Developmental stage-specific spontaneous activity contributes to callosal axon projections

  1. Yuta Tezuka
  2. Kenta M Hagihara
  3. Kenichi Ohki
  4. Tomoo Hirano
  5. Yoshiaki Tagawa

  • Curated by eLife

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    Evaluation Summary:

    The manuscript adds to an emerging story about the role of activity in the formation of callosal connections across the brain. Previous research of the authors' and other labs had shown that overexpressing the potassium channel Kir2.1, which reduces activity levels in the developing cortical network, blocks the formation of callosal connections almost entirely. Here, the authors show that they can use a TET system to switch off the activity of an Kir2.1 to probe when activity might be necessary or sufficient for the formation of callosal connections. The authors find that artificial restoration of activity with DREADS is sufficient to rescue the formation of callosal connections, and that there is a critical period (somewhere between P5-P15) where activity must occur in order for the connections to form within the cortex. Finally, the authors show that when the potassium channel is removed during the critical period, the cortex exhibits activity, but few highly synchronous events. These results indicate that it is activity in general and not specifically highly synchronous activity that is necessary for the final innervation of the callosal cortex.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

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Abstract

The developing neocortex exhibits spontaneous network activity with various synchrony levels, which has been implicated in the formation of cortical circuits. We previously reported that the development of callosal axon projections, one of the major long-range axonal projections in the brain, is activity dependent. However, what sort of activity and when activity is indispensable are not known. Here, using a genetic method to manipulate network activity in a stage-specific manner, we demonstrated that network activity contributes to callosal axon projections in the mouse visual cortex during a ‘critical period’: restoring neuronal activity during that period resumed the projections, whereas restoration after the period failed. Furthermore, in vivo Ca 2+ imaging revealed that the projections could be established even without fully restoring highly synchronous activity. Overall, our findings suggest that spontaneous network activity is selectively required during a critical developmental time window for the formation of long-range axonal projections in the cortex.

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  1. Version published to 10.7554/elife.72435 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    Using Tet-off system, Kir2.1 was expressed (or not) during the key time of callosal development from E15 to P15. Restoring activity either by adding Dox during a critical period from P6 to P15 or using DREADDs from P10-14 could rescue the callosal projection to the cortex, whereas later restoration of activity (with Dox) was not successful. Did this successful rescue lead to normal activity? Calcium imaging in animals with Kir2.1 had low levels of any kind of activity, both highly correlated and low correlation, but P6-13 dox treatment partially restored only low-correlation activity and not high correlation activity at P13. The effects of DREADDs on activity was not similarly measured though it was effective for at least partially restoring the callosal projection.

    Overall this study builds on earlier findings regarding the importance of neuronal activity in the formation of a normal callosal projection, using in utero electroporation which is particularly well suited for this subject. It makes the case very compellingly that near-normal callosal connectivity can be produced if activity is permitted during a critical period window from P6 or P10 to P15, though the exact timing of this window is imprecise because the elimination of Kir expression was not systematically quantified. For transmembrane proteins like channels it can often take many days for protein expression to completely abate.

    We thank the reviewer for their positive evaluation and the constructive comments. Based on the comment on Kir expression, we conducted new experiments using pTRE-Tight2Kir2.1EGFP, with which EGFP signals reflect localization of over-expressed Kir2.1, and examined when the expression of Kir2.1EGFP went down after Dox treatment at P6. At P6 (before Dox treatment), the signals of Kir2.1EGFP (stained with anti-GFP antibody) were observed in the periphery of the soma and along dendrites, implying that Kir2.1EGFP was transported to the cellular membrane. At P10 and P15 (4 days and 9 days after Dox treatment), Kir2.1EGFP signals were not observed in the periphery of the soma and along dendrites. We noted that low-level green signals were observed in the central part of the cell body. These may stem from low-level expression of Kir2.1EGFP in nuclei or cytosol even after Dox treatment. Alternatively, and more likely, these may reflect bleed-through of RFP signals into GFP channel. Overall, we confirmed that Kir2.1 proteins that were localized to the cellular membrane were largely down-regulated. We described these observations in detail in the figure legend of Figure 1-figure supplement 3, and added the result as Figure 1-figure supplement 3.

    I found the quantification of the callosal projection to be rather minimal and the normalization approach not entirely transparent. For example does activity from P10-15 restore the full normal PATTERN of callosal connectivity or merely the density of input overall?

    We thank the reviewer for this comment. Based on the comment, we added analyses of the pattern of callosal projections; the width of callosal axon innervation zone in layers 2/3 and 5, and densitometric line scans across all cortical layers. Our original quantification showed that the density of callosal axons reaching their target layer (i.e. cortical layer 2/3) is almost recovered in P6-P15 DOX condition (Fig1B-D), but new analyses suggest some aspects of callosal axon projections (the width of the innervation zone in layer 2/3 and 5 (Figure 1-figure supplement 4A,B), and lamina specific innervation pattern (Figure 1-figure supplement 4C)) might be only partially recovered. We have added these new results as Figure 1-figure supplement 4. In future study, we would like to assess the effect of the manipulations at finer resolution by 3D morphological reconstruction of axons of individual neurons.

    Also in the discussion it would be nice to more clearly establish whether activity is thought to be maintaining a projection already formed by P10 or permitting the emergence of such a pattern.

    Thank you for the suggestion. We have added thorough discussions about this point as follows. Page 7, lines 198-208:

    “In the previous study, we showed that callosal axons could reach the innervation area almost normally under activity-reduction, and that the effects of activity-reduction became apparent afterwards (Mizuno et al., 2007). Callosal axons elaborate their branches extensively in P10P15 (Mizuno et al., 2010), and axon branching is regulated by neuronal activity (Matsumoto and Yamamoto, 2016). It is likely that activity is required for the processes of formation, rather than the maintenance of the connections already formed by P10, but the current study employed massive labeling of callosal axons which is not suited to clarify this. In addition, the restoration of activity in the Tet-off (Figure 1) or DREADD (Figure 2) experiment may not completely rescue the ramification pattern of individual axons. Single axon tracing experiments (Mizuno et al., 2010; Dhande et al., 2011) would be required to clarify this. Nonetheless, our findings suggest that callosal axons retain the ability, or are permitted, to grow and make region- and lamina-specific projections in the cortex during a limited period of postnatal cortical development under an activity-dependent mechanism.”

    The calcium imaging is a valuable validation of the Kir expression approach, but it the study here appears to overinterpret what may simply be an intermediate level of activity restoration rather than a specific restoration of L events, as it seems that L events would be the most likely to occur under conditions of reduced overall activity. One possibility is that the absence of H events at P13 in the calcium is due to residual Kir expression creating a drag on high level network activation rather than any more complicated change in patterned spontaneous activity/connectivity. The conclusions from this study regarding the permissive role of activity during a critical window and the lack of a requirement for highly correlated activity are valuable, even if somewhat imprecise on both counts. The authors should probably refrain from use of the term patterned activity given that this was measured but not systematically compared to unpatterned spontaneous activity.

    We thank the reviewer for this constructive comment. Based on this comment, we removed the term “patterned activity” throughout the manuscript and revised the title, abstract, introduction, results, and discussion extensively. For example, in the Discussion, we revised as follows.

    “We have shown that the projections could be established even without fully restoring highly synchronous activity (Figure 4). L events, but not H events, were present in P13 cortex after Dox treatment at P6. L events may be sufficient for the formation of callosal projections. Alternatively, any form of activity with certain level(s) (i.e., “sufficiently” high activity with no specific pattern) could be permissive for the formation of callosal connections.”

    Reviewer #2 (Public Review):

    Tezuka et al. use in vivo manipulations of spontaneous activity to identify the activitydependent mechanisms of callosal projection development. Previous research of the authors' and other labs had shown that overexpressing the potassium channel Kir2.1, which reduces activity levels in the developing cortical network, blocks the formation of callosal connections almost entirely.

    The current manuscript corroborates and extends these previous discoveries by:

    1. Demonstrating that the effect of Kir overexpression can be rescued by pharmacogenetic network activation using DREADDs.
    2. Revealing the requirement of network activity for the development of callosal projections during a particular developmental time window and by
    3. Directly relating perturbed callosal development to the actual changes in activity patterns caused by the experimental manipulations.

    Thus, this paper is important for our understanding of the role of neuronal activity in the development of long-range connections in the brain. In addition it provides strong evidence for a role of specific activity patterns in this process.

    In general, the approach is very straightforward and the results clearly interpreted. Nevertheless, there are a few points to consider.

    We thank the reviewer for these positive and supportive comments.

    1. It is not clear in which cortical area(s) the in vivo 2-photon recordings were performed and in how far cortical areas that actually receive/send callosal projections were included or not in the analysis.

    In response to this comment, we revised the text in the method section as follows.

    “We aimed to record spontaneous neuronal activity in putative binocular zones in V1 (2.5 mm lateral of midline and 1 mm anterior of the posterior suture). Since the boundaries between V1 and higher visual areas, AL/LM are not as obvious as those in adult, our recordings likely contained juxtaposed lateral monocular V1 and AL/LM as well.”

    Based on our colleaguesʼ unpublished observations, V1 and AL/LM can be distinguished solely by spontaneous activity patterns even before eye-opening. They also found frequencies of spontaneous activity are similar across mono/binocular regions of V1 and AL/LM (Murakami, Ohki, et al. unpublished). Thus, our results should hold even with the variability in recording sites.

    1. It is not discussed what the duration of the CNO effect is. Do daily injections rescue activity patterns for 24 hours or a significant proportion of this period?

    In response to this critical comment, we revised the text in the method section as follows.

    “A previous study showed that an intraperitoneally injected CNO was effective (in terms of increasing activity) for about 9hrs (Alexander et al., 2009). The “partial rescue” effect we observed (Figure 2) may suggest that activity was not fully restored during 24hrs by our daily CNO injections.”

    Reviewer #3 (Public Review):

    The manuscript by Tezuka adds to an emerging story about the role of activity in the formation of callosal connections across the brain. Here, the authors show that they can use a TET system to switch off the activity of an exogenous potassium channel, in order to probe when activity might be necessary or sufficient for the formation of callosal connections. The authors find that artificial restoration of activity with DREADS is sufficient to rescue the formation of callosal connections, and that there is a critical period (somewhere between P5-P15) where activity must occur in order for the connections to form within the cortex. Finally, the authors show that when the potassium channel is removed during the critical period, the cortex exhibits activity, but few highly synchronous events. These results indicate that it is activity in general and not specifically highly synchronous activity that is necessary for the final innervation of the callosal cortex.

    In general, the study is well done, and the writeup is polished, well summarized. The figures are solid. There are only a few criticisms/suggestions.

    We thank the reviewer for the positive evaluation.

    Major issue: Have the authors demonstrated a requirement for "patterned spontaneous activity"?

    The authors claim variously in the abstract ("a distinct pattern of spontaneous activity") and in the results (pg 6, "our observations indicate that patterned spontaneous activity") and discussion (pg 6, "we demonstrated that patterned spontaneous activity") that it is "patterned" spontaneous activity that is key for the formation of callosal connections. However, when I was reading the paper, I came to the opposite conclusion: that any sufficiently high spontaneous activity is sufficient for the formation of these connections.

    The authors showed that relieving the KIR expression from P5-15 allows the connections to form; however, in Figure 4, the authors show that the nature of the activity produced in the cortex (in terms of mixtures of H and L events) is very different. Nevertheless, the connections can form. Further, the authors showed that increasing activity when KIR is expressed using DREADS restores the connections. The pattern of activity produced by this DREADS + KIR expression is likely to be very different from the pattern of activity of a typically-developing animal. In total, I thought that the authors demonstrated, quite nicely, that it is just the presence of sufficient activity that is key to the innervation of the contralateral cortex. (It's not cell autonomous, as the authors showed before; there seems to be a "sufficient activity" requirement).

    Therefore, I think the authors should remove references to the requirement of patterned activity and instead say something about sufficiently high activity (or some characterization that the authors choose). I think they've shown quite nicely that a specific pattern of the spontaneous activity is not important.

    We thank the reviewer for this very important insight and interpretation. After considering all the currently presented data again, we have come to agree with the interpretation stated by the reviewer. We removed the term “patterned activity” throughout the manuscript and revised the title, abstract, introduction, results, and discussion extensively. Nevertheless, we would not completely discard the possibility that specific patterns of spontaneous activity, such as L-events, could potentially have some active contribution to the development of projection circuits, and would like to further address this in future study.

    For example, in the Discussion, we revised the text as follows.

    “We have shown that the projections could be established even without fully restoring highly synchronous activity (Figure 4). L events, but not H events, were present in P13 cortex after Dox treatment at P6. L events may be sufficient for the formation of callosal projections. Alternatively, any form of activity with certain level(s) (i.e., “sufficiently” high activity with no specific pattern) could be permissive for the formation of callosal connections.”

  3. Version published to 10.1101/2021.07.21.453297v2 on bioRxiv
  4. eLife

    Evaluation Summary:

    The manuscript adds to an emerging story about the role of activity in the formation of callosal connections across the brain. Previous research of the authors' and other labs had shown that overexpressing the potassium channel Kir2.1, which reduces activity levels in the developing cortical network, blocks the formation of callosal connections almost entirely. Here, the authors show that they can use a TET system to switch off the activity of an Kir2.1 to probe when activity might be necessary or sufficient for the formation of callosal connections. The authors find that artificial restoration of activity with DREADS is sufficient to rescue the formation of callosal connections, and that there is a critical period (somewhere between P5-P15) where activity must occur in order for the connections to form within the cortex. Finally, the authors show that when the potassium channel is removed during the critical period, the cortex exhibits activity, but few highly synchronous events. These results indicate that it is activity in general and not specifically highly synchronous activity that is necessary for the final innervation of the callosal cortex.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

  5. eLife

    Reviewer #1 (Public Review):

    Using Tet-off system, Kir2.1 was expressed (or not) during the key time of callosal development from E15 to P15. Restoring activity either by adding Dox during a critical period from P6 to P15 or using DREADDs from P10-14 could rescue the callosal projection to the cortex, whereas later restoration of activity (with Dox) was not successful. Did this successful rescue lead to normal activity? Calcium imaging in animals with Kir2.1 had low levels of any kind of activity, both highly correlated and low correlation, but P6-13 dox treatment partially restored only low-correlation activity and not high correlation activity at P13. The effects of DREADDs on activity was not similarly measured though it was effective for at least partially restoring the callosal projection.

    Overall this study builds on earlier findings regarding the importance of neuronal activity in the formation of a normal callosal projection, using in utero electroporation which is particularly well suited for this subject. It makes the case very compellingly that near-normal callosal connectivity can be produced if activity is permitted during a critical period window from P6 or P10 to P15, though the exact timing of this window is imprecise because the elimination of Kir expression was not systematically quantified. For transmembrane proteins like channels it can often take many days for protein expression to completely abate.

    I found the quantification of the callosal projection to be rather minimal and the normalization approach not entirely transparent. For example does activity from P10-15 restore the full normal PATTERN of callosal connectivity or merely the density of input overall? Also in the discussion it would be nice to more clearly establish whether activity is thought to be maintaining a projection already formed by P10 or permitting the emergence of such a pattern.

    The calcium imaging is a valuable validation of the Kir expression approach, but it the study here appears to overinterpret what may simply be an intermediate level of activity restoration rather than a specific restoration of L events, as it seems that L events would be the most likely to occur under conditions of reduced overall activity. One possibility is that the absence of H events at P13 in the calcium is due to residual Kir expression creating a drag on high level network activation rather than any more complicated change in patterned spontaneous activity/connectivity. The conclusions from this study regarding the permissive role of activity during a critical window and the lack of a requirement for highly correlated activity are valuable, even if somewhat imprecise on both counts. The authors should probably refrain from use of the term patterned activity given that this was measured but not systematically compared to unpatterned spontaneous activity.

  6. eLife

    Reviewer #2 (Public Review):

    Tezuka et al. use in vivo manipulations of spontaneous activity to identify the activity-dependent mechanisms of callosal projection development. Previous research of the authors' and other labs had shown that overexpressing the potassium channel Kir2.1, which reduces activity levels in the developing cortical network, blocks the formation of callosal connections almost entirely.

    The current manuscript corroborates and extends these previous discoveries by:

    1. Demonstrating that the effect of Kir overexpression can be rescued by pharmacogenetic network activation using DREADDs.

    2. Revealing the requirement of network activity for the development of callosal projections during a particular developmental time window and by

    3. Directly relating perturbed callosal development to the actual changes in activity patterns caused by the experimental manipulations.

    Thus, this paper is important for our understanding of the role of neuronal activity in the development of long-range connections in the brain. In addition it provides strong evidence for a role of specific activity patterns in this process.

    In general, the approach is very straightforward and the results clearly interpreted. Nevertheless, there are a few points to consider.

    1. It is not clear in which cortical area(s) the in vivo 2-photon recordings were performed and in how far cortical areas that actually receive/send callosal projections were included or not in the analysis.

    2. It is not discussed what the duration of the CNO effect is. Do daily injections rescue activity patterns for 24 hours or a significant proportion of this period?

  7. eLife

    Reviewer #3 (Public Review):

    The manuscript by Tezuka adds to an emerging story about the role of activity in the formation of callosal connections across the brain. Here, the authors show that they can use a TET system to switch off the activity of an exogenous potassium channel, in order to probe when activity might be necessary or sufficient for the formation of callosal connections. The authors find that artificial restoration of activity with DREADS is sufficient to rescue the formation of callosal connections, and that there is a critical period (somewhere between P5-P15) where activity must occur in order for the connections to form within the cortex. Finally, the authors show that when the potassium channel is removed during the critical period, the cortex exhibits activity, but few highly synchronous events. These results indicate that it is activity in general and not specifically highly synchronous activity that is necessary for the final innervation of the callosal cortex.

    In general, the study is well done, and the writeup is polished, well summarized. The figures are solid. There are only a few criticisms/suggestions.

    Major issue: Have the authors demonstrated a requirement for "patterned spontaneous activity"?

    The authors claim variously in the abstract ("a distinct pattern of spontaneous activity") and in the results (pg 6, "our observations indicate that patterned spontaneous activity") and discussion (pg 6, "we demonstrated that patterned spontaneous activity") that it is "patterned" spontaneous activity that is key for the formation of callosal connections. However, when I was reading the paper, I came to the opposite conclusion: that any sufficiently high spontaneous activity is sufficient for the formation of these connections.

    The authors showed that relieving the KIR expression from P5-15 allows the connections to form; however, in Figure 4, the authors show that the nature of the activity produced in the cortex (in terms of mixtures of H and L events) is very different. Nevertheless, the connections can form. Further, the authors showed that increasing activity when KIR is expressed using DREADS restores the connections. The pattern of activity produced by this DREADS + KIR expression is likely to be very different from the pattern of activity of a typically-developing animal. In total, I thought that the authors demonstrated, quite nicely, that it is just the presence of sufficient activity that is key to the innervation of the contralateral cortex. (It's not cell autonomous, as the authors showed before; there seems to be a "sufficient activity" requirement).

    Therefore, I think the authors should remove references to the requirement of patterned activity and instead say something about sufficiently high activity (or some characterization that the authors choose). I think they've shown quite nicely that a specific pattern of the spontaneous activity is not important.

  8. Version published to 10.1101/2021.07.21.453297v1 on bioRxiv