Ultrastructural sublaminar-specific diversity of excitatory synaptic boutons in layer 1 of the adult human temporal lobe neocortex

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 synpatic 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 experiment 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 realized earlier, but still think it is important.

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 occured 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.

(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 syanpses 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 is inappropriate in this study.

(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.

(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.

(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).
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 estimatation with the possibility of a certain degree of error.

(6) Large interindividual differences in the synapse density should be discussed in the Discussion.

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.

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.

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.

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 synaptic boutons in layer 1 of the human temporal neocortex. They examined the size of each synapse, the macular or perforated appearance, the size of the synaptic active zone, the number and volume of the mitochondria, and the number of synaptic and 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 are 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 increased 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.

We would like to thank reviewer#1 for his very positive comments on our manuscript stating that such data about the fine structure of the human neocortex are are highly relevant.

Weaknesses:

There are several weaknesses in this work. First, the authors should check and review extensively for improvements to the use of English. Second, several additional analyses performed on the existing data could substantially elevate the value of the data presented. Much more information could be gained from the existing data about the functions of the investigated layer, of the cortical column, and about the information processing of the human neocortex. Third, several methodological concerns weaken the conclusions drawn from the results.

We would like to thank the reviewer for his critical and thus helpful comments on our manuscript. We took the first comment of the reviewer concerning the English and have thus improved our manuscript by rephrasing and shortening sentences. Secondly, according to the reviewer several additional analyses should be performed on the existing data, which could substantially elevate the value of the data presented. We will implement some of the suggestions in the improved version of the manuscript where appropriate. We will address a more detailed answer to the reviewer’s queries in her/his suggestions to the authors (see below). However, the reviewer states himself: “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”.

Reviewer #2 (Public review):

Summary:

The study of Rollenhagen et al. examines the ultrastructural features of Layer 1 of the human temporal cortex. The tissue was derived from drug-resistant epileptic patients undergoing surgery, and was selected as far as possible from the epilepsy focus, and as such considered to be non-epileptic. The analyses included 4 patients with different ages, sex, medication, and onset of epilepsy. The manuscript 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, that the L1 synaptic boutons mainly have a single active zone, a very large pool of synaptic vesicles, and are mostly devoid of astrocytic coverage.

Strengths:

The manuscript is well-written and easy to read. The Results section gives a detailed set of figures showing many morphological parameters of synaptic boutons and glial elements. The authors provide comparative data of all the layers examined by them so far in the Discussion. Given that anatomical data in the human brain are still very limited, the current manuscript has substantial relevance. The work appears to be generally well done, the EM and EM tomography images are of very good quality. The analysis is clear and precise.

We would like to thank the reviewer for his very positive evaluation of our paper and the comments that such data have a substantial relevance, in particular in the human neocortex. In contrast to reviewer#1, this reviewer’s opinion is that the manuscript is well written and easy to read.

Weaknesses:

One of the main findings of this paper is that "low degree of astrocytic coverage of L1 SBs suggests that glutamate spillover and as a consequence synaptic cross-talk may occur at the majority of synaptic complexes in L1". However, the authors only quantified the volume ratio of astrocytes in all 6 layers, which is not necessarily the same as the glial coverage of synapses. In order to strengthen this statement, the authors could provide 3D data (that they have from the aligned serial sections) detailing the percentage of synapses that have glial processes in close proximity to the synaptic cleft, that would prevent spillover.

We agree with the reviewer that we only quantified the volume ratio of the astrocytic coverage but not necessarily the percentage of synapses that may or not contribute to the formation of the ‘tripartite’ synapse. As suggested, we will re-analyze our material with respect to the percentage of coverage for individual synaptic boutons in each layer and will implement the results in the improved version of the manuscript. However, since this is a completely new analysis that is time-consuming we would like to ask the reviewer for additional time to perform this task.

A specific statement is missing on whether only glutamatergic boutons were analyzed in this MS, or GABAergic boutons were also included. There is a statement, that they can be distinguished from glutamatergic ones, but it would be useful to state it clearly in the Abstract, Results, and Methods section what sort of boutons were analyzed. Also, what is the percentage of those boutons from the total bouton population in L1?

We would like to thank the reviewer for this comment. Although our title clearly states, we focused on quantitative 3D-models of excitatory synaptic boutons, we will point out that more clearly in the Methods and Result chapters. Our data support recent findings by others (see for example Cano-Astorga et al. 2023, 2024; Shapson-Coe et al. 2024) that have evaluated the ratio between excitatory vs. inhibitory synaptic boutons in the temporal lobe neocortex, the same area as in our study, which was between 10-15% inhibitory terminals but with a significant layer and region specific difference. We will include the excitatory vs. inhibitory ratio and the corresponding citations in the Results section.

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 results 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 partially disagree with the reviewer on this point. Using high-resolution transmission electron microscopy, we measured the distance from the outer-to-outer membrane only on those synaptic vesicles that were round in shape with a clear ring-like structure to avoid double counts and discarded all those that were only partially cut according to criteria developed by Abercrombie (1946) and Boissonnat (1988). We assumed that within a 55±5 nm thick ultrathin section (silver to gray interference contrast) all clear-ring-like vesicles were distributed in this section assuming a vesicle diameter between 25 to 40nm. For large DCVs, double-counts were excluded by careful examination of adjacent images and were only counted in the image where they appeared largest.

In addition, we have measured synaptic vesicles using TEM tomography and came to similar results. We will address this in Material and Methods that both methods were used.

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. Indeed, it is debated whether the morphologically docked vesicles are the ones, that are readily releasable, as further molecular steps, such as proper priming are also a prerequisite for release.

We thank the reviewer for this comment. However, nobody before us tried to define a morphological correlate for the three functionally defined pools of synaptic vesicles since synaptic vesicles normally are distributed over the entire nerve terminal. As already mentioned above, after long and thorough discussions with Profs. Bill Betz, Chuck Stevens, Thomas Schikorski and other experts in this field we tried to define the readily releasable (RRP), recycling (RP) and resting pools by measuring the distance of each synaptic vesicle to the presynaptic density (PreAZ). Using distance as a criterion, we defined the RRP including all vesicles that were located within a distance (perimeter) of 10 to 20 nm from the PreAZ that is less than an average vesicle diameter (between 25 to 40 nm). The RP was defined as vesicles within a distance of 60-200 nm away, still quite close but also rapidly available on demand and the remaining ones beyond 200 nm were suggested to belong to the resting pool. This concept was developed for our first publication (Sätzler et al. 2002) and this approximation since then is very much acknowledged by scientist working in the field of synaptic neuroscience and computational neuroscientist. We were asked by several labs worldwide whether they can use our data of the perimeter analysis for modeling. We agree that our definition of the three pools can be seen as arbitrary but we never claimed that our approach is the truth but nothing as the truth. Concerning the debate whether only docked vesicles or also those very close the PreAZ should constitute the RRP we have a paper in preparation using our perimeter analysis, EM tomography and simulations trying to clarify this debate. Our preliminary results suggest that the size of the RRP should be reconsidered.

Tissue shrinkage due to aldehyde fixation is a well-documented phenomenon that needs compensation when dealing with density values. The authors cite Korogod et al 2015 - which actually draws attention to the problem comparing aldehyde fixed and non-fixed tissue, still the data is non-compensated in the manuscript. Since all the previous publications from this lab are based on aldehyde fixed non-compensated data, and for this sake, this dataset should be kept as it is for comparative purposes, it would be important to provide a scaling factor applicable to be able to compare these data to other publications.

We thank the reviewer for his suggestion. However, for several reasons we did not correct for shrinkage caused by aldehyde fixation. There are papers by Eyre et al. (2007) and the mentioned paper by Korogod et al. 2015 that have demonstrated that cryo-fixation reveals larger numbers of docked synaptic vesicles, a smaller glial volume, and a less intimate glial coverage of synapses and blood vessels compared to chemical fixation. Other structural subelements such as active zone size and shape and the total number of synaptic vesicles remained unaffected. In two further publications Zhao et al. (2012a, b) investigating hippocampal mossy fiber boutons using cryo-fixation and substitutions came to similar results with respect to bouton and active zone size and number and diameter of synaptic vesicles compared to aldehyde-fixation as described by Rollenhagen et al. 2007 for the same nerve terminal. This was one of the reasons not correcting for shrinkage. In addition, all cited papers state that chemical fixation in general provides a much better ultrastructural preservation of tissue samples when compared with cryo-fixation and substitution where optimal preservation is only regional within a block of tissue and therefore less suitable for large-scale ultrastructural analyses as we performed.

Reviewer #3 (Public review):

Summary:

Rollenhagen et 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.

We are very thankful to the reviewer upon his very positive comments about our data analysis and presentation.

Weaknesses:

The study provides only morphological details, these can be useful in the future when combined with functional assessments or computational approaches. The authors emphasize the importance of their findings on astrocytic coverage and suggest important implications for glutamate spillover. However, the percentage of synapses that form tripartite synapses has not been quantified, the authors' functional claims are based solely on volumetric fraction measurements.

We thank the reviewer for his critical comments on our findings concerning the layer-specific astrocytic coverage as also suggested by reviewer#2. As already stated above we will analyze the astrocytic coverage and the layer-specific percentage of astrocytic contribution to the ‘tripartite’ synapse in more detail. We are, however, a bit puzzled about the comment that structural anatomists usually receive that our study only provides morphological details. Our thorough analysis of structural and synaptic parameters of synaptic boutons underlie and might even predict the function of synaptic boutons in a given microcircuit or network and will thus very much improve our understanding and knowledge about the functional properties of these structures, in particular in the human brain where such studies are still quite rare. The main goal of our studies in the human neocortex was the quantitative morphology of synaptic boutons and thus the synaptic organization of the cortical column, layer by layer which to our knowledge is the first such detailed study undertaken in the human brain. Our efforts have set a golden standard in the analysis of synaptic boutons embedded in different microcircuits und is meanwhile internationally very well accepted.

The distinction between excitatory and inhibitory synapses is not clear, they should be analyzed separately.

As already stated above in response to reviewer#1 our study focused on excitatory synaptic boutons since they represent the majority of synapses. However, in the improved version of our manuscript in the Material and Method section we included a paragraph with structural criteria to distinguish excitatory from inhibitory terminals (see also our comment to reviewer#1 concerning this point) including appropriate citations.

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 are also more complex than suggested in the manuscript. The text should better reflect the limitations of the conclusions that can be drawn from the authors' data.

We thank the reviewer for this comment. However, it has been shown by meanwhile numerous publications that the shape and size of the active zone together with the pool of synaptic vesicles and the astrocytic coverage critically determines synaptic transmission and synaptic strength, but can also contribute to the modulation of synaptic plasticity (see also citations within the text). It has been shown that synaptic boutons can switch upon certain stimulation conditions to different modes of release (uni- vs. multiquantal, uni- vs multivesicular release) and from asynchronous to synchronous release leading also to the modulation of synaptic short- and long-term plasticity. To the second comment: When we started with our first paper about the Calyx of Held – principal neuron synapse in the MNTB (Sätzler et al. 2002) we tried to define a morphological correlate for the three functionally defined pools. As already mentioned above in our reply to the other two reviewers, this is rather difficult since synaptic vesicles are normally distributed over the entire nerve terminal. After long and thorough discussions with Bill Betz, Chuck Stevens and other leading scientist in the field of synaptic neuroscience, we together with Bert Sakmann tried to define a morphological correlate for the functionally defined pools using a perimeter analysis. We defined the readily releasable pool as vesicles 10 to 20 nm away from the presynaptic active zone, the recycling pool as those in 60-200 nm distance and the remaining as those belonging to the resting pool. However, it has been shown by capacitance measurements (see for example Hallermann et al 2003), FM1-43 investigations (see for example Henkel et al. 1996) and high-resolution electron microscopy (see for example Schikorski and Stevens 2001; Schikorski 2014) that our estimate of the RRP nearly perfectly matches with the functionally defined pools at hippocampal and cortical synapses (Silver et al. 2003). In addition, in one of our own papers (Rollenhagen et al. 2018) we also estimated the RP functionally from trains of EPSPs using an exponential fit analysis and came to similar results upon its size using the perimeter analysis.

Of course, as stated by the reviewer the scenario could be more complex, using other criteria but we never claimed that our morphologically defined pools are the truth but nothing as the truth but we believe it offers a quite good approximation.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Abstract:

Avoid the numerous abbreviations in the abstract. The paragraph describing the results obtained in this study is too short. Include more results, such as the size of the active zone, the proportion of perforated synapses, the ratio of synapses terminating on dendrites/spines, the percentage of volume occupied by mitochondria, etc. In the last paragraph, compare the layer-specific data to other layers of the neocortex before writing the concluding sentence.

To meet the word limits of the abstract (150 words) defined by eLife we had to use abbreviations. We followed the suggestions by the reviewer and expanded our abstract by adding the proportion of macular vs. perforated active zone and the percentage of mitochondria within an SB. However, we did not include the comparison of structural parameters in the Abstract since this is discussed thoroughly in the MS at other places (see Results and Discussion).

Results:

First of all, wonderful data! Lots of work, very valuable quantitative electron microscopy results.

Main concerns:

Adding several analyses would give much more information about the cortical synaptic organization. It would be very useful to differentiate between excitatory and inhibitory terminals (and give their ratio) and include this information in all different analyses, such as in the SV number, SV pool analysis, mitochondrion analysis, etc., that would give functional information as well. You have all the data for this, and you know how to differentiate between inhibitory and excitatory synapses, it can be done. We could see the possible morphological differences between excitatory and inhibitory synapses (maybe one is larger/has more SVs, etc. than the other). Based on these possible differences conclusions could be drawn about functional hypotheses, such as one or the other is more efficient in inducing postsynaptic potentials, excitation or inhibition is more pronounced in layer 1, etc. Furthermore, looking at the ratio of perforated synapses, we could gain information about the formation of new synapses. Maybe there is a difference between excitatory and inhibitory circuits in this point of view.

To the first point: Since our focus was on excitatory synaptic boutons as already stated in the title we have not analyzed inhibitory SBs. To do so, we have to re-analyze our complete data which is time-consuming and an additional workload. However, we can give a ratio excitatory vs. inhibitory synaptic boutons which was between 10-15% but with layer-specific differences. Our finding are in good agreement with a recent publication in Science by the Lichtman group (Shapson-Coe et al. 2024) and work by the DeFelipe group (Cano-Astorga et al. 2023, 2024) estimating the number of inhibitory boutons in different layers of the temporal lobe neocortex as we did by 10-15%. We included a small paragraph about inhibitory synapses, their percentage and included the citations in our Results section. Concerning the ratio between macular, non-perforated vs. perforated active zones we stated the majority of synaptic boutons were of the macular, non-perforated type (~75%; see improved version of the MS). If perforated, this was found predominantly on the postsynaptic site, but quite rare in L1 SBs. Since GABAergic terminals had only a small or no clearly visible PSD this would be hard to look at.

To the last point, it has been demonstrated that the number of dense core vesicles and their fusion with the presynaptic density could be a critical factor in the build-up of the active zone. In addition, the findings of the Geinismann group suggesting that perforated synapses are more efficient than non-perforated ones is nowadays very controversially discussed since other factors such as size of the active zone (see for example Matz et al. 2010; Holderith et al. 2012) and the astrocytic coverage contribute to synaptic efficacy and strength.

Related to this topic: although in the case of rat CA1 pyramidal cells all inhibitory synapses terminated on dendritic shafts (Megias et al., Neuroscience 2001), please be aware that both excitatory and inhibitory synapses can terminate on both dendritic shafts and spines in humans (inhibitory synapses are though rare on spines, usually less than 10%, but they do exist, see for example Wittner et al, Neuroscience, 2001). Please, define the excitatory/inhibitory nature of the synapses based on morphological features (not on their postsynaptic target), i.e., flattened vesicles and thin postsynaptic density for GABAergic synapses, whereas larger, round vesicles and thick postsynaptic density for glutamatergic synapses. Anyway, the ratio of excitatory and inhibitory synapses on dendrites and spines in the two sublamina would also give useful information about the synaptic organization of the human neocortical layer 1.

We are aware that not all terminals targeting on spines are excitatory, in turn it has been shown that not all terminals on shafts were inhibitory as long thought (Silver et al. 2003). However, as stated by the reviewer their abundancy on spines is rather low. At the moment it is rather unclear which functional impact inhibitory terminals on spines have, despite a local inhibition (see for example Kubota et al. eLife 2015), and thus their role is rather speculative since excitatory synapses are the predominant class on dendritic spines. As already stated above the ratio of excitatory vs. inhibitory terminals is between 10-15% and not significantly different between the two sublaminae. We are willing to add this in the results section (see in the improved version of the manuscript).

(2) About the glial coverage: Please, specify how glial elements were determined. What were the morphological features specific to astroglial processes? In Figure 5, how could we know whether the glial element marked by green is not a spine neck? The lack of morphological features specific to glial processes makes this analysis weak. The most accurate would be to make it with the aid of GFAP staining. I know this is not possible with your existing data, but at least, provide information on how glial processes were identified.

We used the criteria first described by Peters et al. (1991) and Ventura and Harris (1999) identifying astrocytic profiles by their irregular stellate shape, relatively clear cytoplasm, numerous glycogen granules and bundles of intermediate filaments. After more than 20 years of structural investigations, we hope that the reviewers will believe us that we can identify astrocytic processes at the high-resolution TEM level. In some of our publications (Rollenhagen et al. 2007; 2015; 2018; Yakoubi et al. 2019a) we have used glutamine synthetase pre-embedding immunhistochemistry to identify astrocytic processes, but a disadvantage of this method is the reduction of the ultrastructural preservation of the tissue. We have included the criteria to identify astrocytic processes of glial coverage in our manuscript together with the two citations (see improved version of the manuscript).

(3) The authors state that the total number of SVs was very variable. How was the distribution of the number of SVs? Homogenous distribution suggests that different types of synapses cannot be distinguished based on their morphological features, whereas distribution with more than one peak would suggest that different types of synapses are present in L1, and that they can be differentiated by their appearance (number of SVs, for example). This might be also related to the type of synapse (i.e., excitatory or inhibitory). The same applies to the number of RP and resting pool SVs.

To look for differences in structural and synaptic parameters that can further classify synaptic boutons we have performed a hierarchical cluster and multivariance analysis. However, it turned out that according to structural and functional parameters no further classification into subtypes could be done.

(4) The authors should check and review extensively for improvements to the use of English. The Results and Discussion sections contain many sentences which are not easy to understand. They have either a too complicated structure, or they are incomplete and hard to follow. Few examples: "The RRP/PreAZ at p20 nm criterium was on average 19.05 {plus minus} 17.23 SVs (L1a: 25.04 {plus minus} 21.09 SVs and L1b: 13.07 {plus minus} 13.87SVs) and thus nearly 2-fold larger for L1a." If you take out the parenthesis, the sentence has no meaning. "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." Rephrase these types of sentences, please.

We partially agree with the reviewer. We have improved our manuscript by rephrasing and shortening sentences.

Other suggestions:

(1) Put the synaptic density part after the description of the neuronal and synaptic composition part, it is more logical this way (i.e., first qualitative description, the distinction between sublayers, then quantitative data). Please write down in the description of the neuronal and synaptic composition part how L1a and L1b were differentiated (see also my comment on Figure 1).

We agree with the reviewer and did the change according to the suggestion. For a better understanding, we have also expanded the neuronal and synaptic description of the two sublaminae in L1.

(2) Introduce a list of abbreviations at the beginning, that would help.

It is quite unusual to provide a list of abbreviations in eLife. However, when used first the full meaning of the abbreviations is now given.

(3) What is cleft width? Usually, it refers to the distance between the pre- and the postsynaptic membrane, but here, I think it refers to the size (diameter) of the active zone. Please, clarify in the Result section (as it appears earlier than the Methods section, where it is explained). I would probably use the expression "synaptic cleft size" instead of "synaptic cleft width" to avoid misunderstanding.

We thank the reviewer for the suggestion and used synaptic cleft size for better clarity and have transferred the sentence from the Material and Methods to the Results section.

(4) The description of the different SVs (RRP, RP, etc.) is not clear in lines 236-242. What does it mean, that RRP vesicles are located {less than or equal to}10 nm and {less than or equal to}20 nm from the active zone? Explain, why the two different distance criteria were used. Furthermore, how were the vesicles located at p20-p60 defined? Why were these vesicles not considered in the determination of the different pools?

As stated in the public review to the reviewers concern we have tried to define a morphological correlate to the three functionally defined pools. After thorough discussions, with leading scientists in the field of synaptic neuroscience we have decided to use the distance of individual vesicles from the PreAZ and sort vesicles upon these criteria. One can argue that this approach is random, however, these distance criteria were described by Rizzoli and Betz (2004, 2005) and Denker and Rizzoli (2010). As also stated in the public review there is still a controversial discussion whether only docked or omega-shaped SVs constitute the RRP. 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.

(5) Please, explain how the number of docked vesicles can be 3x larger in L1b, than the number of vesicles located at p10? Docked vesicles are the closest (with the membrane touching the PreAZ)... if this comes from the fact that another pool of boutons was used for the EM tomography analysis, then the entire pool of boutons analyzed, then it means that the selection of boutons for the EM tomography is highly biased. This also implies that EM tomography data are most probably not valid for the entire L1b. The difference might also come from the different ratios of dendrite/spine synapses included in the two different analyses. In this case, it would be helpful to distinguish between synapses terminating on dendrites/spines and analyse them separately (same as for inhibitory/excitatory, which is not exactly the same as dendrite/spine!). Different n numbers of synapses are given in the text (n=25, 25, 25 25) and in Table 2 (n=91, 98, 87, and 84) for the analysis of the docked vesicles, please, correct this.

This is a correct value and thus there is a nearly 3-fold difference. The TEM tomography was carried out on the same blocks that have been used for our 3D-volume reconstructions. To carry out TEM tomography we had to use thicker sections (250 nm) to look for complete SBs as we also did in our serial sections, but of course, we could not quantify the same SBs. The completeness of SBs was one of our main criteria to reconstruct structural and synaptic parameters. The second was that the synaptic cleft was cut perpendicular. Only SBs that met these criteria were chosen for further quantitative analysis. In this respect we are of course biased in both methods.

Secondly, as already stated we did not quantify inhibitory terminals in serial sections. However, we did not find significant differences between shaft vs. spine synapses.

Finally, in Table 2 the total number of ‘docked’ SVs is given analyzed from the total number of SBs analyzed.

Discussion:

Please include the recent findings of human L1 neurons, including the "rosehip" cells in the L1 neuronal network, see Boldog et al., Nat Neurosci 2018. It would be also useful to consider in the discussion the human-specific cortical synchrony and integration phenomena derived from in vitro data (Mansvelder, Lein, Tamas, Wittner, Larkum, Huberfeld labs, etc.), and how the synaptic morphology can be related to these.

We thank the reviewer and include the reference in our chapter functional significance.

Figures and Tables:

Figure 1: In the legend, it is written that CR cells are marked by an asterisk, but on the figure it is marked by arrowheads. H: I would put the dashed line slightly lower, just above the two neuronal cell bodies. Now it looks like in the middle of the astrocytic layer. One of the asterisks marking the CR cell is not above the nucleus of that cell. I: the gabaergic neuron is outside of the framed area. I would delete the frame, anyway, the arrowheads and the asterisk are enough to show what the authors want to show.

We have changed the Figure according to the suggestions raised by the reviewer.

Figure 3: The transparent yellow is not visible. It is a bit disturbing that the contours of the boutons are not visible, I would make the transparent yellow stronger (less transparent). The SVs in green/magenta will be still visible.

We wanted to highlight the internal subelements of SBs and thus made the covering transparent but we think it is still visible.

Figure 6C: The data concerning other layers than L1 are most probably taken from other publications of the research group. One is cited (for L6), but not the others. Please correct this, or if not, then write this in the Results and Methods.

We changed the citation in the improved version of the manuscript. We overlooked that the values for L4 and L5 were already published in Schmuhl-Giesen et al. 2022.

Table 1: What does central and lateral cleft width mean in Table 1? Furthermore, please, give the name for abbreviations CV and IQR in Tables 1 and 2.

The measurements of the synaptic cleft are now described in detail in the Results section. We now have given the full names for CV and IQR in the legends of tables 1 and 2.

Supplemental Figures 1 and 2: Why Hu01 and Hu02 are twice? What is the difference? Based on the figure legend, it is L1a and L1b? If yes, please, indicate on the figure or in the legend.
Supplemental Table 1: What is TLE in the case of Hu_04? If it is temporal lobe epilepsy, then why age at epilepsy onset is missing?

Yes, Hu01 and Hu02 were selected for both L1a and L1b in separate serial sections preparations each. We indicated this now in the figure legend. Concerning Hu_04, unfortunately we do not have any further information about the medical background of the patient.

Supplemental Table 1 (Patient table), that there are many abbreviations explained which do not appear in the table (lBAZ: Brivaracetam CBZ: Carbamazepine; CLB: Clobazam; ESL: Eslicarbazepin; GGL: Ganglioglioma, etc.), please check and correct.

We have removed the unnecessary abbreviations.

Other minor suggestions:

What is Pr? Please, give the name a first appearance (line 368).

We explained Pr (release probability) when used for the first time.

Give the name for t-LDT, please (lines 442-443).

We explained t-LTD (timing-dependent long-term depression) when used for the first time.

Typo in line 169: DCW instead of DCV (dense core vesicle), DCV is used in the figure legends.

We changed DCW to DCV.

Typo in line 190: Yokoubi instead of Yakoubi (reference).

We changed Yokoubi to Yakoubi.

Typo in line 237: Rizzoloi instead of Rizzoli (reference).

We changed Rizzoloi to Rizzoli.

Line 229-230: One reference is not inserted properly - Piccolo and Bassoon.

The reference of Schoch and Gundelfinger and Murkherjee to the build-up of the active zone and the role of DCV containing Piccolo and Bassoon are properly cited in the text.

Typo in line 398: exit instead of exist.

Corrected

Typo in line 700: Reynolds (1063) instead of 1963.

Corrected

Reviewer #2 (Recommendations for the authors):

Abstract:

The last sentence seems far-fetched, and unrelated to the manuscript. How mostly single active zone boutons can "mediate, integrate and synchronize contextual and cross-modal information, enabling flexible and state-dependent processing of feedforward sensory inputs from other layers of the cortical column"? Which of the anatomical findings of the manuscript led to these conclusions?

According to the review by Schuman et al. (2021) layer 1 is regarded as a layer that mediate, integrate and synchronize contextual and cross-modal information, enabling flexible and state-dependent processing of feedforward sensory inputs from other layers of the cortical column to which the structural quantitative 3D- models of SBs contribute since they are an integral element connecting neurons and building networks.

I am also puzzled by the authors' statement in more than one place of the manuscript that "L1a can be characterized as a predominantly astrocytic sublamina". If the L1 contains the lowest measured volume ratio of glial processes (Figure 6), then this description does not seem to hold. Please rephrase.

The reviewer is right and we rephrased the sentences for more clarity in the improved version of our manuscript.

Results:

The authors find large inter-patient variability in the synapse density at L1, which raises the issue of what were the criteria to include certain patients in the analyses. Apparently, these are different from the ones analysed in their previous papers, and all the provided parameters were different (sex, age, medication, onset of epilepsy), and any of them can result in altered synapse density.

First, we have not used all patients for this study. Secondly, it was not possible to use all patients for all six layers.

It would be useful to add a panel for Figure 1 with synapse density across the different layers, as they provide this data in the Discussion.

We implemented a Supplementary Table 1 with the synaptic density values over all layers compared in the Discussion.

I cannot find Source Data 1 in the manuscript although it is referred to in more than 1 place (e.g. page 5 line 100).

Source data were uploaded when our manuscript was submitted directly to eLife as Supplemental Material. However, as stated by bioRxiv ‘any Supplemental Materials associated with this manuscript have not been transferred to bioRxiv to avoid the posting of potentially sensitive information’ all source data have not been uploaded to the preprint server.

Page 5 line 100 the correct value is 7.3*107 or rather 108?

We corrected the value in the improved version of the MS.

It would be nice to put the synapse density values into context by comparing them to e.g. mouse, rat, or monkey data.

Since we are working on the human temporal lobe neocortex we avoided to compare those data with those estimated in experimental animals. In addition as discussed by DeFelipe et al. (1999) different methods were used to quantify synaptic density in experimental animals so these results are difficult to compare.

Page 5 Line 117 CR-cells stands for Cayal-Retzius cells?

CR-cells is the abbreviation for Cajal-Retzius cells.

Page 6 Line 146 repeated sentence.

We deleted the repeated sentence.

Page 7 Line 154 "file-scale TEM" ??

We replaced file-scale by fine-scale.

Page 7 Line 164 "GABAergic synapses identified by the smaller more spherical SVs". With this fixation condition, GABAergic vesicles are more ovoid than glutamatergic ones. What were the criteria to distinguish them?

To our knowledge in meanwhile numerous publications using the same fixation inhibitory terminals contain more spherical and smaller and not roundish synaptic vesicles and showed no clear prominent PSDs as described in our paper. We have addressed that more clearly in the results section of the improved version of the MS.

Page 8 line 197 "The majority (~98%) of SBs in L1a and L1b had only a single (Figures 2C-E, 3A-C, E) at most two or three AZs" is in striking contrast with the other statement from page 7 Line 163 "Numerous SBs in both sublaminae were seen to establish either two or three synaptic contacts on the same spine or dendrite". Which of these statements is valid? Please provide exact quantification for this statement and decide which one is true.

It is true that the majority of synaptic boutons had a single active zone. However, for example on a spine not only a single but also two or three SBs can be found. We have rephrased this sentence for more clarity.

Page 9 Line 206 "L1 AZs did not show a large variability in size as indicated by the low SD, CV, and variance (Table 1)" Is this inter-patient variance of mean values? As in Supplementary Figure 1, both the SBs volume and PreAZ area show large variability in a given patient sample. Only the inter-patient variability of mean values seems low. Please state it clearly throughout the MS for other datasets as well.

For clarity concerning the variability between patients and structural parameters we have generated box plots (Suppl. Figures 1 and 2).

Page 9 Line 208 data is on Figure 5A and not 8A.

We thank the reviewer and corrected the citation of the Figure

Page 12 Line 295 how can the number of docked vesicles for L1b be larger than the one measured by the perimeter p10 nm? This later should contain the docked and PreAZ membrane proximal pool as well. This difference is even larger if we assume, that at EM tomography only partial AZs were analysed in a 200 nm thick section, not the entire AZ as for the perimeter measurement. Can the authors provide density estimates by dividing the docked / p10 nm vesicle numbers with the AZ area and comparing them?

This is a result comparing both methods. To the second concern: As stated in the text only synaptic boutons were the active zone can be followed from the beginning to its end and were the synaptic cleft was cut perpendicular were included in the TEM tomography sample as we also did in our 3D-volume reconstructions.

Methods:

Page 25 Line 624 While the PSD area can be equivocally measured, due to the dense appearance of the PSD on the EM images, the PreAZ is more difficult to outline due to lack of evident anatomical markers except the synaptic cleft (the dense material is much thinner). That is why in many publications the PreAZ area is considered to be identical to the PSD area. What are the anatomical criteria used here for the PreAZ? Why do the authors correct the PSD area, which is easy to measure with the PreAZ area that is much less certain to outline?

As stated in material and Methods both the pre- and postsynaptic densities are not defined by placing a closed contour in both densities because one can’t be certain that the dense accumulation of particles defining both areas since the impregnation (staining) and contrast of both structures critically depends on the uranyl and lead staining which could led to misinterpretation due to different staining results. That’s why we have drawn a contour line from the beginning to the end of the presynaptic density and extrapolated that for the postsynaptic density (for details see Material and Methods). In our samples both the pre- and postsynaptic densities were always clearly visible in those boutons further analyze.

Page 26 Line 640 vesicle density measurement: All the synaptic vesicles that are in the 50 nm thick section in their entirety are missed, and there are methods based on EM tomography to correct these estimations. One can not assume, that the error caused by "double counts" of vesicles cancels for the lost ones. There are stereological methods to estimate both types of error please include them and correct the values.

We would like to point out that the whole body of our work to structural analysis of vesicle pools is based on image data stemming from transmission electron microscopy (TEM) generating a projection of the entire volume of the ultra-thin section and NOT from scanning electron microscopy (SEM) where only a small volume close to the surface of the section would be captured. Operating in TEM mode ensures that no vesicle is missed only because it is embedded in its entirety in the section as postulated by the reviewer. Hence, EM tomography, which is basically a TEM operating from different incident angles in relation to the specimen or section, does not provide any advantage in detecting these vesicles. It does, however, help to better position a 3D object within the section volume itself and therefore allows to detect objects that could overlap from one viewing angle by using another angle. As the average vesicle diameter is of similar size compared to the section thickness, the possibility of a complete overlap to happen, however, is almost zero. And as we only count clear ring-like structures, a stereological correction factor calculated according to Abercrombie (1946) would underestimate real counts (see also Saetzler et al. 2002). 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.

Page 27 Line 664 and 665 "sections" are still tissue blocks, as sectioning comes after if the process is correctly written. Please correct.

We have corrected this according to the reviewer’s comment.

Page 43 Figure 4 D Data for L1b is missing, only the correlation line is visible.

Corrected in a new Figure.

Page 44 Figure 5 C arrowheads are in the correct places? Some of them do not seem to point to the edge of the synapse.

We carefully checked the Figure and adjusted the arrowheads.

Figure 5 E lower arrowhead labels something, that is difficult to identify but does not seem to be a vesicle.

We agree with the reviewer on this point and changed the figure accordingly.

Figure 5 F, the upper vesicle is at least 10 nm apart from the PreAZ membrane. Did the authors consider it as docked (indicated with arrowhead, according to the legend it labels docked vesicles)?

We agree with the reviewer on this point and changed the figure accordingly.

Page 45 Figure 6 B one of the 2 synaptic boutons (sb), sb2 has a tangential active zone that precludes the identification of the pre- and post-synaptic membranes, still 2 "docked vesicles" are labeled. How were they classified as docked? Please remove these tangential synapses from the dataset, as membranes can not be identified.

The reviewer is right that the active zone is tangentially cut, however, the two vesicles are associated with the AZ. In addition, we did not use this AZ for vesicle data analysis.

Page 46 Line 1124 interneuron axon labelled in green not brown.

Corrected as suggested by the reviewer.

Line 1129 SStC is missing.

Changed according to the reviewer’s comment.

Page 48 Table 2 Number of docked vesicles Median values are rounded to integer values? If yes why?

The statistic package used rounded to the given values.

Page 51 Supplementary Table 1 Hu_04 Histopathology, what does TLE stands for?

TLE: temporal lobe epilepsy. We included the abbreviation in the legend of Supplementary Table1, that is now table 2.

Reviewer #3 (Recommendations for the authors):

(1) Reanalysis of astrocytic coverage based on the % of synapses that form tripartite synapses.

We have reanalyzed the data concerning this point (new Figure 6D).

(2) Segregation of excitatory and inhibitory synapses.

We have now included a paragraph in our results section to distinguish between excitatory and inhibitory synapses.

(3) Better explanation of the limits of the study to assess functional parameters.

We disagree with the reviewer on this point and have not included an explanation concerning the limits of this study.