Histomorphometrical Analyses of the Mouse Suprachiasmatic Nucleus

Materials and reagents

Reagents

1. Cholera toxin subunit B (Recombinant), Alexa Fluor^TM 555 conjugate (Invitrogen^TM, catalog number: C34776)

Equipment

1. Zeiss Axioskop 50 equipped with color Axiocam 208

2. Objective Plan-APOCHROMAT 10×/0.32 (Zeiss, catalog number: 4440630)

3. Objective Plan-APOCHROMAT 10× (Zeiss, catalog number: 420340-9901)

4. Zeiss AxioImager M2 microscope equipped with a motorized stage, an AxioCam MRm and the ApoTome imaging system (any comparable system can be used)

Software and datasets

1. AxioVision software, Zeiss version 4.5, Carl Zeiss AG, Germany,

https://www.micro-shop.zeiss.com/en/us/system/software-axiovision+software-products/1007/

2. NIH ImageJ software with the Fiji image processing package, https://imagej.net

3. Microsoft^® Excel for Mac (Version 16.106.1)

4. GraphPad Prism version 10.0.0 for Mac, GraphPad Software, www.graphpad.com

5. Zen software, Carl Zeiss AG, https://www.zeiss.com/microscopy/en/products/software/zeiss-zen.html

6. Zen Lite software, Carl Zeiss AG, https://www.zeiss.com/microscopy/us/products/software/zeiss-zen-lite.html

Note: A free copy of Zen Lite can be attained by creating a MyZEISS account to download the ZEISS Microscopy Installer using the link above. This is not just a viewer for the CZI files but it allows performing image acquisition, processing and analysis.

General notes

The SCN is an oblong structure, with three anatomically identifiable subdivisions: anterior, middle, and posterior, based on both shape and functional connectivity. In most of our work, we focused on the largest subdivision, the mid-SCN (Figure 1) but have also taken measurements spanning from the most anterior to the most posterior extent of the nucleus. To obtain and combine these measurements from multiple mouse brains, it is important to identify the (five to seven) sections that contain the largest cross-sectional area of the nucleus (i.e., the mid-SCN); hence, we typically stain between 10 and 15 consecutive sections paired on the rostral (anterior) to caudal (posterior) axis per animal for a total of 5–6 mice per experimental group.

Accurate delineation of the SCN borders presents methodological challenges due to the gradual transitions in cell density between the SCN and the surrounding hypothalamic region (Figure 1B, C; Supplementary Figure 1). The left and right mid-SCN are identified in Nissl- or DAPI-stained coronal brain sections by location, based on the morphology of the third ventricle and the optic chiasm, as well as cell density (Figure 1B, C). Because the borders of the DAPI- or Nissl-defined SCN are not sharply demarcated and somewhat arbitrary, measurements are performed independently by two observers masked to the experimental groups. Inter-observer consistency can be verified by having two or more observers score the same material independently and quantifying how closely their measurements agree; their training should continue until the intraclass correlation coefficient (ICC) approaches values equal or greater than 0.75. We note that the SCN lacks morphological features that would allow unambiguous and objective identification of its borders; accordingly, successful implementation of these protocols relies on proper training, extensive practice and experience (see Supplementary Figure 1 for some examples of outlined SCN). In a like manner, quantification of retinal afferent fiber density and spatial distribution requires consistent region-of-interest (ROI) selection and standardized image analysis parameters. Variability in image acquisition, scaling, and analysis workflows can significantly affect morphometric and densitometric measurements.

Procedure

The procedures for the injections of the cholera toxin, brain harvesting, tissue processing and Nissl staining can be found in the supplementary methods file as well as in prior publications included in the validation section [14–16,19].

The protocols below provide a detailed step-by-step workflow to: 1) obtain measures of the area, perimeter, height and width of the SCN identified by either Nissl or DAPI staining in consecutive mouse coronal brain sections (20 or 50 μm thickness, respectively; Figure 1B, C) [14–16,19]; 2) analyze the light input to the SCN via the retino-hypothalamic pathway using the neurotracer cholera toxin (β-subunit) conjugated to Alexa Fluor^TM 555 [19] in consecutive coronal brain sections (50 μm thickness).

A. Image acquisition of the middle SCN

1. Acquire images (2D) of the sections containing the SCN (from rostral to caudal). We use the Zen Zeiss software and a Zeiss AxioImager M2 microscope equipped with a motorized stage and an AxioCam MRm for fluorescently stained sections or a Zeiss Axioskop equipped with a color Axiocam if Nissl-stained; comparable imaging systems can be used.

2. In the Acquisition mode tab of Zen, activate the channels for all the fluorophores [Cyanine3 (Cy3) for the cholera toxin and DAPI in our case]. The SCN is identified by location, shape and cell density in the ventral (inferior) part of the coronal brain sections using a 10× objective (Figure 1) to include both left and right SCN in one image.

3. In the same tab, select Live, focus and determine the exposure time for all the channels by clicking on set exposure for the sections of each animal in all the experimental groups included in a set; then, choose the lowest value and keep it constant while imaging all the sections of the animals in that set. Determine the exposure time for the second set, the third and so on until the images for all the sets are acquired. See in the note below what we consider to be “a set”.

4. Capture images of the left and right SCN from all the sections, since it will be easier to identify and select the images with the largest nuclei by opening them side by side. Save the files as “name.czi.”

Table 1. Parameters used for image acquisition

Notes:

1. “A set” is composed of one animal per experimental group or condition (wild-type control, wild-type treated, mutant control, mutant treated) and usually, we aim for at least 5 sets, i.e., the number of independent samples or biological replicates per experimental group or condition is n = 5. All brains in “a set” are usually processed (frozen, cut, and stained) together (see Supplementary Methods).

2. It is good practice to minimize the exposure time for fluorescent images to avoid acquiring overexposed images or bleaching the sections. The time usually does not differ much between the sets of animals (Table 1); nevertheless, we determine/measure the exposure time for each set. This is a delicate, albeit time-consuming step.

Figure 1. Representative images of the suprachiasmatic nucleus (SCN) in mouse coronal brain sections. (A) Immunofluorescent staining of two of the main neuronal cell types present in the SCN: vasoactive-intestinal peptide (VIP)-positive cells (green) are identifiable in the ventrolateral part of the SCN with long processes projecting into the dorsal part; arginine vasopressin (AVP)-positive cells (magenta) are present in the dorsomedial part of the SCN. (B) DAPI- or (C) Nissl-stained coronal sections containing the SCN. In these images, the cell density characteristic of the SCN can be seen in comparison to the rest of the anterior hypothalamus. The location of the third ventricle and of the optic chiasm is shown in relation to the SCN. Note that all the images shown in this and subsequent figures were acquired using a 10× objective and the scale bars are equal to 100 μm.

B. Morphometrical analyses of the SCN using Zeiss AxioVision software

1. To estimate the area, perimeter, height and width of the left and right SCN, convert the .czi files (Figure 1B, C) to .zvi to be opened in the AxioVision software (Zeiss). This is one of the options given by ZEN under save as or export.

2. Launch the AxioVision software and open the .zvi file.

3. Select and apply the correct scaling to the image, so the measurements will be in the correct (“real”) units. This can be done (Figure 2) by either opening it from the Measure menu (Figure 2A; it will open in a separate window) or clicking on Scalings (magenta box in Figure 2A) in the Work area menu (ctrl W to open the latter); the menu will open below (Figure 2B).

4. Select the proper objective, click on Activate and then on Apply to Image.

Figure 2. Using the Zeiss AxioVision Software for histomorphometrical analyses of the suprachiasmatic nucleus (SCN). (A) After opening the image in AxioVision, step 1 is to apply the correct scaling (magenta box). (B) The Properties dialog window can be opened from the menu at the bottom of the image (green box) or by right-clicking on the image.

5. Open the Properties window. There are several ways to open this:

a. From the View Menu.

b. From the shortcut in the toolbar menu at the bottom of the image window (green box in Figure 2B).

c. By right-clicking on the image to open the shortcut menu (Figure 2B).

6. In Properties (Figure 3A), click on the Display tab and select Linear for default display if using Nissl-stained sections or Min/Max for fluorescence (DAPI). Alternatively, both the linear and the Min/Max icons (magenta box; Figure 3A) can be found in the toolbar below the image window. These settings provide the optimal contrast and brightness to visualize the nuclei’s shape and borders, mostly based on cell density. Use the icon with the magnifier selected in the toolbar below the image (Figure 3A, B) to resize it to fit in the window. Shortcuts to zoom in and out are also present.

7. Keep the Properties window open; details and information about the image can be found in the other tabs.

8. Find and select the proper drawing tool in the Measure menu (Figure 3A). More tools can be added as needed; these can also be present as a toolbar on the top ribbon of the AxioVision window.

Figure 3. Using the Zeiss AxioVision Software for histomorphometrical analyses of the suprachiasmatic nucleus (SCN). (A) Select and apply the linear or Min/Max correction for an optimal display of the image from either the first tab of the Properties window, Display, or the bottom menu (magenta box). The drawing tools are found in the Measure menu at the top. (B) The second tab of the Properties window, Attributes, contains a series of options to modify the appearance of the drawn shapes (outlines, lines, etc.) used for the analyses. The menu at the bottom of the image contains a series of useful shortcuts, including those to resize the image.

9. To measure the cross-sectional area and perimeter: select the Outline or Outline spline tool from the measure menu (Figure 3A) and draw the shape of the SCN (Figure 3B) by carefully following its border based on the difference in cell density between the nucleus and the lateral hypothalamus (see also Figure 1B, C and Supplementary Figure 1).

10. To measure the height and the width of the SCN: select and use the Length tool to draw a line from dorsal to ventral (or vice versa) at the tallest part (Figure 3B; green line) or from lateral to medial (or vice versa) at the widest part (Figure 3B; yellow line), respectively.

Notes:

1. The lines and shapes drawn are considered Annotations on top of the image and can be temporarily removed by right-clicking on the image and selecting/deselecting View Annotations (Figure 2B, right inset).

2. Enlarging the image makes it difficult to see the borders of the SCN; conversely, the nucleus becomes more evident when zooming out.

11. In Properties, the Attributes tab (Figure 3B) contains the options to change the color, thickness, appearance or type of lines as well as the font, color and size of the text (Figure 3B). In this window, select the Annotation to be formatted, then choose the settings in the menus below. It is also possible to select whether to display and justify any text with the annotations.

12. Visualize the values obtained in Measurement, the last tab of the Properties window (Figure 4A), and extract them by clicking on Create table on the left side of this window. The table generated opens in a new window (Figure 4B); save it as name.csv. Alternatively, copy and paste the values needed (area, perimeter, length) into an Excel spreadsheet. These values are linked to the image, i.e., clicking on a specific annotation/shape in the image will highlight the associated value in the table and vice versa (Figure 4C).

Figure 4. Using the Zeiss AxioVision Software for histomorphometrical analyses of the suprachiasmatic nucleus (SCN). (A) The last tab of the Properties window, Measurement, contains the values obtained. (B) These can be exported by creating a Data Table and saving it as .csv. (C) Clicking on a value in the Measurement tab highlights the associated shape/annotation in the image and vice versa.

C. Retinal–SCN connectivity

Anomalies or deficits in the retinal afferent fibers to the SCN can impair the ability of the organism to respond to photic cues and properly entrain and synchronize to the light cycle. The fluorescent signal of the retinal innervation of the SCN is strongest in the ventral aspect, where the retino-hypothalamic fibers reach the nuclei. Hence, we describe two methods of analysis carried out on the images of five consecutive sections per animal containing the mid-SCN: 1) analysis of the relative intensity of the cholera toxin fluorescent ipRGC processes performed separately in both the left and right whole SCN, and 2) examination of the distribution of the cholera toxin fluorescent signal for each left and right ventral SCN separately.

1. Quantification of the relative intensity of cholera toxin fluorescent ipRGC fibers in the whole SCN by scanning densitometry using the Fiji image processing package of ImageJ:

a. Launch Fiji/ImageJ.

b. Open the .czi file(s) by dragging and dropping into ImageJ (Figure 5A).

Note: Do not try to open the file from the FILE > OPEN menu, as it will not work.

Figure 5. Using Fiji/ImageJ to detect deficits in the retino-hypothalamic pathway. (A) The .czi files of the images acquired with the Zeiss Zen software can be opened by Drag & Drop. (B) Stack Viewing window for standard images on the left (green box) and Z-Stack images on the right (magenta box). The grayscale option allows for optimal visualization of fluorescent images.

c. The drag & drop triggers the opening of the window-menu Bio-Formats Import Options (Figure 5B), before the image is opened. In this menu, depending on the type of images [Z-stacks vs. single plane (2D) images] to be analyzed, check that the following are selected:

i. Single plane images: Standard image (Figure 5B, green box).

ii. Z-stack images: Hyperstack (Figure 5B, magenta box).

iii. Autoscale

iv. Color Options: Greyscale; although often the image automatically opens in black and white; if it does not, go back and check this selection.

v. Split Channels: Each channel will open in a separate window, so the DAPI image can be seen side-by-side with the cholera toxin (or another marker) image. This is very helpful to move and reposition the ROI as needed.

vi. Split Focal Planes: to be selected if the images are Z-stacks, to analyze each frame in sequence; these will open in separate windows.

vii. Click Ok in this and the next menu, where you are asked to choose a stack. Images will open.

Note: These settings will also work for older versions of Fiji.

d. Upon opening, the images are usually quite dark, thus adjust their brightness by Image > Adjust > Brightness/Contrast (Figure 6). The B&C menu will open in a separate window. Select Auto; no other adjustment is needed (Figure 6, green box).

e. To set up one ROI for the left SCN and one for the right SCN: open the ROI Manager from the Analyze menu > Tools > ROI Manager (Figure 7). The ROI menu will open in a separate window.

Figure 6. Using Fiji/ImageJ to detect deficits in the retino-hypothalamic pathway. Steps to adjust the brightness and contrast of the images, since upon opening, these are typically completely dark. Selecting Auto (green box) is enough to provide a good visualization of the images.

f. Select the freehand tool (Figure 7, purple box).

g. Using the DAPI image, outline the entire left SCN (Figure 7, turquoise outline), click ADD in the ROI Manager (Figure 7, cyan box); repeat for the right SCN (Figure 7, yellow outline), and click ADD.

h. The ROIs can be found in the left window of the ROI manager (Figure 7, cyan box), rename and save in a dedicated folder by selecting More > Save > Rename as… (Figure 7, green box). Click on More > Open and navigate to the folder where the ROIs were saved to open them for subsequent sessions.

i. Before beginning the measurements, select the scale units (microns) in the Specify window (Figure 7, yellow box), which contains further information on the ROI, such as the size and position. Modify the color, thickness and appearance of the line of the ROIs in the Properties (Figure 7, turquoise box) pop-up window.

Notes:

1. The left and right ROIs will be slightly different in size; still, they must be of fixed size and kept constant for all the images of the samples of the project.

2. To draw the ROI, we use the DAPI image containing the largest SCN. This is identified by aligning on the screen the consecutive images of all the samples containing the central portion of the SCN and then, besides visually identifying it, by measuring those that appear with the largest cross-sectional area.

j. To superimpose the ROI on the image of the SCN showing the cholera toxin tracer (Figure 8): select the image, then the ROI in the window of the ROI manager, reposition it if and as needed by using the arrows on the keyboard, to make sure that the entire SCN is within the ROI. Click on Update to save any changes to the position (Figure 7, magenta box). Caution should be taken to not change the size or shape of the ROIs when repositioning them.

Note: It is helpful to have the cholera toxin and corresponding DAPI image open side-by-side when superimposing the ROIs, as there will be some small variations in the position of the SCN among the images due to uneven mounting and image acquisition, thus the need to reposition it from image to image. The thickness of the optic chiasm is a good landmark to limit and minimize this inconsistency when capturing the images.

Figure 7. Using Fiji/ImageJ to detect deficits in the retino-hypothalamic pathway. Steps to draw the region of interest (ROI) for the left and right suprachiasmatic nucleus (SCN) using the freehand tool (purple box). These should be of fixed size, and great care should be taken not to inadvertently modify the size and shape if repositioned. The colored boxes indicate different commands/functions in the ROI Manager; Cyan box: click to Add the newly drawn ROI to the ROI Manager; Magenta box: Update to apply any change made to the ROI; Green box: More, opens a pop-up menu with multiple functions (open, save, specify); Turquoise box: Properties, to modify the appearance of the ROIs.

k. To add the parameters to include in the analysis, go to Measure > Set Measurements (Figure 8, turquoise box), and in the pop-up window, select Mean gray value and Integrated density.

l. To quantify the fluorescent signal, select Measure on the Analyze menu or in the ROI manager (Figure 7) as well as directly by Command/Control M. The Results window will open (Figure 8). Repeat for all the images to be analyzed.

m. To save the results, select File > Save as in the results window (Figure 8, green box) or right-click on this window; it will be saved as a .csv file.

Notes:

1. The mean gray value gives a measure of the average brightness of the pixels within the ROI, whilst the integrated density is measured as the Mean gray values × Area of the ROI. Both are measured from at least 5 consecutive sections per animal. The values derived from the left and right SCN of each section are averaged to obtain one value per section; the values from all the sections per animal are then averaged to obtain one value per animal.

2. If the analysis is performed on Z-stack images, the mean gray value and integrated density are acquired from each of the most central frames (likely 10 to 15). The values obtained for each Z-stack frame of the left SCN and those for the right SCN are averaged separately to obtain one value per side; the values for the left and right SCN are averaged to obtain one value per image; all these are averaged to obtain one value per animal.

Figure 8. Using Fiji/ImageJ to detect deficits in the retino-hypothalamic pathway. Steps to measure by scanning densitometry the relative intensity of the cholera toxin signal in the entire suprachiasmatic nucleus (SCN). Turquoise box: Set Measurements, to choose the parameters to include in the analysis; Green box: File, to open the menu to export and save the measurements obtained.

2. Quantification of the fluorescent intensity signal distribution of the neuroanatomical tracer cholera toxin in the ventral SCN using the Profile Plot Plugin of Fiji/ImageJ:

a. Begin by following all the steps described in C1 from a to e (Figures 5, 6 and 7).

b. Select the rectangle shape (Figure 9, purple box) and set up the ROIs for the left and the right SCN using the cholera toxin images.

Note: The Plot Profile only works with this shape.

Figure 9. Using Fiji/ImageJ to detect deficits in the retino-hypothalamic pathway. Steps to set up the ROI using the Rectangle tool (Purple box) and obtain Plot Profiles of the distribution of the cholera toxin fluorescence intensity in the ventral suprachiasmatic nucleus (SCN). Orange box: click to Add the new ROI to the ROI Manager; Turquoise box: More > Plot Defaults, this pop-up window contains information on the size and type of plot.

c. Draw a rectangular box to include the ventral part of either the left or right SCN and click Add in the ROI Manager (Figure 9, orange box). Save it directly from the ROI manager window (Figure 7, More > save > location/folder).

d. Carefully move the ROI to the other ventral SCN, position it properly, click add (Figure 9, orange box) in the ROI Manager window. The second ROI will be added. Save it.

Notes:

1. Make sure that the shapes enclose at best all the afferent fibers in the ventral SCN. The ROIs might not be perfectly aligned in some of the images but a bit offset due to uneven mounting.

2. The ROI for the left and right SCN should be of the same fixed size (width × height).

3. The measures (width and height) of the ROIs can be found in ROI manager > More > Specify (Figure 7).

e. To superimpose the ROI on the image: select the image and then the ROI in the window of the ROI manager; reposition it, if necessary, by using the arrows on the keyboard. Make sure not to change its size or shape! Click Update to save the change (Figure 7, magenta box) as needed; often, all the images for the same sample have the same alignment.

f. To generate a column plot profile: go to ANALYZE > PLOT PROFILE (or Command/Control K, Figure 9). A window with the plot of the intensity (gray value) will open (Figure 9).

g. The default is the horizontal plot; to change these settings, open an image, superimpose the ROI and obtain a plot profile.

h. In the menu at the bottom of the window of the plot, select More > Plot Defaults > Vertical (Figure 9, turquoise boxes). The new settings will remain unchanged for all the images to be assessed in that session. These should be checked at the beginning of each session.

Note: In the horizontal plot, the average intensity is measured along the vertical y-axis or column. This can be described as a series of “lines or columns” that measures the staining intensity parallel to the height (Figure 10A, magenta arrows) of the rectangle moving lengthwise (lateral to medial for the left and medial to lateral for the right SCN; Figure 10A, cyan arrows); then, a column average plot is generated (Figures 9 and 10B). In this, the x-axis represents the horizontal distance through the SCN, whilst the y­axis represents the average pixel intensity per vertical line within the rectangular box (Figure 10B).

Figure 10. Using Fiji/ImageJ to detect deficits in the retino-hypothalamic pathway. (A)Schematic of the directionality for the measurement of the average intensity using the horizontal plot. The magenta arrows depict the direction of the densitometric scanning parallel to the short side (height) of the rectangle ROI and moving lengthwise (cyan arrows): lateral to medial for the left suprachiasmatic nucleus (SCN) and medial to lateral for the right SCN. (B) Examples of plots obtained for the left (blue trace) and right (green trace) SCN, in which the opposite position of the peaks of intensity for the cholera toxin signal in the most lateral parts of the nuclei can be appreciated. Deep violet box: Data, opens a menu with several functions to save the plots or add a Fit function. (C) Steps to extrapolate the values generated by the plots. Turquoise box: List; clicking on this will open the window with the numerical values of the plot.

i. Once the plots are obtained for several, if not for all, the images, before exporting the obtained values, in the Plot window, select Data (Figure 10B, deep violet box) > Add Fit to identify the polynomial that allows for the best estimation and alignment of the intensity peaks on the x-axis for all the left and right SCN.

j. Once the best fit is identified and applied to all plot profiles, save the data for each plot: click on List (Figure 10C, turquoise box) and a window with the values will open. To save all the values, go to File > save as (Figure 10C) or copy directly to an Excel spreadsheet.

We performed all the subsequent analyses of the plot profiles for the left and right SCN separately to obtain a single curve per side, per animal by averaging the profiles of the 5 sections. In our latest study [19], the fifth-order polynomial curves were found to be the best fit to estimate the position of the intensity peak on the x-axis; the original y-axis values were aligned using this position in Excel and averaged arithmetically to obtain one profile (either left or right).

Data analysis

Data analyses are performed using Microsoft Excel and GraphPad Prism, as reported in [16,19]. The statistical tests to be used should be determined based upon the experimental design, the number of biological replicates/groups, and variables.

Validation of protocol

This protocol (or part of it) was previously used in the following peer-reviewed research articles:

1. Li et al. [14] Circadian rhythm disruption in a mouse model of Rett syndrome circadian disruption in RTT. Neurobiology of Disease. (Figure 3)

2. Kuljis et al. [15] Sex Differences in Circadian Dysfunction in the BACHD Mouse Model of Huntington’s Disease. PLoS ONE. (Figure 3, Tables 5 and 6)

3. Lee et al. [16] Sleep/Wake Disruption in a Mouse Model of BLOC-1 Deficiency. Frontiers in Neuroscience. (Figures 5 and 6)

4. Wang et al. [19] Scheduled feeding improves behavioral outcomes and reduces inflammation in a mouse model of fragile X syndrome. eLife. (Figure 5, Table 4)

5. Wang et al., [25] Melatonin treatment of repetitive behavioral deficits in the Cntnap2 mouse model of autism spectrum disorder. Neurobiology of Disease. (Figure S7)

6. Lee et al. [27] Sex-dimorphic effects of biogenesis of lysosome-related organelles complex-1 deficiency on mouse perinatal brain development. Journal of Neuroscience Research. (Table 3, Figures 2 and 3)

In [15], we reported sex differences in the size of the SCN in male and female wild-type mice, with the females having a smaller SCN (81%) than their male counterparts, as well as a significantly reduced SCN in male (87%), but not female, BACHD mice, a model of Huntington’s Disease, as compared to male wild types. MeCp2-null and BLOC-1-deficient male mice, two models of neurodevelopmental disabilities, displayed opposite phenotypes with, respectively a significantly smaller (14%) and larger (10%) SCN in comparison to their wild-type counterparts [14,16]. Based on our published results, we can infer that expected values for the area, height, and width of the mid-SCN in wild-type animals should be around 425,000–500,000 μm², 300μm, and 275 μm, respectively. Variability in the values obtained for the area (+/-100000) has been observed across the five studies; likely, these measurements may vary depending on the number of the sections containing the mid-SCN (5 vs. 7) used for the analysis, the type of staining (Nissl vs. DAPI staining) as well as the age of the animals.

Supplementary information

The following supporting information can be downloaded here:

1. Supplementary methods

2. Supplementary Figure 1: Examples of outlined suprachiasmatic nuclei (SCN) in Nissl- (A) or DAPI- (B) stained sections.

Acknowledgments

Conceptualization, E.C.D., C.A.G.; Investigation, F.Y.L., O.N.H., S.A.M.B.V.; Writing—Original Draft, S.A.M.B.V., C.A.G.; Writing—Review & Editing, O.N.H., E.C.D., C.S.C., C.A.G.; Supervision, E.C.D., C.A.G..

This work was supported in part by a grant from the National Institute of General Medical Sciences (RO1GM112942) to E.C.D. and C.A.G.; the core equipment used in this study was supported by the National Institute of Child Health Development (NICHD) under Award Number P50HD103557. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

The protocols were originally described and validated in [16] and [19]. These protocols were developed, modified, or derived from [14], [15] and [26].

The following figure was created using BioRender: Graphical overview, https://BioRender.com/sar1pvw.

Competing interests

The authors declare that they have no competing interests.

Ethical considerations

All experimental procedures were approved by the UCLA Animal Research Committee (protocol ARC-2020-009) and conformed to guidelines from the UCLA Division of Laboratory Animal Medicine (DLAM) and the National Institutes of Health (NIH).

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