Author response:
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.
