Unveiling the cell biology of hippocampal neurons with dendritic axon origin

  1. Yuhao Han
  2. Roland Thuenauer
  3. Kay Grünewald
  4. Marina Mikhaylova

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Abstract

In mammalian axon-carrying-dendrite (AcD) neurons, the axon emanates from a basal dendrite, instead of the soma, to create a privileged route for action potential generation at the axon initial segment (AIS). However, it is unclear how such unusual morphology is established and whether the structure and function of the AIS in AcD neurons is preserved. Here, we show that the AcD neurons follow an intrinsically encoded developmental program where a single precursor neurite first gives rise to the axon and then to the AcD. The AIS possesses a similar cytoskeletal architecture as the canonical AIS that stems from the soma, and similarly functions as a trafficking barrier to retain axon-specific molecular composition. However, unlike soma-derived AIS, the AIS of AcD neurons does not undergo homeostatic-plasticity, contains less cisternal organelles and receives fewer inhibitory inputs. These distinct features of the AIS could account for the higher intrinsic excitability of AcD neurons.

Highlights

  • The development of AcD neurons is intrinsically driven and does not depend on specific connectivity patterns

  • AcD neurons generate the axon and AcD from the same precursor neurite

  • The stem dendrite of an AcD neuron displays axon-like microtubule organization

  • Similar to the AIS emerging from the soma, the AIS of AcD neurons can selectively filter dendritic cargo

  • The AIS of AcD neurons does not undergo chronic homeostatic plasticity and receives fewer inhibitory inputs

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    Reply to the reviewers

    Reviewer_01

    Major comments:

    1. The authors cite that acetylated and tyrosinated microtubules have different spatial and compartmental distribution in dendrites and axons and investigate the distribution in the AIS of nonAcD cells and AcD cells, as well as the stem dendrites. However, they just show one example of two different cells (Figure 2D and E) without any statistical analysis. Either, they should remove this part or provide a thorough quantification. Reply: The spatial and compartmentalized distribution of stable and dynamic MTs in the dendrites and axons of nonAcD neurons has been extensively studied and reviewed (see Kapitein & Hoogenraad, 2011; Katrukha et al., 2021; Tas et al., 2017 for reference). However, the organization of the MT cytoskeleton in AcD neurons is still unknown. Here, we provide the very first evidence on the distribution of tyrosinated and acetylated MTs in AcD neurons, as well as data on MT orientations. We agree with the reviewer that to make our results on the spatial organization of these post-translational modifications in AcD neurons more complete, we need to provide a more thorough quantification analysis.

    To achieve this, we plan to perform immunostainings on DIV10 neurons using antibodies against tyrosinated (tyr) and acetylated (ac-) tubulin to label dynamic and stable MTs, respectively. Subsequently, we will conduct high-resolution 3D confocal imaging and measure fluorescent intensity to illustrate the abundance and staining patterns of tyr- and ac- MTs in the axons and dendrites of AcD neurons. Since the spatial distribution of tyr- and ac-MTs is distinguishable with confocal microscopy, we will retain STED examples in the figures but conduct new analyses on confocal imaging data. We will measure the total fluorescent intensity of tyr- and ac- MTs in different compartments of AcD neurons and normalize it to the size of the measured area. We will then compare the normalized intensity values between the axons and dendrites of AcD neurons to examine whether there is a specific distribution pattern of stable and dynamic MTs. We will analyse at least 3 independent primary culture preparations with a minimum of 30 cells. Using the same dataset, we will also quantify the percentage of AcD neurons with ac-MTs specifically elongating into the axon compared to AcD.

    The authors use EGFP-Rab3A vesicle to investigate anterograde transport at the axon and dendrites. They find a slightly faster transport of these vesicles at the AIS of AcD cells and conclude the axonal cargos in general are transported faster across the AIS in AcD cells. In my opinion, this generalization based on one type of vesicle is too farfetched.

    Reply: The Rab3A protein is associated with pre-synaptic vesicles that are transported by KIF1A and KIF1Bβ, members of the kinesin-3 family, towards pre-synaptic buttons (see Guedes-Dias & Holzbaur, 2019; Niwa et al., 2008 for reference). Since KIF1A and KIF1Bβ are common motor proteins that mediate MT-based transport of different types of vesicles (e.g., synaptic vesicles and dense-core vesicles, see Carabalona et al., 2016; Helmer & Vallee, 2023 for reference), we reasoned that Rab3A should be a representative marker for an axonal cargo. However, this indeed does not rule out whether the faster trafficking effect we saw is specific to presynaptic vesicles, as different types of vesicles tend to recruit different modulators that could lead to different trafficking features.

    To address this question, we will perform a live-imaging experiment including two additional organelle marker proteins, Neuropeptide Y (NPY) and Lysosome-associated membrane protein 1 (Lamp1). NPY is transported into the axon via KIF1A and KIF1Bβ-mediated dense-core vesicles (see Helmer & Vallee, 2023; Lipka et al., 2016 for reference). Lamp1 is associated with lysosomes and a range of endocytic organelles that recruit both kinesin-1 and kinesin-3, and are transported into both axons and dendrites (as reviewed in Cabukusta & Neefjes, 2018). By introducing two additional types of vesicles, we should be able to answer whether AcD neurons, in general, tend to transport cargoes into the axon faster than nonAcD neurons.

    __Minor comments: __

    In the introduction, the authors describe how synaptic inputs are received at the dendrites and propagated to the soma in the form of membrane depolarizations. They should add 'excitatory' to synaptic inputs or also describe the impact of inhibitory synaptic inputs at the dendrites.

    In my opinion, Figure 2 could be presented in a slightly better way. The lower part of panel A better fits to panel B, which is next to the upper part of panel A. I understand that the authors systematically present their data first for nonAcD cells and then for AcD cells. However, in this special case it is a little bit more difficult to read the current figure in that order. The results displayed in Figure 4 are presented in a slightly confusing order. The authors jump from 4D to 4G, then to 4I and 4E, 4H, 4F. Similarly, 4M and N are addressed before 4O and P to finally get to 4K and L. It would be beneficial to present and address the data in a stringent way.

    Reply: Thank you for the suggestions on how to improve the data representation in the figures. We will change Figures 2 and 4 and make adjustments in the text upon revision since we also plan to include additional data.

    Reviewer_02

    Major comments:

    1. The authors suggest that there is reduced Na+ channel density at AcD AIS compared to other AIS arising from the cell body. This is not convincing. Immunostaining for Na+ channels is notoriously difficult and sensitive to fixation since the epitopes of the anti-Pan Nav antibodies are highly sensitive to fixation. In addition, this is based on immunofluorescence intensity quantification. Since the mechanism of localization is through binding to AnkG, the authors should also measure other AIS proteins like AnkG, b4 spectrin, and Nfasc. Do these change? If all uniformly change I would be much more inclined to accept the conclusion. If they do not change, it still doesn't rule out the concern about fixation conditions and slight differences in the cultures. The authors indicate there is about a 40% reduction in fluorescence intensity. That is quite large. This big difference should also be confirmed in brain sections. Reply: The potential fixation issue and antibody sensitivity on Na+ channel staining are indeed valid considerations, and we are aware of them. However, it should be noted that we used pan-Na+ channel antibodies that were previously characterised and widely used in literature (see Solé et al., 2019; Yang et al., 2020 for references). Furthermore, our samples underwent the same fixation and staining protocol, and comparable numbers of AcD and nonAcD neurons were imaged from the same preparation and coverslip for each experiment. Imaging settings were also kept constant. Any loss of Na+ channel staining at the AIS due to fixation should affect both neuron types and therefore our conclusion is justified. Nevertheless, the reviewer's point regarding other AIS components is valid and will be investigated further in the revised manuscript.

    Following the reviewer's suggestion to further strengthen our conclusion, we will measure the intensity of AnkG, βIV-spectrin, and neurofascin in DIV21 AcD and nonAcD neurons. We will compare a minimum of 3 independent cultures, each containing at least 10 cells of each type per culture.

    We agree with the reviewer that confirming observed differences in Na+ channel staining using brain slices would be beneficial. However, conducting such experiments presents several challenges. Firstly, one approach could involve immunostaining with antibodies against AIS marker AnkG, in combination with somatodendritic marker MAP2 and pan-Nav. However, this method lacks the advantage of clearly identifying neuronal morphology as seen in dissociated cultures, making the outcome unclear and difficult for analysis and interpretation. Alternatively, the use of Thy1-GFP rats, where a subset of neurons is labelled with GFP, could allow for morphological studies. Unfortunately, we do not have access to this rat line, and the process of importing it, obtaining permits, and establishing a colony is beyond the timeframe for manuscript revision. Additionally, while pan-Nav antibodies have shown reliability in dissociated cultures, their efficacy in tissue staining is less certain. We could provide example images upon request. Secondly, endogenously labelling of Na+ channels is another option, but remains a significant challenge. Recent developments in endogenous labelling, such as the CRISPR/Cas9-based method using pORANGE by Fréal et al. (Fréal et al., 2023), and the generation of Scn1a-GFP transgenic mice by Yamagata et al. (Yamagata et al., 2023), offer potential solutions. However, the labelling efficiency of pORANGE is uncertain, and both methods are time-consuming and cannot be completed within the three-month revision period.

    As an alternative, we propose emphasising that our results are based on in vitro experiments and discussing the advantages and limitations of this approach in the discussion section.

    The analysis of inhibitory synapse differences at the AIS are also not compelling - this is a limitation of the culture system. The authors have no control over the density of inhibitory neurons in the culture well. This interaction is not intrinsic to the AcD neuron, but rather a feature of neuron-neuron interactions which should only be modelled in the animal.

    Reply: The reviewer is correct in pointing out that establishing inhibitory synapses at the AIS is not an intrinsic feature of AcD neurons; it depends on the network and should be modelled in animals. We will include this limitation of the cell culture model in the discussion section in the revised manuscript. We also understand the reviewer's concern that the lower amount of inhibitory synapses at AcD neuron AIS might be due to uneven density of inhibitory neurons between cultures. Nonetheless, assuming that the number of inhibitory neurons is constant between preparations, it is an interesting observation that AcD neurons form fewer inhibitory synapses at the AIS. This may be related to the features of the AIS and its morphology and should be further investigated.

    To make our study more comprehensive and also address the reviewer's concern regarding the presence of inhibitory neurons, we will perform immunostainings in dissociated cultures (40.000 cells per 18 mm coverslip, same as in experiments with synapse quantification) with antibodies against pCaMKIIa, an excitatory neuron marker, and GAD1, a marker for inhibitory neurons. Then, we will quantify the density of inhibitory neurons in the culture. We will perform measurements from 3-6 independent cultures by analysing large fields of view in different areas of a coverslip (20-30 neurons per area) to determine if the density of inhibitory neurons varies between cultures as well as preparations. Furthermore, as also requested by reviewer 4, we will perform new immunostainings where pre- and post-synaptic markers (VGAT and Gephyrin) will be included in the same sample together with the AIS (AnkG or Neurofascin) and dendritic marker (MAP2). Synapses that contain pre- and post-synaptic components will be analysed and included in the revised version of the manuscript.

    Finally, the major limitation of this study is that it is performed in vitro. Surprisingly, the authors actually argue this is a feature of their system. While it is true some of the questions can be addressed perfectly well in vitro, many cannot. In the first paragraph of the results the authors state an advantage of their system is that there are no microenvironments to influence the development of the AcDs. I'm afraid I view this as a drawback. The authors suggest this is an opportunity to examine intrinsic mechanisms of development - true, but it also foregoes the opportunity to determine if the outcomes are different from what occurs in vivo. To this point, the authors report that only 15-20% of the population of hippocampal neurons in culture are AcD neurons. But in their introduction they cite other literature indicating 50% of hippocampal neurons in vivo are AcD neurons - this suggests that the environment of the hippocampus in vivo influences whether a neuron becomes an AcD neuron or not.

    Reply: The reviewer is right in pointing that the in vivo environment could indeed affect AcD neuron development, and we also find this to be a very interesting topic to investigate in the future. Even more intriguingly, as shown in a preprint by Lehmann et al. (doi: https://doi.org/10.1101/2023.07.31.551236), network activity stimulates neurons to acquire AcD morphology. While it is true that the impact of the microenvironment on AcD neuron development cannot be studied in dissociated cultures, our in vitro data undoubtedly support the fact that hippocampal neurons can intrinsically develop into AcD morphology independent of the in vivo environment. As also mentioned in the next point, our statement "...their development must be driven by genetically encoded factors rather than specific..." might sound too definitive and therefore eliminate possible effects from the microenvironment. We will revise this part. Although it is highly desirable to move cell biological studies from neuronal cell cultures to tissue, to date, it is still very challenging to perform many of experiments which we did in this study in slices or living animals due to a lack of appropriate technologies and tools. We are convinced that many basic biological questions can be and should be studied in simplified culturing models because they are truly fundamental, they should also be reproducible in these models.

    To address the reviewer's question regarding the percentage difference between our data and the previous study by Thome et al. (2014), several factors should be considered. First, as noted by the reviewer, our results were obtained from an in vitro system, which is not directly comparable to the in vivo model system used in Thome et al.'s study (Thome et al., 2014). Second, the age of the neurons quantified in our developmental experiments is DIV5 and DIV7. This young age disparity could contribute to the percentage difference, as Thome et al. analyzed neurons from P28-35 adult animals, where 50% of the AcD neuron population was observed, specifically in the CA1 region. Third, it's important to note that in other hippocampal regions, the percentage of AcD neurons is lower (approximately 20-30%). Since our hippocampal primary cultures contain neurons from all hippocampal regions, this may have averaged out our quantification of AcD neuron percentage. Additionally, in the study by Benavides-Piccione et al. (Benavides-Piccione et al., 2020), they reported 20% AcD neurons in the CA1 region of hippocampi isolated from 8-week-old mouse pups, a number similar to what we observed in vitro. Interestingly, Thome et al. reported that in P8 pups, AcD neuron population in hippocampal CA1 region is 30%. This number increased to 50% in adult animals at age of P28-35, suggesting there is perhaps an age dependent increase of AcD neuron population. This could be an additional reason of why we only saw 15-20% of AcD neurons in our in vitro system, regardless of the in vivo environment.

    In the revised version, we will clarify these points in the introduction and discussion sections. Additionally, we will quantify the proportion of AcD neurons in mature DIV21 dissociated hippocampal cultures and compare it to DIV7 cultures to assess whether there is an increase in the AcD population over time. We believe that this experiment, combined with the explanations provided above, will sufficiently address the reviewer's question. However, it is important to acknowledge that the establishment of neuronal networks in vitro differ from those in vivo. Therefore, there may be potential differences in the outcomes.

    I appreciated the balanced discussion of whether this is a stochastic or genetically programmed process. This could have been emphasized earlier in the results since the authors invoke the concept that "...their development must be driven by genetically encoded factors rather than specific...". The authors have not shown this and cannot show it in this system. Indeed, as stated in point 4 above, I think their data argue against a simple genetic program.

    Reply: As suggested by the reviewer and noted in point 4, we will revise the section on AcD neuron development in our manuscript to emphasize that hippocampal neurons may adopt AcD morphology through genetic or stochastic mechanisms. While we acknowledge that environmental and activity factors may also influence this process, particularly in mature neurons, our study focuses on developing neurons where genetic and stochastic factors are likely to be predominant. This conclusion is supported by the observation that neurons develop into AcD morphology in vitro, where environmental and activity patterns do not mimic those of in vivo systems.

    Indeed, our current manuscript does not explore genetic factors involved in AcD neuron development. To address this question, one approach could be to label AIS markers endogenously in dissociated cultures using the PORANGE method (see Willems et al., 2020 for reference) or utilize AnkG-GFP transgenic mice (Fréal et al., 2023; Thome et al., 2023) along with a volume marker like mRuby or GFP. This would allow for the identification of AcD and nonAcD neurons in vivo and in vitro, followed by single-cell transcriptomics analysis to uncover potential genetic factors. Subsequently, candidate genes could be manipulated to demonstrate their essential role in AcD neuron development. However, such experiments require significant time and resources beyond the scope of our current revision timeframe. Nonetheless, this question presents an exciting direction for future research.

    Reviewer 3

    Major comments:

    1. The authors classify neurons into axon-carrying dendrite (AcD) and non-AcD neurons by measuring the stem dendrite length (> 3 µm). I could not find the validity for this cut-off. The non-AcD neurons in Fig. 6B appear more AcD to this reviewer, and, in addition, other researchers have proposed a third category of 'shared root' neurons (doi: 10.7554/eLife.76101). For purposes of reproducibility and transparency, please provide first a comprehensive overview of the entire population of morphologies (i.e. all cells in control conditions). The distances from the soma could be plotted in histogram (etc.) and authors may want to think about independent supporting evidence for the cut-off to classify AcD and non-AcD neurons. Reply: Concerning the validity of AcD neuron classification, we did measure the length of the stem dendrite, as shown in Figure S4G, with an average distance of around 10 µm. However, we admit that this information is presented relatively late in the manuscript. To address the reviewer's criticism, in the revised version, we will include a supplementary figure displaying a gallery of representative images of both AcD and nonAcD neurons analyzed in our study (please refer to Hodapp et al., 2022; Fig S1 C&D; Fig S3 as an example). Given the sample size of AcD and nonAcD neurons in our study, including all images would result in a very large figure (for example, Figure 1: DIV5: 83 AcD neurons out of 427 cells, DIV7: 47 AcD neurons out of 387 cells). We will only show representative examples of AcD neurons in the gallery. Additionally, as suggested, we will plot the length of the stem dendrite (or axon distance) of AcD neurons as a histogram to demonstrate that the AcD neurons included in our study indeed have a stem dendrite longer than 3 µm. To further validate the used classification method, we will measure the diameter of the stem dendrite in all analyzed AcD neurons and then compare the distance between the soma and the start of the axon in each analyzed AcD neuron to the diameter of its stem dendrite. As described by Hodapp et al. (Hodapp et al., 2022; Fig S1A), AcD neurons are expected to have a stem dendrite longer than their diameter.

    We have considered having independent evidence to support the classification of nonAcD and AcD neurons. However, the method used by Thome et al. and Wahle et al. for AcD and nonAcD neuron classification is well established and widely accepted (see Thome et al., 2014; Wahle et al., 2022 for references). Similar standards were also employed by Benavides-Piccione et al. (Benavides-Piccione et al., 2020). Introducing independent evidence could potentially raise further doubts, so we have chosen to maintain consistency with previous studies.

    As for the "shared root" neurons described by Wahle et al., we did not analyze this category separately and included them in the nonAcD subtype. Nonetheless, it is an interesting direction to explore in the future. For completeness, we will discuss this point in the revised manuscript.

    Related to point #1 the primary hippocampal neuron system is excellent for cell biological questions but comes with the drawback of imaginative morphologies including neurons with multiple axons and AISs. It is not mentioned here but literature indicates up to 20% of neurons have two axons (e.g. doi: 10.1007/s12264-017-0169-3, 10.1083/jcb.200707042). How did the authors classify the double axon cells? Since the main hypothesis is the existence of an intrinsic program for AcD neurons (p. 5 top), the two axons from one neuron should develop similarly. The authors can easily test this with the data.

    Reply: We appreciate the reviewer's comment regarding the choice of the model system for this type of study. Indeed, as they pointed out, in primary cultures, some neurons develop more than one axon. Since we did not find any supporting evidence from the literature reporting that hippocampal neurons have multiple axons in vivo, we only analyzed neurons with one axon for both AcD and nonAcD neurons. We will clarify this in our method section of the revised manuscript.

    Some interpretations about function are not correct and the authors should reconsider these. A role of cisternal organelles on neuronal excitability remains to be demonstrated (and see doi.org/10.1002/cne.21445 showing there is none). In addition, the statement that lower fluorescence intensity of Pan-Nav1 is indicating reduced excitability is flawed. Antibody staining does not scale linearly with voltage-gated sodium channel density and since the AIS of AcD neurons is further from the soma it is most likely smaller in diameter which may account for apparent fluorescent differences. For biophysical reasons (for details I refer to 10.3389/fncel.2019.00570, 10.1016/j.conb.2018.02.016 and 10.7554/eLife.53432) smaller diameter axons will be easier to depolarize by depolarizing voltage-gated channels or excitatory synapses. Finally, in AcD neurons the AIS distance from the soma poses all sorts of interesting cable properties with the soma and the local dendritic membrane and the electrotonic properties alone suffice to make these neurons more excitable.

    Reply: The reviewer brings up very valid and important points that we will address in the revised manuscript. First, we will rephrase and adjust our interpretations regarding the functions of the cisternal organelle in the AIS. As also mentioned by reviewer #2, we are aware that antibody staining does not properly reflect Na+ channel density. As discussed above, we will also measure other AIS proteins that anchor Na+ channels to see if there are any correlations in fluorescence intensity between them and Nav1. We agree with the reviewer that AcD neuron's AIS could have a smaller diameter, resulting in fewer Na+ channels. Indirect evidence is already available in the study of Benavides-Piccione et al., showing a smaller axon diameter in AcD neurons compared to nonAcD neurons in both human and mouse brain sections (Figure S4). To test this in our model system, we propose to measure the AIS diameter in AcD neurons. If this is indeed the case, we will indicate it in our revised manuscript and edit the section on Na+ channels.

    Exploring the biophysical properties of the AIS and axons of AcD neurons is indeed a highly interesting direction to pursue and is the project in its own. It would necessitate the use of computational modeling approaches, which require considerable time and resources that are not feasible within the timeframe of this revision.

    Comparing AcD and non-AcD neurons for AIS plasticity is an excellent idea but the present statistical design is not suitable for answering this question. The authors should directly compare non-AcD and AcD neurons within a two-way ANOVA design, asking the question whether the independent variable axon type is significantly different and interacts with plasticity.

    Related points: 'AIS distance' in Figure 7 seems to refer to something else than distance from soma (Figure 1). Please clarify. What were the absolute distances from the soma for the AcD neurons and was this dependent on treatment?

    Reply: We appreciate reviewer's comment and in the revised version we will perform the analysis using two-way ANOVA.

    Regarding the terminology and definitions used in our manuscript, the "AIS distance" refers to the measurement between the start of the AIS and the axon initiating point, as depicted in Figure S4 of the manuscript. We adopted this parameter from the previous study by Grubb et al. (Grubb & Burrone, 2010), ensuring consistency in our investigation of AIS plasticity. For AcD neurons, where the axon branches out from the dendrite, we defined the AIS distance as the length between the start of the AIS and the border of the stem dendrite, as illustrated in Figure S4B.

    In Figure 1, the term "distance from soma" represents the length of stem dendrite and used for AcD and nonAcD neuron classification. As shown in Figure S4G, the absolute distance from the soma for AcD neurons is approximately 10 µm and remains consistent across treatments. We will explain these points more clearly in the revised manuscript.

    Minor comments:

    1. At p. 7 is stated that "The percentage of none-AcD forming collaterals at DIV1 is much lower than for AcD neurons" but statistical support is lacking. The conclusion in the next line is that "AcD neurons follow consensus development". That is puzzling given the difference just mentioned before. Please clarify. Reply: We will provide statistical support for comparing collateral formation between nonAcD and AcD neurons at DIV1.

    Regarding the second point concerning consensus development, we were referring to the general developmental sequence of AcD neurons, as described by Dotti et al. (see Dotti et al., 1988 for reference), where neurons typically first establish an axon and then dendrites. This sequence is not necessary related to collateral formation, which indeed differs between nonAcD and AcD neurons. The ability to form collaterals may come from local differences in microtubule (MT) and actin dynamics at AcD neuron precursor axons, but it does not alter the fact that AcD neurons initially establish an axon and subsequently dendrites. We will clarify it in the revised manuscript.

    A study not cited in this manuscript showed distinct dendritic morphologies (doi: 10.1073/pnas.1607548113) and AcD interneurons are different for their axonal arborization (doi: 10.1242/dev.202305). Differences in growth of branch arborization could hint to subtypes. Are the AcD and non-AcD neurons different in their adult morphology? A detailed account of the axonal and dendritic trees would strengthen the data.

    Reply: Thank you for pointing this out. We will include this citation. In the study by Hodapp et al., it was shown that AcD and nonAcD neurons exhibit similar dendritic morphology and do not differ in spine density, number of dendritic branches, and total dendritic length. However, in hippocampal AcD neurons, the AcD occupies 35% of the total basal dendrite length, which is larger than basal dendrites in nonAcD neurons, suggesting that AcD neurons do possess specific features in their dendritic trees.

    Regarding the axons of AcD neurons, there is currently no detailed study available, and it would be more appropriate to investigate neuronal connectivity through tracing studies in animals rather than in primary cultures. Therefore, this question falls outside the scope of the current manuscript.

    Some key references are not included here, and a number of these are mentioned above. In the context of the detailed MT and Rab3A vesicle and cargo transport studies, please acknowledge some of the pioneering work of Alan Peters revealing the ultrastructure of axons emerging from dendrites. See Figs. 5-7 in Peters, Proskauer and Kaiserman-Abramof IR., J Cell Biol 39:604 (1968). What is the identity of the neurons? It makes a difference if the cells are interneurons or pyramidal neurons, CA1 or CA3-like. For plasticity experiments the authors uses cells as independent measurements, but this is inflating the power. How many cultures were used?

    Reply: Thank you for pointing this out; we will include the suggested references in the revised manuscript. In our study, we focused on excitatory neurons from the hippocampus. We distinguished neuron types morphologically or with the inhibitory neuron marker GAD1. Identifying CA1, CA2, CA3, and DG subtypes in dissociated culture is more challenging, and this would be an interesting avenue to explore in an in vivo system. Here, we focused on fundamental cell biology aspects related to the AIS structure and its trafficking barrier function, which should be similar in all these neuron types. While there may be subtype-specific differences in AIS plasticity, investigating this is beyond the scope of our manuscript.

    For the plasticity experiments, we used a total of 3 independent cultures, from which we collected a comparable number of neurons. In response to the reviewer's concern, we will also plot the mean of each culture to illustrate the variability of our data points.

    Reviewer 4

    Major comments:

    1. A general limitation of this study is the low N for some critical experiments. In several experiments, individual cells become an N, therefore boosting the power of the analysis when in reality, due to the known heterogeneity of AIS length, position, and general cell morphology in vitro, the aim should be to compare means across animals / preparations, each consisting of a comparable number of individual cells. This is especially important for the analyses of COs, axo-axonic synapses and channel expression at the AIS. Reply: We would like to mention that this is a cell biological study where neurons are grown in dissociated cultures. To prepare one such culture, we typically use hippocampi from 6-8 E18 rat embryos, which are then mixed in one suspension before plating. The cells are then plated on coverslips in a 12-well plate format. When referring to replicates, for all experiments except for the longitudinal study of 5-day-long time-lapse imaging of developmental sequences (Figure 1), we used between 3 to 6 independent preparations. From each preparation, we took a comparable number of cells derived from 4-6 different coverslips. For each experiment, we measured more than a hundred cells, which is standard practice in the field. To address the issue with individual measurements, in the revised manuscript, we will additionally plot the means of each independent preparation.

    Such critical parameters as e.g. synaptic innervation at the AIS are investigated in a way that does not support the clear statements given, e.g. "The AIS of AcD neurons receives fewer inhibitory inputs" (Highlights statement) or "AcD neurons have less inhibitory synapses at the AIS" (header of Fig. 6). The overall number of analyzed cells is low (3 and 4 preparations, respectively and approximately 50-cells for each marker). The combination of a pre- and postsynaptic marker for inhibitory / excitatory neurons is a solid decision, but the analysis is not done based on the close approximation of these markers, in 3D, along an AIS, but rather in maxIPs and without any regard of whether pre-and postsynaptic markers are actually close to each other not. The expression of these markers alone just points towards the epitopes being expressed, but are they localized to each other in such a manner that they could form bona fide synapses? The methods are not totally clear on the image depth (tile scans with 5 µm in z will not provide the detail of information to resolve synapses, so how did the authors address the subcellular analysis here and for the CO and VGSCs?). And generally, were Nyquist conditions taken into consideration throughout the study? This can be clarified in text and does not require additional experiments.

    Reply: The overall number of cells for quantifying inhibitory synapses along the AIS was approximately 80 cells for each synaptic marker. To clarify this, we will indicate the number of cells in the figure legend of our revised manuscript and will additionally plot mean values across independent preparations.

    In the current manuscript, our main goal was to provide an initial quantitative measurement of AIS features in AcD neurons to see if they differ from nonAcD neurons. Hence, maxIPs are sufficient for this purpose as they summarize the 3D information. To make our study more comprehensive, following the reviewer's suggestion, we will conduct additional experiments to co-label pre- and post-inhibitory synapses at the AIS with VGAT and gephyrin, respectively. Then, we will image samples in 3D to measure the density as well as the distance between pre- and post-synapses at the AIS of AcD neurons and compare them to nonAcD neurons.

    The Nyquist condition was taken into consideration throughout the study. The pixel size of our data collection was 0.081 µm for the laser scanning microscope, as indicated in our methods section. Given the optical setup of our microscope and the fluorophores used to label target proteins (information available in the methods section of our manuscript), the acceptable Nyquist lateral sampling size (or pixel size, in other words) for confocal images is between 0.083 to 0.093 µm and 0.2 µm in the z-plane. In our data collection for laser scanning confocal images, the z-step size was 0.5 µm (see methods section of our manuscript), which is indeed undersampling the data. However, this should not significantly affect our analysis based on maxIPs. The new stainings with matched pre- and post-synaptic markers will be imaged with a smaller z-step (0.2 µm) and then reconstructed in 3D.

    The chapter on AIS plasticity is certainly an interesting addition to the study, but is a bit superficial, yet reaches strong conclusions ("More importantly, it further indicates that the AIS of AcD neurons is insensitive to activity changes"). This is based on un-physiological concentrations of KCl, and certainly not on network manipulation that truly tests synaptic activity. It also comes back to the 1st point above. A suggestion would be to edit the conclusion.

    Reply: KCl treatment globally depolarizes the membrane potential of neurons, leading to an increase in intracellular calcium via voltage-sensitive calcium channels as well as NMDA and AMPA receptors (Rienecker et al., 2020). This protocol has been used in several initial studies describing the plasticity of the AIS (see Evans et al., 2013, 2017; Grubb & Burrone, 2010; Jamann et al., 2021; Muir & Kittler, 2014; Wefelmeyer et al., 2015 for references). Moreover, as shown by Evans et al. and Grubb et al. (see Evans et al., 2013; Grubb & Burrone, 2010 for references), AIS plasticity is not abolished by TTX, which blocks Na+ channels, but is prevented by L-type calcium channel blockers. This suggests that the occurrence of AIS plasticity is independent of action potentials but more sensitive to calcium-related pathways downstream of membrane potential depolarization and post-synaptic activation. Hence, we believe our results are indicative of how the AIS would react when calcium signaling pathways are altered by activity levels. To address the reviewer's concern, we will focus our conclusion more on membrane potential depolarization and calcium signalling and edit out statements.

    As discussed above in response to reviewer #3, the quantification of AIS plasticity includes 3 independent preparations, comprising approximately 200 neurons in total. To prevent inflation of statistical power in the analysis, we will also plot the means and standard error of the mean (SEM) for each independent experiment and assess whether any differences persist.

    The rationale behind looking at the cisternal organelle (CO) in this study is outlined in the Introduction, where the authors state that "...... and is responsible for calcium handling". What is "calcium-handling" and where is the evidence cited? Furthermore, in the Results, they state that "...both compounds (VGSCs and COs) are critical for the AIS to regulate neuronal excitability". While this is the case for VGSCs, there is no conclusive evidence in the literature whether of not the CO is "critical" for neuronal excitability. In fact, a number of neurons have no CO in the AIS (as much as 50% of all AIS in mouse primary visual cortex for example do not express synpo at the AIS at all, Schlüter et al., 2017). The CO can therefore not be as critical for AP initiation as the authors state. Furthermore, the authors state that "AIS plasticity in excitatory neurons is triggered by calcium signaling". While certainly shown and adequately cited here, other factors (independent of calcium) can also play a role, therefore this statement is a bit absolute and should be edited accordingly.

    Reply: Thank you for constructive editorial suggestions. Regarding the first question on calcium handling, we were referring to Ca2+ storage and release mechanisms. Benedeczky et al. already showed the existence of SERCA-type Ca2+ pumps at the membrane of the cisternal organelle (CO) to demonstrate the involvement of Ca2+ sequestering/storage by the CO at the AIS (Benedeczky et al., 1994). Although indirect, Sánchez-Ponce et al. showed the presence of IP3R, which promotes Ca2+ release from internal storage, at the AIS and partially colocalizes with synaptodin (Sánchez-Ponce et al., 2011). This is also the same case for the Ca2+-binding protein annexin 6. Together, this evidence indicates a putative role of the CO in regulating Ca2+ dynamics (storage/release) at the AIS. Since Ca2+ levels have a significant impact on action potential generation and timing at the AIS (see Bender & Trussell, 2009; Yu et al., 2010 for references), and therefore should be strictly regulated, it is likely that the CO at the AIS is important for regulating neuronal excitability by controlling Ca2+ dynamics. However, as mentioned by the reviewer, there are no conclusive pieces of evidence showing the relationship between the CO and neuron excitability regulation. We will edit our statement accordingly.

    In contrast to the findings of Schlüter et al. (Schlüter et al., 2019), which were conducted in the mouse primary visual cortex, Sánchez-Ponce et al. showed that nearly 90% of hippocampal neurons contain synaptopodin, the CO marker protein, at the AIS. Furthermore, Schlüter et al. also demonstrated that in the other 50% of neurons containing COs at the AIS, the COs change size during visual deprivation, and their presence correlates with AIS length changes as well as eye-opening. These observations do suggest that COs are related to neuronal activity. However, this correlation and the formation of COs may be specific to neuro subtypes or require certain triggers. This is another interesting direction to explore, and we will include it in the discussion of the revised manuscript.

    Regarding the last point on Ca2+ and AIS plasticity, we were not excluding other factors that could potentially participate in AIS plasticity and will also discuss it in the revised version.

    The Introduction ends with the rationale of the study, namely that the authors seek to ....."provide a detailed characterization of the AIS, including its structural and functional properties....". Structure is investigated, but function is limited to the barrier function of the AIS. Since the authors provide no electrophysiology that would really dissect AIS function, I suggest to rephrase this part and focus on transport.

    Reply: As suggested, we will certainly emphasize the cargo barrier function of the AIS in AcD neurons in our introduction. But we would like to keep the term "AIS function", because it has already been nicely demonstrated electrophysiologically by previous studies that the plasticity effect of the AIS is very important for maintaining cellular homeostasis.

    The Discussion is more a list of future plans than a context to current data. The authors could move some of the new questions they identify into an "outlook" section at the end? Also, again have a critical look at the literature that is cited and which statements are accurate.

    For example, the 2nd phrase in the Discussion states that is was shown that AcD neurons have a "role in memory consolidation", referenced to Hodapp et al., 2022. However, that paper does not provide direct evidence of such a role for AcD neurons. The statement "Collectively, our data provide new insights into the development of AcD neurons and demonstrate that there are differences in AIS functionality between AcD and nonAcD neurons", is not correct. AIS function was not investigated outside of the axonal barrier, and here, the AcD and nonAcD cells do not differ. Also, although the Discussion is geared towards excitatory / glutamatergic neurons, it has been shown by others that interneurons show an even stronger trend to exhibit AcD morphology (work by the Wahle lab and others). This is not clear from the current text (also compare "...AcD neurons being a different subtype if pyramidal neuron").

    Further original publications should be included in the paragraph highlighting patch-clamp recordings (see above). In the same context, the statement "...showed that rapid AID plasticity occurs mainly in hippocampal dentate gyrus cells but not in principal excitatory neurons" is not accurate (see Kim, Kuba, Jamann and others). Generally, the Introduction and Discussion would benefit from a very clear distinction between studies done in vitro versus those done ex vivo or in vivo. This needs to be stated in the Abstract as well.

    Methods: For the imaging of synapses, the CO and VGSCs, it is not clear to me from the methods whether Nyquist conditions were applied to produce data that can support the quantification of nanoscale structures. Basing the analysis and interpretation of channel expression on fluorescence intensity profiles is problematic (variance in staining quality from samples to sample, lack of an internal standard). This should be noted in the text. In the text, the first two references given for "Induction of plasticity" do not reference the correct papers.

    Reply: Thank you for the valuable suggestions; we will incorporate them into the revised version of the manuscript. The structure will undoubtedly benefit from these improvements. We will also have a further look into our interpretation of the literatures as well as citations during our revision time frame.

    Regarding methods, as stated in response to the second point raised by this reviewer, we ensured that the Nyquist condition was adhered to throughout the study. The pixel size, z-step size, and optical setup of the microscopes used were already indicated in our methods section. With respect to Na+ channel staining, we were indeed aware of the potential issues posed by the experimental setup, and we will explicitly mention this in our revised manuscript. Additionally, we plan to measure other AIS scaffolding and membrane proteins that anchor Na+ channels to assess for potential changes, which could indirectly support our Na+ channel staining results.

    Finally, the text is lacking a discussion of limitations of the study, especially from a methodological point of view. In the Abstract/Summary already, the authors could point out that this is a pure in vitro study. Interestingly, to this day, AIS relocation during plasticity events has only been shown in cell culture systems, and not in vivo. Therefore, this needs to be put into context here - the chosen system is great for the type of imaging approach presented here, but may look at a type of AIS plasticity that is not seen in vivo.

    Reply: These are very good points. We will include the limitations of the study in the discussion. Indeed, due to technical and methodological challenges, the relocation of the AIS has not yet been demonstrated using animal models. However, in the study by Wefelmeyer et al. (Wefelmeyer et al., 2015), a similar relocation of the AIS resulting from chronic stimulation was observed in hippocampal organotypic slices, and it was accompanied by reduced excitability of neurons. Furthermore, in the same study, neurons with axons/AIS originating from basal dendrites were also mentioned. However, the measurement of chronic AIS plasticity in their study was not performed based on different classes of neuron types. Hence, our work complements their results. Given that the network connectivity of organotypic slices is much closer to real physiological conditions, it is likely that similar plastic adaptations could occur in vivo.

    __Minor comments __

    1. How does intrinsic neuronal activity play into developmental programs in vitro? Electrical activity in maturing neurons is a major part of how networks are shaped, and cells differentiate. This is not genetically encoded per se, but has been shown to be a major driving force of neuronal development in vivo. Is this reflected in the culture setting in any way? And have the authors considered testing early changes in activity patterns in their cultures to see whether AcDs and nonAcDs develop in similar percentages? To clarify, I am not asking for additional experiments. Reply: It is indeed a valid point that activity can influence neuronal morphology. Lehmann et al. (pre-print, doi: https://doi.org/10.1101/2023.07.31.551236) have recently demonstrated that increased network activity leads to more excitatory principal neurons adopting AcD morphology. However, our developmental data were collected from DIV0 to DIV5, an age at which dissociated neurons do not yet form functional excitatory synapses. Therefore, it is highly unlikely that network activity plays a role in shaping AcD neuron development during this early stage.

    The authors may want to add a bit of a technical discussion on the choice of KCl and TTX as triggers for plasticity, especially at the non-physiological concentrations offered here and elsewhere (15 mM KCl).

    Reply: We appreciate the reviewer for pointing this out. We will add this in our revised manuscript.

    Some key statements would benefit from citing the appropriate original literature (some examples would be the original work by Kole, Bender and Brette on the role of the AIS in AP initiation; original work by D'Este and Letterier on the dendritic and axonal scaffold using nanoscopy; work by Kim, Kuba and Jamann on AIS plasticity in vitro and in vivo that is critical for a more informed discussion of AIS plasticity here, and others)

    Reply: These are very good points, we will make suggested edits in the revised version.

    In the Introduction, the authors word their text explicitly for excitatory neurons. However, AIS plasticity has also been observed in interneurons (work by the Grubb lab for example), and axo-axonic synapses are in fact not all inhibitory - this is in important factor to consider given the embryonic state of the culture material. Does the DIV maturation reflect how axo-axonic synapses "switch" from excitatory to inhibitory in vivo (also see work of the Burrone lab)? Can the conclusions form the paper really be drawn based on this type of system?

    Reply: The AIS plasticity was indeed also observed in inhibitory interneurons (see Chand et al., 2015 for reference) and show opposite phenotypes compared to excitatory neurons. Also related to major comment #5, we did take the potential influence of AcD interneurons on the outcome of AIS plasticity experiment into consideration. Therefore, we also did a control experiment where inhibitory interneurons were labelled with GAD1 after chronic KCl treatment and these neurons were excluded from the analysis. Consistently, we got the same results that excitatory AcD neurons do not undergo chronic AIS plasticity. We will include this data in our revised manuscript. Further, in our current manuscript, we decided to focus on excitatory AcD neurons not only because they are the major functional unit in neuronal circuits, but also because the majority of the electrophysiological features were studied in excitatory AcD neurons. But we agree with the reviewer that AcD interneuron is definitely an interesting subject for follow up research in the future.

    As mentioned by the reviewer, Pan-Vazquez et al. (Pan-Vazquez et al., 2020) nicely showed that axo-axonic synapses made by GABAergic Chandelier cells (ChCs) depolarise neurons in brain slices obtained from P12-18 animals. But this effect is reversed in slices obtained from older animals (>>P40). Of note, their results were based on cortical neurons but not hippocampal neurons, hence cell type specificity should be considered. More importantly, previous study reported that this conversion or switch of GABAergic interneurons from excitatory to inhibitory occurs on hippocampal neurons in P12-13 animals (Leinekugel et al., 1995). In dissociated hippocampal neurons from E18 rat embryos, this switch of GABAergic interneurons takes place on DIV9-11 and completes on DIV19, which should have a comparable neuronal developmental stage as the P12-13 in in vivo system (see Ganguly et al., 2001 for reference). Therefore, the conclusion could be drawn in an in vitro system, but it certainly needs to be validated in in vivo system.

    The authors state that "less COs account for higher intrinsic excitability". Why is that the case?

    Reply: According to Yu et al. and Bender et al., Ca2+ transient at the AIS regulates the generation of action potentials (APs). For instance, reducing Ca2+ transient at the AIS by blocking Ca2+ channels with either mibefradil (a T-type Ca2+ channel antagonist) or Ni2+ (which blocks R- and T-type channels) decreased the number of spikelets evoked by EPSP-like current injection and delayed the timing of spike generation (please see Bender & Trussell, 2009 for details). Therefore, we speculate that Ca2+ transients are less affected when there are fewer cisternal organelles (COs) at the AIS, which could have a more direct impact on AP initiation. However, this is just our hypothesis, and there is indeed no direct evidence showing that COs regulate Ca2+ dynamics. We will discuss this in the revised manuscript.

    Last but not least, some very recent studies on AcD biology (Stevens, Thome, Lehmann, Wahle) is available online also on preprint servers and may provide additional support for the current study.

    Reply: We will check these pre-prints and include relevant information into the revised version.

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    Referee #4

    Evidence, reproducibility and clarity

    Summary

    Han and colleagues present cell biological data on the development of axon-carrying dendrite (AcD) neurons - cells, in which the axon emerges from a dendrite and not as is more common, from the soma. This particular morphological feature is not new; Cajal already described AcD neurons and since then, their existence was demonstrated in a number of CNS and PNS neurons and across species. However, more recent work from the rodent hippocampus ex vivo and in vivo has pointed towards an interesting functional consequence of AcD morphology, in that these cells can circumvent perisomatic inhibition during network oscillations. Han and colleagues therefore investigate one of the core questions that arises from these previous studies: How is the AcD configuration achieved during development?

    The authors utilize an in vitro model (isolated hippocampal neurons from E18 rat) and investigate core parameters of axonal maturation, especially the cytoskeleton, membrane-associated proteins and intra-axonal calcium stores of the axon initial segment (AIS), where action potentials are generated and which contributes to neuronal polarity at different days in vitro (= maturation states). Using a combination of immunofluorescence, confocal, spinning disk and STED microscopy, and plasticity protocols, the authors present evidence indicating that during development in vitro, AcD neurons follow an intrinsically encoded developmental program, the AIS in nonAcD and AcD cells has comparable cytoskeletal features and retains cellular polarity in both configurations. Using culture conditions to elicit AIS plasticity, the authors then find differences in that AcD neurons do not seem to undergo AIS plasticity and generally show a reduced number of intra-axonal calcium stores. Also, AcD neurons are shown to have fewer axo-axonic synapses at the AIS than nonAcD neurons.

    Major comments

    This study aims at investigating an important question in AcD biology, and uses an easily-accessible model system (E18 rat derived hippocampal neurons in vitro). In this, the study follows previous work in vitro, and nicely reproduces some data, which is a strength of this current study in my opinion. That said, the system is, by nature, artificial and the emergence of axons in vitro often deviates from data obtained in vivo.

    Throughout the manuscript, the authors often draw clear-cut conclusions which require a far more critical reflection of what their model can actually accomplish. Thus, a number of statements are not supported by the data (see below). The presentation of the data in the Supplements needs to reflect data distribution, which they currently do not. Likewise, showing S.E.M. instead of S.D. needs to be looked at critically. Otherwise, the data and methods are presented in such a way that they can be reproduced. The quality of the micrographs and videos is excellent and convey the main messages of the study in a very accessible way. I do not see the need for additional experiments, but would ask the authors to critically look at the following issues:

    1. A general limitation of this study is the low N for some critical experiments. In several experiments, individual cells become an N, therefore boosting the power of the analysis when in reality, due to the known heterogeneity of AIS length, position, and general cell morphology in vitro, the aim should be to compare means across animals / preparations, each consisting of a comparable number of individual cells. This is especially important for the analyses of COs, axo-axonic synapses and channel expression at the AIS.
    2. Such critical parameters as e.g. synaptic innervation at the AIS are investigated in a way that does not support the clear statements given, e.g. "The AIS of AcD neurons receives fewer inhibitory inputs" (Highlights statement) or "AcD neurons have less inhibitory synapses at the AIS" (header of Fig. 6). The overall number of analyzed cells is low (3 and 4 preparations, respectively and approximately 50-cells for each marker). The combination of a pre- and postsynaptic marker for inhibitory / excitatory neurons is a solid decision, but the analysis is not done based on the close approximation of these markers, in 3D, along an AIS, but rather in maxIPs and without any regard of whether pre-and postsynaptic markers are actually close to each other not. The expression of these markers alone just points towards the epitopes being expressed, but are they localized to each other in such a manner that they could form bona fida synapses? The methods are not totally clear on the image depth (tile scans with 5 µm in z will not provide the detail of information to resolve synapses, so how did the authors address the subcellular analysis here and for the CO and VGSCs?). And generally, were Nyquist conditions taken into consideration throughout the study? This can be clarified in text and does not require additional experiments.
    3. The chapter on AIS plasticity is certainly an interesting addition to the study, but is a bit superficial, yet reaches strong conclusions ("More importantly, it further indicates that the AIS of AcD neurons is insensitive to activity changes"). This is based on unphysiological concentrations of KCl, and certainly not on network manipulation that truly tests synaptic activity. It also comes back to the 1st point above. A suggestion would be to edit the conclusion.
    4. The rationale behind looking at the cisternal organelle (CO) in this study is outlined in the Introduction, where the authors state that "...... and is responsible for calcium-handling". What is "calcium-handling" and where is the evidence cited? Furthermore, in the Results, they state that "...both compounds (VGSCs and COs) are critical for the AIS to regulate neuronal excitability". While this is the case for VGSCs, there is no conclusive evidence in the literature whether of not the CO is "critical" for neuronal excitability. In fact, a number of neurons have no CO in the AIS (as much as 50% of all AIS in mouse primary visual cortex for example do not express synpo at the AIS at all, Schlüter et al., 2017). The CO can therefore not be as critical for AP initiation as the authors state. Furthermore, the authors state that "AIS plasticity in excitatory neurons is triggered by calcium signaling". While certainly shown and adequately cited here, other factors (independent of calcium) can also play a role, therefore this statement is a bit absolute and should be edited accordingly.
    5. The Introduction ends with the rationale of the study, namely that the authors seek to ....."provide a detailed characterization of the AIS, including its structural and functional properties....". Structure is investigated, but function is limited to the barrier function of the AIS. Since the authors provide no electrophysiology that would really dissect AIS function, I suggest to rephrase this part and focus on transport.
    6. The Discussion is more a list of future pans than a context to current data. The authors could move some of the new questions they identify into an "outlook" section at the end? Also, again have a critical look at the literature that is cited and which statements are accurate. For example, the 2nd phrase in the Discussion states that is was shown that AcD neurons have a "role in memory consolidation", referenced to Hodapp et al., 2022. However, that paper does not provide direct evidence of such a role for AcD neurons. The statement "Collectively, our data provide new insights into the development of AcD neurons and demonstrate that there are differences in AIS functionality between AcD and nonAcD neurons", is not correct. AIS function was not investigated outside of the axonal barrier, and here, the AcD and nonAcD cells do not differ. Also, although the Discussion is geared towards excitatory / glutamatergic neurons, it has been shown by others that interneurons show an even stronger trend to exhibit AcD morphology (work by the Wahle lab and others). This is not clear from the current text (also compare "...AcD neurons being a different subtype if pyramidal neuron"). Further original publications should be included in the paragraph highlighting patch-clamp recordings (see above). In the same context, the statement "...showed that rapid AID plasticity occurs mainly in hippocampal dentate gyrus cells but not in principal excitatory neurons" is not accurate (see Kim, Kuba, Jamann and others). Generally, the Introduction and Discussion would benefit from a very clear distinction between studies done in vitro versus those done ex vivo or in vivo. This needs to be stated in the Abstract as well.

    Methods: For the imaging of synapses, the CO and VGSCs, it is not clear to me from the methods whether Nyquist conditions were applied to produce data that can support the quantification of nanoscale structures. Basing the analysis and interpretation of channel expression on fluorescence intensity profiles is problematic (variance in staining quality from samples to sample, lack of an internal standard). This should be noted in the text. In the text, the first two references given for "Induction of plasticity" do not reference the correct papers.

    Finally, the text is lacking a discussion of limitations of the study, especially from a methodological point of view. In the Abstract/Summary already, the authors could point out that this is a pure in vitro study. Interestingly, to this day, AIS relocation during plasticity events has only been shown in cell culture systems, and not in vivo. Therefore, this needs to be put into context here - the chosen system is great for the type of imaging approach presented here, but may look at a type of AIS plasticity that is not seen in vivo.

    Minor comments

    1. How does intrinsic neuronal activity play into developmental programs in vitro? Electrical activity in maturing neurons is a major part of how networks are shaped, and cells differentiate. This is not genetically encoded per se, but has been shown to be a major driving force of neuronal development in vivo. Is this reflected in the culture setting in any way? And have the authors considered testing early changes in activity patterns in their cultures to see whether AcDs and nonAcDs develop in similar percentages? To clarify, I am not asking for additional experiments.
    2. The authors may want to add a bit of a technical discussion on the choice of KCl and TTX as triggers for plasticity, especially at the non-physiological concentrations offered here and elsewhere (15 mM KCl).
    3. Some key statements would benefit from citing the appropriate original literature (some examples would be the original work by Kole, Bender and Brette on the role of the AIS in AP initiation; original work by D'Este and Letterier on the dendritic and axonal scaffold using nanoscopy; work by Kim, Kuba and Jamann on AIS plasticity in vitro and in vivo that is critical for a more informed discussion of AIS plasticity here, and others)
    4. In the Introduction, the authors word their text explicitly for excitatory neurons. However, AIS plasticity has also been observed in interneurons (work by the Grubb lab for example), and axo-axonic synapses are in fact not all inhibitory - this is in important factor to consider given the embryonic state of the culture material. Does the DIV maturation reflect how axo-axonic synapses "switch" from excitatory to inhibitory in vivo (also see work of the Burrone lab)? Can the conclusions form the paper really be drawn based on this type of system?
    5. The second header in Results is not clearly formulated. What is meant by "consensus developmental sequence"?
    6. The authors state that "less COs account for higher intrinsic excitability". Why is that the case?
    7. Last but not least, some very recent studies on AcD biology (Stevens, Thome, Lehmann, Wahle) is available online also on preprint servers and may provide additional support for the current study.

    Referees cross-commenting

    In my opinion, the comments by the other three reviewers are clear, insightful and supportive of / complementary to my own. There are some strong leads within the revisions that the authors hopefully find helpful in the preparation of their final manuscript. I have no doubt that the final publication will be viewed as an important and significant finding in the field of axon onset biology.

    Significance

    The study by Han and colleagues addresses a timely and relevant question and provides excellent quality imaging data (fixed cells and live imaging), as well as convincing superresolution. The authors also provide a solid methods section that will aid others in repeating these experiments. The central question of how AcD neurons develop is of great interest and the study highlights novel findings especially regarding the detailed analysis of axonal and dendritic transport features in AcD cells. The authors also point out a number of questions that arise from their data and that can provide helpful insight for other researchers in the field. The study has limitations in that it is a pure in vitro study, and some data are based on a low sample number as well as superficial expression analysis (synapses, channels). A number of conclusions made by the authors are therefore not really supported by the current data. The discussion would benefit from a more detailed analysis of the current literature in the field and needs critical reflection on what the shown data really support in form of a section on "limitations".

    The study is of broader interest for numerous research fields (developmental neurobiology, axon biology, AIS biology, neuronal plasticity). Given the focus that AcD neurons have received recently in the field of learning and memory consolidation, this study also provides interesting future questions for researchers with a background in network function and behavior.

    Reviewer's expertise: Developmental neurobiology, axonal plasticity, AcD neuron morphology and development, in vivo rodent behavior, human slices, confocal and superresolution microscopy, patch-clamp electrophysiology

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    Referee #3

    Evidence, reproducibility and clarity

    In this manuscript Han and colleagues report about structural and functional studies on the development of axons originating from dendrites. Leveraging the primary hippocampal neuron preparation, they investigated fundamental cell biological questions including microtubule organization, cargo transport and the early neurite development. I am impressed by the timelapse movies with live AIS labels providing, to the best of my knowledge, the first glance into the development of an axon emerging from a dendrite. The study is technically very good, a pleasure to read, and the results are well described. While conclusions about structure are well supported by their data, the claims about 'function' are weak and speculative. I have listed some issues and by improving clarity the study could become a valuable resource for the field.

    1. The authors classify neurons into axon-carrying dendrite (AcD) and non-AcD neurons by measuring the stem dendrite length (> 3 µm). I could not find the validity for this cut-off. The non-AcD neurons in Fig. 6B appear more AcD to this reviewer, and, in addition, other researchers have proposed a third category of 'shared root' neurons (doi: 10.7554/eLife.76101). For purposes of reproducibility and transparency, please provide first a comprehensive overview of the entire population of morphologies (i.e. all cells in control conditions). The distances from the soma could be plotted in histogram (etc.) and authors may want to think about independent supporting evidence for the cut-off to classify AcD and non-AcD neurons.
    2. Related to point #1 the primary hippocampal neuron system is excellent for cell biological questions but comes with the drawback of imaginative morphologies including neurons with multiple axons and AISs. It is not mentioned here but literature indicates up to 20% of neurons have two axons (e.g. doi: 10.1007/s12264-017-0169-3, 10.1083/jcb.200707042). How did the authors classify the double axon cells? Since the main hypothesis is the existence of an intrinsic program for AcD neurons (p. 5 top), the two axons from one neuron should develop similarly. The authors can easily test this with the data.
    3. Some interpretations about function are not correct and the authors should reconsider these. A role of cisternal organelles on neuronal excitability remains to be demonstrated (and see doi.org/10.1002/cne.21445 showing there is none). In addition, the statement that lower fluorescence intensity of Pan-Nav1 is indicating reduced excitability is flawed. Antibody staining does not scale linearly with voltage-gated sodium channel density and since the AIS of AcD neurons is further from the soma it is most likely smaller in diameter which may account for apparent fluorescent differences. For biophysical reasons (for details I refer to 10.3389/fncel.2019.00570, 10.1016/j.conb.2018.02.016 and 10.7554/eLife.53432) smaller diameter axons will be easier to depolarize by depolarizing voltage-gated channels or excitatory synapses. Finally, in AcD neurons the AIS distance from the soma poses all sorts of interesting cable properties with the soma and the local dendritic membrane and the electrotonic properties alone suffice to make these neurons more excitable.
    4. Comparing AcD and non-AcD neurons for AIS plasticity is an excellent idea but the present statistical design is not suitable for answering this question. The authors should directly compare non-AcD and AcD neurons within a two-way ANOVA design, asking the question whether the independent variable axon type is significantly different and interacts with plasticity. Related points: 'AIS distance' in Figure 7 seems to refer to something else than distance from soma (Figure 1). Please clarify. What were the absolute distances from the soma for the AcD neurons and was this dependent on treatment?

    Minor comments

    At p. 7 is stated that "The percentage of none-AcD forming collaterals at DIV1 is much lower than for AcD neurons" but statistical support is lacking. The conclusion in the next line is that "AcD neurons follow consensus development". That is puzzling given the difference just mentioned before. Please clarify. A study not cited in this manuscript showed distinct dendritic morphologies (doi: 10.1073/pnas.1607548113) and AcD interneurons are different for their axonal arborization (doi: 10.1242/dev.202305). Differences in growth of branch arborization could hint to subtypes. Are the AcD and non-AcD neurons different in their adult morphology? A detailed account of the axonal and dendritic trees would strengthen the data.

    Some key references are not included here, and a number of these are mentioned above. In the context of the detailed MT and Rab3A vesicle and cargo transport studies, please acknowledge some of the pioneering work of Alan Peters revealing the ultrastructure of axons emerging from dendrites. See Figs. 5-7 in Peters, Proskauer and Kaiserman-Abramof IR., J Cell Biol 39:604 (1968).

    What is the identity of the neurons? It makes a difference if the cells are interneurons or pyramidal neurons, CA1 or CA3-like.

    For plasticity experiments the authors uses cells as independent measurements, but this is inflating the power. How many cultures were used?

    Referees cross-commenting

    When reading the other reviews I feel they are constructive and providing sufficient conceptual and technical insight to prepare a revision. Although some concerns are overlapping, with 4 independent review reports perhaps not all issues can be addressed within the estimated time frame of 3 months.

    Significance

    That axons originate from dendrites dates back to the 19th century drawings of Ramón y Cajal but today most textbook and schematic drawings of the neuron still show polarized axons and dendrites both emerging from the soma. Since a few years this specific morphological arrangement begins to receive attention and many fundamental cell biological questions remain to be answered. Leveraging the primary hippocampal neuron preparation, the authors use technically clever experiments to generate new insight into the microtubule organization, cargo transport and the early neurite development. The live imaging of fluorescent labelled axon initial segments is elegant, and an important conclusion is that the stem process, carrying dendrite and axon, grows at a later stage in development. Limitations of the primary neurons should be discussed, however, and the functional consequences of positioning axons on dendrites are not as simple as described by the authors. The study could become a valuable resource for those working in basic research, providing new technical directions.

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    Referee #2

    Evidence, reproducibility and clarity

    The manuscript by Han et al. investigates the properties of AIS along axon carrying dendrites (AcDs). These are enigmatic structures with at present poorly defined features. Han et al. work to further characterize the nature of these AIS. Overall, the data are mostly compelling and the reveal that AIS at AcDs are mostly like those of AIS arising from the cell body. Many features were examined and shown not to differ. There were a few instances where the authors claim differences, and this reviewer is not convinced - see comments below. Overall, I think with bit more careful examination of the main differences this could be a nice descriptive paper reporting features of AIS along AcDs.

    Major questions:

    1. The authors suggest that there is reduced Na+ channel density at AcD AIS compared to other AIS arising from the cell body. This is not convincing. Immunostaining for Na+ channels is notoriously difficult and sensitive to fixation since the epitopes of the anti-Pan Nav antibodies are highly sensitive to fixation. In addition, this is based on immunofluorescence intensity quantification. Since the mechanism of localization is through binding to AnkG, the authors should also measure other AIS proteins like AnkG, b4 spectrin, and Nfasc. Do these change? If all uniformly change I would be much more inclined to accept the conclusion. If they do not change, it still doesn't rule out the concern about fixation conditions and slight differences in the cultures. The authors indicate there is about a 40% reduction in fluorescence intensity. That is quite large. This big difference should also be confirmed in brain sections.
    2. The analysis of inhibitory synapse differences at the AIS are also not compelling - this is a limitation of the culture system. The authors have no control over the density of inhibitory neurons in the culture well. This interaction is not intrinsic to the AcD neuron, but rather a feature of neuron-neuron interactions which should only be modeled in the animal.
    3. Finally, this reviewer is also skeptical of the chronic plasticity changes in AIS 'distance.' The authors claim their results are consistent with prior reports, but they see about a 1.5 um shift. Prior studies (Grubb et al.) report 15-17 um change - a full order of magnitude larger than what is reported here. The authors also show no differences in other previously described changes at the AIS. Together with the other results showing AcD neurons and non- AcD neuron AIS are mostly the same, the conclusion that one behaves differently is not compelling with the tiny shift reported.
    4. Finally, the major limitation of this study is that it is performed in vitro. Surprisingly, the authors actually argue this is a feature of their system. While it is true some of the questions can be addressed perfectly well in vitro, many cannot. In the first paragraph of the results the authors state an advantage of their system is that there are no microenvironments to influence the development of the AcDs. I'm afraid I view this as a drawback. The authors suggest this is an opportunity to examine intrinsic mechanisms of development - true, but it also foregoes the opportunity to determine if the outcomes are different from what occurs in vivo. To this point, the authors report that only 15-20% of the population of hippocampal neurons in culture are AcD neurons. But in their introduction they cite other literature indicating 50% of hippocampal neurons in vivo are AcD neurons - this suggests that the environment of the hippocampus in vivo influences whether a neuron becomes an AcD neuron or not.
    5. I appreciated the balanced discussion of whether this is a stochastic or genetically programmed process. This could have been emphasized earlier in the results since the authors invoke the concept that "...their development must be driven by genetically encoded factors rather than specific...". The authors have not shown this and cannot show it in this system. Indeed, as stated in point 4 above, I think their data argue against a simple genetic program.

    Significance

    interesting subject, timely as features of AIS are of great interest now - especially as a relatively new form of neuronal plasticity. Highly descriptive paper, but emphasizes in this reviewer's opinion that AcD neurons and non AcD neurons have AIS that are essentially the same.

  5. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    The manuscript by Han et al. describes the structural and functional differences between pyramidal cells in which the axon emanates from a basal dendrite (axon-carrying dendrite cell, AcD cell) and cells with a 'canonical', i.e. somatic origin of the axon (nonAcD cells). They investigate how pyramidal neurons develop into AcD or nonAcD cells during cell development and characterize the cytoskeletal architecture in the two cell classes. Additionally, they examine whether and how axon initial segments, the most important structure for action potential generation, change upon varying activity of the neuron.

    The major claims of the paper are:

    i) The formation into an AcD or nonAcD cell is intrinsically encoded by a developmental program.

    ii) The cytoskeletal structure of AcD and nonAcD cells is similar. However, the stem dendrite inherent only to AcD cells is structurally more similar to an axon than to a dendrite

    iii) Axon initial segments of AcD cells contain less cisternal organelles and show less homeostatic plasticity The authors make use of primary cell cultures from rat hippocampus which are a standard model to investigate developmental questions of single cells and neuronal networks. The manuscript is well structured and in general reads well and the data and conclusions are convincing. I have only a few major comments.

    Major comments:

    The authors cite that acetylated and tyrosinated microtubules have different spatial and compartmental distribution in dendrites and axons and investigate the distribution in the AIS of nonAcD cells and AcD cells, as well as the stem dendrites. However, they just show one example of two different cells (Figure 2D and E) without any statistical analysis. Either, they should remove this part or provide a thorough quantification. The authors use EGFP-Rab3A vesicle to investigate anterograde transport at the axon and dendrites. They find a slightly faster transport of these vesicles at the AIS of AcD cells and conclude the axonal cargos in general are transported faster across the AIS in AcD cells. In my opinion, this generalization based on one type of vesicle is too far-fetched. As stated above, the manuscript is well structured and generally reads well. However, throughout the text there are always small mistakes that should be corrected by careful proofreading. Examples are

    Page 6, last paragraph: ...AcD neurons generated [a] collateral...

    Page 24, last paragraph: ... line was then drew [drawn] along ...

    Page 24, last paragraph: Neurons with ... was consider [were considered] as ...

    Page 25, first paragraph: Antibodies ... was [were]

    Page 41, (E) Percentage of AcD neurons [that] generate [a] collateral or bifurcate

    Minor comments:

    In the introduction, the authors describe how synaptic inputs are received at the dendrites and propagated to the soma in the form of membrane depolarizations. They should add 'excitatory' to synaptic inputs or also describe the impact of inhibitory synaptic inputs at the dendrites.

    In my opinion, Figure 2 could be presented in a slightly better way. The lower part of panel A better fits to panel B, which is next to the upper part of panel A. I understand that the authors systematically present their data first for nonAcD cells and then for AcD cells. However, in this special case it is a little bit more difficult to read the current figure in that order.

    The results displayed in Figure 4 are presented in a slightly confusing order. The authors jump from 4D to 4G, then to 4I and 4E, 4H, 4F. Similarly, 4M and N are addressed before 4O and P to finally get to 4K and L. It would be beneficial to present and address the data in a stringent way.

    Significance

    General assessment:

    This study addresses a very important and timely question about structural and functional cell diversity of cortical pyramidal neurons. The specific function of AcD cells is currently mostly unknown, which is astonishing given their abundance of 15-50% of pyramidal neurons in cortical structures.

    Advance:

    This study presents a significant step forward in comprehending the structural and functional relationship of signal computation in single neurons.

    Audience:

    The study will be important for a wide readership working on very different levels including cellular, network, and behavioral neuroscience.

  6. Version published to 10.1101/2023.09.14.557747v2 on bioRxiv
  7. Version published to 10.1101/2023.09.14.557747v1 on bioRxiv