Expansion microscopy reveals nano-scale insights into the human neuromuscular junction

Abstract

The neuromuscular junction (NMJ) is a specialized synapse that relays signals from the lower motor neuron to the skeletal muscle. Here, we detail the development and application of expansion microscopy (ExM) as a highly accessible, relatively cheap, powerful, and reproducible tool with which to obtain high-resolution insights into the subcellular structure and function of NMJs from whole-mount preparations, previously only achievable using super-resolution microscopy. ExM is equally applicable to both mouse and human tissue samples, facilitating high-resolution comparative analyses. Qualitative and quantitative analysis of ExM images reveals significant differences in the distribution of acetylcholine receptors, synaptic vesicles, and voltage-gated Na⁺ 1.4 (NaV1.4) channels between human and mouse NMJs that are not readily observable using conventional confocal microscopy. We conclude that ExM offers a cost-effective and adaptable approach to facilitate nano-scale imaging of the NMJ.

Keywords: CP: Imaging; CP: Neuroscience; comparative anatomy; expansion microscopy; neuromuscular junction; super-resolution; synapse.

Graphical abstract

Figure 1

Human and mouse NMJs imaged using ExM (A) Representative confocal micrographs showing examples of expanded NMJs (≈4×) from mouse (left) and human (right) muscle biopsies stained with α-bungarotoxin to reveal AChRs (cyan) and immunohistochemical labeling of SV2 to reveal presynaptic vesicles (magenta). Note the level of detail that can be observed with regard to the precise distribution and morphological arrangement of each label: AChRs exhibited distinctive patterns between human and mouse NMJs and SV2-labeled synaptic vesicles exhibited a more punctate distribution at the human NMJ. (B) Representative confocal micrographs showing examples of expanded NMJs (≈4×) from mouse (left) and human (right) muscle biopsies stained with α-bungarotoxin to reveal AChRs (cyan) and immunohistochemical labeling of NaV1.4 to reveal Na channels (magenta). Again, note the level of detail that can be observed with regard to the precise distribution and morphological arrangement of each label: in human NMJs, the boundaries of Nav1.4 rims extended further beyond the AChRs compared to those in mice. Images in (A) and (B) were acquired with a 20× objective and treated with a deconvolution algorithm. The scale bars (5 μm) correspond to pre-expansion dimensions.

Figure 2

Pre- and post-expansion analysis of NMJs Comparison of representative confocal micrographs of NMJs, pre- and post-expansion. (A) Examples of mouse (left) and human (right) NMJs pre- and post-expansion (top and bottom, respectively). Note: these are examples of different NMJs, as it was not possible to image the same NMJs before and after the expansion process. NMJ samples were labeled and imaged identically: α-bungarotoxin for AChRs (cyan) and NaV1.4 for Na channels (magenta) as two distinct dual-pseudo colors; images were acquired with a 20× objective. Scale bars (4 μm) correspond to pre-expansion dimensions. (B) Quantitative analysis of NMJ endplate area as a core morphological variable pre- and post-expansion in both mouse (top) and human (bottom) NMJs revealed an expansion factor of ∼4× with ExM. Bar charts represent mean ± SD; each data point represents an individual NMJ (mouse pre-ExM n = 16 and post-ExM n = 17, while human pre-ExM n = 21 and post-ExM n = 17). Unpaired t test (two-tailed), ∗∗∗∗p < 0.0001.

Figure 3

Increased resolution of NMJ structure using ExM compared to standard confocal microscopy Representative examples showing the superiority of 4×ExM to standard confocal microscopy with respect to revealing NMJ ultrastructure. (A) shows representative examples of raw confocal images of AChRs at a single human NMJ, acquired using a 60× objective, whereas (B) is an image of AChRs at an expanded (4×) NMJ captured using a 20× objective. Scale bars: 4 μm (in biological units = ∼1 μm pre-expansion). ExM images revealed significantly more details due to the increased spatial separation of fluorophores in the ExM protocol. When zooming in on a single bouton (bottom images), the expanded NMJs maintained good image quality and resolution, allowing for the identification and tracing of details that were not possible with raw confocal images, which lost resolution and became blurred upon zooming in. Scale bars in zoomed-in images: 2 μm (in biological units: 500 nm).

Figure 4

Comparison of different deconvolution techniques for use on ExM images Series of images illustrating the effect of deconvolution on images of expanded (≈4×) human NMJs. The left images show unprocessed raw images, with middle columns showing the same images after applying the classical maximum likelihood estimation (CMLE) deconvolution algorithm based on the theoretical point spread function (PSF) and the right columns showing the same images processed using an identical deconvolution algorithm but with the precise experimental PSF. NMJs were stained with α-bungarotoxin for AChRs (in cyan) and SV2 for presynaptic vesicles (magenta). Notably, the deconvolution process led to a substantial enhancement in image contrast, noise reduction, and resolution in visualizing nano-scale structural details. Scale bars: 4 μm, (biological unit = 1 μm pre-expansion).

Figure 5

ExM reveals differences in AChR distribution between human and mouse NMJs (A–C) Quantitative analysis of the morphological characteristics of AChR stripes at human and mouse NMJs imaged using ExM data based on biological data obtained from post-expansion dimensions normalized to the expansion factor. Statistical analysis (unpaired t test) revealed a significant increase in the distance between AChR strips (A), AChR strip width (B), and AChR strip length (C) at the human NMJ compared to mice. Bar charts are presented as mean ± SEM; each data point represents an average of total AChR stripes from a single NMJ (≈35 individual AChR stripes per NMJ in human and ≈35 individual AChR stripes per NMJ in mouse). Total number of human NMJs = 16 (individual AChR stripe n ≈ 500). Mouse NMJ number = 17 (individual AChR stripe n ≈ 700). ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. (A′–C′) Absolute dimension data obtained from ExM images, prior to normalization to the expansion factor. Total number of human NMJs = 16 (individual AChR stripes n ≈ 500). Total number of mouse NMJs = 17 (individual AChR stripes n ≈ 700). Distance between AChR strips (A′), AChR strip width (B′), and AChR strip length (C′). Bar charts show mean ± SD; each data point represents an individual AChR stripe. Unpaired t test, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Figure 6

Differential alignment of pre- and postsynaptic structures at human and mouse NMJs revealed using ExM Representative micrographs of ≈4×ExM NMJs revealing AChR distribution (cyan) versus SV2 distribution (magenta) in mouse (A) and human (B). At mouse NMJs, qualitative analysis of the distribution of the two markers suggested a homogeneous distribution of SV2 (e.g., synaptic vesicles) over the entire region of AChRs at the endplate, which was supported by quantitative analysis of the fluorescence intensity profiles (Pearson correlation of 0.8064) (i). In contrast, discernible puncta of SV2 labeling were evident at human NMJs, resulting in a greater spatial dissociation between the SV2 and AChR signals in the fluorescence intensity profiles (Pearson correlation of 0.5104) (ii). Scale bars: 4 μm.

Figure 7

Differential alignment of NaV1.4 channels and AChRs at human and mouse NMJs revealed using ExM Representative micrographs of ≈4×ExM NMJs revealing AChR distribution (cyan) versus NaV1.4 distribution (magenta) in mouse (A) and human (B). At mouse NMJs, qualitative analysis of the distribution of the two markers suggested a close matching of AChRs and NaV1.4 channels, which was supported by quantitative analysis of the fluorescence intensity profiles (Pearson correlation of 0.8519) (i). In contrast, NaV1.4 labeling was observed to extend well beyond the AChR domain at human NMJs, resulting in a greater spatial dissociation between the NaV1.4 and AChR signals in the fluorescence intensity profiles (Pearson correlation of 0.6548) (ii). Scale bars: 4 μm.