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. 2022 Aug 12;17(8):e0271799. doi: 10.1371/journal.pone.0271799

Isolation, cryo-laser scanning confocal microscope imaging and cryo-FIB milling of mouse glutamatergic synaptosomes

Prerana Gogoi 1, Momoko Shiozaki 2, Eric Gouaux 1,3,*
Editor: Sang H Lee4
PMCID: PMC9374259  PMID: 35960737

Abstract

Ionotropic glutamate receptors (iGluRs) at postsynaptic terminals mediate the majority of fast excitatory neurotransmission in response to release of glutamate from the presynaptic terminal. Obtaining structural information on the molecular organization of iGluRs in their native environment, along with other signaling and scaffolding proteins in the postsynaptic density (PSD), and associated proteins on the presynaptic terminal, would enhance understanding of the molecular basis for excitatory synaptic transmission in normal and in disease states. Cryo-electron tomography (ET) studies of synaptosomes is one attractive vehicle by which to study iGluR-containing excitatory synapses. Here we describe a workflow for the preparation of glutamatergic synaptosomes for cryo-ET studies. We describe the utilization of fluorescent markers for the facile detection of the pre and postsynaptic terminals of glutamatergic synaptosomes using cryo-laser scanning confocal microscope (cryo-LSM). We further provide the details for preparation of lamellae, between ~100 to 200 nm thick, of glutamatergic synaptosomes using cryo-focused ion-beam (FIB) milling. We monitor the lamella preparation using a scanning electron microscope (SEM) and following lamella production, we identify regions for subsequent cryo-ET studies by confocal fluorescent imaging, exploiting the pre and postsynaptic fluorophores.

Introduction

Glutamate released from the presynaptic terminal acts upon the postsynaptic ionotropic glutamate-receptor ion channels (iGluRs) that include the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid), NMDA (N-methyl-D-aspartic acid) and kainate receptors, causing the influx of cations (Na+, K+ and Ca2+) and resulting in excitatory synaptic transmission [1,2]. iGluRs are mostly concentrated in the postsynaptic density (PSD) and are anchored by an intricate web of specialized protein molecules that regulate their trafficking and modulate their expression and functional properties, influencing synaptic plasticity [3–6]. In the “lateral” dimension of a synapse, AMPA receptors (AMPAR) and NMDA receptors (NMDAR) are arranged in a distinctive subsynaptic distribution to align with the presynaptic release site, which in turn, influences receptor activation [7]. Within the synapse, AMPARs and NMDARs are organized into subregions of higher receptor density termed nanodomains or nanoclusters [8–10]. A typical hippocampal synapse contains one to three nanodomains, 80–100 nm in diameter, with an estimated ~25 receptors per nanocluster and ~100 receptors per synapse [9–11]. However, depending on the brain region and synapse size, the number and size of the nanodomain varies [12]. AMPAR nanodomains are localized at the PSD periphery and broadly distributed across the synapse, while NMDAR nanodomains occupy the central region of the PSD. [11,13–17]. A visual insight into the arrangement of the iGluRs in the postsynaptic terminal, in conjunction with the presynaptic terminal, would contribute towards understanding the molecular basis of synaptic transmission.

One attractive model for studying synapses are pinched-off synaptic nerve terminals, known as synaptosomes [18,19]. Typically, synaptosomes are ~0.5–1 μm in diameter and consist of re-sealed presynaptic and postsynaptic nerve terminals with the ability to retain functional properties such as membrane potential and depolarization-induced neurotransmitter release [20–23]. A re-sealed presynaptic compartment encloses the contents of the nerve terminal such as synaptic vesicles, mitochondria and cytoskeleton. The postsynaptic termini within a synaptosome carries a portion of the postsynaptic membrane along with the postsynaptic density (PSD). Most importantly, the postsynaptic membranes bear receptors including iGluRs, along with a set of scaffold proteins that constitute the PSD and hold the receptors in position [24–32]. Density gradient centrifugation using either sucrose, Ficoll or Percoll have been popularly used for isolating synaptosomes. These methods are especially useful for nerve terminals on dendritic spines and their application results in synaptosomes containing all the neurotransmitter types [33–38]. Over time, attempts have been made to reduce the preparation time in order to minimize synaptosomal shrinkage and mechanical damage and to increase viability and functional integrity [37–42].

Synaptosomes can be employed as an experimental system for gaining insight into the structural organization of iGluRs at the PSD using present-day structure determination techniques. Recent developments in the field of cryo-electron tomography (ET) makes it an attractive tool to elucidate the biological structures such as glutamatergic synaptosomes in their near-to-native state [43–46]. Vitreous sectioning of mammalian synapses in organotypic slices or in dissociated primary neuronal cultural have been applied to image synapses using cryo-ET [47–49]. However, vitreous sections suffer from substantial compression artifacts and primary neuronal cultures tend to grow into thick areas which are difficult to image. While a previous study was successful in performing cryo-ET of cultured hippocampal neurons in distinguishing excitatory and inhibitory synapses [50], methods to visualize synapses derived from native brain tissue may allow for additional insights into the structure and organization of synaptic zones. Another recent study demonstrated the advantages of utilizing a synaptosomal preparation [46]. However, no studies of cultured neurons or of synaptosomes have exploited fluorescent markers to unambiguously identify GluA2-containing glutamatergic synapses. To address this issue, we have developed a workflow to prepare artifact-free thin (~100–200 nm) dual fluorescently-labelled glutamatergic synaptosomes on cryo-electron microscopy (cryo-EM) grids by utilizing cryo-focused ion beam (cryo-FIB) milling [51–53]. We utilized a knock‐in mouse line that expresses a fully functional fluorescently (mVenus) labelled vesicular glutamate transporter-1 (vGLUT1), a specific presynaptic marker for glutamatergic synapses [54]. For identification of post synapses, we utilized a well characterized GluA2 subunit specific antibody fragment, 15F1 Fab, tagged with mCherry (15F1 Fab-mCherry) [55,56].

Here we detail a workflow for the preparation of glutamatergic synaptosomes using three different methods of density gradient centrifugation for subsequent cryo-ET studies (Fig 1). Irrespective of the density gradient centrifugation method used, the preparation time of synaptosomes to cryo-EM grid preparation can be completed in ~4–5 hrs. Cryo-confocal fluorescence microscopy was employed to identify glutamatergic synaptosomes. Subsequently, fluorescence guided cryo-focused ion beam (cryo-FIB) milling was performed for rendering lamellae suitable for cryo-ET studies.

Fig 1. Workflow for the preparation of cryo-FIB milled glutamatergic synaptosomes for cryo-ET studies.

Fig 1

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Materials and methods

The protocol described in this peer-reviewed article is published on protocols.io dx.doi.org/10.17504/protocols.io.kxygxz5mkv8j/v1 and is included for printing as S1 File with this article.

Expected results

We utilized fluorescence-detection size-exclusion chromatography (FSEC) [57] to confirm the presence of glutamatergic synaptosomes in the retrieved fraction after sucrose, Ficoll or Percoll density gradient centrifugation. In all instances, the presence of the vGLUT1-mVenus and 15F1 Fab-mCherry bound AMPAR in the synaptosome preparation were indicated by fluorescence signals in the mVenus (λex: 510 nm, λem: 535 nm) and mCherry (λex: 580 nm, λem: 610 nm) channels, respectively. The elution times of the vGLUT1 (~1850 sec) and AMPA (~1450 sec) correspond to their expected molecular weights, ~175 and ~600 kDa, respectively (Fig 2).

Fig 2. FSEC analysis of isolated glutamatergic synaptosomes.

Fig 2

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Detection of (A) vGLUT1-mVenus and (B) AMPAR bound to 15F1 Fab-mCherry in isolated synaptosomes using Venus (λex: 510 nm, λem: 530 nm) and mCherry (λex: 580 nm, λem: 610 nm) channels, respectively, via FSEC.

Cryo-EM grids of glutamatergic synaptosomes prepared using a sucrose density gradient had a distinct drawback as compared to synaptosomes prepared using either Ficoll or Percoll density gradient. Synaptosomes prepared using sucrose density gradient tend to form aggregates after application on cryo-EM grids (Fig 3A–3C). A similar event could be observed for undiluted synaptosome samples prepared using Ficoll or Percoll density gradients. However, a 50-fold dilution of the synaptosomes prepared by Ficoll or Percoll density gradient results in a homogeneous distribution on cryo-EM grids (Fig 3D–3I). The presence of glutamatergic synaptosomes is marked by the presence of overlapping green and red fluorescence signals on the EM grids (Fig 3J and 3K).

Fig 3.

Fig 3

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Cryo-LSM images of glutamatergic synaptosomes on cryo-EM grids isolated using (A-C) sucrose, (D-F) Ficoll and (G-I) Percoll density gradient centrifugation. Fluorescence signals of vGLUT1-mVenus and AMPAR-15F1 Fab-mCherry at the pre and postsynaptic compartments of synaptosomes are in the green and red channel, respectively. Zoomed-in images of glutamatergic synaptosomes prepared using (J) Ficoll and (K) Percoll density gradient centrifugation corresponding to areas enclosed in cyan dashed box in (F) and (I). Glutamatergic synaptosomes are highlighted with white arrows. Scale bar in (A-I): 5 μm; scale bar in (J,K): 2 μm.

Further, we filtered the synaptosomes after density gradient centrifugation by passing through a 1 μm filter using a thermobarrel extruder. The filtered synaptosomes were applied on cryo-EM grids and subsequently imaged under cryo-LSM. Glutamatergic synaptosomes subjected to filtration appeared to be more monodisperse, with a uniform distribution (Fig 4A–4D).

Fig 4. Cryo-LSM images of glutamatergic synaptosomes on cryo-EM grids passed through 1 μm filter using a thermobarrel extruder.

Fig 4

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Fluorescence signals from green, red and both green & red channels corresponding to (A) vGLUT1-mVenus, (B) AMPAR-15F1 Fab-mCherry and (C) both vGLUT1-mVenus & AMPAR-15F1 Fab-mCherry. (D) Zoomed-in area enclosed in cyan dashed box in (C) with glutamatergic synaptosomes highlighted with white arrows. Scale bar in (A-C): 5 μm and scale bar in (D): 2 μm.

Cryo-FIB milling of synaptosomes resulted in lamellae with a thickness range of ~100–200 nm with sample area of ~3–6 μm (Fig 5A–5C). To examine the presence of fluorescent signals associated with glutamatergic synaptosome on the milled lamella, the grids were imaged using cryo-LSM. Interestingly, the fluorescence signal from glutamatergic synaptosomes corresponding to vGLUT1-mVenus and 15F1 Fab-mCherry bound to AMPAR could be detected, indicating the successful preparation of cryo-FIB milled lamellae of glutamatergic synaptosomes (Fig 6A–6F). To further confirm the presence of synaptosome on lamella, cryo-ET imaging was performed on a FIB-milled lamella and the tomogram was reconstructed. The reconstructed tomogram revealed a typical synaptosome (diameter: < 1 μm) with a presynaptic terminal associated to a much smaller postsynaptic compartment separated by a synaptic cleft of ~ 20 nm. The presynaptic and postsynaptic membranes had a smooth and continuous appearance without any visible signs of aggregation (Fig 6G).

Fig 5. Cryo-SEM images of synaptosomes at different stages of FIB-milling.

Fig 5

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(A) Cryo-SEM images of the whole cryo-EM grid with the selected squares for FIB-milling highlighted with cyan sphere. (B) Representative cryo-SEM image of a lamella preparation during rough milling. The lamella lies between the two milling patterns shown as yellow bars. Lateral micro-expansion joints [58], marked with yellow arrow, are created on both side of the lamella (only shown for the right-hand side) (C) Representative image of a polished synaptosome lamella with the sample area that can be imaged using cryo-ET enclosed in cyan dashed box. The platinum (Pt) gas injection system (GIS) layer is marked with yellow double arrow. Scale bar: 5 μm.

Fig 6.

Fig 6

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Cryo-LSM images of glutamatergic synaptosomes in two cryo-FIB-milled lamellae, (A-C) lamella 1 and (D-F) lamella 2. Fluorescence signal from green, red and both green & red channels corresponding to (A) vGLUT1-mVenus, (B) AMPAR-15F1 Fab-mCherry and (C) overlay of vGLUT1-mVenus and AMPAR-15F1 Fab-mCherry, respectively. Glutamatergic synaptosomes are highlighted with white arrows. Scale bar: 5 μm.

The workflow presented here describes the conditions to prepare glutamatergic synaptosomes using density gradient centrifugation followed by preparation of lamellae using cryo-FIB milling suitable for cryo-ET imaging. A comparison was drawn among synaptosomes prepared by sucrose, Ficoll and Percoll to identify the conditions most suitable for cryo-EM grid set up and subsequent lamella preparation. Even though a similar amount of time is required, synaptosomes prepared using sucrose density gradient centrifugation tend to form aggregates on the cryo-EM grids. On the other hand, synaptosomes prepared using Ficoll or Percoll appear to be monodispersed upon dilution. Moreover, synaptosomes isolated by Percoll have an added advantage of involving less preparation time. Using fluorescently labelled vGLUT1 and AMPAR for the pre and postsynaptic compartments aided in distinguishing glutamatergic synaptosomes as well as acted as a guide to identify target areas or glutamatergic synaptosomes during cryo-FIB milling. The ability to obtain the fluorescence signal during cryo-LSM imaging can further be exploited for screening lamellae with glutamatergic synaptosomes and for picking targets after alignment during cryo-ET imaging.

Supporting information

S1 Fig. Typical appearance of centrifuge tubes after density gradient centrifugation.

(A) Typical appearance of centrifuge tubes after sucrose density gradient centrifugation. The fraction F2 at the interface between 0.8 and 1.2 M sucrose corresponds to synaptosomes. A similar result is observed for Ficoll density gradient centrifugation with the synaptosome fraction (F2) lying between 8 and 14% Ficoll. In case of sucrose and Ficoll density gradient centrifugation, fractions F1 and F3 represent myelin & membranes and extrasynaptosomal mitochondria, respectively [33,36]. (B) Typical appearance after Percoll density gradient centrifugation. Fraction F4 at the interface between 15 and 23% Percoll corresponds to synaptosomes. Fractions F1, F2, F3 and F5 contain membranes, myelin & membranes, synaptosomes along with membrane vesicles and extrasynaptosomal mitochondria, respectively [38].

(TIF)

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S1 File

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Acknowledgments

We thank the Janelia Research Campus for Aquilos cryo-FIB-SEM use, A. Goehring and A. Matsui for maintaining vGLUT1-mVenus mouse colony, A. Matsui for guidance and advice on cryo-LSM, Y. Zhao and J. Yu for providing 15F1 Fab-mCherry plasmid, Y. Zhao for initiating the project, J. Elferich for advice on cryo-EM grid preparation for cryo-FIB milling, R. Hallford for help with manuscript preparation and Gouaux laboratory members for helpful discussions.

Data Availability

All relevant data are within the paper and its Supporting information files.

Funding Statement

This work was supported by the NIH (NINDS) grant 2R01NS038631 to E.G. and E.G. is an investigator with the Howard Hughes Medical Institute. The funders had and will not have a role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Sang H Lee

28 Mar 2022

PONE-D-22-00522Isolation, cryo-laser scanning confocal microscope imaging and cryo-FIB milling of mouse glutamatergic synaptosomesPLOS ONE

Dear Dr. Gouaux,

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Academic Editor

PLOS ONE

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[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature?

Reviewer #1: Yes

Reviewer #2: No

**********

2. Has the protocol been described in sufficient detail?

Descriptions of methods and reagents contained in the step-by-step protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample sizes and replication needed to ensure that the data are robust and reproducible.

Reviewer #1: Yes

Reviewer #2: Partly

**********

3. Does the protocol describe a validated method?

The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data.

Reviewer #1: No

Reviewer #2: No

**********

4. If the manuscript contains new data, have the authors made this data fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: N/A

**********

5. Is the article presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please highlight any specific errors that need correcting in the box below.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript by Gogoi et al. described the workflow for the preparation of glutamatergic synaptosomes for cryo-electron tomography (cryo-ET) analysis. To identify the glutamatergic synaptosomes, the authors took advantage of the knock-in mouse brain in which presynaptic vGLUT1-mVenus is expressed and the GluA2 specific antibody fragment tagged with mCherry (15F1 Fab-mCherry). After density gradient centrifugation, the authors identified glutamatergic synaptosomes by cryo-confocal fluorescence microscopy, which visualizes the presynaptic vGLUT1-mVenus and postsynaptic AMPAR-15F1 Fab-mCherry-positive synaptosomes. Subsequently, cryo-FIB milling was used for rendering lamellae for cryo-ET analysis. Although cryo-ET studies are now topical and timely issues and the manuscript is clearly written, the actual cryo-ET data (i.e., cryo-ET imaging of this synaptosomes) is missing. So, the readers cannot evaluate this protocol.

Major comments

1. The authors should add the representative cryo-ET images of glutamatergic synaptosomes acquired by this method.

2. The authors should cite the two key references in the cryo-ET studies of synapses and discuss them.

(1) Rubén Fernández-Busnadiego. Cryo-Electron Tomography of the Mammalian Synapse.

Methods Mol Biol. 2018;1847:217-224. doi: 10.1007/978-1-4939-8719-1_16.

(2) Chang-Lu Tao et al. Differentiation and Characterization of Excitatory and Inhibitory Synapses by Cryo-electron Tomography and Correlative Microscopy.

J Neurosci 2018 Feb 7;38(6):1493-1510.doi: 10.1523/JNEUROSCI.1548-17.2017.

Minor comment

The authors should spell out “FSEC” in the line 111 and “Pt GIS” in the lien 161.

Reviewer #2: This brief protocol describes a synaptoneurosome preparation for cryoET studies. The data would be better suited as part of a research article, at present it is unclear whether this workflow will permit structural characterisation of synaptic receptor complexes.

Other comments

1. It would be helpful to present a workflow schematic in the main text figure, as it is currently not described with sufficient clarity.

2. In the “Cryo-FIB milling of glutamatergic synaptosomes” section, fluorescent light microscope images were used for the guidance of FIB milling. An overlay image of FLM and SEM would be needed to demonstrate the alignment.

3. In main text line 138, the authors mentioned filtering of synaptosomes by using a thermobarrel extruder, was this sample used for the following EM grids preparation and FIB milling? Please clarify

4. a TEM image of a representative lamella and an its overlay with the corresponding light microscope image should be added to figure 5.

5. The authors state that by following their procedure the final lamella can reach 100-200 nm thickness, a section of a reconstructed tomogram or other evidence should be added to support this statement.

6. In S1 Fig, the authors mention the existence of other components in density gradient fractions ( “myelin & membranes and extrasynaptosomal mitochondria”). Western blot or other experimental evidence is needed to support the identification of those components.

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2022 Aug 12;17(8):e0271799. doi: 10.1371/journal.pone.0271799.r002

Author response to Decision Letter 0

12 May 2022

Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript by Gogoi et al. described the workflow for the preparation of glutamatergic synaptosomes for cryo-electron tomography (cryo-ET) analysis. To identify the glutamatergic synaptosomes, the authors took advantage of the knock-in mouse brain in which presynaptic vGLUT1-mVenus is expressed and the GluA2 specific antibody fragment tagged with mCherry (15F1 Fab-mCherry). After density gradient centrifugation, the authors identified glutamatergic synaptosomes by cryo-confocal fluorescence microscopy, which visualizes the presynaptic vGLUT1-mVenus and postsynaptic AMPAR-15F1 Fab-mCherry-positive synaptosomes. Subsequently, cryo-FIB milling was used for rendering lamellae for cryo-ET analysis. Although cryo-ET studies are now topical and timely issues and the manuscript is clearly written, the actual cryo-ET data (i.e., cryo-ET imaging of this synaptosomes) is missing. So, the readers cannot evaluate this protocol.

Major comments

1. The authors should add the representative cryo-ET images of glutamatergic synaptosomes acquired by this method.

We thank the reviewer for the suggestion and we have added a reconstructed tomogram of synaptosomes acquired by this method to the revised version of the manuscript.

2. The authors should cite the two key references in the cryo-ET studies of synapses and discuss them.

(1) Rubén Fernández-Busnadiego. Cryo-Electron Tomography of the Mammalian Synapse.

Methods Mol Biol. 2018;1847:217-224. doi: 10.1007/978-1-4939-8719-1_16.

(2) Chang-Lu Tao et al. Differentiation and Characterization of Excitatory and Inhibitory Synapses by Cryo-electron Tomography and Correlative Microscopy.

J Neurosci 2018 Feb 7;38(6):1493-1510.doi: 10.1523/JNEUROSCI.1548-17.2017.

As suggested by the reviewer, both the references are discussed in the revised version of the manuscript as suggested by the reviewer.

Minor comment

The authors should spell out “FSEC” in the line 111 and “Pt GIS” in the lien 161.

“FSEC” and “Pt GIS” have been spelled out in the revised version of the manuscript.

Reviewer #2: This brief protocol describes a synaptoneurosome preparation for cryoET studies. The data would be better suited as part of a research article, at present it is unclear whether this workflow will permit structural characterisation of synaptic receptor complexes.

Other comments

1. It would be helpful to present a workflow schematic in the main text figure, as it is currently not described with sufficient clarity.

We thank the reviewer for the suggestion and we have incorporated a workflow schematic in the main text (Fig 1).

2. In the “Cryo-FIB milling of glutamatergic synaptosomes” section, fluorescent light microscope images were used for the guidance of FIB milling. An overlay image of FLM and SEM would be needed to demonstrate the alignment.

Although fluorescence light images were used to guide the FIB-milling, we were unable to discern synaptosome-like features in the SEM images, simply due to the constraints of the imaging mode. Thus, we do not believe that an overlay of the FLM and SEM images would constructively augment the manuscript. What we did do, however, was to perform fluorescence imaging on the FIB-milled lamella, to confirm that our FLM guided milling was successful, as evidenced by the presence of adjacent or overlapping red and green fluorescence (Fig 6).

3. In main text line 138, the authors mentioned filtering of synaptosomes by using a thermobarrel extruder, was this sample used for the following EM grids preparation and FIB milling? Please clarify

In the revised version of the manuscript, we have mentioned that the filtered synaptosomes were used for the preparation of EM grids.

4. a TEM image of a representative lamella and an its overlay with the corresponding light microscope image should be added to figure 5.

TEM images were acquired on the lamella for which overlapping green and red fluorescence signal were detected in the cryo-LSM. However, we have not employed any tools or other ways to overlay the images.

5. The authors state that by following their procedure the final lamella can reach 100-200 nm thickness, a section of a reconstructed tomogram or other evidence should be added to support this statement.

The thickness of the lamella was measured during cryo FIB milling using the AutoTEM 2.0 software and monitored using SEM. To further support the preparation of successful synaptosome lamellae, we have added a reconstructed tomogram to the revised version of the manuscript.

6. In S1 Fig, the authors mention the existence of other components in density gradient fractions ( “myelin & membranes and extrasynaptosomal mitochondria”). Western blot or other experimental evidence is needed to support the identification of those components.

In this study, the steps for density gradient separation of synaptosomes from other components has been performed and similar results were reproduced as described in previous studies (Gray and Whittaker, 1962; Cotman and Matthews, 1971; Dunkley et al., 2008). All these studies have been cited appropriately (S1 Fig legend). Given the extensive prior documentation, we respectively assert that further analysis is not a productive use of time or resources.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file. (31.4KB, docx)

Decision Letter 1

Sang H Lee

8 Jul 2022

Isolation, cryo-laser scanning confocal microscope imaging and cryo-FIB milling of mouse glutamatergic synaptosomes

PONE-D-22-00522R1

Dear Dr. Gouaux,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Sang H Lee, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature?

Reviewer #1: Yes

**********

2. Has the protocol been described in sufficient detail?

Descriptions of methods and reagents contained in the step-by-step protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample sizes and replication needed to ensure that the data are robust and reproducible.

Reviewer #1: Yes

**********

3. Does the protocol describe a validated method?

The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data.

Reviewer #1: Yes

**********

4. If the manuscript contains new data, have the authors made this data fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the article presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please highlight any specific errors that need correcting in the box below.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors suitably responded to all my comments. Now, the paper will provide readers with a useful workflow for the preparation glutamatergic synaptosomes for Cryo-ET analysis.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

**********

Acceptance letter

Sang H Lee

4 Aug 2022

PONE-D-22-00522R1

Isolation, cryo-laser scanning confocal microscope imaging and cryo-FIB milling of mouse glutamatergic synaptosomes

Dear Dr. Gouaux:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Sang H Lee

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. Typical appearance of centrifuge tubes after density gradient centrifugation.

    (A) Typical appearance of centrifuge tubes after sucrose density gradient centrifugation. The fraction F2 at the interface between 0.8 and 1.2 M sucrose corresponds to synaptosomes. A similar result is observed for Ficoll density gradient centrifugation with the synaptosome fraction (F2) lying between 8 and 14% Ficoll. In case of sucrose and Ficoll density gradient centrifugation, fractions F1 and F3 represent myelin & membranes and extrasynaptosomal mitochondria, respectively [33,36]. (B) Typical appearance after Percoll density gradient centrifugation. Fraction F4 at the interface between 15 and 23% Percoll corresponds to synaptosomes. Fractions F1, F2, F3 and F5 contain membranes, myelin & membranes, synaptosomes along with membrane vesicles and extrasynaptosomal mitochondria, respectively [38].

    (TIF)

    Click here for additional data file. (229.2KB, tif)
    S1 File

    (PDF)

    Click here for additional data file. (98.9KB, pdf)
    Attachment

    Submitted filename: Response to Reviewers.docx

    Click here for additional data file. (31.4KB, docx)

    Data Availability Statement

    All relevant data are within the paper and its Supporting information files.

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