The rapid developmental rise of somatic inhibition disengages hippocampal dynamics from self-motion

  1. Robin F Dard
  2. Erwan Leprince
  3. Julien Denis
  4. Shrisha Rao Balappa
  5. Dmitrii Suchkov
  6. Richard Boyce
  7. Catherine Lopez
  8. Marie Giorgi-Kurz
  9. Tom Szwagier
  10. Théo Dumont
  11. Hervé Rouault
  12. Marat Minlebaev
  13. Agnès Baude
  14. Rosa Cossart
  15. Michel A Picardo

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    Evaluation Summary:

    This study investigates hippocampal dynamics over the course of early postnatal development with respect to spontaneous movements. Pioneering in vivo imaging in the hippocampus of neonatal mice, the authors find evidence for an abrupt developmental transition in this neural activity at the end of the first postnatal week in rodents and contribute to the understanding of how cognitive functions could emerge from the immature brain.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

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Abstract

Early electrophysiological brain oscillations recorded in preterm babies and newborn rodents are initially mostly driven by bottom-up sensorimotor activity and only later can detach from external inputs. This is a hallmark of most developing brain areas, including the hippocampus, which, in the adult brain, functions in integrating external inputs onto internal dynamics. Such developmental disengagement from external inputs is likely a fundamental step for the proper development of cognitive internal models. Despite its importance, the developmental timeline and circuit basis for this disengagement remain unknown. To address this issue, we have investigated the daily evolution of CA1 dynamics and underlying circuits during the first two postnatal weeks of mouse development using two-photon calcium imaging in non-anesthetized pups. We show that the first postnatal week ends with an abrupt shift in the representation of self-motion in CA1. Indeed, most CA1 pyramidal cells switch from activated to inhibited by self-generated movements at the end of the first postnatal week, whereas the majority of GABAergic neurons remain positively modulated throughout this period. This rapid switch occurs within 2 days and follows the rapid anatomical and functional surge of local somatic GABAergic innervation. The observed change in dynamics is consistent with a two-population model undergoing a strengthening of inhibition. We propose that this abrupt developmental transition inaugurates the emergence of internal hippocampal dynamics.

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  1. Version published to 10.7554/elife.78116 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    We thank the reviewer for a very constructive evaluation of our work and for a fair summary of its main strengths. We have addressed her/his main concerns as follows:

    1. The experiments involve an invasive neurosurgical procedure used to perform hippocampal imaging, which removes the ipsilateral overlying somatosensory cortex, and it is not possible to evaluate from the data provided that this surgery does not disrupt network function, especially given the focus on movement-related activity patterns.

    We thank the reviewer for bringing up this important issue. Indeed, our experimental access to early hippocampal activity with 2-photon calcium imaging relies on a quite invasive procedure. However, the many control experiments we have performed indicate that early hippocampal dynamics were not significantly altered by the surgery. First, our extracellular electrophysiological recordings from a sample of 6 mice (ranging from P6 to P11, Figure 1- figure supplement 1C) show that the frequency of early sharp waves (eSW) was slightly but not significantly reduced in the ipsilateral hemisphere compared to the contralateral one. Of note, a similar “non-significant” decrease had been previously reported by another group (Graf et al 2021 Fig S6C). As suggested by the reviewer, we can speculate that this slight decrease may result from a reduction of the sensory feedback re-afference originating from the right limbs. Indeed, we observed that movements of the right limbs (contralateral to the window implant) elicited a slightly smaller response than those from the left limbs. This observation has been added to Figure 1 - Supplement 1E and described in the results (lines 128-134) and discussion (lines 314-320).

    We have performed additional control experiments using EMG nuchal electrodes in two pups aged P5 and P6. We observed that, an hour following the surgery (corresponding to the recovery time in our experimental procedure), the composition of the sleep-wake cycle (with 70 to 80 % of active sleep) was comparable to previous reports (Jouvet-Mounier, 1969, Fig 4). This quantification was added to Figure 1- figure supplement 1B (lines 82-86).

    1. State-dependent parameters are not adequately described, controlled, and examined quantitatively to ensure that data from similar behavioral states is being used for analysis across ages. Network activity from wakefulness, REM/active sleep and NREM/quiet sleep should not be presumed to be indistinguishable.

    We would like to point out that our analysis across ages focused on the population response following animal movements, and not across all behavioral states. That said, it is true that two types of movements can be distinguished, namely the twitches and the complex ones. To take this behavioral heterogeneity into account, we have now separately quantified the hippocampal activation following twitches (movement during active sleep) and complex movement (during wakefulness). We show in Figure 2 - figure supplement 1B that the hippocampal response to twitches and complex movements is similar across ages. Thus, even if the amount of time spent in each behavioral state is modified over the developmental period that we have studied, we are pretty confident that it does not impact the transition we have described in the relationship between animal movements and hippocampal activity. Additionally, we were able to combine in one P5 mouse pup 2p-imaging with nuchal EMG recordings and separately computed the PMTH for movements observed during REM or wakefulness (Figure 2 - figure supplement 1C). We show that CA1 hippocampal neurons were activated time-locked to movement in both behavioral states, with only the amplitude of the population response differing between wakefulness than during REM. This point is now included in the result section (lines 148-152) and discussed (lines 324-327).

    1. Currently employed statistics are not rigorous, unified, or sensitive, and do not support all of the authors' claims. Data shown suggest potentially significant changes that have not been identified due to suboptimal statistical approach and/or underpowering.

    We obviously agree with this reviewer that rigorous statistics should be employed and can certify that the data analyzed in the submitted manuscript was carefully examined following that principle. We feel that his/her strong criticism regarding that point was not fully justified. In particular, we do not understand why statistical tests should be “unified” across different figures of the paper. Rather, statistical tests should be adapted to the sample size and distribution. Of course, the same tests were used for similar datasets. This revised manuscript now contains further description and justification of all the tests included in every figure panels.

    1. The authors use an artificial neural network approach to infer cell classification (pyramidal cell vs. interneuron). From the data provided, it is not possible to adequately evaluate whether these 'inferred' interneurons represent the same population as conventionally labeled interneurons.

    We thank the reviewer for this important remark and apologize for the lack of detailed description of our method to ‘infer’ interneurons. This method was previously published (Denis et al., 2020), and designed to identify interneurons from their calcium fluorescence signals in the absence of a reporter. Most importantly, this cell type classifier was trained and tested on a dataset in which interneurons were labeled using a reporter mouse line (GAD 76-Cre). This dataset is included in this article. This means that all the ‘labelled’ interneurons included here were also used for the training and the test dataset. As for the activity classifier, the training and test data sets covered all the developmental ages used in the study. Thus, the previously published statistics (accuracy/sensitivity) of this classifier should well account for the present analysis. This method is now described in better detail in the results (line 183) and methods parts (lines 616-619). We now also illustrate in the figures how this classifier can infer interneurons with 91% precision (split up of prediction vs ground truth in test data are reported from Denis et al) and that these ‘infered’ interneurons are activated with movement just as genetically ‘labeled’ interneurons (Figure 3 - figure supplement 1B-E).

    1. Functional GABAergic activity is not assessed across development (only at P9-10), limiting mechanistic conclusions that can be drawn.

    We thank the reviewer for this comment that reveals some lack of clarity in the previous description of our experiments. Indeed, functional GABAergic activity was also assessed before P9, however, given that there are no GABAergic axons in the CA1 pyramidal layer at early stages (for both CCK cf. Morozov and Freund 2003, and prospective PV cells cf. Figure 4A,B), there is no signal to be measured either. We have now added a new figure (Figure 4 - figure supplement 1) to clarify this point. In agreement with our Syt2 longitudinal quantification, we show, using tdTomato expression in the Gad67cre driver mouse line, that GABAergic perisomatic innervation is only visible after p9. This matches as well our attempted imaging experiments using axon enriched GCaMP in mice before P9.

    1. The present analyses are almost exclusively focused on movement-related epochs, substantially limiting conclusions that can be drawn as to what neural dynamics are actually occurring during epochs that the authors propose comprise internal representations.

    We agree with this reviewer that our study is focusing on movement-related episodes and that we are not assessing hippocampal representations, especially since the pups are recorded in conditions that minimize external environmental influences. Still, we observe that there is a switch in the distribution of spontaneous activity in CA1 after P9, with most activity occurring outside from the synchronous calcium events and detached from movement. The exact nature of this activity remains to be studied, however, it is most likely not evoked by extrinsic phasic inputs and rather represents local dynamics. We have now removed reference to ‘internal representations” or “internal models” in the two previous instances of use i(abstract and discussion) and replaced them, when possible by “self-referenced” representations alluding to self-generated-movement-triggered activity.

    Reviewer #2 (Public Review):

    The study by Dard et al aims to uncover the post-natal emergence of mature network dynamics in the hippocampus, with a particular focus on how pyramidal cells and interneurons change their response to spontaneous limb movement. Several previous studies have investigated this topic using electrophysiology, but this study is the first to utilize 2-photon calcium imaging, enabling the recording of hundreds of individual neurons, and discrimination between pyramidal cell and interneuron activity. The aims of the study are of broad interest to all neuroscientists studying development (including neurodevelopmental disorders) and the basic science of network dynamics.

    The main conclusions of the study are that (1) in early life, most pyramidal cell activity occurs in bursts synchronized to spontaneous movement, (2) by P12, pyramidal cell activity is largely desynchronized from spontaneous movement, and indeed movement triggers an inhibition in the pyramidal network (approximately 2-4sec following movement), (3) unlike pyramidal cells, interneuron activity remains positively modulated by movement, throughout the period P1-P12, (4) the changes in pyramidal cell activity are achieved by means of increases in perisomatic inhibition, between P8 and P10.

    It should be noted that conclusion (1) and to some extent conclusion (2) have already been reported, by previous studies using electrophysiology (as clearly acknowledged by the authors).

    A principal strength of this manuscript is the extremely high quality of the data that the authors are able to use in support of (1) and (2), with very large numbers of neurons being analyzed to clearly delineate the relationship between neural activity and movement. The finding that pyramidal cells become inhibited following movement is novel, I believe. Furthermore, this study offers the first description of the development of interneuron activity, in this experimental context.

    The main weakness of the manuscript is that the authors cannot provide direct functional evidence for the conclusion (4). As shown by the analysis in support of conclusion (3), interneuron activity with respect to movement does not actually change during the developmental period being studied, making it prima facie unlikely that this is the cause of changes in pyramidal network responses to movement. To overcome this, the study describes the activity of GABA-ergic axon terminals in the pyramidal cell layer at P9-10, but it appears that due to technical problems this was not possible in younger animals. It, therefore, remains unknown if the functional inhibitory inputs to pyramidal cells are changing over the ages studied.

    We thank this reviewer for acknowledging the broad interest of the study, its novelty, and the high quality of our dataset. The main concern raised by this reviewer (lack of axonal activity experiments in younger pups) was in fact a misunderstanding of the experiments performed and we apologize for this lack of clarity. Reviewer #2 is correct in that the relationship between interneuron activity and movement does not change over the developmental period studied. However, we have only included GABAergic axonal imaging after P9, not due to a technical problem but rather because there are no GABAergic axons in the pyramidal layer before (we see GABAergic neurites only outside the layer). We have now dedicated a new supplementary figure (Figure 4 - figure supplement 1) to explain why we could not image GABAergic axons in the pyramidal cell layer at earlier developmental stages.

    The study does describe increases in the protein synaptotagmin-2, in the pyramidal cell layer, between P3 and P11, but in my opinion, this molecular evidence for increases in perisomatic inhibition does not match the (very high) standards of neuronal function/activity reported elsewhere in the manuscript.

    In the absence of parvalbumin expression in early development, synaptotagmin-2 has been described as the best marker of prospective PV boutons in the cortex (Someijer et al. 2012). This molecular marker has been used in other studies (Modol et al. Neuron 2020, Sigal et al. PNAS 2019). We respectfully disagree with this reviewer, and think that quantification from immunohistochemistry experiments is as high of a standard as functional imaging as it is the only way to describe the anatomical structure of active neuronal processes.

    Reviewer #3 (Public Review):

    Dard and colleagues use both in vivo calcium imaging and computational modelling to explore the relationship between the early movement of CA1 hippocampal activity in neonatal mice.

    The manuscript represents a significant technical advance in that the authors have pioneered the use of multiphoton imaging to record activity in the hippocampus of awake neonates. Overall the presentation of the data is convincing although I would recommend a number of tweaks to the figures and the inclusion of some raw data to better direct and inform non-expert readers. I also believe that the assessment of long-range inputs using pseudo-rabies virus should be present in the main body of the manuscript as opposed to supplemental material. The computational modeling supports their idea but does not exclude other possibilities. Further, it is not clear to what extent the strengthening of local excitatory input onto the interneurons - the dominant route of recurrent input in the hippocampus, is important; something that the authors acknowledge in the discussion.

    Overall, I believe the paper adds to our knowledge of the timeline of development and further identified the postnatal day (P)9-P10 window as important in emergent cortical processing. The fact that this is linked to an increase in GABAergic innervation has implications for our understanding of both normal and dysfunctional brain development.

    We thank the reviewer for his constructive comments and helpful suggestions. As suggested, this revised version now includes some raw-data and better descriptions to guide non-expert readers. Regarding the inclusion of rabies-tracing experiments in the main part of the MS, we would like to state here that there are still a number of limitations with the use of this method during development (incubation time, spatial precision of the injection site, etc. ) that limit the interpretation and quantification of the results. As a result, we have decided to remain only qualitative, focusing on identifying the brain regions that could send projections onto CA1 pyramidal cells and interneurons. We believe that this type of description is more suited for a supplementary figure than a principal figure, but will be happy to change this, if the reviewer and editors think otherwise.

  3. eLife

    Evaluation Summary:

    This study investigates hippocampal dynamics over the course of early postnatal development with respect to spontaneous movements. Pioneering in vivo imaging in the hippocampus of neonatal mice, the authors find evidence for an abrupt developmental transition in this neural activity at the end of the first postnatal week in rodents and contribute to the understanding of how cognitive functions could emerge from the immature brain.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    In this manuscript, Dard et al. investigate hippocampal dynamics over the course of early postnatal development. They find evidence for an abrupt developmental transition in this neural activity at the end of the first postnatal week in rodents and postulate that it is related to the emergence of internal representations. This work is interesting as it explores the developmental expression of neural activity patterns and contributes to the understanding of how cognitive functions could emerge from the immature brain. Additional methodological and statistical analysis is necessary to support the suggested conclusions of this work.

    Strengths:
    The authors employ in vivo imaging of the hippocampus in developing mice and merge these techniques with cell labeling to try to establish clues as to the mechanisms of the neural activity patterns they observe. This work provides information in a relatively understudied field and could help to provide developmental timelines and cellular mechanisms for the maturation of neural networks.

    Weaknesses:

    1. The experiments involve an invasive neurosurgical procedure used to perform hippocampal imaging, which removes the ipsilateral overlying somatosensory cortex, and it is not possible to evaluate from the data provided that this surgery does not disrupt network function, especially given the focus on movement-related activity patterns.
    2. State-dependent parameters are not adequately described, controlled, and examined quantitatively to ensure that data from similar behavioral states is being used for analysis across ages. Network activity from wakefulness, REM/active sleep and NREM/quiet sleep should not be presumed to be indistinguishable.
    3. Currently employed statistics are not rigorous, unified, or sensitive, and do not support all of the authors' claims. Data shown suggest potentially significant changes that have not been identified due to suboptimal statistical approach and/or underpowering.
    4. The authors use an artificial neural network approach to infer cell classification (pyramidal cell vs. interneuron). From the data provided, it is not possible to adequately evaluate whether these 'inferred' interneurons represent the same population as conventionally labeled interneurons.
    5. Functional GABAergic activity is not assessed across development (only at P9-10), limiting mechanistic conclusions that can be drawn.
    6. The present analyses are almost exclusively focused on movement-related epochs, substantially limiting conclusions that can be drawn as to what neural dynamics are actually occurring during epochs that the authors propose comprise internal representations.

    Overall:
    The authors aimed to demonstrate a shift in hippocampal neural activity from primarily responding to external stimuli (i.e. body movements) to manifesting internal network dynamics. They identify local GABAergic innervation as a likely candidate mechanism for this shift. While interesting, the current analytic methods used are insufficient to fully support the authors' claims.

  5. eLife

    Reviewer #2 (Public Review):

    The study by Dard et al aims to uncover the post-natal emergence of mature network dynamics in the hippocampus, with a particular focus on how pyramidal cells and interneurons change their response to spontaneous limb movement. Several previous studies have investigated this topic using electrophysiology, but this study is the first to utilize 2-photon calcium imaging, enabling the recording of hundreds of individual neurons, and discrimination between pyramidal cell and interneuron activity. The aims of the study are of broad interest to all neuroscientists studying development (including neurodevelopmental disorders) and the basic science of network dynamics.

    The main conclusions of the study are that (1) in early life, most pyramidal cell activity occurs in bursts synchronized to spontaneous movement, (2) by P12, pyramidal cell activity is largely desynchronized from spontaneous movement, and indeed movement triggers an inhibition in the pyramidal network (approximately 2-4sec following movement), (3) unlike pyramidal cells, interneuron activity remains positively modulated by movement, throughout the period P1-P12, (4) the changes in pyramidal cell activity are achieved by means of increases in perisomatic inhibition, between P8 and P10.

    It should be noted that conclusion (1) and to some extent conclusion (2) have already been reported, by previous studies using electrophysiology (as clearly acknowledged by the authors).

    A principal strength of this manuscript is the extremely high quality of the data that the authors are able to use in support of (1) and (2), with very large numbers of neurons being analyzed to clearly delineate the relationship between neural activity and movement. The finding that pyramidal cells become inhibited following movement is novel, I believe. Furthermore, this study offers the first description of the development of interneuron activity, in this experimental context.

    The main weakness of the manuscript is that the authors cannot provide direct functional evidence for the conclusion (4). As shown by the analysis in support of conclusion (3), interneuron activity with respect to movement does not actually change during the developmental period being studied, making it prima facie unlikely that this is the cause of changes in pyramidal network responses to movement. To overcome this, the study describes the activity of GABA-ergic axon terminals in the pyramidal cell layer at P9-10, but it appears that due to technical problems this was not possible in younger animals. It, therefore, remains unknown if the functional inhibitory inputs to pyramidal cells are changing over the ages studied. The study does describe increases in the protein synaptotagmin-2, in the pyramidal cell layer, between P3 and P11, but in my opinion, this molecular evidence for increases in perisomatic inhibition does not match the (very high) standards of neuronal function/activity reported elsewhere in the manuscript.

  6. eLife

    Reviewer #3 (Public Review):

    Dard and colleagues use both in vivo calcium imaging and computational modelling to explore the relationship between the early movement of CA1 hippocampal activity in neonatal mice.

    The manuscript represents a significant technical advance in that the authors have pioneered the use of multiphoton imaging to record activity in the hippocampus of awake neonates. Overall the presentation of the data is convincing although I would recommend a number of tweaks to the figures and the inclusion of some raw data to better direct and inform non-expert readers. I also believe that the assessment of long-range inputs using pseudo-rabies virus should be present in the main body of the manuscript as opposed to supplemental material.

    The computational modelling supports their idea but does not exclude other possibilities. Further, it is not clear to what extent the strengthening of local excitatory input onto the interneurons - the dominant route of recurrent input in the hippocampus, is important; something that the authors acknowledge in the discussion.

    Overall, I believe the paper adds to our knowledge of the timeline of development and further identified the postnatal day (P)9-P10 window as important in emergent cortical processing. The fact that this is linked to an increase in GABAergic innervation has implications for our understanding of both normal and dysfunctional brain development.

  7. Version published to 10.1101/2021.06.08.447542v2 on bioRxiv
  8. Version published to 10.1101/2021.06.08.447542v1 on bioRxiv