Structural development and dorsoventral maturation of the medial entorhinal cortex

Thank you for submitting your work entitled "Structural Development and Dorsoventral Maturation of the Medial Entorhinal Cortex" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Howard Eichenbaum as the Reviewing Editor and Eve Marder as the Senior Editor.

The following individuals involved in review of your submission have agreed to reveal their identity: Thomas Van Groen, Andre Fenton, and Rosamund Langston (peer reviewers).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

This paper provides important new insights into the developmental circuitry of the medial entorhinal and parasubiculum circuitry, of high importance to considerable current research on the functional organization of these brain areas.

Essential revisions:

The reviewers had several general concerns about explanation of the findings and many recommendations for improvement of the data presentation. These are detailed below.

Reviewer #1:

The manuscript by Ray and Brecht is quite interesting and relatively well written. The Introduction, Methods and Discussion are fine, even if the Discussion is a bit short. The Methods are good, but could be more detailed. The Results, however, need to be substantially revised or improved. One minor issue is that "superficial layers" need to be defined, even if most people know those are layers I to III, another issue are the tangential sections, that should be more clearly explained in the text. If a cortical area changes in size, by definition neuronal density decreases, this needs to be more clearly stated and discussed. The biggest issue is the presentation of the figures in the Results. Figure 1 shows changes in the size of the superficial layers, but also includes the deep layers. Figure 2: the autocorrelation images need to be the same size as the insets of the immunohistochemical staining images to be acceptable and interpretable. The figure orientation is needed in Figure 2A; what is where? For instance, what is the band of bright staining on the right side of the image? The acetylcholinesterase activity staining is of low quality (at least in the images provided). Similarly, in Figure 3, images showing the sections at equal magnification would be helpful (orientation?). Figure 5 is of too low quality to be interpretable, doublecortin is supposed to stain neurons, which is not very visible in this figure. Taken together, this will make the data more understandable for readers; in the current version the images are not exceedingly helpful.

Reviewer #2:

General assessment:

This is a clear and valuable study, describing the anatomical and developmental expression of calbindin and other molecular markers of pyramidal cells in the MEC and parasubiculum across developmental time from late gestation to young adulthood. These cells are important elements of the microcircuitry that generates spatially-tuned discharge. Whether the histochemically identified Calbindin⁺ cells correspond to functionally defined grid cells and/or border cells is controversial, which makes the present study an important contribution to the debate. The debate is crucial to notions of how the hippocampal-entorhinal system generates and computes information about space from the different functional components that have been identified such as distances, locations, directions, borders and speed. The development of the functional cell classes has been studied in recent years to constrain the theories, but which functionally-identified cells correspond to which histochemical and morphological cell classes is still unclear and clarification of this issue will provide important knowledge of the circuit wiring diagram that will drive theories of neural computation that are grounded in structure-function relationships. In summary, this manuscript is an important contribution.

Summary of concerns:

The manuscript is clearly written and the studies appear to be done carefully, and the procedures are straightforward. The data look clear and the interpretations of the measurements are not controversial. There are however a number of improvements I will suggest that will make the report more accessible to the general readership of eLife.

1) The analyses and interpretation of the histochemical images depend strongly on knowledge of the anatomical topography of the region, which non-specialists will not be. Whether the sections are tangential or parasaggital, and knowledge of the precise cutting angles is important even for the specialist. Consequently, it would be valuable to provide a 3-D model of the brain or just the cortical region with indications of the tangential and parasaggital planes and to use these on the Figures as a short-hand to help orient the reader. I know this is asking too much, but I will mention it to make my point: a 3-D CLARITY image of the immunolabeled cells would go a long way towards showing this very cool grid-like organization to the non-specialist and specialist alike. A 3-D model could accomplish the same.

2) I was disappointed not to see analysis of the stellate ocean cells, the coexistence of which in the region, but outside the pyramidal patches, is the source of the controversy. The authors should explain in the manuscript why parallel analyses were not performed.

3) I like the Discussion, which is appropriately driven by the findings. Again, for the general readership of eLife, I suggest the Discussion be expanded to more explicitly include the differential hippocampal and neocortical connectivity of the stellate and pyramidal cells, a discussion of what is known about their function properties, and why it is important to understanding how information about space is computed.

Reviewer #3:

Although structural maturation has been shown for the hippocampus this was lacking in the literature for entorhinal cortex despite the huge recent interest in the spatially selective cell types found there, so this paper is timely and of high theoretical importance.

The Introduction focuses on Layer II of MEC and is very brief – a little more attention to the cell types of Layer III and PaS and their electrophysiological characteristics, and what makes them different to the Layer II neurons would be nice since quite a lot of the results focus on these and not on layer II.

Also how does one identify stellate neurons and their developmental profile, and why is this not done? Also why are deeper layers of MEC not analysed? These are not suggestions for more experiments, just requests to explain briefly why they were not included here.

A bit more information about wolframin would be good – the authors initially state it is co-localised with calbindin but from their results this is not always the case?

The figures are mainly well explained, and the layout of the individual images, with the data presented graphically, is intuitive and aesthetically pleasing. The images clearly depict what is described and the mean data that is shown in the graphs.

Figure titles and/or images should include whether they depict MEC or PaS (e.g. not clear from Figure 5 where it is referred to in the text as being MEC layer II AND PaS as far as I read.)

Discuss relevance of wolframin vs. calbindin and why they appear separately in PaS but are co-localised in pyramidal cells of MEC layer 2.

In the Discussion it is a very interesting theory that the dorso-ventral structural development profile may reflect the maturation of the range of the spatial navigation system however this would be quite difficult to study since coincident with this is the fact that rats explore further after their eyes open at the end of the second postnatal week and it would be difficult to manipulate this behaviour to occur earlier, although similar things have been achieved with experiments to open rat pups' eyelids earlier.

The following theory suggested in the fourth paragraph of the Discussion that the patchy doublecortin co-localised with calbindin may reflect different functional maturation of border and grid cells requires a little more explanation, I did not see any obvious link.

Overall the paper provides much-needed high-quality solid anatomical evidence for dorso-ventral structural maturation occurring in the MEC and PaS, similar to that seen in the hippocampus. This data should feed into various models of neuronal spatial representations and presents the possibility that subsets of grid cells may mature before others so maps could potentially be formed of smaller spaces before larger ones, and a full complement of grid cells may not be necessary to see grid-like firing, a hypothesis which is testable and intriguing.

Essential revisions:

The reviewers had several general concerns about explanation of the findings and many recommendations for improvement of the data presentation. These are detailed below. Reviewer #1:

The manuscript by Ray and Brecht is quite interesting and relatively well written. The Introduction, Methods and Discussion are fine, even if the Discussion is a bit short. The Methods are good, but could be more detailed.

We thank the reviewer for appreciating the manuscript. We agree with the reviewer’s assessment about the brevity of certain sections of the manuscript and have performed a significant revision to all sections of the manuscript to result in a more detailed and comprehensive revised manuscript.

The Results, however, need to be substantially revised or improved. One minor issue is that "superficial layers" need to be defined, even if most people know those are layers I to III, another issue are the tangential sections, that should be more clearly explained in the text.

We agree with the reviewer’s assessment and have implemented their suggestion.

Changes:

Results, first paragraph – We have addressed the definition of superficial layer.

Video 1 – We have added a novel schematic video that illustrates the tangential sectioning process and provides the reader with a clear perspective of how the tangential sections are obtained from the rodent brain, and how the structures seen in tangential sections relate to the brain in situ.

If a cortical area changes in size, by definition neuronal density decreases, this needs to be more clearly stated and discussed. The biggest issue is the presentation of the figures in the Results. Figure 1 shows changes in the size of the superficial layers, but also includes the deep layers.

We agree with this criticism and thank the reviewer for the suggestion to include variation in depth in deep layers of the MEC with increasing age.

Changes:

Discussion, fourth paragraph – We discuss the effect of increasing brain size on neuronal density.

Figure 1D and Results, first paragraph – We have updated Figure 1D and the Results to include changes in size in the deep layers of MEC.

Figure 2: the autocorrelation images need to be the same size as the insets of the immunohistochemical staining images to be acceptable and interpretable. The figure orientation is needed in Figure 2A; what is where? For instance, what is the band of bright staining on the right side of the image?

We thank the reviewer for pointing out this oversight.

Changes:

Figure 2A-F – We have ensured that the inset of the immunohistochemical stain and the autocorrelation images are of the same size. The scale bar in the autocorrelation image is half the length of the one in the immunohistochemical image, since the autocorrelation denotes twice the length and breadth of the immunohistochemical image.

Figure legends (all relevant figures) – We have denoted the orientation in one panel of every figure, and now we state in the figure legends that it applies to all the other panels unless explicitly noted.

Figure legend (Figure 2) – We mention in the figure legend that the bright green band pointed out by the reviewer denotes the parasubiculum.

The acetylcholinesterase activity staining is of low quality (at least in the images provided). Similarly, in Figure 3, images showing the sections at equal magnification would be helpful (orientation?).

We agree with the reviewer’s criticism and have changed the layout of the figure to address the issue.

Changes:

Figure 2G-1 – We have changed the layout of the figure to enable better visualization and co-localization of acetylcholinesterase activity and calbindin immunoreactivity.

Figure legends (all relevant figures) – In Figure 3 and elsewhere, as noted above, we have updated the legend to state that the orientation illustrated in one panel applies to the entire figure unless explicitly stated.

Figure 5 is of too low quality to be interpretable, doublecortin is supposed to stain neurons, which is not very visible in this figure. Taken together, this will make the data more understandable for readers; in the current version the images are not exceedingly helpful.We agree with the referee’s criticism, about the need for a higher magnification image to visualize doublecortin localization in a single neuron. However, since other reviewers found the layout of these images quite intuitive, we decided not to change Figure 5 (now Figure 6) and instead added a novel Figure 7 to address this issue.

Changes:

Figure 7 – We have now added a novel Figure 7, as a result of our new experiments, which shows at higher magnification the co-localization of doublecortin in the cell membrane of the cells, with calbindin.

This figure also illustrates reelin which mark stellate cells in layer 2 of MEC, in response to queries raised by the other reviewers and quantifies the differential co-localization of doublecortin with calbindin and reelin during early postnatal development.

Reviewer #2:

Summary of concerns: The manuscript is clearly written and the studies appear to be done carefully, and the procedures are straightforward. The data look clear and the interpretations of the measurements are not controversial. There are however a number of improvements I will suggest that will make the report more accessible to the general readership of eLife. 1) The analyses and interpretation of the histochemical images depend strongly on knowledge of the anatomical topography of the region, which non-specialists will not be. Whether the sections are tangential or parasaggital, and knowledge of the precise cutting angles is important even for the specialist. Consequently, it would be valuable to provide a 3-D model of the brain or just the cortical region with indications of the tangential and parasaggital planes and to use these on the Figures as a short-hand to help orient the reader. I know this is asking too much, but I will mention it to make my point: a 3-D CLARITY image of the immunolabeled cells would go a long way towards showing this very cool grid-like organization to the non-specialist and specialist alike. A 3-D model could accomplish the same. We thank the reviewer for the idea of a 3D model, and have included a novel schematic video (Video 1) to address these concerns.

Changes:

Video 1 – We have added this video, which illustrates the tangential sectioning process and provides the reader with a clear perspective of how the tangential sections are obtained from the rodent brain, and how the grid-like structures seen in tangential sections relate to the brain in situ.

2) I was disappointed not to see analysis of the stellate ocean cells, the coexistence of which in the region, but outside the pyramidal patches, is the source of the controversy. The authors should explain in the manuscript why parallel analyses were not performed. This comment is similar to the criticism of another reviewer and we think the omission of stellate cell development was a major flaw in the previous version of our manuscript. We have performed a series of experiments to address this concern, and now provide a detailed perspective of the development of stellate cells.

Changes:

Figure 2—figure supplement 1 – New figure, illustrating the spatial layout of reelin+ stellate cells during development.

Figure 7 – We have added this new figure, illustrating the co-localization of doublecortin with pyramidal but not stellate cells during early postnatal development.

We have also made appropriate changes in the Abstract, Results and Discussion sections of the manuscript to reflect these novel results.

In addition, we found an increasing expression of reelin in layer 3 neurons with development, which we have highlighted in new Figure 4 and new Figure 5—figure supplement 1.

3) I like the Discussion, which is appropriately driven by the findings. Again, for the general readership of eLife, I suggest the Discussion be expanded to more explicitly include the differential hippocampal and neocortical connectivity of the stellate and pyramidal cells, a discussion of what is known about their function properties, and why it is important to understanding how information about space is computed. We thank the reviewer for the appreciation and the idea to highlight the differential connectivity profiles of pyramidal and stellate cells and its impact in spatial information processing.

Changes:

Discussion, seventh paragraph – Discussion on different projection patterns of pyramidal and stellate cells.

Reviewer #3:

Although structural maturation has been shown for the hippocampus this was lacking in the literature for entorhinal cortex despite the huge recent interest in the spatially selective cell types found there, so this paper is timely and of high theoretical importance. The Introduction focuses on Layer II of MEC and is very brief – a little more attention to the cell types of Layer III and PaS and their electrophysiological characteristics, and what makes them different to the Layer II neurons would be nice since quite a lot of the results focus on these and not on layer II. We thank the reviewer for pointing out this oversight, and we have expanded the Introduction to include the characteristics of cells in layer 3 and parasubiculum in addition to layer 2.

Changes:

Introduction, fourth paragraph – We have included an introduction on layer 3 and the functional characteristics of the cells found there.

Introduction, fifth paragraph – We have included an introduction on parasubicuulum and the functional characteristics of the cells found there.

Also how does one identify stellate neurons and their developmental profile, and why is this not done? Also why are deeper layers of MEC not analysed? These are not suggestions for more experiments, just requests to explain briefly why they were not included here. This comment is similar to a query raised by another reviewer, and we have performed a series of novel experiments to explore the development of stellates by using the extracellular matrix protein Reelin. We have performed a series of experiments to address this concern, and now provide a detailed perspective of the development of stellate cells. For deep layers, we added the development of their thickness during development, congruent with the recommendations of another reviewer.

Changes:

Figure 2—figure supplement 1 – New figure, illustrating the spatial layout of reelin+ stellate cells during development.

Figure 7 – New figure, illustrating the co-localization of doublecortin with pyramidal but not stellate cells during early postnatal development.

Figure 1D; Results, first paragraph– We have updated Figure 1D and the Results to include changes in size in the deep layers of MEC.

We have also made appropriate changes in the Abstract, Results and Discussion sections of the manuscript to reflect these novel results.

In addition, we found an increasing expression of reelin in layer 3 neurons with development, which we have highlighted in new Figure 4 and new Figure 5—figure supplement 1.

A bit more information about wolframin would be good – the authors initially state it is co-localised with calbindin but from their results this is not always the case? We have now expanded our discussion on wolframin on its co-localization with calbindin in layer 2 of MEC but not the developed parasubiculum.

Changes:

Discussion, fifth paragraph – We expanded the Discussion to include how studies target specific cell-types using their molecular profiles, and how experiments towards understanding the specific roles of these proteins might provide insights in understanding the functional differences in these cells.

The figures are mainly well explained, and the layout of the individual images, with the data presented graphically, is intuitive and aesthetically pleasing. The images clearly depict what is described and the mean data that is shown in the graphs.

We thank the reviewer for the appreciation of the figures.

Figure titles and/or images should include whether they depict MEC or PaS (e.g. not clear from Figure 5 where it is referred to in the text as being MEC layer II AND PaS as far as I read.) We thank the reviewer for pointing out this oversight, and have now corrected the figure title and legend.

Discuss relevance of wolframin vs. calbindin and why they appear separately in PaS but are co-localised in pyramidal cells of MEC layer 2.As noted above, we provide a more comprehensive discussion on wolframin and calbindin, and their possible functions.

Changes:

Discussion, fifth paragraph – Discussion on the roles of wolframin and calbindin.

In the Discussion it is a very interesting theory that the dorso-ventral structural development profile may reflect the maturation of the range of the spatial navigation system however this would be quite difficult to study since coincident with this is the fact that rats explore further after their eyes open at the end of the second postnatal week and it would be difficult to manipulate this behaviour to occur earlier, although similar things have been achieved with experiments to open rat pups' eyelids earlier. We thank the reviewer for finding the theory interesting, and have now discussed in greater detail how it can be tested by the early eyelid opening experiments, as pointed out by the reviewer.

Changes:

Discussion, sixth paragraph – Discussion on possible experiments to test dorso-ventral maturation hypothesis.

The following theory suggested in the fourth paragraph of the Discussion that the patchy doublecortin co-localised with calbindin may reflect different functional maturation of border and grid cells requires a little more explanation, I did not see any obvious link. We apologize for not having adequately clarified this point, and have now addressed this issue in greater detail by performing a series of experiments to simultaneously analyze the co-localization of the immature neuronal marker doublecortin, with pyramidal cell marker calbindin, and stellate cell marker reelin in layer 2 of MEC. We find a differential neuronal maturation profile, which indicates that the structural development of pyramidal and stellate cells, closely mirrors the differential functional maturation profiles of grid and border cells respectively.

Changes:

Figure 7 – New figure, illustrating the co-localization of doublecortin with pyramidal and not stellate cells during early postnatal development.

We have also made appropriate changes in the Abstract, Results and Discussion sections of the manuscript to reflect these novel results.

Overall the paper provides much-needed high-quality solid anatomical evidence for dorso-ventral structural maturation occurring in the MEC and PaS, similar to that seen in the hippocampus. This data should feed into various models of neuronal spatial representations and presents the possibility that subsets of grid cells may mature before others so maps could potentially be formed of smaller spaces before larger ones, and a full complement of grid cells may not be necessary to see grid-like firing, a hypothesis which is testable and intriguing.

We thank the reviewer for the comprehensive summary of our manuscript, which mirrors our enthusiasm.