishnan, L.S., Heer, C., Cherian, C., Sheffield, M.E. Reward expectation extinction restructures and degrades CA1 spatial maps through loss of a dopaminergic reward proximity signal. Nat Commun 13, 6662 (2022).

    The authors should explain and justify the use of a longer linear track (3m, as opposed to 2m in the DAT-cre mice) in the LC axon recording experiments.

    We appreciate the reviewer's insightful comment regarding the use of a longer linear track (3m, as opposed to 2m in the DAT-cre mice) in the LC axon recording experiments. The choice of a 3m track for LC axon recordings was made to align with a previous experiment from our lab (Dong et al., 2021), in which mice were exposed to a novel 3m track while CA1 pyramidal cell populations were recorded. In that study, we detailed the time course of place field formation within the novel track. Our current hypothesis is that LC axons signal novelty, and we aimed to investigate whether the time course of LC axon activity aligns with the time course of place field formation. This hypothesis, and the potential role of LC axons in facilitating plasticity for new place field formation, is further discussed in the Discussion section of our paper.

    For the VTA axon recordings, we utilized a 2m track, consistent with another recent study from our lab (Krishnan et al., 2022), where reward expectation was manipulated, and CA1 pyramidal cell populations were recorded. By matching the track length to this prior study, we aimed to explore how VTA dopaminergic inputs to CA1 might influence CA1 population dynamics along the track under conditions of varying reward expectations.

    We acknowledge that using different track lengths for LC and VTA recordings introduces a variable that could potentially confound direct comparisons. To address this, we normalized the track lengths for our LC versus VTA comparison analysis. This normalization allowed us to directly compare patterns of activity across the two types of axons by adjusting the data to a common scale, thereby ensuring that any observed differences or similarities are attributable to the intrinsic properties of the axons rather than differences in track lengths. By doing so, we could assess relative changes in activity levels at matched spatial bins.

    Although the experiences of the animals on the different track lengths are not identical, our observations suggest that LC and VTA axon signals are not majorly influenced by variations in track length. LC axons are associated with velocity and a pre-motion initiation signal, neither of which are affected by track length. VTA axons, which also correlate with velocity, can be compared to LC axon velocity signals because mice reach maximal velocity very quickly a long the track, well before the end of the 2m track. The range of velocities are therefore capture on both track lengths. While VTA axons exhibit ramping activity as they approach the reward zone—a signal potentially modulated by track length—LC axons do not show such ramping to reward signals. Thus, a comparison across different track lengths is justified for this aspect of our analysis.

    To further enhance the rigor of our comparisons between axon dynamics recorded on 2m and 3m tracks, we conducted an additional analysis plotting axon activity by time to reward and actual (un-normalized) distance from reward (Supplementary Figure 4). This analysis revealed very similar signals between the two sets of axons, supporting our initial conclusions.

    We thank the reviewer for raising this important point and hope that our detailed explanation and additional analysis address their concern.

    Krishnan, L.S., Heer, C., Cherian, C., Sheffield, M.E. Reward expectation extinction restructures and degrades CA1 spatial maps through loss of a dopaminergic reward proximity signal. Nat Commun 13, 6662 (2022).

    Dong, C., Madar, A. D. & Sheffield, M.E. Distinct place cell dynamics in CA1 and CA3 encode experience in new environments. Nat Commun 12, 2977 (2021).

    Reviewer #2 (Public Review):

    Summary:

    The authors used 2-photon Ca2+-imaging to study the activity of ventral tegmental area (VTA) and locus coeruleus (LC) axons in the CA1 region of the dorsal hippocampus in head-fixed male mice moving on linear paths in virtual reality (VR) environments.

    The main findings were as follows:

    - In a familiar environment, the activity of both VTA axons and LC axons increased with the mice's running speed on the Styrofoam wheel, with which they could move along a linear track through a VR environment.

    - VTA, but not LC, axons showed marked reward position-related activity, showing a ramping-up of activity when mice approached a learned reward position.

    - In contrast, the activity of LC axons ramped up before the initiation of movement on the Styrofoam wheel.

    - In addition, exposure to a novel VR environment increased LC axon activity, but not VTA axon activity.

    Overall, the study shows that the activity of catecholaminergic axons from VTA and LC to dorsal hippocampal CA1 can partly reflect distinct environmental, behavioral, and cognitive factors. Whereas both VTA and LC activity reflected running speed, VTA, but not LC axon activity reflected the approach of a learned reward, and LC, but not VTA, axon activity reflected initiation of running and novelty of the VR environment.

    I have no specific expertise with respect to 2-photon imaging, so cannot evaluate the validity of the specific methods used to collect and analyse 2-photon calcium imaging data of axonal activity.

    Strengths:

    (1) Using a state-of-the-art approach to record separately the activity of VTA and LC axons with high temporal resolution in awake mice moving through virtual environments, the authors provide convincing evidence that the activity of VTA and LC axons projecting to dorsal CA1 reflect partly distinct environmental, behavioral and cognitive factors.

    (2) The study will help a) to interpret previous findings on how hippocampal dopamine and norepinephrine or selective manipulations of hippocampal LC or VTA inputs modulate behavior and b) to generate specific hypotheses on the impact of selective manipulations of hippocampal LC or VTA inputs on behavior.

    Weaknesses:

    (1) The findings are correlational and do not allow strong conclusions on how VTA or LC inputs to dorsal CA1 affect cognition and behavior. However, as indicated above under Strengths, the findings will aid the interpretation of previous findings and help to generate new hypotheses as to how VTA or LC inputs to dorsal CA1 affect distinct cognitive and behavioral functions.

    (2) Some aspects of the methodology would benefit from clarification.

    First, to help others to better scrutinize, evaluate, and potentially to reproduce the research, the authors may wish to check if their reporting follows the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines for the full and transparent reporting of research involving animals (https://arriveguidelines.org/). For example, I think it would be important to include a sample size justification (e.g., based on previous studies, considerations of statistical power, practical considerations, or a combination of these factors). The authors should also include the provenance of the mice. Moreover, although I am not an expert in 2-photon imaging, I think it would be useful to provide a clearer description of exclusion criteria for imaging data.

    We thank the reviewer for helping us formalize the scientific rigor of our study. There are ten ARRIVE Guidelines and we have addressed most of them in our study already. However, there is an opportunity to add detail. We have listed below all ten points and how we have addressed each one (and point out any new additions):

    (1) Experimental design - we go into great depth explaining the experimental set-up, how we used the autofluorescent blebs as imaging controls, how we controlled for different sample sizes between the two populations, and the statistical tests used for comparisons. We also carefully accounted for animal behavior when quantifying and describing axon dynamics both in the familiar and novel environments.

    (2) Sample size - we state both the number of ROIs and mice for each analysis. We have now also added the number of mice we observed specific types of activity in.

    (3) Inclusion/exclusion criteria - The following has now been added to the Methods section: Out of the 36 NET-Cre mice injected, 15 were never recorded from for either failing to reach behavioral criteria, or a lack of visible expression in axons. Out of the 54 DAT-Cre mice injected, imaging was never conducted in 36 of them for lack of expression or failing to reach behavioral criteria. Out of the remaining 21 NET-CRE, 5 were excluded for heat bubbles, z-drift, or bleaching, while 10 DAT-Cre were excluded for the same reasons. This was determined by visually assessing imaging sessions, followed by using the registration metrics output by suite2p. This registration metric conducted a PCA on the motion-corrected ROIs and plotted the first PC. If the PC drifted largely, to the point where no activity was apparent, the video was excluded from analysis.

    (4) Randomization - Already included in the paper is a description of random downsampling of LC axons to make statistical comparisons with VTA axons. LC axons were selected pseudo-randomly (only one axon per imaging session) to match VTA sampling statistics. This randomization was repeated 1000 times and comparisons were made against this random distribution.

    (5) Blinding-masking - no blinding/masking was conducted as no treatments were given that would require this. We will include this statement in the next version.

    (6) Outcomes - We defined all outcomes measured, such as those related to animal behavior and axon signaling.

    (7) Statistical methods - None of the reviewers had any issues regarding our description of statistical methods, which we described in great detail in this version of the paper.

    (8) Experimental animals - We have now described that DAT- Cre mice were obtained through JAX labs, and NET-Cre mice were obtained from the Tonegawa lab (Wagatsuma et al. 2017). This was absent in the initial version of the paper.

    (9) Experimental procedure - Already listed in great detail in Methods section.

    (10) Results - Rigorously described in detail for behaviors and related axon dynamics.

    Wagatsuma, Akiko, Teruhiro Okuyama, Chen Sun, Lillian M. Smith, Kuniya Abe, and Susumu Tonegawa. “Locus Coeruleus Input to Hippocampal CA3 Drives Single-Trial Learning of a Novel Context.” Proceedings of the National Academy of Sciences 115, no. 2 (January 9, 2018): E310–16. https://doi.org/10.1073/pnas.1714082115.

    Second, why were different linear tracks used for studies of VTA and LC axon activity (from line 362)? Could this potentially contribute to the partly distinct activity correlates that were found for VTA and LC axons?

    We thank the reviewer for pointing this out and giving us a chance to address it directly. A detailed response to this is written above for a similar comment from reviewer 1.

    Third, the authors seem to have used two different criteria for defining immobility. Immobility was defined as moving at <5 cm/s for the behavioral analysis in Figure 3a, but as <0.2 cm/s for the imaging data analysis in Figure 4 (see legends to these figures and also see Methods, from line 447, line 469, line 498)? I do not understand why, and it would be good if the authors explained this.

    This is a typo leftover from before we converted velocity from rotational units of the treadmill to cm/s. This has now been corrected.

    (3) In the Results section (from line 182) the authors convincingly addressed the possibility that less time spent immobile in the novel environment may have contributed to the novelty-induced increase of LC axon activity in dorsal CA1 (Figure 4). In addition, initially (for the first 2-4 laps), the mice also ran more slowly in the novel environment (Figure 3aIII, top panel). Given that LC and VTA axon activity were both increasing with velocity (Figure 1F), reduced velocity in the novel environment may have reduced LC and VTA axon activity, but this possibility was not addressed. Reduced LC axon activity in the novel environment could have blunted the noveltyinduced increase. More importantly, any potential novelty-induced increase in VTA axon activity could have been masked by decreases in VTA axon activity due to reduced velocity. The latter may help to explain the discrepancy between the present study and previous findings that VTA neuron firing was increased by novelty (see Discussion, from line 243). It may be useful for the authors to address these possibilities based on their data in the Results section, or to consider them in their Discussion.

    We appreciate the reviewer's insightful comment regarding the potential impact of decreased velocity on novelty responses in LC and VTA axons. The decreased velocity in the novel environment could lead to a diminished novelty response in LC axons and could mask a subtle novelty signal in VTA axons. We have now included the following points in our discussion:

    “In addition, as noted above, on average we did observe a velocity associated signal in VTA axons. When mice were exposed to the novel environment their velocity initially decreased. This would be expected to reduce the average signal across the VTA axon population relative to the higher velocity in the familiar environment. It is possible that this decrease could somewhat mask a subtle novelty induced signal in VTA axons. Therefore, additional experiments should be conducted to investigate the heterogeneity of these axons and their activity under different experimental conditions during tightly controlled behavior.”

    “As discussed above, the slowing down of animal behavior in the novel environment could have decreased LC axon activity and reduced the magnitude of the novelty signal we detected during running. The novelty signal we report here may therefore be an under estimate of it's magnitude under matched behavioral settings.”

    However, it is important to note that although VTA axons, on average, showed activity modulated by velocity in a familiar rewarded environment, this relationship was largely due to the activity of two VTA axons that were strongly modulated by velocity, indicating heterogeneity within the VTA axon population in dCA1. We have highlighted this point in the discussion. We also discuss that:

    “It is possible that some VTA DA inputs to dCA1 respond to novel environments, and the small number of axons recorded here are not representative of the whole population.”

    (4) Sensory properties of the water reward, which the mice may be able to detect, could account for reward-related activity of VTA axons (instead of an expectation of reward). Do the authors have evidence that this is not the case? Occasional probe trials, intermixed with rewarded trials, could be used to test for this possibility.

    Mice receive their water reward through a water spout that is immobile and positioned directly in front of their mouth. Water delivery is triggered by a solenoid when the mice reach the end of the virtual track. Therefore, because the water spout is immobile and the water reward is not delivered until they reach the end of the track, there is nothing for the mice to detect during their run. We have added clarifications about the water spout to the Methods and Results sections, along with appropriate discussion points.

    Additionally, we note that the ramping activity of VTA axons is still present on the initial laps with no reward (Krishnan et al., 2022), indicating that this activity is not directly related to the presence or absence of water but is instead associated with the animal’s reward expectation.

    We thank the reviewer for raising this point and hope that these clarifications address their concern.

    Reviewer #3 (Public Review):

    Summary:

    Heer and Sheffield provide a well-written manuscript that clearly articulates the theoretical motivation to investigate specific catecholaminergic projections to dorsal CA1 of the hippocampus during a reward-based behavior. Using 2-photon calcium imaging in two groups of cre transgenic mice, the authors examine the activity of VTA-CA1 dopamine and LC-CA1 noradrenergic axons during reward seeking in a linear track virtual reality (VR) task. The authors provide a descriptive account of VTA and LC activities during walking, approach to reward, and environment change. Their results demonstrate LC-CA1 axons are activated by walking onset, modulated by walking velocity, and heighten their activity during environment change. In contrast, VTA-CA1 axons were most activated during the approach to reward locations. Together the authors provide a functional dissociation between these catecholamine projections to CA1. A major strength of their approach is the methodological rigor of 2-photon recording, data processing, and analysis approaches. These important systems neuroscience studies provide solid evidence that will contribute to the broader field of learning and memory. The conclusions of this manuscript are mostly well supported by the data, but some additional analysis and/or experiments may be required to fully support the author's conclusions.

    Weaknesses:

    (1) During teleportation between familiar to novel environments the authors report a decrease in the freezing ratio when combining the mice in the two experimental groups (Figure 3aiii). A major conclusion from the manuscript is the difference in VTA and LC activity following environment change, given VTA and LC activity were recorded in separate groups of mice, did the authors observe a similar significant reduction in freezing ratio when analyzing the behavior in LC and VTA groups separately?

    In response to the comment regarding the freezing ratios during teleportation between familiar and novel environments, we have analyzed the freezing ratios and lap velocities of DAT-Cre and NET-Cre mice separately (Fig. 3Aiii). Our analysis shows that the mean lap velocities of both groups overlap in the familiar environment and significantly decrease on the first lap of the novel environment (Fig. 3iii, top). For subsequent laps, the velocities in both groups are not statistically significantly different from the familiar environment lap velocities.

    Freezing ratios also show a statistically significant decrease on the first lap of the novel environment compared to the familiar environment in both groups (Fig. 3iii, bottom). In the NETCRE mice, the freezing ratios remain statistically lower in subsequent laps, while in the DATCRE mice, the following laps show a similar trend but without statistical significance. This lack of statistical significance in the DAT-CRE mice is likely due to their already lower freezing ratios in the familiar environment. Overall, the data demonstrate similar behavioral responses in the two groups of mice during the switch from the familiar to the novel environment.

    (2) The authors satisfactorily apply control analyses to account for the unequal axon numbers recorded in the LC and VTA groups (e.g. Figure 1). However, given the heterogeneity of responses observed in Figures 3c, 4b and the relatively low number of VTA axons recorded (compared to LC), there are some possible limitations to the author's conclusions. A conclusion that LC-CA1 axons, as a general principle, heighten their activity during novel environment presentation, would require this activity profile to be observed in some of the axons recorded in most all LC-CA1 mice.

    We agree with the reviewer’s point. To address this issue, when downsampling LC axons to compare to VTA axons, we matched the sampling statistics of the VTA axons/mice by only selecting one LC axon from each mouse to match the VTA dataset.

    Additionally, we have now included the number of recording sessions and the number of mice in which we observed each type of activity. This information has been added to further clarify and support our conclusions.

    Additionally, if the general conclusion is that VTA-CA1 axons ramp activity during the approach to reward, it would be expected that this activity profile was recorded in the axons of most all VTA-CA1 mice. Can the authors include an analysis to demonstrate that each LC-CA1 mouse contained axons that were activated during novel environments and that each VTA-CA1 mouse contained axons that ramped during the approach to reward?

    As above, we have now added the number of mice that had each activity type we report in the paper here.

    (3) A primary claim is that LC axons projecting to CA1 become activated during novel VR environment presentation. However, the experimental design did not control for the presentation of a familiar environment. As I understand, the presentation order of environments was always familiar, then novel. For this reason, it is unknown whether LC axons are responding to novel environments or environmental change. Did the authors re-present the familiar environment after the novel environment while recording LC-CA1 activity?

    While we did not vary the presentation order of familiar and novel environments, we recorded the activity of LC axons in some mice when exposed to a dark environment (no VR cues) prior to exposure to the familiar environment. Our analysis of this data demonstrates that LC axons are also active following abrupt exposure to the familiar environment.

    We have added a new figure showing this response (Supplementary Figure 5A) and expanded on our original discussion point that LC axon activity generally correlates with arousal, as this result also supports that interpretation.

    We thank the reviewer for highlighting this important consideration. It certainly helps with the interpretation regarding what LC axons generally encode.

    >Recommendations for the authors:

    Reviewer #1 (Recommendations For The Authors):

    In addition to what has been described in the public review, I have the following recommendations:

    The sample size of DA axon recordings should be increased with the use of a single GCaMP for valid conclusions to be made about the lack of novelty-inducted activity in these axons.

    We have increased the n of VTA GCaMP6s axons in the familiar environment by including two axons that were recorded in the familiar rewarded condition. We have also conducted an analysis comparing GCaMPs versus GCaMP7b, which is discussed in detail above.

    Regarding the concerns about valid conclusions of novelty-induced activity in VTA axons, we have added a comment in the discussion to tone down our conclusions regarding the lack of a novelty signal in the VTA axons. This valid concern is discussed in detail above.

    The title is currently very generic, and non-informative. I recommend the use of more specific language in describing the type of behavior under investigation. It is not clear to the reviewer why 'learning' is included here.

    Original title: “Distinct catecholaminergic pathways projecting to hippocampal CA1 transmit contrasting signals during behavior and learning”

    To make it more specific to the experiments conducted here, we have changed the title to this:

    New title: “Distinct catecholaminergic pathways projecting to hippocampal CA1 transmit contrasting signals during navigation in familiar and novel environments”

    Error noted in Figure 4C legend - remove reference to VTA ROIs.

    The reference to VTA ROIs has been removed from the figure legend

    Reviewer #2 (Recommendations For The Authors):

    (1) The concluding sentence of the Abstract could be more specific: which distinct types of information are reflected/'signaled'/'encoded' by LC and VTA inputs to dorsal CA1?

    The abstract has been adjusted accordingly. The new sentence is more specific: “These inputs encode unique information, with reward information in VTA inputs and novelty and kinematic information in LC inputs, likely contributing to differential modulation of hippocampal activity during behavior and learning.”

    (2) Line 46/47: The study by Mamad et al. (2017) did not quite show that VTA dopamine input to dorsal CA1 'drives place preference'. To my understanding, the study showed that suppression of VTA dopamine signaling in a specific place caused avoidance of this place and that VTA dopamine signaling modulated hippocampal place-related firing. So, please consider rephrasing.

    Corrected, thanks for pointing this out.

    (3) Legend to Figure 3AIII: 'Each lap was compared to the first lap in F . . .' Could you clarify if 'F' refers to the 'familiar environment?

    Figure legend has been changed accordingly

    (4) Line 176: '36 LC neurons' - should this not be '36 imaged axon terminals in dorsal CA1' or something along these lines?

    This reference has been changed to “LC axon ROIs”

    (5) Line 353: Why was water restriction started before the hippocampal window implant, if behavioral training to run for water reward only started after the implant? Please clarify.

    A sentence was added to the methods to explain that this was done to reduce bleeding and swelling during the hippocampal window implantation.

    (6) Line 377: '. . . which took 10-14 days (although some mice never reached this threshold).' How many mice did not reach the criterion within 14 days? I think it is not accurate to say the mice 'never' reached the threshold, as they were only tested for a limited period of time.

    We have added details of how many mice were excluded from each group and the reason why they were excluded.

    (7) Exclusion criteria for imaging data: The authors state (from line 402): 'Imaging sessions with large amounts of drift or bleaching were excluded from analysis (8 sessions for NET mice, 6 sessions for LC Mice).' What exactly were the quantitative exclusion criteria? Were these defined before the onset of the study or throughout the study?

    Imaging sessions were first qualitatively assessed by looking for disappearance or movement of structures in the Z-plane throughout the imaging FOV. Additionally, following motion correction in suite2p, we used the registration metrics, which plots the first Principle Component of the motion corrected images, to assess for drift, bleaching, or heat bubbles. If this variable increased or decreased greatly throughout a session, to the point where any apparent activity was not visible in the first PC, the dataset was excluded. We have added these exclusion criteria to the methods section.

    Reviewer #3 (Recommendations For The Authors):

    Please provide a justification or rationale for having two different criteria for immobility (< 5cm/sec) and freezing (<0.2 cm/sec). If VTA and LC axon activities are different between these two velocities, please provide some commentary on this difference.

    This is a typo leftover from before we converted velocity from rotational units to cm/s.

  14. Version published to 10.1101/2023.11.29.569214v4 on bioRxiv
  15. Version published to 10.1101/2023.11.29.569214v3 on bioRxiv
  16. eLife

    Author Response

    Reviewer #1 (Public Review):

    Summary:

    Heer and Sheffield used 2 photon imaging to dissect the functional contributions of convergent dopamine and noradrenaline inputs to the dorsal hippocampus CA1 in head-restrained mice running down a virtual linear path. Mice were trained to collect water rewards at the end of the track and on test days, calcium activity was recorded from dopamine (DA) axons originating in the ventral tegmental area (VTA, n=7) and noradrenaline axons from the locus coeruleus (LC, n=87) under several conditions. When mice ran laps in a familiar environment, VTA DA axons exhibited ramping activity along the track that correlated with distance to reward and velocity to some extent, while LC input activity remained constant across the track, but correlated invariantly with velocity and time to motion onset. A subset of recordings taken when the reward was removed showed diminished ramping activity in VTA DA axons, but no changes in the LC axons, confirming that DA axon activity is locked to reward availability. When mice were subsequently introduced to a new environment, the ramping to reward activity in the DA axons disappeared, while LC axons showed a dramatic increase in activity lasting 90 s (6 laps) following the environment switch. In the final analysis, the authors sought to disentangle LC axon activity induced by novelty vs. behavioral changes induced by novelty by removing periods in which animals were immobile and established that the activity observed in the first 2 laps reflected novelty-induced signal in LC axons.

    Strengths:

    The results presented in this manuscript provide insights into the specific contributions of catecholaminergic input to the dorsal hippocampus CA1 during spatial navigation in a rewarded virtual environment, offering a detailed analysis of the resolution of single axons. The data analysis is thorough and possible confounding variables and data interpretation are carefully considered.

    Weaknesses:

    Aspects of the methodology, data analysis, and interpretation diminish the overall significance of the findings, as detailed below.

    The LC axonal recordings are well-powered, but the DA axonal recordings are severely underpowered, with recordings taken from a mere 7 axons (compared to 87 LC axons). Additionally, 2 different calcium indicators with differential kinetics and sensitivity to calcium changes (GCaMP6S and GCaMP7b) were used (n=3, n=4 respectively) and the data pooled. This makes it very challenging to draw any valid conclusions from the data, particularly in the novelty experiment. The surprising lack of novelty-induced DA axon activity may be a false negative. Indeed, at least 1 axon (axon 2) appears to be showing a novelty-induced rise in activity in Figure 3C. Changes in activity in 4/7 axons are also referred to as a 'majority' occurrence in the manuscript, which again is not an accurate representation of the observed data.

    The reviewer points out a weakness in the analysis of VTA axons in our dataset. The relatively low n (currently 7) comes from the fact that VTA axons in the CA1 region of the hippocampus are very sparse and very difficult to record from (due to their sparsity and the low level of baseline fluorescence inherent in long range axon segments). This is the reason they have not been recorded from in any other lab outside of our lab. LC axons, on the other hand, are more abundant in CA1. In the paper when comparing VTA versus LC axons we deal with the mismatch in n by downsampling the LC axons to match the VTA axons and repeated this 1000 times to create a distribution. However, because the VTA axon n is relatively low, it is possible that we have not sampled the VTA axon population sufficiently and therefore have a biased population in our dataset. The issue is that it takes months for the baseline expression of GCaMP to reach sufficient levels to be able to record from VTA axons, and it is typical to find only a single axon in a FOV per animal. There are additional reasons why mice and/or axon recordings do not reach criteria and cannot be included in the dataset (these exclusion criteria are reported in the Methods section). For instance, out of the 54 DAT-Cre mice injected, images were never conducted in 36 for lack of expression or because mice failed to reach behavioral criteria. Another 11 mice were excluded for heat bubbles that developed during imaging, z-drift of the FOV, or bleaching of the GCaMP signal.

    However, we do have n=2 additional VTA axon recordings that we will add to the dataset to bring the n up from 7 to 9. We plan on re-analyzing the data with n=9 VTA axons and making comparisons to down-sampled LC axons as described above. This boost in n will increase the power of our VTA axon analysis. To more formally test whether this is sufficient for statistical tests, we plan to utilize the G*power power-analysis tool to compute statistical power for each of the different tests we use. We will report this in the next version of the paper. However, the n=2 additional axons were nor recorded in the novel environment, so the next version will remain at n=7 for the novel environment analysis. We agree with the reviewer that the lack of the novelty induced DA axon activity may be a false negative, and so we will adjust the description of our results and discussion accordingly.

    During the data collection of VTA axon activity we tried two variants of GCaMP: 6s and 7b, to see if one would increase the success rate of finding and recording from VTA axons. Given the long time-course of these experiments and the low yield in success, we pooled the GCaMP variants together to increase statistical power. Because the 2 additional VTA DA axons that were recorded from expressed GCaMP6s, the next version of the paper will have n=5 GCaMP6s, and n=4 GCaMP7b VTA DA axons, which will allow us to compare the activity of the two sensors in the familiar environment. The reviewer correctly pointed out that the sensors themselves could confound our results, and so they should not be pooled unless we can show they do not produce different signals in the axons. We will make this comparison and report the findings in the next version of the paper. If we find no significant differences, we will pool the data. If differences are detected, we will keep these axons separate for subsequent analysis and comparisons to LC axons.

    The authors conducted analysis on recording data exclusively from periods of running in the novelty experiment to isolate the effects of novelty from novelty-induced changes in behavior. However, if the goal is to distinguish between changes in locus coeruleus (LC) axon activity induced by novelty and those induced by motion, analyzing LC axon activity during periods of immobility would enhance the robustness of the results.

    This is indeed true, and this suggested analysis could further support our conclusions regarding the LC novelty signal. For the next version of the paper, we will use the periods of immobility to analyze and isolate any novelty induced activity in LC axons. However, following exposure to the novel environment, mice spend much less time immobile, therefore there may not be sufficient periods of immobility close in time to the exposure to the novel environment (which is when the novelty signal occurs). We plan to analyze mouse behavior during the early exposure to the novel environment for immobility and check whether we have enough of this behavior to perform the suggested analysis.

    The authors attribute the ramping activity of the DA axons to the encoding of the animals' position relative to reward. However, given the extensive data implicating the dorsal CA1 in timing, and the remarkable periodicity of the behavior, the fact that DA axons could be signalling temporal information should be considered.

    This is a very good point. We agree that the VTA DA axons could be signaling temporal information, as we have previously shown that these axons also exhibit ramping activity when you average their activity by time to reward (Krishnan et. al., 2022). We will conduct this analysis on this dataset. We have not, however, conducted any experiments designed to separate out time from distance, such as the experiments conducted in Kim et. al., 2020. Therefore, we cannot determine whether this is due to proximity in space to reward or time to reward. We will clarify in our text that by proximity, we mean either place or time, and cannot conclude which feature of the experience drives the VTA axon signal.

    Krishnan, L.S., Heer, C., Cherian, C., Sheffield, M.E. Reward expectation extinction restructures and degrades CA1 spatial maps through loss of a dopaminergic reward proximity signal. Nat Commun 13, 6662 (2022).

    Kim, HyungGoo R., Athar N. Malik, John G. Mikhael, Pol Bech, Iku Tsutsui-Kimura, Fangmiao Sun, Yajun Zhang, et al. A Unified Framework for Dopamine Signals across Timescales. Cell 183, no. 6 (2020).

    The authors should explain and justify the use of a longer linear track (3m, as opposed to 2m in the DAT-cre mice) in the LC axon recording experiments.

    LC axon activity was recorded on a 3m track to match the track length from an experiment we recently published (Dong et al., 2021) in which mice were exposed to a novel 3m track while populations of CA1 pyramidal cells were recorded. In that paper we described the time course of place field formation on the novel track. We wanted to test if LC axons signaled novelty (as we hypothesized) and whether the time course of LC axon activity matched the time course of place field formation. We briefly discuss this in the Discussion section of this paper and hypothesize that LC axons in CA1 could open a window of plasticity in which new place fields can form.

    VTA axons were recorded on a 2m track (same VR tracks as LC axons were recorded on) to match another recent paper from our lab in which reward expectation was manipulated (Krishnan et al, 2022). In that study CA1 populations of pyramidal cells were recorded during the reward expectation experiment. To match the experience during recordings of VTA axons in CA1 to test how reward expectation may influence axon signaling along the track, we also used a 2m track. The idea was to check how VTA dopaminergic inputs to CA1 may influence CA1 population dynamics along the track.

    Although the tracks were identical for LC and VTA recordings for both the familiar and novel tracks in terms of visual cues and design, the track lengths are different (simply modulated by gain control of the rotary encoder). To account for this we normalized the lengths for our comparison analysis. This normalization allows for a direct comparison of the patterns of activity across the two types of axons, controlling for the potential confound introduced by the different track lengths. By adjusting the data to a common scale, we could assess the relative changes in activity levels at matched spatial bins, ensuring that any observed differences or similarities are due to the intrinsic properties of the axons rather than differences in track lengths. However, the different lengths do make the animal’s experience slightly different. This is somewhat offset by the observations in our study that none of the LC or VTA axon signals would be expected to be majorly influenced by variations in track length. For instance, LC axons are associated with velocity and a pre-motion initiation signal, neither of which would be influenced by track length. VTA axons are also associated with velocity, which would not influence a direct comparison to LC axon velocity signals as mice reach maximal velocity very rapidly along the track. VTA axons do ramp up in activity as they approach the reward zone, and this signal could be modulated by track length (or maybe not if the signal is encoding time to reward rather than distance). However, LC axons show no ramping to reward signals, so a comparison across axons recorded on different track lengths for this analysis is justified.

    However, to add rigor to comparisons of axon dynamics recorded along 2m and 3m tracks, we plan to plot axon activity of both sets of axons by time to reward, and actual (un-normalized) distance from reward.

    Krishnan, L.S., Heer, C., Cherian, C., Sheffield, M.E. Reward expectation extinction restructures and degrades CA1 spatial maps through loss of a dopaminergic reward proximity signal. Nat Commun 13, 6662 (2022).

    Dong, C., Madar, A. D. & Sheffield, M.E. Distinct place cell dynamics in CA1 and CA3 encode experience in new environments. Nat Commun 12, 2977 (2021).

    Reviewer #2 (Public Review):

    Summary:

    The authors used 2-photon Ca2+-imaging to study the activity of ventral tegmental area (VTA) and locus coeruleus (LC) axons in the CA1 region of the dorsal hippocampus in head-fixed male mice moving on linear paths in virtual reality (VR) environments.

    The main findings were as follows:

    • In a familiar environment, the activity of both VTA axons and LC axons increased with the mice's running speed on the Styrofoam wheel, with which they could move along a linear track through a VR environment.
    • VTA, but not LC, axons showed marked reward position-related activity, showing a ramping-up of activity when mice approached a learned reward position.
    • In contrast, the activity of LC axons ramped up before the initiation of movement on the Styrofoam wheel.
    • In addition, exposure to a novel VR environment increased LC axon activity, but not VTA axon activity.

    Overall, the study shows that the activity of catecholaminergic axons from VTA and LC to dorsal hippocampal CA1 can partly reflect distinct environmental, behavioral, and cognitive factors. Whereas both VTA and LC activity reflected running speed, VTA, but not LC axon activity reflected the approach of a learned reward, and LC, but not VTA, axon activity reflected initiation of running and novelty of the VR environment.

    I have no specific expertise with respect to 2-photon imaging, so cannot evaluate the validity of the specific methods used to collect and analyse 2-photon calcium imaging data of axonal activity.

    Strengths:

    (1) Using a state-of-the-art approach to record separately the activity of VTA and LC axons with high temporal resolution in awake mice moving through virtual environments, the authors provide convincing evidence that the activity of VTA and LC axons projecting to dorsal CA1 reflect partly distinct environmental, behavioral and cognitive factors.

    (2) The study will help a) to interpret previous findings on how hippocampal dopamine and norepinephrine or selective manipulations of hippocampal LC or VTA inputs modulate behavior and b) to generate specific hypotheses on the impact of selective manipulations of hippocampal LC or VTA inputs on behavior.

    Weaknesses:

    (1)The findings are correlational and do not allow strong conclusions on how VTA or LC inputs to dorsal CA1 affect cognition and behavior. However, as indicated above under Strengths, the findings will aid the interpretation of previous findings and help to generate new hypotheses as to how VTA or LC inputs to dorsal CA1 affect distinct cognitive and behavioral functions.

    (2) Some aspects of the methodology would benefit from clarification.
    First, to help others to better scrutinize, evaluate, and potentially to reproduce the research, the authors may wish to check if their reporting follows the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines for the full and transparent reporting of research involving animals (https://arriveguidelines.org/). For example, I think it would be important to include a sample size justification (e.g., based on previous studies, considerations of statistical power, practical considerations, or a combination of these factors). The authors should also include the provenance of the mice. Moreover, although I am not an expert in 2-photon imaging, I think it would be useful to provide a clearer description of exclusion criteria for imaging data.

    We thank the reviewer for helping us formalize the scientific rigor of our study. There are ten ARRIVE Guidelines and we have addressed most of them in our study already. However, there is an opportunity to add detail. We have listed below all ten points and how we have or will address each one.

    (1) Experimental design - we go into great depth explaining the experimental set-up, how we used the autofluorescent blebs as imaging controls, how we controlled for different sample sizes between the two populations, and the statistical tests used for comparisons. We also carefully accounted for animal behavior when quantifying and describing axon dynamics both in the familiar and novel environments.

    (2)Sample size - We state both the number of ROIs and mice for each analysis. Wherever we state how many axons had a certain kind of activity, we will also state the number of mice we saw this activity in. For the next version of the paper, we plan to conduct a power analysis using G*power to assess the power of our sample sizes for statistical analysis.

    (3) Inclusion/exclusion criteria - Out of the 36 NET-Cre mice injected, 15 were never recorded for either failing to reach behavioral criteria, or a lack of visible expression in axons. Out of the 54 DAT-Cre mice injected, images were never conducted in 36 for lack of expression or failing to reach behavioral criteria. Out of the remaining 21 NET-CRE, 5 were excluded for heat bubbles, z-drift, or bleaching, while 11 DAT-Cre were excluded for the same reasons. This was determined by visually assessing imaging sessions, followed by using the registration metrics output by suite2p. This registration metric conducted a PCA on the motion-corrected ROIs and plotted the first PC. If the PC drifted largely, to the point where no activity was apparent, the video was excluded from analysis.

    (4) Randomization - Already included in the paper is a description of random down sampling of LC axons to make statistical comparisons with VTA axons. LC axons were selected pseudo-randomly (only one axon per imaging session) to match VTA sampling statistics. This randomization was repeated 1000 times and comparisons were made against this random distribution.

    (5) Blinding-masking - no blinding/masking was conducted as no treatments were given that would require this. We will include this statement in the next version.

    (6) Outcomes - We defined all outcomes measured, such as those related to animal behavior and related axon signaling.

    (7) Statistical methods - None of the reviewers had any issues regarding our description of statistical methods, which we described in detail in this version of the paper.

    (8) Experimental animals - We described that DAT- Cre mice were obtained through JAX labs, and NET-Cre mice were obtained from the Tonegawa lab (Wagatsuma et al. 2017)

    (9) Experimental procedure - Already listed in detail in Methods section.

    (10) Results - Rigorously described in detail for behaviors and related axon dynamics.

    Wagatsuma, Akiko, Teruhiro Okuyama, Chen Sun, Lillian M. Smith, Kuniya Abe, and Susumu Tonegawa. “Locus Coeruleus Input to Hippocampal CA3 Drives Single-Trial Learning of a Novel Context.” Proceedings of the National Academy of Sciences 115, no. 2 (January 9, 2018): E310–16. https://doi.org/10.1073/pnas.1714082115.

    Second, why were different linear tracks used for studies of VTA and LC axon activity (from line 362)? Could this potentially contribute to the partly distinct activity correlates that were found for VTA and LC axons?

    A detailed response to this is written above for a similar comment from reviewer 1.

    Third, the authors seem to have used two different criteria for defining immobility. Immobility was defined as moving at <5 cm/s for the behavioral analysis in Figure 3a, but as <0.2 cm/s for the imaging data analysis in Figure 4 (see legends to these figures and also see Methods, from line 447, line 469, line 498)? I do not understand why, and it would be good if the authors explained this.

    This is an error leftover from before we converted velocity from rotational units of the treadmill to cm/s. This will be corrected in the next version of the paper.

    (3) In the Results section (from line 182) the authors convincingly addressed the possibility that less time spent immobile in the novel environment may have contributed to the novelty-induced increase of LC axon activity in dorsal CA1 (Figure 4). In addition, initially (for the first 2-4 laps), the mice also ran more slowly in the novel environment (Figure 3aIII, top panel). Given that LC and VTA axon activity were both increasing with velocity (Figure 1F), reduced velocity in the novel environment may have reduced LC and VTA axon activity, but this possibility was not addressed. Reduced LC axon activity in the novel environment could have blunted the noveltyinduced increase. More importantly, any potential novelty-induced increase in VTA axon activity could have been masked by decreases in VTA axon activity due to reduced velocity. The latter may help to explain the discrepancy between the present study and previous findings that VTA neuron firing was increased by novelty (see Discussion, from line 243). It may be useful for the authors to address these possibilities based on their data in the Results section, or to consider them in their Discussion.

    This is a great point. The decreased velocity in the novel environment could lead to a diminished novelty response in LC axons. We will add a discussion point on this in the next version. This could also be the case for VTA axons, so will add a discussion point that the lack of novelty signaling seen in VTA axons could be due to reduced velocity masking this signal.

    (4) Sensory properties of the water reward, which the mice may be able to detect, could account for reward-related activity of VTA axons (instead of an expectation of reward). Do the authors have evidence that this is not the case? Occasional probe trials, intermixed with rewarded trials, could be used to test for this possibility.

    Mice receive their water reward through a waterspout that is immobile and positioned directly in front of their mouth (which is also immobile as they are head fixed) and water delivery is triggered by a solenoid when the mice reach the end of the virtual track. Therefore, because the waterspout remains in the same place relative to the mouse, and the water reward is not delivered until they reach the end of the virtual track, there is nothing for the mice to detect. We will update the paper to make this clearer.

    Additionally, on the initial laps with no reward, the ramping activity is still present (Krishnan et al, 2022) indicating this activity is not directly related to the presence/absence of water but is instead caused by reward expectation.

    Reviewer #3 (Public Review):

    Summary:

    Heer and Sheffield provide a well-written manuscript that clearly articulates the theoretical motivation to investigate specific catecholaminergic projections to dorsal CA1 of the hippocampus during a reward-based behavior. Using 2-photon calcium imaging in two groups of cre transgenic mice, the authors examine the activity of VTA-CA1 dopamine and LC-CA1 noradrenergic axons during reward seeking in a linear track virtual reality (VR) task. The authors provide a descriptive account of VTA and LC activities during walking, approach to reward, and environment change. Their results demonstrate LC-CA1 axons are activated by walking onset, modulated by walking velocity, and heighten their activity during environment change. In contrast, VTA-CA1 axons were most activated during the approach to reward locations. Together the authors provide a functional dissociation between these catecholamine projections to CA1. A major strength of their approach is the methodological rigor of 2-photon recording, data processing, and analysis approaches. These important systems neuroscience studies provide solid evidence that will contribute to the broader field of learning and memory. The conclusions of this manuscript are mostly well supported by the data, but some additional analysis and/or experiments may be required to fully support the author's conclusions.

    Weaknesses:

    (1) During teleportation between familiar to novel environments the authors report a decrease in the freezing ratio when combining the mice in the two experimental groups (Figure 3aiii). A major conclusion from the manuscript is the difference in VTA and LC activity following environment change, given VTA and LC activity were recorded in separate groups of mice, did the authors observe a similar significant reduction in freezing ratio when analyzing the behavior in LC and VTA groups separately?

    In response to this comment, we will analyze the freezing ratios in DAT-Cre and NET-Cre mice separately. However, other members of the lab have seen the same result in other mouse strains (See Dong et al. 2021), so we do not expect to see a difference (but it is certainly worth checking).

    (2) The authors satisfactorily apply control analyses to account for the unequal axon numbers recorded in the LC and VTA groups (e.g. Figure 1). However, given the heterogeneity of responses observed in Figures 3c, 4b and the relatively low number of VTA axons recorded (compared to LC), there are some possible limitations to the author's conclusions. A conclusion that LC-CA1 axons, as a general principle, heighten their activity during novel environment presentation, would require this activity profile to be observed in some of the axons recorded in most all LC-CA1 mice.

    We agree with the reviewer’s point here. To help avoid this problem, when downsampling LC axons to compare to VTA axons, we matched the sampling statistics of the VTA axons/mice (i.e. only one LC axon was taken from each mouse to match the VTA dataset).

    However, in the next version of the paper we will also report the number of mice that we see a significant novel response in. We will also add the number of mice with significant activity for each of the measures in the familiar environment (e.g. how many mice had axons positively correlated with velocity).

    Additionally, if the general conclusion is that VTA-CA1 axons ramp activity during the approach to reward, it would be expected that this activity profile was recorded in the axons of most all VTA-CA1 mice. Can the authors include an analysis to demonstrate that each LC-CA1 mouse contained axons that were activated during novel environments and that each VTA-CA1 mouse contained axons that ramped during the approach to reward?

    As stated above, we will add the number of mice that had each activity type we reported here.

    (3) A primary claim is that LC axons projecting to CA1 become activated during novel VR environment presentation. However, the experimental design did not control for the presentation of a familiar environment. As I understand, the presentation order of environments was always familiar, then novel. For this reason, it is unknown whether LC axons are responding to novel environments or environmental change. Did the authors re-present the familiar environment after the novel environment while recording LC-CA1 activity?

    This is an important point to address. While we never varied the presentation order of the familiar vs novel environments, we did record the activity of LC axons in some of the mice in a dark environment (no VR cues) prior to exposure to the familiar environment. We will look at these axons to address whether they respond to initial exposure to the familiar environment. This will allow us to check whether they are responding to environmental change or novelty. We will add this analysis to the next version of the paper.

  17. Version published to 10.7554/elife.95213.1 on eLife
  18. eLife

    eLife assessment

    This study presents important findings on the differential activity of noradrenergic and dopaminergic input to dorsal hippocampus CA1 in head-fixed mice traversing a runway in a virtual environment that is familiar or novel. While the data appear to be rigorously analysed, and the observed divergence in the dynamics of activity in the dopaminergic and noradrenergic axons is solid, there are some methodological concerns that mean the strength of evidence is currently incomplete.

  19. eLife

    Reviewer #1 (Public Review):

    Summary:

    Heer and Sheffield used 2 photon imaging to dissect the functional contributions of convergent dopamine and noradrenaline inputs to the dorsal hippocampus CA1 in head-restrained mice running down a virtual linear path. Mice were trained to collect water rewards at the end of the track and on test days, calcium activity was recorded from dopamine (DA) axons originating in the ventral tegmental area (VTA, n=7) and noradrenaline axons from the locus coeruleus (LC, n=87) under several conditions. When mice ran laps in a familiar environment, VTA DA axons exhibited ramping activity along the track that correlated with distance to reward and velocity to some extent, while LC input activity remained constant across the track, but correlated invariantly with velocity and time to motion onset. A subset of recordings taken when the reward was removed showed diminished ramping activity in VTA DA axons, but no changes in the LC axons, confirming that DA axon activity is locked to reward availability. When mice were subsequently introduced to a new environment, the ramping to reward activity in the DA axons disappeared, while LC axons showed a dramatic increase in activity lasting 90 s (6 laps) following the environment switch. In the final analysis, the authors sought to disentangle LC axon activity induced by novelty vs. behavioral changes induced by novelty by removing periods in which animals were immobile and established that the activity observed in the first 2 laps reflected novelty-induced signal in LC axons.

    Strengths:

    The results presented in this manuscript provide insights into the specific contributions of catecholaminergic input to the dorsal hippocampus CA1 during spatial navigation in a rewarded virtual environment, offering a detailed analysis of the resolution of single axons. The data analysis is thorough and possible confounding variables and data interpretation are carefully considered.

    Weaknesses:

    Aspects of the methodology, data analysis, and interpretation diminish the overall significance of the findings, as detailed below.

    The LC axonal recordings are well-powered, but the DA axonal recordings are severely underpowered, with recordings taken from a mere 7 axons (compared to 87 LC axons). Additionally, 2 different calcium indicators with differential kinetics and sensitivity to calcium changes (GCaMP6S and GCaMP7b) were used (n=3, n=4 respectively) and the data pooled. This makes it very challenging to draw any valid conclusions from the data, particularly in the novelty experiment. The surprising lack of novelty-induced DA axon activity may be a false negative. Indeed, at least 1 axon (axon 2) appears to be showing a novelty-induced rise in activity in Figure 3C. Changes in activity in 4/7 axons are also referred to as a 'majority' occurrence in the manuscript, which again is not an accurate representation of the observed data.

    The authors conducted analysis on recording data exclusively from periods of running in the novelty experiment to isolate the effects of novelty from novelty-induced changes in behavior. However, if the goal is to distinguish between changes in locus coeruleus (LC) axon activity induced by novelty and those induced by motion, analyzing LC axon activity during periods of immobility would enhance the robustness of the results.

    The authors attribute the ramping activity of the DA axons to the encoding of the animals' position relative to reward. However, given the extensive data implicating the dorsal CA1 in timing, and the remarkable periodicity of the behavior, the fact that DA axons could be signalling temporal information should be considered.

    The authors should explain and justify the use of a longer linear track (3m, as opposed to 2m in the DAT-cre mice) in the LC axon recording experiments.

  20. eLife

    Reviewer #2 (Public Review):

    Summary:

    The authors used 2-photon Ca2+-imaging to study the activity of ventral tegmental area (VTA) and locus coeruleus (LC) axons in the CA1 region of the dorsal hippocampus in head-fixed male mice moving on linear paths in virtual reality (VR) environments.

    The main findings were as follows:

    - In a familiar environment, the activity of both VTA axons and LC axons increased with the mice's running speed on the Styrofoam wheel, with which they could move along a linear track through a VR environment.
    - VTA, but not LC, axons showed marked reward position-related activity, showing a ramping-up of activity when mice approached a learned reward position.
    - In contrast, the activity of LC axons ramped up before the initiation of movement on the Styrofoam wheel.
    - In addition, exposure to a novel VR environment increased LC axon activity, but not VTA axon activity.

    Overall, the study shows that the activity of catecholaminergic axons from VTA and LC to dorsal hippocampal CA1 can partly reflect distinct environmental, behavioral, and cognitive factors. Whereas both VTA and LC activity reflected running speed, VTA, but not LC axon activity reflected the approach of a learned reward, and LC, but not VTA, axon activity reflected initiation of running and novelty of the VR environment.

    I have no specific expertise with respect to 2-photon imaging, so cannot evaluate the validity of the specific methods used to collect and analyse 2-photon calcium imaging data of axonal activity.

    Strengths:

    (1) Using a state-of-the-art approach to record separately the activity of VTA and LC axons with high temporal resolution in awake mice moving through virtual environments, the authors provide convincing evidence that the activity of VTA and LC axons projecting to dorsal CA1 reflect partly distinct environmental, behavioral and cognitive factors.

    (2) The study will help a) to interpret previous findings on how hippocampal dopamine and norepinephrine or selective manipulations of hippocampal LC or VTA inputs modulate behavior and b) to generate specific hypotheses on the impact of selective manipulations of hippocampal LC or VTA inputs on behavior.

    Weaknesses:

    (1) The findings are correlational and do not allow strong conclusions on how VTA or LC inputs to dorsal CA1 affect cognition and behavior. However, as indicated above under Strengths, the findings will aid the interpretation of previous findings and help to generate new hypotheses as to how VTA or LC inputs to dorsal CA1 affect distinct cognitive and behavioral functions.

    (2) Some aspects of the methodology would benefit from clarification.
    First, to help others to better scrutinize, evaluate, and potentially to reproduce the research, the authors may wish to check if their reporting follows the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines for the full and transparent reporting of research involving animals (https://arriveguidelines.org/). For example, I think it would be important to include a sample size justification (e.g., based on previous studies, considerations of statistical power, practical considerations, or a combination of these factors). The authors should also include the provenance of the mice. Moreover, although I am not an expert in 2-photon imaging, I think it would be useful to provide a clearer description of exclusion criteria for imaging data.
    Second, why were different linear tracks used for studies of VTA and LC axon activity (from line 362)? Could this potentially contribute to the partly distinct activity correlates that were found for VTA and LC axons?
    Third, the authors seem to have used two different criteria for defining immobility. Immobility was defined as moving at <5 cm/s for the behavioral analysis in Figure 3a, but as <0.2 cm/s for the imaging data analysis in Figure 4 (see legends to these figures and also see Methods, from line 447, line 469, line 498)? I do not understand why, and it would be good if the authors explained this.

    (3) In the Results section (from line 182) the authors convincingly addressed the possibility that less time spent immobile in the novel environment may have contributed to the novelty-induced increase of LC axon activity in dorsal CA1 (Figure 4). In addition, initially (for the first 2-4 laps), the mice also ran more slowly in the novel environment (Figure 3aIII, top panel). Given that LC and VTA axon activity were both increasing with velocity (Figure 1F), reduced velocity in the novel environment may have reduced LC and VTA axon activity, but this possibility was not addressed. Reduced LC axon activity in the novel environment could have blunted the novelty-induced increase. More importantly, any potential novelty-induced increase in VTA axon activity could have been masked by decreases in VTA axon activity due to reduced velocity. The latter may help to explain the discrepancy between the present study and previous findings that VTA neuron firing was increased by novelty (see Discussion, from line 243). It may be useful for the authors to address these possibilities based on their data in the Results section, or to consider them in their Discussion.

    (4) Sensory properties of the water reward, which the mice may be able to detect, could account for reward-related activity of VTA axons (instead of an expectation of reward). Do the authors have evidence that this is not the case? Occasional probe trials, intermixed with rewarded trials, could be used to test for this possibility.

  21. eLife

    Reviewer #3 (Public Review):

    Summary:

    Heer and Sheffield provide a well-written manuscript that clearly articulates the theoretical motivation to investigate specific catecholaminergic projections to dorsal CA1 of the hippocampus during a reward-based behavior. Using 2-photon calcium imaging in two groups of cre transgenic mice, the authors examine the activity of VTA-CA1 dopamine and LC-CA1 noradrenergic axons during reward seeking in a linear track virtual reality (VR) task. The authors provide a descriptive account of VTA and LC activities during walking, approach to reward, and environment change. Their results demonstrate LC-CA1 axons are activated by walking onset, modulated by walking velocity, and heighten their activity during environment change. In contrast, VTA-CA1 axons were most activated during the approach to reward locations. Together the authors provide a functional dissociation between these catecholamine projections to CA1. A major strength of their approach is the methodological rigor of 2-photon recording, data processing, and analysis approaches. These important systems neuroscience studies provide solid evidence that will contribute to the broader field of learning and memory. The conclusions of this manuscript are mostly well supported by the data, but some additional analysis and/or experiments may be required to fully support the author's conclusions.

    Weaknesses:

    (1) During teleportation between familiar to novel environments the authors report a decrease in the freezing ratio when combining the mice in the two experimental groups (Figure 3aiii). A major conclusion from the manuscript is the difference in VTA and LC activity following environment change, given VTA and LC activity were recorded in separate groups of mice, did the authors observe a similar significant reduction in freezing ratio when analyzing the behavior in LC and VTA groups separately?

    (2) The authors satisfactorily apply control analyses to account for the unequal axon numbers recorded in the LC and VTA groups (e.g. Figure 1). However, given the heterogeneity of responses observed in Figures 3c, 4b and the relatively low number of VTA axons recorded (compared to LC), there are some possible limitations to the author's conclusions. A conclusion that LC-CA1 axons, as a general principle, heighten their activity during novel environment presentation, would require this activity profile to be observed in some of the axons recorded in most all LC-CA1 mice. Additionally, if the general conclusion is that VTA-CA1 axons ramp activity during the approach to reward, it would be expected that this activity profile was recorded in the axons of most all VTA-CA1 mice. Can the authors include an analysis to demonstrate that each LC-CA1 mouse contained axons that were activated during novel environments and that each VTA-CA1 mouse contained axons that ramped during the approach to reward?

    (3) A primary claim is that LC axons projecting to CA1 become activated during novel VR environment presentation. However, the experimental design did not control for the presentation of a familiar environment. As I understand, the presentation order of environments was always familiar, then novel. For this reason, it is unknown whether LC axons are responding to novel environments or environmental change. Did the authors re-present the familiar environment after the novel environment while recording LC-CA1 activity?

  22. Version published to 10.1101/2023.11.29.569214v2 on bioRxiv
  23. Version published to 10.1101/2023.11.29.569214v1 on bioRxiv