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  1. Author Response

    Reviewer #1 (Public Review):

    Redman and colleagues employed microprisms and two-photon optical imaging to track separately the structure of dorsal CA1 pyramidal neurons or the activity patterns of dorsal Dentate Gyrus, CA3, CA2 and CA1 pyramidal neurons, longitudinally in live mice. First, they carried out a characterization of the optical properties of their system. Second, they performed an example tracking of dendritic spines in the apical aspect of dorsal CA1 pyramidal neurons. Finally, they characterized differences in spatial coding along the tri-synaptic pathway, in the same animals. The main focus of the manuscript is technological and the authors show interesting data to support their technique, which I believe will be of relevance to neuroscientists interested in the hippocampal formation.

    Strengths.

    While using microprisms to achieve a "side" view of neurons in specific brain areas is not new per se [see Chia et al., J. Neurophysiol. (2009), Andermann et al., Neuron (2013), Low et al., PNAS (2014) etc.] the authors were able to visualize activity of a large neuronal circuit such as the hippocampal trisynaptic pathway - for the first time - in the same animal exploring an environment. This is not only a technical feat but it opens new scientific avenues to study how information is transformed at different stages within the hippocampus, as such I think this will be of broad interest for people in the field. In addition, the authors demonstrated imaging of dendritic spines in the apical aspect of pyramidal neurons but limited to dorsal CA1 due to the labelling density of the transgenic mouse line they decided to use. Despite the fact that imaging apical dendritic spines in dorsal CA1 has been shown earlier [see Schmid et al., Neuron (2016) and Ulivi et al., JoVE (2019)], the use of the micro periscope greatly increases the flexibility of these sort of experiments by enabling tracking of large portion (both apically and basally) of the dendritic arbors of dorsal CA1 pyramidal neurons.

    Thank you for the positive comments. We have clarified that apical CA1 dendrites have been imaged in previous work as you point out, just not along the somatodendritic axis (lines 127-130). We have also clarified that we were able to image CA2 and CA3 spines as well (only DG exhibited the increased labeling density in Thy1-GFP-M mice; lines 130-132).

    Weaknesses.

    While the data are sufficient to demonstrate the technique, the conceptual advance of the paper is very narrow. The findings on spatial coding differences in different hippocampal subregions - namely a nonuniform distribution of spatial information in the different hippocampal subregions - do not add new knowledge but largely confirm the literature. The results on the dynamics of apical dendritic spines of pyramidal neurons in dorsal CA1 seem to confirm previous work, but the interpretation of these results differs fundamentally. In fact both papers cited by the authors (Attardo et al., and Pfeiffer et al.,) come to the conclusion that dendritic spines on basal dendrites of CA1 pyramidal neurons are highly unstable, at least by comparison to other neocortical areas. The authors seem to ignore this discrepancy. However, this discrepancy has importance also to the characterization of the technique the authors developed. In fact, the optical resolution of the system strongly affects the ability to resolve neighboring spines - especially at the high density of dorsal CA1 - and thus it has a direct effect on the measures of synaptic stability [Attardo et al., Nature, (2015)]. The authors duly report lateral and axial resolutions for their micro periscopes and both are lower than the ones of Attardo and Pfeiffer, thus the authors should consider the effects of this difference on the interpretation of their data.

    We agree that the advance described in this manuscript is more methodological than conceptual. We do have other studies in progress that will be of greater conceptual interest. However, we believe the technique is of sufficient interest to the field that it is worth publishing the methodological approach and characterization as soon as possible.

    We have also addressed the comparison with Attardo et al. and Pfeiffer et al. mentioned by the reviewer. We actually agree with the previous work that dendritic spines in CA1 show a high degree of instability compared to cortex, finding ~15% spine addition and ~13% spine subtraction between consecutive days (Fig. 3H, I), similar to single-day turnover rates observed in Attardo et al. and other papers. Despite the high turnover rate, the fraction of experimentally observed spines that persist across 8-10 days plateaus around 75-80%, indicating that there is a substantial fraction of apical spines that remain stable in the face of ongoing daily turnover. This was also observed in basal dendrites by Attardo et al. (with similar survival fractions) and Pfeiffer et al. (albeit with lower survival fractions), so we would not necessarily characterize this as a discrepancy. We have clarified these points in the manuscript (lines 157, 162-168, 331-332).

    The reviewer pointed out that some previous studies used super-resolution microscopy to detect smaller structures and reduce optical merging. This would be an excellent extension of our work, as in principle super-resolution microscopy could be used with the implanted microperiscopes. Although the survival fractions we observed were similar to Attardo et al., they were higher than Pfeiffer et al., possibly due to the predicted effects of optical merging. We have updated the text to note that our results may inflate the degree of stability due to resolution limitations (lines 165-68, 335-340).

    Reviewer #2 (Public Review):

    Strengths

    The Hippocampus is a key brain region for episodic and spatial memory. The major Hippocampal subregions: Dentate Gyrus (DG), CA3, and CA1 have predominantly been investigated independently due to technical limitations that only allow one subregion to be recorded from at a time. In this paper the authors developed a new method that allows DG, CA3, and CA1 to be imaged simultaneously in the same mouse during behavior with a 2-photon microscope. This method will allow investigation of the interactions between Hippocampal subregions during memory processes - a critical yet unexplored area of Hippocampal research. This method therefore provides a new tool that will help provide insight into the complex functions of the Hippocampus during behavior.

    This method also provides high resolution optical access to deep dendritic structures that have been out of reach with existing methods. The authors demonstrate they can measure the structure of single spines on distal apical dendrites of CA1 cells. They track populations of spines and quantify spine changes, spines loss, and spine appearance. Spine turnover is thought to be a key process in how the Hippocampus encodes and consolidates memories, and this method provides a means to quantify spine dynamics over very long time periods (months) and can be used to study spine dynamics in CA3 and DG.

    We appreciate the comments.

    Weaknesses

    This method requires the implantation of a relatively large glass microperiscope that cuts through part of the Septal end of the Hippocampus. This is a necessary step to image transversally and observe all the major subregions simultaneously. This is an unfortunate limitation as it damages the very circuits being investigated. The authors attempt to address this by measuring the functional properties of Hippocampal cells, such as their place field features, and claim they are similar to those measured with other methods that do not damage the Hippocampus. However, it is very likely the implant-induced damage is affecting the imaged cells in some way, so caution should be taken when using this method. The authors are very aware of this and briefly discuss the issue. In addition, the authors observe damaged adjacent to face of the glass microperiscope that extends to ~300 um from the face. This area should therefore be avoided when imaging the Hippocampus through the microperiscope.

    We agree. This will be important for the interpretation of experiments using the microperisope approach. For many experiments, electrophysiology or traditional CA1 imaging approaches might be preferable to avoid damage to the hippocampal structure. We have tried to be straightforward about these caveats in our discussion. However, we believe the capability of imaging the transverse hippocampal circuit will allow a number of experiments that are currently intractable, and that the benefits will outweigh the caveats in these cases.

    Reviewer #3 (Public Review):

    Redman et al. describe a novel approach for long-term cellular and sub-cellular resolution functional and structural imaging of the transverse hippocampal circuit in mice. The authors discuss their procedure for implanting a glass microperiscope and show data that clearly support their ability to simultaneously record from neurons within the DG, CA3, and CA1 subregions of the hippocampus. They offer optical characterization demonstrating sufficient resolution to image at the cellular and subcellular level, which is further supported by experimental data characterizing changes in morphology of CA1 apical dendritic spines. Finally, neurons are recorded from as mice engage in navigation behavior, allowing authors to characterize spatial properties of hippocampal cells and relate findings to prior work in the field.

    The ability to image from multiple hippocampal subregions simultaneously is a great technical achievement, sure to advance study of the hippocampal circuit. In particular, this approach will likely have tremendous application for addressing the question of how neural representations dynamically change across the hippocampal subfields during initial encoding of novel contexts or later during retrieval of familiar. While the feasibility and utility of this preparation is supported by the data, further characterization of recorded cells will aid the comparison of data collected using this imaging approach to data previously collected with other methodologies.

    Thank you for the comments, we have addressed the specific concerns below.

    1. Further measures could be taken to more thoroughly evaluate the impact of the implant on cell health. While authors evaluate glial markers, it is not obvious how long after implant these measurements were taken. Additionally, authors could characterize cell responses of neurons recorded proximal to and more distal to their implant to further evaluate implant effect on cell health.

    Good points. We have added the date post implantation for the histology samples (Figure 1F caption). To address the second point, we added additional experiments characterizing functional response properties as a function of depth (Figure S7). We did not find systematic changes in place field width or place cell spatial information, as a function of imaging depth (lines 220-224; Figure S7A, B). We did however find a significant relationship between the decay constant for the fitted transients and depth, with cells close ( 130 um) to the surface of the microperiscope face exhibiting slower decay (Figure S7C). This appeared to be due to a small fraction of cells exhibiting longer decay times closer to the microperiscope face. As a result, we advise only imaging neurons >150 um from the microperiscope face (lines 224-226).

    1. More in-depth analysis of place cells will aid the comparison of data collected using this novel approach to previously published data. For instance, trial-by-trial data and clearer descriptions of inclusion criteria will allow readers a more detailed understanding of observed place cells.

    We have included example place cells with individual trial data (Figure 5C) and have added additional discussion and detail on our selection process for identifying place cells (lines 207-209, 663-666, 674676). In the revised manuscript, we further increased the stringency of our place cell criteria so that none of the cells with time shuffled responses pass the criteria. It should be noted that our place cells were not as reliable as those recorded in the presence of reward (Go et al, 2021). We chose to forgo reward to help ensure that the neurons were responding to spatial location and not to other task variables, but this likely reduced response reliability (see Krishnan et al, bioRxiv; Pettit et al, 2022). We have added discussion of this issue to the manuscript (lines 307-318).

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  2. Evaluation Summary:

    The authors develop a new technique allowing simultaneous imaging of hippocampal subfields in behaving mice. This paper will be of interest to the large number of neuroscientists who study the hippocampal circuit, and more broadly to those interested in methods to enable high-resolution in vivo imaging across depths in the 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 #2 and Reviewer #3 agreed to share their name with the authors.)

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  3. Reviewer #3 (Public Review):

    Redman et al. describe a novel approach for long-term cellular and sub-cellular resolution functional and structural imaging of the transverse hippocampal circuit in mice. The authors discuss their procedure for implanting a glass microperiscope and show data that clearly support their ability to simultaneously record from neurons within the DG, CA3, and CA1 subregions of the hippocampus. They offer optical characterization demonstrating sufficient resolution to image at the cellular and subcellular level, which is further supported by experimental data characterizing changes in morphology of CA1 apical dendritic spines. Finally, neurons are recorded from as mice engage in navigation behavior, allowing authors to characterize spatial properties of hippocampal cells and relate findings to prior work in the field.

    The ability to image from multiple hippocampal subregions simultaneously is a great technical achievement, sure to advance study of the hippocampal circuit. In particular, this approach will likely have tremendous application for addressing the question of how neural representations dynamically change across the hippocampal subfields during initial encoding of novel contexts or later during retrieval of familiar. While the feasibility and utility of this preparation is supported by the data, further characterization of recorded cells will aid the comparison of data collected using this imaging approach to data previously collected with other methodologies.

    1. Further measures could be taken to more thoroughly evaluate the impact of the implant on cell health. While authors evaluate glial markers, it is not obvious how long after implant these measurements were taken. Additionally, authors could characterize cell responses of neurons recorded proximal to and more distal to their implant to further evaluate implant effect on cell health.

    2. More in-depth analysis of place cells will aid the comparison of data collected using this novel approach to previously published data. For instance, trial-by-trial data and clearer descriptions of inclusion criteria will allow readers a more detailed understanding of observed place cells.

    Was this evaluation helpful?
  4. Reviewer #2 (Public Review):

    Strengths

    The Hippocampus is a key brain region for episodic and spatial memory. The major Hippocampal subregions: Dentate Gyrus (DG), CA3, and CA1 have predominantly been investigated independently due to technical limitations that only allow one subregion to be recorded from at a time. In this paper the authors developed a new method that allows DG, CA3, and CA1 to be imaged simultaneously in the same mouse during behavior with a 2-photon microscope. This method will allow investigation of the interactions between Hippocampal subregions during memory processes - a critical yet unexplored area of Hippocampal research. This method therefore provides a new tool that will help provide insight into the complex functions of the Hippocampus during behavior.

    This method also provides high resolution optical access to deep dendritic structures that have been out of reach with existing methods. The authors demonstrate they can measure the structure of single spines on distal apical dendrites of CA1 cells. They track populations of spines and quantify spine changes, spines loss, and spine appearance. Spine turnover is thought to be a key process in how the Hippocampus encodes and consolidates memories, and this method provides a means to quantify spine dynamics over very long time periods (months) and can be used to study spine dynamics in CA3 and DG.

    Weaknesses

    This method requires the implantation of a relatively large glass microperiscope that cuts through part of the Septal end of the Hippocampus. This is a necessary step to image transversally and observe all the major subregions simultaneously. This is an unfortunate limitation as it damages the very circuits being investigated. The authors attempt to address this by measuring the functional properties of Hippocampal cells, such as their place field features, and claim they are similar to those measured with other methods that do not damage the Hippocampus. However, it is very likely the implant-induced damage is affecting the imaged cells in some way, so caution should be taken when using this method. The authors are very aware of this and briefly discuss the issue. In addition, the authors observe damaged adjacent to face of the glass microperiscope that extends to ~300 um from the face. This area should therefore be avoided when imaging the Hippocampus through the microperiscope.

    Was this evaluation helpful?
  5. Reviewer #1 (Public Review):

    Redman and colleagues employed microprisms and two-photon optical imaging to track separately the structure of dorsal CA1 pyramidal neurons or the activity patterns of dorsal Dentate Gyrus, CA3, CA2 and CA1 pyramidal neurons, longitudinally in live mice. First, they carried out a characterization of the optical properties of their system. Second, they performed an example tracking of dendritic spines in the apical aspect of dorsal CA1 pyramidal neurons. Finally, they characterized differences in spatial coding along the tri-synaptic pathway, in the same animals. The main focus of the manuscript is technological and the authors show interesting data to support their technique, which I believe will be of relevance to neuroscientists interested in the hippocampal formation.

    Strengths.
    While using microprisms to achieve a "side" view of neurons in specific brain areas is not new per se [see Chia et al., J. Neurophysiol. (2009), Andermann et al., Neuron (2013), Low et al., PNAS (2014) etc.] the authors were able to visualize activity of a large neuronal circuit such as the hippocampal tri-synaptic pathway - for the first time - in the same animal exploring an environment. This is not only a technical feat but it opens new scientific avenues to study how information is transformed at different stages within the hippocampus, as such I think this will be of broad interest for people in the field. In addition, the authors demonstrated imaging of dendritic spines in the apical aspect of pyramidal neurons but limited to dorsal CA1 due to the labelling density of the transgenic mouse line they decided to use. Despite the fact that imaging apical dendritic spines in dorsal CA1 has been shown earlier [see Schmid et al., Neuron (2016) and Ulivi et al., JoVE (2019)], the use of the micro periscope greatly increases the flexibility of these sort of experiments by enabling tracking of large portion (both apically and basally) of the dendritic arbors of dorsal CA1 pyramidal neurons.

    Weaknesses.
    While the data are sufficient to demonstrate the technique, the conceptual advance of the paper is very narrow. The findings on spatial coding differences in different hippocampal subregions - namely a non-uniform distribution of spatial information in the different hippocampal subregions - do not add new knowledge but largely confirm the literature. The results on the dynamics of apical dendritic spines of pyramidal neurons in dorsal CA1 seem to confirm previous work, but the interpretation of these results differs fundamentally. In fact both papers cited by the authors (Attardo et al., and Pfeiffer et al.,) come to the conclusion that dendritic spines on basal dendrites of CA1 pyramidal neurons are highly unstable, at least by comparison to other neocortical areas. The authors seem to ignore this discrepancy. However, this discrepancy has importance also to the characterization of the technique the authors developed. In fact, the optical resolution of the system strongly affects the ability to resolve neighboring spines - especially at the high density of dorsal CA1 - and thus it has a direct effect on the measures of synaptic stability [Attardo et al., Nature, (2015)]. The authors duly report lateral and axial resolutions for their micro periscopes and both are lower than the ones of Attardo and Pfeiffer, thus the authors should consider the effects of this difference on the interpretation of their data.

    Was this evaluation helpful?