Author response:
The following is the authors’ response to the previous reviews
Public Reviews:
Reviewer #1 (Public review):
Summary:
The Authors investigated the anatomical features of the excitatory synaptic boutons in layer 1 of the human temporal neocortex. They examined the size of the synapse, the macular or the perforated appearance and the size of the synaptic active zone, the number and volume of the mitochondria, the number of the synaptic and the dense core vesicles, also differentiating between the readily releasable, the recycling and the resting pool of synaptic vesicles. The coverage of the synapse by astrocytic processes was also assessed, and all the above parameters were compared to other layers of the human temporal neocortex. The Authors conclude that the subcellular morphology of the layer 1 synapses is suitable for the functions of the neocortical layer, i.e. the synaptic integration within the cortical column. The low glial coverage of the synapses might allow the glutamate spillover from the synapses enhancing synaptic crosstalk within this cortical layer.
Strengths:
The strengths of this paper are the abundant and very precious data about the fine structure of the human neocortical layer 1. Quantitative electron microscopy data (especially that derived from the human brain) are very valuable, since this is a highly time- and energy consuming work. The techniques used to obtain the data, as well as the analyses and the statistics performed by the Authors are all solid, strengthen this manuscript, and mainly support the conclusions drawn in the discussion.
Comments on latest version:
The corrected version of the article titled “Ultrastructural sublaminar specific diversity of excitatory synaptic boutons in layer 1 of the adult human temporal lobe neocortex" has been improved thanks to the comments and suggestions of the reviewers. The Authors implemented several of my comments and suggestions. However, many of them were not completed. It is understandable that the Authors did not start a whole new series of experiments investigating inhibitory synapses (as it was a misunderstanding affecting 2 reviewers from the three). But the English text is still very hard to understand and has many mistakes, although I suggested to extensively review the use of English. Furthermore, my suggestion about avoiding many abbreviations in the abstract, analyse and discuss more the perforated synapses, the figure presentation (Figure 3) and including data about the astrocytic coverage in the Results section were not implemented. My questions about the number of docked vesicles and p10 vesicles, as well as about the different categories of the vesicle pools have not been answered neither. Many other minor comments and suggestions were answered, corrected and implemented, but I think it could have been improved more if the Authors take into account all of the reviewers' suggestions, not only some of them. I still have several main and minor concerns, with a few new ones as well I did not realize earlier, but still think it is important.
We would like to thank the reviewer for the comments.
- We worked on the English again and tried to improve the language.
- We avoided to use too many abbreviations in the Abstract and reduced them to a minimum.
- We included a small paragraph about non-perforated vs. perforated active zones in both the Results and Discussion sections. However, since the majority of active zones in all cortical layers of the human TLN were of the macular type, we concluded that it is not relevant to describe their function in more detail.
- In Figure 3 A-C we added contour lines to the boutons to make their outlines more visible.
- We completed the data about the astrocytic coverage in the Results section (see also below).
- Concerning the vesicle pools please see below.
Main concerns:
(1) Epileptic patients:
As all patients were epileptic, it is not correct to state in the abstract that non-epileptic tissue was investigated. Even if the seizure onset zone was not in the region investigated, seizures usually invade the temporal lobe in TLE. If you can prove that no spiking activity occurred in the sample you investigated and the seizures did not invade that region, then you can write that it is presumably non-epileptic. I would suggest to write “L1 of the human temporal lobe neocortical biopsy tissue". See also Methods lines 608-612. Write only “non-epileptic" or “non-affected" if you verified it with EcoG. If this was the case, please write a few sentences about it in the Methods.
We rephrased Material and Methods concerning this point and added that patients were monitored with EEG, MRI and multielectrode recordings. In addition, we stated that the epileptic focus was always far away from the neocortical tissue samples. Furthermore, we added a small paragraph that functional studies using the same methodology have shown that neocortical access tissue samples taken from epilepsy surgery do not differ in electrophysiological properties and synaptic physiology when compared with acute slice preparations in experimental animals and we quoted the relevant papers.
We hope that the reviewer is now convinced that our tissue samples can be regarded as non-affected.
(2) About the inhibitory/excitatory synapses.
Since our focus was on excitatory synaptic boutons as already stated in the title we have not analyzed inhibitory SBs. Now, I do understand that only excitatory synapses were investigated. Although it was written in the title, I did not realized, since all over the manuscript the Authors were writing synapses, and were distinguishing between inhibitory and excitatory synapses in the text and showing numerous excitatory and inhibitory synapses on Figure 2 and discussing inhibitory interneurons in the Discussion as well. Maybe this was the reason why two reviewers out of the three (including myself) thought you investigated both types of synapses but did not differentiated between them. So, please, emphasize in the Abstract (line 40), Introduction (for ex. line 92-97) and the Discussion (line 369) that only excitatory synaptic boutons were investigated.
As this paper investigated only excitatory synaptic boutons, I think it is irrelevant to write such a long section in the Discussion about inhibitory interneurons and their functions in the L1 of the human temporal lobe neocortex. Same applies to the schematic drawing of the possible wiring of L1 (Figure 7). As no inhibitory interneurons were examined, neither the connection of the different excitatory cells, only the morphology of single synaptic boutons without any reference on their origin, I think this figure does not illustrate the work done in this paper. This could be a figure of a review paper about the human L1, but is inappropriate in this study.
We followed the reviewer’s suggestion and pointed out explicitly that we only investigated excitatory synaptic boutons. We also changed the Discussion and focused more on circuitry in L1 and the role of CR-cells.
(3) Perforated synapses
The findings of the Geinismann group suggesting that perforated synapses are more efficient than non-perforated ones is nowadays very controversially discussed” I did not ask the Authors to say that perforated synapses are more efficient. However, based on the literature (for ex. Harris et al, 1992; Carlin and Siekievitz, 1982; Nieto-Sampedro et al., 1982) the presence of perforated synapses is indeed a good sign of synapse division/formation - which in turn might be coupled to synaptic plasticity (Geinisman et al, 1993), increased synaptic activity (Vrensen and Cardozo, 1981), LTP (Geinisman et al, 1991, Harris et al, 2003), pathological axonal sprouting (Frotscher et al, 2006), etc. I think it is worth mentioning this at least in the Discussion.
We agree with the reviewer and added a small paragraph in the Results section about the two types of AZs in L1 of the human TLN. We pointed out that there are both types, macular non-perforated and perforated AZs, but the majority in all layers were of the non-perforated type. In the Discussion we added some paper pointing out the role of perforated synapses.
(4) Question about the vesicle pools
Results, Line 271: Still not understandable, why the RRP was defined as {less than or equal to}10 nm and {less than or equal to}20nm. Why did you use two categories? One would be sufficient (for example {less than or equal to}20nm). Or the vesicles between 10 and 20nm were considered to be part of RRP? In this case there is a typo, it should be {greater than or equal to}10 nm and {less than or equal to}20nm.
The answer of the Authors was to my question raised: We decided that also those very close within 10 and 20 nm away from the PreAZ, which is less than a SV diameter may also contribute to the RRP since it was shown that SVs are quite mobile.
This does not clarify why did you use two categories. Furthermore, I did not receive answer (such as Referee #2) for my question on how could you have 3x as many docked vesicles than vesicles {less than or equal to}10nm. The category {less than or equal to}10nm should also contain the docked vesicles. Or if this is not the case, please, clarify better what were your categories.
We thank the reviewer for pointing out that mentioning two distance criteria (p10 and p20) to define one physiological entity (RRP) is somewhat confusing and we acknowledge that the initial response to the reviewers falls short of explaining this choice. This is indeed only understandable in the context of the original paper by Sätzler et al. 2002, where these criteria were first introduced. We therefore referenced this publication more prominently in the paragraph in question.
So to explain this, we first would like to clarify the definition of the two RRP classification criteria used (p10 and p20), which has caused some confusion amongst the reviewers as to which vesicles where included or not:
- p10 criterion: p£10 nm (SVs have a minimum distance less than or equal to 10 nm from the PreAZ), including ‘docked’ vesicles which have a distance of zero or less (p0)
- p20 criterion: p£20 nm (SVs have a minimum distance less than or equal to 20 nm from the PreAZ), including vesicles of the p10 criterion.
As mentioned, these criteria were introduced first in Sätzler et al. 2002 looking at the Calyx of Held synapse. In that paper, we tried to establish a morphological correlate to existing physiological measurements, which included the RRP. As there is no known marker that would allow to discriminate between vesicles that contribute to the RRP anatomically, we looked at existing physiological experiments such as Schneggenburger et al. 1999; Wu and Borst 1999; Sun and Wu 2001 and compared their total numbers to our measurements. As the number of docked vesicles (p0, see above) was on the lower side of these physiological estimates, we also looked at vesicles close to the AZ, which we think could be recruited within a short time (£ 10 msec). Comparing with existing literature, we found that at p20 we get pool sizes comparable to midrange estimates of reported RRP sizes. In order to account for the variability of the observed physiological pool sizes, we reported all three measurements (p0, p10, p20) not only in the original Calyx of Held, but in all subsequent studies of different CNS synapses of our group since then.
As it remains uncertain if such correlate indeed exists, we therefore followed the suggestion to rephrase RRP and RP to putative RRP and putative RP (see also Rollenhagen et al. 2007). We thank both reviewers for pointing out this omission.
Concerning the difference between ‘docked’ vesicles and vesicles within the p10 perimeter criterion. First of all, the reviewer is right in saying that the category p10 ({less than or equal to}10nm) should also contain the docked vesicles (see above). The fact to have 3x as many ‘docked’ vesicles in our TEM tomography than in the p10 distance analysis could be partly explained, on the one hand, by a very high variability between patients (as expressed by the high SD, table 1) and, on the other hand, by a high intraindividual synaptic bouton variability. In both sublayers, there is a huge difference in the number of vesicles within the p10 criterion of individual synaptic boutons ranging from 0 to ~40 with a mean value of ~1 to ~4 (calculated per patient), the upper level being close to the values calculated with TEM tomography for the ‘docked’ vesicles.
(5) Astrocytic coverage
On Fig. 6 data are presented on the astrocytic coverage derived from L1 and L4. In my previous review I asked to include this in the text of the Results as well, but I still do not see it. It is also lacking from the Results how many samples from which layer were investigated in this analysis. Only percentages are given, and only for L1 (but how many patients, L1a and/or L1b and/or L4 is not provided). In contrast, Figure 6 and Supplementary Table 2 (patient table) contains the information that this analysis has been made in L4 as well. Please, include this information in the text as well (around lines 348-360).
In our previous revised version, we had included the values shown in Fig. 6 for both L1 and L4 in the Results section (L4: lines 352 – 355: ‘The findings in L1…’). However, we agree with the reviewer and have now also added the number of patients and synapses investigated (now lines 359 – 365).
About how to determine glial elements. I cannot agree with the Authors that glial elements can be determined with high certainty based only on the anatomical features of the profiles seen in the EM. “With 25 years of experience in (serial) EM work" I would say, that glial elements can be very similar to spine necks and axonal profiles.
All in all, if similar methods were used to determine the glial coverage in the different layers of the human neocortex, than it can be compared (I guess this is the case). However, I would say in the text that proper determination would need immunostaining and a new analysis. This only gives an estimation with the possibility of a certain degree of error.
We do not entirely agree with the reviewer on this point. As stated in the text, there are structural criteria to identify astrocytic elements (see citations quoted). These golden standard criteria are commonly used also by other well-known groups (DeFelipe and co-workers, Francisco Clasca and co-workers; Michael Frotscher the late and co-workers etc.). However, in a past paper about astrocytic coverage of synaptic complexes in L5 of the human TLN, immunohistochemistry against glutamine synthetase, a key enzyme in astrocytes, was carried out to describe the coverage. This experiment supports our findings in the other cortical layers of the human TLN. As the reviewer might know, immunohistochemistry always led to a reduction in ultrastructural preservation, so we decided not to use immunohistochemistry for the further publications of the other cortical layers. We added a short notice on this in the Material and Methods section.
(6) Large interindividual differences in the synapse density should be discussed in the Discussion.
As suggested by the reviewer we have included a sentence in the Discussion that interindividual differences can be either related to differences in age, gender and the use of different methodology as suggested by DeFelipe and co-workers (1999)
Reviewer #2 (Public review):
Summary:
The study of Rollenhagen et al examines the ultrastructural features of Layer 1 of human temporal cortex. The tissue was derived from drug-resistant epileptic patients undergoing surgery, and was selected as further from the epilepsy focus, and as such considered to be non-epileptic. The analyses has included 4 patients with different age, sex, medication and onset of epilepsy. The MS is a follow-on study with 3 previous publications from the same authors on different layers of the temporal cortex:
Layer 4 - Yakoubi et al 2019 eLife
Layer 5 - Yakoubi et al 2019 Cerebral Cortex,
Layer 6 - Schmuhl-Giesen et al 2022 Cerebral Cortex
They find, the L1 synaptic boutons mainly have single active zone a very large pool of synaptic vesicles and are mostly devoid of astrocytic coverage.
Strengths:
The MS is well written easy to read. Result section gives a detailed set of figures showing many morphological parameters of synaptic boutons and surrounding glial elements. The authors provide comparative data of all the layers examined by them so far in the Discussion. Given that anatomical data in human brain are still very limited, the current MS has substantial relevance.
The work appears to be generally well done, the EM and EM tomography images are of very good quality. The analyses is clear and precise.
Weaknesses:
The authors made all the corrections required, answered most of my concerns, included additional data sets, and clarified statements where needed.
My remaining points are:
Synaptic vesicle diameter (that has been established to be ~40nm independent of species) can properly be measured with EM tomography only, as it provides the possibility to find the largest diameter of every given vesicle. Measuring it in 50 nm thick sections result in underestimation (just like here the values are ~25 nm) as the measured diameter will be smaller than the true diameter if the vesicle is not cut in the middle, (which is the least probable scenario). The authors have the EM tomography data set for measuring the vesicle diameter properly.
We thank the reviewer for the helpful comments. We followed the recommendation to measure the vesicle diameter using our TEM tomography tilt series, but came to similar results concerning this synaptic parameter. As stated in our Material and Methods section, we only counted (measured) clear ring-link structures according to a paper by Abercrombie (1963). Since our results are similar for both methods, we do believe that our measurements are correct. Even random single measurements on the original 3D tilt-series yielded comparable results (Lübke and co-workers, personal observation). Furthermore, our results are within ranges, although with high variability, also described by other groups (see discussion lines 436 - 449). We therefore hope that the reviewer will now accept our measurements.
It is a bit misleading to call vesicle populations at certain arbitrary distances from the presynaptic active zone as readily releasable pool, recycling pool and resting pool, as these are functional categories, and cannot directly be translated to vesicles at certain distances. Even it is debated whether the morphologically docked vesicles are the ones, that are readily releasable, as further molecular steps, such as proper priming is also a prerequisite for release.
It would help to call these pools as "putative" correlates of the morphological categories.
We followed the suggestion by the reviewer and renamed our vesicle pools as putative RRP, putative RP and putative resting pools.
Reviewer #3 (Public review):
Summary:
Rollenhagen at al. offer a detailed description of layer 1 of the human neocortex. They use electron microscopy to assess the morphological parameters of presynaptic terminals, active zones, vesicle density/distribution, mitochondrial morphology and astrocytic coverage. The data is collected from tissue from four patients undergoing epilepsy surgery. As the epileptic focus was localized in all patients to the hippocampus, the tissue examined in this manuscript is considered non-epileptic (access) tissue.
Strengths:
The quality of the electron microscopic images is very high, and the data is analyzed carefully. Data from human tissue is always precious and the authors here provide a detailed analysis using adequate approaches, and the data is clearly presented.
Weaknesses:
The text connects functional and morphological characteristics in a very direct way. For example, connecting plasticity to any measurement the authors present would be rather difficult without any additional functional experiments. References to various vesicle pools based on the location of the vesicles is also more complex than it is suggested in the manuscript. The text should better reflect the limitations of the conclusions that can be drawn from the authors' data.
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
Astrocytic coverage
On Fig. 6 data are presented on the astrocytic coverage derived from L1 and L4. In my previous review I asked to include this in the text of the Results as well, but I still do not see it. It is also lacking from the Results how many samples from which layer were investigated in this analysis. Only percentages are given, and only for L1 (but how many patients, L1a and/or L1b and/or L4 is not provided). In contrast, Figure 6 and Supplementary Table 2 (patient table) contains the information that this analysis has been made in L4 as well. Please, include this information in the text as well (around lines 348-360).
See above.
About how to determine glial elements. I cannot agree with the Authors that glial elements can be determined with high certainty based only on the anatomical features of the profiles seen in the EM. “With 25 years of experience in (serial) EM work" I would say, that glial elements can be very similar to spine necks and axonal profiles. Please, see the photos below, out of the 16 circled profiles (2nd picture, very similar to each other) only 3 belong to an astroglial cell (last picture, purple profiles-purple cell), 10 are spines/spine necks/small caliber dendrites of pyramidal cells, 3 are axonal profiles (last but one picture, blue profiles, marked with arrows on the right side). If you follow in your serial sections those elements which you think are glial processes and indeed they are attached to a confidently identifiable glial cell, I agree, it is a glial process. But identifying small, almost empty profiles without any specific staining, from one single EM section, as glial process is very uncertain. Please, check the database of the Allen Institute made from the V1 visual cortex of a mouse. It is a large series of EM sections where they reconstructed thousands of neurons, astroglial and microglial cells. It is possible to double click on the EM picture on a profile and it will show the cell to which that profile belongs. https://portal.brain-map.org/connectivity/ultrastructural-connectomics Pictures included here: https://elife-rp.msubmit.net/eliferp_files/2024/11/25/00132644/02/132644_2_attach_21_29456_convrt.pdf
All in all, if similar methods were used to determine the glial coverage in the different layers of the human neocortex, than it can be compared (I guess this is the case). However, I would say in the text that proper determination would need immunostaining and a new analysis. This only gives an estimation with the possibility of a certain degree of error.
As stated above, we carried out glutamine synthetase immunohistochemistry in L5 of the human TLN and came to the same results. However, we added a sentence on this in the chapter on astrocytic coverage in the Material and Methods section. Additionally, we modified this chapter according to the reviewer’s suggestion.
Minor comments
Introduction: Last sentence is not understandable (lines 101-103), please rephrase. (contribute to understand or contribute in understanding or contribute to the understanding of..., but definitely not contribute to understanding). The authors should check and review extensively for improvements to the use of English, or use a program such as Grammarly.
Results: Grammar (line 107): L1 in the adult mammalian neocortex represents a relatively...
Line 173: “Some SBs in both sublaminae were seen to establish either two or three SBs on the same spine, spines 173 of other origin or dendritic shafts." - Some SBs established two or three SBs? I would write Some SBs established two or three synapses on...
Line 243: “The synaptic cleft size were slightly, but non-significantly different"
Line 260: “DCVs play an important role in endo- and exocytosis, the build-up of PreAZs by releasing Piccolo and Bassoon (Schoch and Gundelfinger 2006; Murkherjee et al. 2010)," - please, correct this.
We have done corrections as suggested by the reviewer.
Line 374: No point at the end of the last phrase.
Discussion:
Lines 400-404: “The majority of SBs in L1 of the human TLN had a single at most three AZs that could be of the non perforated macular or perforated type comparable with results for other layers in the human TLN but by ~1.5-fold larger than in rodent and non-human primates." - What is comparable with the other layers, but different from animals? Please rephrase this sentence, it is not understandable. I already mentioned this sentence in my previous review, but nothing happened.
Lines 435-437: “Remarkably, the total pool sizes in the human TLN were significantly larger by more than 6-fold (~550 SVs/AZ), and ~4.7-fold (~750 SVs/AZ;) than those in L4 and L5 (Yakoubi et al. 2019a, b; see also Rollenhagen et al. 2018) in rats." Please rethink what you wished to say and compare to the sentence meaning. I think you wanted to compare human TLN L1 pool size to L4 and L5 in the human TLN (Yakoubi 2019a and b) and to rat (Rollenhagen 2018). Instead, you compared all layers of the human TLN to L4 and L5 in rats (with partly wrong references). Please rephrase this. Lines 483-484: “Astrocytes serve as both a physical barrier to glutamate diffusion and as mediate neurotransmitter uptake via transporters".
This sentence is grammatically incorrect, please rephrase.
We corrected the sentences as suggested by the reviewer.
Methods:
In the text, there are only 4 patients (lines 603-604), but in the supplementary table there are 9 patients (5 new included for L4 astrocytic coverage). Please, correct it in the text.
Lines 608-609: “neocortical access tissue samples were resected to control the seizures for histological inspection by neuropathologists." - What is the meaning of this? Please, rephrase.
We thank the reviewer for the comment and included the 5 patients used for L4 to the Material and Methods section, as well as in the Results section.
The reviewer is right, and we rephrased and corrected the sentence concerning the inspection by neuropathologists.
Figures
Figures 5B: The legend says “SB (sb) synapsing on a stubby spine (sp) with a prominent spine apparatus (framed area) and a thick dendritic segment (de) in L1b" - In my opinion this is not one synaptic bouton, but two. Clearly visible membranes separate them, close to the spine.
Supplemental Table 2 (patient table). If there is no information about Hu_04 patient's epilepsy, please write N/A (=non available) instead of - (which means it does not exist).
The reviewer is right, and we corrected the figure and the legend, as well as the table accordingly.
Reviewer #2 (Recommendations for the authors):
The authors addressed almost all of my concern, only this one remained:
If there is, however, relevant literature on "methods based on EM tomography" and "stereological methods to estimate both types of error" (over- and underestimates) that we are missing out on, we would appreciate the reviewer providing us with the corresponding references so that we can include such calculations in our paper.
There is a very detailed new study on calculating correction for TEM 2D 3D, Rothman et al 2023 PLOS One. That addresses most of these issues.
We thank the reviewer for drawing our attention to the publication by Rothman et al. 2023, which is a very detailed and comprehensive study looking at accurately estimating distributions of 3D size and densities of particles from 2D measurements using – amongst others – ET and TEM images as well as synaptic vesicles for validating their method. However, we do not see how this would be relevant to the reported mean diameters and their corresponding variances. And even if we would have reported on vesicle size/diameter distributions (referred to as G(d) in Rothmann et al. 2023), the authors themselves state that “… the results from our ET and TEM image analysis highlight the difficulty in computing a complete G(d) of MFT vesicles due to their small size…
