Pre- and postsynaptic nanostructures increase in size and complexity after induction of long-term potentiation.

Synapses, specialized contact sites between neurons, are the fundamental elements of neuronal information transfer. Synaptic plasticity involves changes in synaptic morphology and the number of neurotransmitter receptors, and is thought to underlie learning and memory. However, it is not clear how these structural and functional changes are connected. We utilized time-lapse super-resolution STED microscopy of organotypic hippocampal brain slices and cultured neurons to visualize structural changes of the synaptic nano-organization of the postsynaptic scaffolding protein PSD95, the presynaptic scaffolding protein Bassoon, and the GluA2 subunit of AMPA receptors by chemically induced long-term potentiation (cLTP) at the level of single synapses. We found that the nano-organization of all three proteins increased in complexity and size after cLTP induction. The increase was largely synchronous, peaking at ∼60 min after stimulation. Therefore, both the size and complexity of individual pre- and post-synaptic nanostructures serve as substrates for tuning and determining synaptic strength.

Structural changes in perineuronal nets and their perforating GABAergic synapses precede motor coordination recovery post stroke.

BACKGROUND: Stroke remains one of the leading causes of long-term disability worldwide, and the development of effective restorative therapies is hindered by an incomplete understanding of intrinsic brain recovery mechanisms. Growing evidence indicates that the brain extracellular matrix (ECM) has major implications for neuroplasticity. Here we explored how perineuronal nets (PNNs), the facet-like ECM layers surrounding fast-spiking interneurons, contribute to neurological recovery after focal cerebral ischemia in mice with and without induced stroke tolerance. METHODS: We investigated the structural remodeling of PNNs after stroke using 3D superresolution stimulated emission depletion (STED) and structured illumination (SR-SIM) microscopy. Superresolution imaging allowed for the precise reconstruction of PNN morphology using graphs, which are mathematical constructs designed for topological analysis. Focal cerebral ischemia was induced by transient occlusion of the middle cerebral artery (tMCAO). PNN-associated synapses and contacts with microglia/macrophages were quantified using high-resolution confocal microscopy. RESULTS: PNNs undergo transient structural changes after stroke allowing for the dynamic reorganization of GABAergic input to motor cortical L5 interneurons. The coherent remodeling of PNNs and their perforating inhibitory synapses precedes the recovery of motor coordination after stroke and depends on the severity of the ischemic injury. Morphological alterations in PNNs correlate with the increased surface of contact between activated microglia/macrophages and PNN-coated neurons. CONCLUSIONS: Our data indicate a novel mechanism of post stroke neuroplasticity involving the tripartite interaction between PNNs, synapses, and microglia/macrophages. We propose that prolonging PNN loosening during the post-acute period can extend the opening neuroplasticity window into the chronic stroke phase.

Quantitative Fluorescence Analysis Reveals Dendrite-Specific Thalamocortical Plasticity in L5 Pyramidal Neurons during Learning.

High-throughput anatomic data can stimulate and constrain new hypotheses about how neural circuits change in response to experience. Here, we use fluorescence-based reagents for presynaptic and postsynaptic labeling to monitor changes in thalamocortical synapses onto different compartments of layer 5 (L5) pyramidal (Pyr) neurons in somatosensory (barrel) cortex from mixed-sex mice during whisker-dependent learning (Audette et al., 2019). Using axonal fills and molecular-genetic tags for synapse identification in fixed tissue from Rbp4-Cre transgenic mice, we found that thalamocortical synapses from the higher-order posterior medial thalamic nucleus showed rapid morphologic changes in both presynaptic and postsynaptic structures at the earliest stages of sensory association training. Detected increases in thalamocortical synaptic size were compartment specific, occurring selectively in the proximal dendrites onto L5 Pyr and not at inputs onto their apical tufts in L1. Both axonal and dendritic changes were transient, normalizing back to baseline as animals became expert in the task. Anatomical measurements were corroborated by electrophysiological recordings at different stages of training. Thus, fluorescence-based analysis of input- and target-specific synapses can reveal compartment-specific changes in synapse properties during learning.SIGNIFICANCE STATEMENT Synaptic changes underlie the cellular basis of learning, experience, and neurologic diseases. Neuroanatomical methods to assess synaptic plasticity can provide critical spatial information necessary for building models of neuronal computations during learning and experience but are technically and fiscally intensive. Here, we describe a confocal fluorescence microscopy-based analytical method to assess input, cell type, and dendritic location-specific synaptic plasticity in a sensory learning assay. Our method not only confirms prior electrophysiological measurements but allows us to predict functional strength of synapses in a pathway-specific manner. Our findings also indicate that changes in primary sensory cortices are transient, occurring during early learning. Fluorescence-based synapse identification can be an efficient and easily adopted approach to study synaptic changes in a variety of experimental paradigms.

Super-Resolution Microscopy Opens New Doors to Life at the Nanoscale.

Super-resolution fluorescence microscopy holds tremendous potential for discovery in neuroscience. Much of the molecular machinery and anatomic specializations that give rise to the unique and bewildering electrochemical activity of neurons are nanoscale by design, ranging somewhere between 1 nm and 1 µm. It is at this scale where most of the unknown and exciting action is and where cell biologists flock to in their dreams, but it was off limits for light microscopy until recently. While the optical principles of super-resolution microscopy are firmly established by now, the technology continues to advance rapidly in many crucial areas, enhancing its performance and reliability, and making it more accessible and user-friendly, which is sorely needed. Indeed, super-resolution microscopy techniques are nowadays widely used for visualizing immunolabeled protein distributions in fixed or living cells. However, a great potential of super-resolution microscopy for neuroscience lies in shining light on the nanoscale structures and biochemical activities in live-tissue settings, which should be developed and harnessed much more fully. In this review, we will present several vivid examples based on STED and RESOLFT super-resolution microscopy, illustrating the possibilities and challenges of nano-imaging in vivo to pique the interest of tech-developers and neurobiologists alike. We will cover recent technical progress that is facilitating in vivo applications, and share new biological insights into the nanoscale mechanisms of cellular communication between neurons and glia.

A Clathrin light chain A reporter mouse for in vivo imaging of endocytosis.

Clathrin-mediated endocytosis (CME) is one of the best studied cellular uptake pathways and its contributions to nutrient uptake, receptor signaling, and maintenance of the lipid membrane homeostasis have been already elucidated. Today, we still have a lack of understanding how the different components of this pathway cooperate dynamically in vivo. Therefore, we generated a reporter mouse model for CME by fusing eGFP endogenously in frame to clathrin light chain a (Clta) to track endocytosis in living mice. The fusion protein is expressed in all tissues, but in a cell specific manner, and can be visualized using fluorescence microscopy. Recruitment to nanobeads recorded by TIRF microscopy validated the functionality of the Clta-eGFP reporter. With this reporter model we were able to track the dynamics of Alexa594-BSA uptake in kidneys of anesthetized mice using intravital 2-photon microscopy. This reporter mouse model is not only a suitable and powerful tool to track CME in vivo in genetic or disease mouse models it can also help to shed light into the differential roles of the two clathrin light chain isoforms in health and disease.

In vivo super-resolution of the brain - How to visualize the hidden nanoplasticity?

Super-resolution fluorescence microscopy has entered most biological laboratories worldwide and its benefit is undisputable. Its application to brain imaging, for example in living mice, enables the study of sub-cellular structural plasticity and brain function directly in a living mammal. The demands of brain imaging on the different super-resolution microscopy techniques (STED, RESOLFT, SIM, ISM) and labeling strategies are discussed here as well as the challenges of the required cranial window preparation. Applications of super-resolution in the anesthetized mouse brain enlighten the stability and plasticity of synaptic nanostructures. These studies show the potential of in vivo super-resolution imaging and justify its application more widely in vivo to investigate the role of nanostructures in memory and learning.

Environmental enrichment enhances patterning and remodeling of synaptic nanoarchitecture as revealed by STED nanoscopy.

Synaptic plasticity underlies long-lasting structural and functional changes to brain circuitry and its experience-dependent remodeling can be fundamentally enhanced by environmental enrichment. It is however unknown, whether and how the environmental enrichment alters the morphology and dynamics of individual synapses. Here, we present a virtually crosstalk-free two-color in vivo stimulated emission depletion (STED) microscope to simultaneously superresolve the dynamics of endogenous PSD95 of the post-synaptic density and spine geometry in the mouse cortex. In general, the spine head geometry and PSD95 assemblies were highly dynamic, their changes depended linearly on their original size but correlated only mildly. With environmental enrichment, the size distributions of PSD95 and spine head sizes were sharper than in controls, indicating that synaptic strength is set more uniformly. The topography of the PSD95 nanoorganization was more dynamic after environmental enrichment; changes in size were smaller but more correlated than in mice housed in standard cages. Thus, two-color in vivo time-lapse imaging of synaptic nanoorganization uncovers a unique synaptic nanoplasticity associated with the enhanced learning capabilities under environmental enrichment.

Gephyrin-Lacking PV Synapses on Neocortical Pyramidal Neurons.

Gephyrin has long been thought of as a master regulator for inhibitory synapses, acting as a scaffold to organize γ-aminobutyric acid type A receptors (GABAARs) at the post-synaptic density. Accordingly, gephyrin immunostaining has been used as an indicator of inhibitory synapses; despite this, the pan-synaptic localization of gephyrin to specific classes of inhibitory synapses has not been demonstrated. Genetically encoded fibronectin intrabodies generated with mRNA display (FingRs) against gephyrin (Gephyrin.FingR) reliably label endogenous gephyrin, and can be tagged with fluorophores for comprehensive synaptic quantitation and monitoring. Here we investigated input- and target-specific localization of gephyrin at a defined class of inhibitory synapse, using Gephyrin.FingR proteins tagged with EGFP in brain tissue from transgenic mice. Parvalbumin-expressing (PV) neuron presynaptic boutons labeled using Cre- dependent synaptophysin-tdTomato were aligned with postsynaptic Gephyrin.FingR puncta. We discovered that more than one-third of PV boutons adjacent to neocortical pyramidal (Pyr) cell somas lack postsynaptic gephyrin labeling. This finding was confirmed using correlative fluorescence and electron microscopy. Our findings suggest some inhibitory synapses may lack gephyrin. Gephyrin-lacking synapses may play an important role in dynamically regulating cell activity under different physiological conditions.

Multi-label in vivo STED microscopy by parallelized switching of reversibly switchable fluorescent proteins.

Despite the tremendous success of super-resolution microscopy, multi-color in vivo applications are still rare. Here we present live-cell multi-label STED microscopy in vivo and in vitro by combining spectrally separated excitation and detection with temporal sequential imaging of reversibly switchable fluorescent proteins (RSFPs). Triple-label STED microscopy resolves pre- and postsynaptic nano-organizations in vivo in mouse visual cortex employing EGFP, Citrine, and the RSFP rsEGP2. Combining the positive and negative switching RSFPs Padron and Dronpa-M159T enables dual-label STED microscopy. All labels are recorded quasi-simultaneously by parallelized on- and off-switching of the RSFPs within the fast-scanning axis. Depletion is performed by a single STED beam so that all channels automatically co-align. Such an addition of a second or third marker merely requires a switching laser, minimizing setup complexity. Our technique enhances in vivo STED microscopy, making it a powerful tool for studying multiple synaptic nano-organizations or the tripartite synapse in vivo.

Stable but not rigid: Chronic in vivo STED nanoscopy reveals extensive remodeling of spines, indicating multiple drivers of plasticity.

Excitatory synapses on dendritic spines of pyramidal neurons are considered a central memory locus. To foster both continuous adaption and the storage of long-term information, spines need to be plastic and stable at the same time. Here, we advanced in vivo STED nanoscopy to superresolve distinct features of spines (head size and neck length/width) in mouse neocortex for up to 1 month. While LTP-dependent changes predict highly correlated modifications of spine geometry, we find both, uncorrelated and correlated dynamics, indicating multiple independent drivers of spine remodeling. The magnitude of this remodeling suggests substantial fluctuations in synaptic strength. Despite this high degree of volatility, all spine features exhibit persistent components that are maintained over long periods of time. Furthermore, chronic nanoscopy uncovers structural alterations in the cortex of a mouse model of neurodegeneration. Thus, at the nanoscale, stable dendritic spines exhibit a delicate balance of stability and volatility.

The murine ortholog of Kaufman oculocerebrofacial syndrome protein Ube3b regulates synapse number by ubiquitinating Ppp3cc.

Kaufman oculocerebrofacial syndrome (KOS) is a severe autosomal recessive disorder characterized by intellectual disability, developmental delays, microcephaly, and characteristic dysmorphisms. Biallelic mutations of UBE3B, encoding for a ubiquitin ligase E3B are causative for KOS. In this report, we characterize neuronal functions of its murine ortholog Ube3b and show that Ube3b regulates dendritic branching in a cell-autonomous manner. Moreover, Ube3b knockout (KO) neurons exhibit increased density and aberrant morphology of dendritic spines, altered synaptic physiology, and changes in hippocampal circuit activity. Dorsal forebrain-specific Ube3b KO animals show impaired spatial learning, altered social interactions, and repetitive behaviors. We further demonstrate that Ube3b ubiquitinates the catalytic γ-subunit of calcineurin, Ppp3cc, the overexpression of which phenocopies Ube3b loss with regard to dendritic spine density. This work provides insights into the molecular pathologies underlying intellectual disability-like phenotypes in a genetically engineered mouse model.

In vivo STED microscopy: A roadmap to nanoscale imaging in the living mouse.

Superresolution microscopy techniques are now widely used, but their application in living animals remains a challenging task. The first superresolution imaging in a live vertebrate was demonstrated with STED microscopy in the visual cortex of an anaesthetized mouse. Here, we explain the requirements for a simple but robust in vivo STED microscope as well as the surgical preparation of the cranial window and the mounting of the mouse in detail. We have developed a mounting stage with a heating plate to keep the mouse body temperature stable and that can be adjusted to the optical axis of the microscope. We have optimised the design to avoid inducing thermal drift, which is critical for nanoscale imaging. STED microscopy with a resolution of 60 nm requires special cranial window preparation to avoid motion artefacts. We have implemented a drain tube to reduce the fluid between the glass window and the surface of the brain, which has been identified as the main cause for the motion artefacts. Together, these advances in the preparation allow the use of a simple intraperitoneal anaesthesia and make the previously used venous infusion and artificial respiration obsolete.

Investigating the feasibility of channelrhodopsin variants for nanoscale optogenetics.

Optogenetics has revolutionized the study of circuit function in the brain, by allowing activation of specific ensembles of neurons by light. However, this technique has not yet been exploited extensively at the subcellular level. Here, we test the feasibility of a focal stimulation approach using stimulated emission depletion/reversible saturable optical fluorescence transitions-like illumination, whereby switchable light-gated channels are focally activated by a laser beam of one wavelength and deactivated by an overlapping donut-shaped beam of a different wavelength, confining activation to a center focal region. This method requires that activated channelrhodopsins are inactivated by overlapping illumination of a distinct wavelength and that photocurrents are large enough to be detected at the nanoscale. In tests of current optogenetic tools, we found that ChR2 C128A/H134R/T159C and CoChR C108S and C108S/D136A-activated with 405-nm light and inactivated by coillumination with 594-nm light-and C1V1 E122T/C167S-activated by 561-nm light and inactivated by 405-nm light-were most promising in terms of highest photocurrents and efficient inactivation with coillumination. Although further engineering of step-function channelrhodopsin variants with higher photoconductances will be required to employ this approach at the nanoscale, our findings provide a framework to guide future development of this technique.

In vivo STED microscopy visualizes PSD95 sub-structures and morphological changes over several hours in the mouse visual cortex.

The post-synaptic density (PSD) is an electron dense region consisting of ~1000 proteins, found at the postsynaptic membrane of excitatory synapses, which varies in size depending upon synaptic strength. PSD95 is an abundant scaffolding protein in the PSD and assembles a family of supercomplexes comprised of neurotransmitter receptors, ion channels, as well as signalling and structural proteins. We use superresolution STED (STimulated Emission Depletion) nanoscopy to determine the size and shape of PSD95 in the anaesthetised mouse visual cortex. Adult knock-in mice expressing eGFP fused to the endogenous PSD95 protein were imaged at time points from 1 min to 6 h. Superresolved large assemblies of PSD95 show different sub-structures; most large assemblies were ring-like, some horse-shoe or figure-8 shaped, and shapes were continuous or made up of nanoclusters. The sub-structure appeared stable during the shorter (minute) time points, but after 1 h, more than 50% of the large assemblies showed a change in sub-structure. Overall, these data showed a sub-morphology of large PSD95 assemblies which undergo changes within the 6 hours of observation in the anaesthetised mouse.

Quantitative optical nanophysiology of Ca2+ signaling at inner hair cell active zones.

Ca2+ influx triggers the release of synaptic vesicles at the presynaptic active zone (AZ). A quantitative characterization of presynaptic Ca2+ signaling is critical for understanding synaptic transmission. However, this has remained challenging to establish at the required resolution. Here, we employ confocal and stimulated emission depletion (STED) microscopy to quantify the number (20-330) and arrangement (mostly linear 70 nm × 100-600 nm clusters) of Ca2+ channels at AZs of mouse cochlear inner hair cells (IHCs). Establishing STED Ca2+ imaging, we analyze presynaptic Ca2+ signals at the nanometer scale and find confined elongated Ca2+ domains at normal IHC AZs, whereas Ca2+ domains are spatially spread out at the AZs of bassoon-deficient IHCs. Performing 2D-STED fluorescence lifetime analysis, we arrive at estimates of the Ca2+ concentrations at stimulated IHC AZs of on average 25 µM. We propose that IHCs form bassoon-dependent presynaptic Ca2+-channel clusters of similar density but scalable length, thereby varying the number of Ca2+ channels amongst individual AZs.

SRpHi ratiometric pH biosensors for super-resolution microscopy.

Fluorescence-based biosensors have become essential tools for modern biology, allowing real-time monitoring of biological processes within living cells. Intracellular fluorescent pH probes comprise one of the most widely used families of biosensors in microscopy. One key application of pH probes has been to monitor the acidification of vesicles during endocytosis, an essential function that aids in cargo sorting and degradation. Prior to the development of super-resolution fluorescence microscopy (nanoscopy), investigation of endosomal dynamics in live cells remained difficult as these structures lie at or below the ~250 nm diffraction limit of light microscopy. Therefore, to aid in investigations of pH dynamics during endocytosis at the nanoscale, we have specifically designed a family of ratiometric endosomal pH probes for use in live-cell STED nanoscopy.Ratiometric fluorescent pH probes are useful tools to monitor acidification of vesicles during endocytosis, but the size of vesicles is below the diffraction limit. Here the authors develop a family of ratiometric pH sensors for use in STED super-resolution microscopy, and optimize their delivery to endosomes.

In vivo mouse and live cell STED microscopy of neuronal actin plasticity using far-red emitting fluorescent proteins.

The study of proteins in dendritic processes within the living brain is mainly hampered by the diffraction limit of light. STED microscopy is so far the only far-field light microscopy technique to overcome the diffraction limit and resolve dendritic spine plasticity at superresolution (nanoscopy) in the living mouse. After having tested several far-red fluorescent proteins in cell culture we report here STED microscopy of the far-red fluorescent protein mNeptune2, which showed best results for our application to superresolve actin filaments at a resolution of ~80 nm, and to observe morphological changes of actin in the cortex of a living mouse. We illustrate in vivo far-red neuronal actin imaging in the living mouse brain with superresolution for time periods of up to one hour. Actin was visualized by fusing mNeptune2 to the actin labels Lifeact or Actin-Chromobody. We evaluated the concentration dependent influence of both actin labels on the appearance of dendritic spines; spine number was significantly reduced at high expression levels whereas spine morphology was normal at low expression.

Lens-based fluorescence nanoscopy.

The majority of studies of the living cell rely on capturing images using fluorescence microscopy. Unfortunately, for centuries, diffraction of light was limiting the spatial resolution in the optical microscope: structural and molecular details much finer than about half the wavelength of visible light (~200 nm) could not be visualized, imposing significant limitations on this otherwise so promising method. The surpassing of this resolution limit in far-field microscopy is currently one of the most momentous developments for studying the living cell, as the move from microscopy to super-resolution microscopy or 'nanoscopy' offers opportunities to study problems in biophysical and biomedical research at a new level of detail. This review describes the principles and modalities of present fluorescence nanoscopes, as well as their potential for biophysical and cellular experiments. All the existing nanoscopy variants separate neighboring features by transiently preparing their fluorescent molecules in states of different emission characteristics in order to make the features discernible. Usually these are fluorescent 'on' and 'off' states causing the adjacent molecules to emit sequentially in time. Each of the variants can in principle reach molecular spatial resolution and has its own advantages and disadvantages. Some require specific transitions and states that can be found only in certain fluorophore subfamilies, such as photoswitchable fluorophores, while other variants can be realized with standard fluorescent labels. Similar to conventional far-field microscopy, nanoscopy can be utilized for dynamical, multi-color and three-dimensional imaging of fixed and live cells, tissues or organisms. Lens-based fluorescence nanoscopy is poised for a high impact on future developments in the life sciences, with the potential to help solve long-standing quests in different areas of scientific research.

Masked rhodamine dyes of five principal colors revealed by photolysis of a 2-diazo-1-indanone caging group: synthesis, photophysics, and light microscopy applications.

Caged rhodamine dyes (Rhodamines NN) of five basic colors were synthesized and used as "hidden" markers in subdiffractional and conventional light microscopy. These masked fluorophores with a 2-diazo-1-indanone group can be irreversibly photoactivated, either by irradiation with UV- or violet light (one-photon process), or by exposure to intense red light (λ∼750 nm; two-photon mode). All dyes possess a very small 2-diazoketone caging group incorporated into the 2-diazo-1-indanone residue with a quaternary carbon atom (C-3) and a spiro-9H-xanthene fragment. Initially they are non-colored (pale yellow), non-fluorescent, and absorb at λ=330-350 nm (molar extinction coefficient (ε)≈10(4) M(-1) cm(-1)) with a band edge that extends to about λ=440 nm. The absorption and emission bands of the uncaged derivatives are tunable over a wide range (λ=511-633 and 525-653 nm, respectively). The unmasked dyes are highly colored and fluorescent (ε=3-8×10(4) M(-1) cm(-1) and fluorescence quantum yields (ϕ)=40-85% in the unbound state and in methanol). By stepwise and orthogonal protection of carboxylic and sulfonic acid groups a highly water-soluble caged red-emitting dye with two sulfonic acid residues was prepared. Rhodamines NN were decorated with amino-reactive N-hydroxysuccinimidyl ester groups, applied in aqueous buffers, easily conjugated with proteins, and readily photoactivated (uncaged) with λ=375-420 nm light or intense red light (λ=775 nm). Protein conjugates with optimal degrees of labeling (3-6) were prepared and uncaged with λ=405 nm light in aqueous buffer solutions (ϕ=20-38%). The photochemical cleavage of the masking group generates only molecular nitrogen. Some 10-40% of the non-fluorescent (dark) byproducts are also formed. However, they have low absorbance and do not quench the fluorescence of the uncaged dyes. Photoactivation of the individual molecules of Rhodamines NN (e.g., due to reversible or irreversible transition to a "dark" non-emitting state or photobleaching) provides multicolor images with subdiffractional optical resolution. The applicability of these novel caged fluorophores in super-resolution optical microscopy is exemplified.