Epitope-preserving magnified analysis of proteome (eMAP)

Park, Joha; Khan, Sarim; Yun, Dae Hee; Ku, Taeyun; Villa, Katherine L.; Lee, Jiachen E.; Zhang, Qiangge; Park, Juhyuk; Feng, Guoping; Nedivi, Elly; Chung, Kwanghun

doi:10.1126/sciadv.abf6589

Cited by 27 publications

(39 citation statements)

References 49 publications

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“…Expansion Microscopy (ExM) (Chen et al, 2015; Gallagher and Zhao, 2021), which physically expands tissue samples ∼4 to 20-fold by embedding them in swellable hydrogels, has resolved subcellular protein distributions of synaptic targets in mouse brain tissue (Chen et al, 2015; Tillberg et al, 2016; Ku et al, 2016; Chozinski et al, 2016; Chang et al, 2017; Murakami et al, 2018; Truckenbrodt et al, 2019; Park et al, 2019; Shen et al, 2020; Campbell et al, 2021; Gao et al, 2021; Park et al, 2021; Damstra et al, 2022), as well as map RNA transcripts within axons and dendrites in the brain (Alon et al, 2021). However, while the expansion-related decrowding avoids fluorescence quenching, ExM has traditionally capitalized almost exclusively on antibody labelings, and no ExM method has demonstrated correlative molecular and contextual imaging with synaptic resolution so far.…”

Section: Introductionmentioning

confidence: 99%

All-optical visualization of specific molecules in the ultrastructural context of brain tissue

M’Saad

Kasula

Kondratiuk

et al. 2022

Preprint

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SummaryUnderstanding the molecular anatomy and neural connectivity of the brain requires imaging technologies that can map the 3D nanoscale distribution of specific proteins in the context of brain ultrastructure. Light and electron microscopy (EM) enable visualization of either specific labels or anatomical ultrastructure, but combining molecular specificity with anatomical context is challenging. Here, we present pan-Expansion Microscopy of tissue (pan-ExM-t), an all-optical mouse brain imaging method that combines ∼24-fold linear expansion of biological samples with fluorescent pan-staining of protein densities (providing EM-like ultrastructural context), and immunolabeling of protein targets (for molecular imaging). We demonstrate the versatility of this approach by imaging the established synaptic markers Homer1, Bassoon, PSD-95, Synaptophysin, the astrocytic protein GFAP, myelin basic protein (MBP), and anti-GFP antibodies in dissociated neuron cultures and mouse brain tissue sections. pan-ExM-t reveals these markers in the context of ultrastructural features such as pre and postsynaptic densities, 3D nanoarchitecture of neuropil, and the fine structures of cellular organelles. pan-ExM-t is adoptable in any neurobiological laboratory with access to a confocal microscope and has therefore broad applicability in the research community.Highlightspan-ExM-t visualizes proteins in the context of synaptic ultrastructureLipid labeling in pan-ExM-t reveals organellar and cellular membranesAll-optical, easily accessible alternative to correlative light/electron microscopyHigh potential for high throughput connectomics studies

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Section: Introductionmentioning

confidence: 99%

All-optical visualization of specific molecules in the ultrastructural context of brain tissue

M’Saad

Kasula

Kondratiuk

et al. 2022

Preprint

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“…1.0 mm coronal slices of primary visual cortex were cut on a vibratome and kept in PBS at 4°C until the day of processing. Slices were then incubated in eMAP hydrogel monomer solution 53 (30% acrylamide [A9099, MilliporeSigma, St. Louis, MO, USA], 10% sodium acrylate [408220, MilliporeSigma], 0.1% bis-acrylamide [161-0142, Bio-Rad Laboratories, Hercules, CA, USA], and 0.03% VA-044 (w/v) [Wako Chemicals, Richmond, VA, USA] in PBS), protected from light, at 4°C overnight. For gelation, slices were mounted between glass slides in eMAP solution and sealed in a 50 mL conical tube with nitrogen gas at positive pressure of 10-12 psi at 37°C for 3 hours.…”

Section: Methodsmentioning

confidence: 99%

A dendritic mechanism for balancing synaptic flexibility and stability

Yaeger

Vardalaki

Brown

et al. 2022

Preprint

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The modification of synaptic weights is critical for learning, while synaptic stability is required to maintain acquired knowledge. Single neurons have thousands of synapses within their dendritic arbors, and how the weights of specific inputs change across experience is poorly understood. Here we report that dendritic compartments receiving input from different presynaptic populations acquire distinct synaptic plasticity and integration rules across maturation. We find that apical oblique dendrites of layer 5 pyramidal neurons in adult mouse primary visual cortex receive direct monosynaptic projections from the dorsal lateral geniculate nucleus (dLGN), linearly integrate input, and lack synaptic potentiation. In contrast, basal dendrites, which do not receive dLGN input, exhibit NMDA receptor (NMDAR)-mediated supralinear integration and synaptic potentiation. Earlier in development, during thalamic input refinement, oblique and basal dendrites exhibited comparable NMDAR-dependent properties. Oblique dendrites gain mature properties with visual experience, and over the course of maturation, spines on oblique dendrites develop higher AMPA/NMDA ratios relative to basal dendrites. Our results demonstrate that cortical neurons possess dendrite-specific integration and plasticity rules that are set by the activity of their inputs. The linear, non-plastic nature of mature synapses on oblique dendrites may stabilize feedforward sensory processing while synaptic weights in other parts of the dendritic tree remain plastic, facilitating robust yet flexible cortical computation in adults.

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“…The screening has further extended the list of synaptic proteins present in different types of synapses (e.g., VGluT1 vs. VGluT2, GABA B R1, mGluR5, or different receptors) that can allow detection of synapses coming from different neuron types or brain regions with ExM. The very recent modification of MAP, denoted epitopepreserving MAP or eMAP, was optimized to achieve maximal preservation of antigenicity in mouse and marmoset brain tissue, thus increasing success rates of staining with synaptic antibodies to more than 94% (Park et al, 2021).…”

Section: State-of-the-art Exm Methods For the Optical Connectomementioning

confidence: 99%

“…Requirements for customized reagents prevented wide adaptation of iExM. Protein retention with the iterative expansion concept was realized in ExR (Sarkar et al, 2020 ), tetra-gel-based ExM (Gao et al, 2021 ), and eMAP (Park et al, 2021 ). For example, ExR, exhibiting effective resolution comparable to that of iExM, is compatible with commercially available antibodies applied to the expanded samples.…”

Section: Introductionmentioning

confidence: 99%

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Towards a Comprehensive Optical Connectome at Single Synapse Resolution via Expansion Microscopy

Sneve¹,

Piatkevich

2022

Front. Synaptic Neurosci.

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Mapping and determining the molecular identity of individual synapses is a crucial step towards the comprehensive reconstruction of neuronal circuits. Throughout the history of neuroscience, microscopy has been a key technology for mapping brain circuits. However, subdiffraction size and high density of synapses in brain tissue make this process extremely challenging. Electron microscopy (EM), with its nanoscale resolution, offers one approach to this challenge yet comes with many practical limitations, and to date has only been used in very small samples such as C. elegans, tadpole larvae, fruit fly brain, or very small pieces of mammalian brain tissue. Moreover, EM datasets require tedious data tracing. Light microscopy in combination with tissue expansion via physical magnification—known as expansion microscopy (ExM)—offers an alternative approach to this problem. ExM enables nanoscale imaging of large biological samples, which in combination with multicolor neuronal and synaptic labeling offers the unprecedented capability to trace and map entire neuronal circuits in fully automated mode. Recent advances in new methods for synaptic staining as well as new types of optical molecular probes with superior stability, specificity, and brightness provide new modalities for studying brain circuits. Here we review advanced methods and molecular probes for fluorescence staining of the synapses in the brain that are compatible with currently available expansion microscopy techniques. In particular, we will describe genetically encoded probes for synaptic labeling in mice, zebrafish, Drosophila fruit flies, and C. elegans, which enable the visualization of post-synaptic scaffolds and receptors, presynaptic terminals and vesicles, and even a snapshot of the synaptic activity itself. We will address current methods for applying these probes in ExM experiments, as well as appropriate vectors for the delivery of these molecular constructs. In addition, we offer experimental considerations and limitations for using each of these tools as well as our perspective on emerging tools.

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Epitope-preserving magnified analysis of proteome (eMAP)

Abstract: eMAP preserves epitopes by purely physical tissue-hydrogel hybridization in swellable tissue-gel networks.

Cited by 27 publications

References 49 publications

All-optical visualization of specific molecules in the ultrastructural context of brain tissue

All-optical visualization of specific molecules in the ultrastructural context of brain tissue

A dendritic mechanism for balancing synaptic flexibility and stability

Towards a Comprehensive Optical Connectome at Single Synapse Resolution via Expansion Microscopy

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