Imaging neuronal structure dynamics using 2‐photon super‐resolution patterned excitation reconstruction microscopy

Visualizing fine neuronal structures deep inside strongly light‐scattering brain tissue remains a challenge in neuroscience. Recent nanoscopy techniques have reached the necessary resolution but often suffer from limited imaging depth, long imaging time or high light fluence requirements. Here, we present two‐photon super‐resolution patterned excitation reconstruction (2P‐SuPER) microscopy for 3‐dimensional imaging of dendritic spine dynamics at a maximum demonstrated imaging depth of 130 μm in living brain tissue with approximately 100 nm spatial resolution. We confirmed 2P‐SuPER resolution using fluorescence nanoparticle and quantum dot phantoms and imaged spiny neurons in acute brain slices. We induced hippocampal plasticity and showed that 2P‐SuPER can resolve increases in dendritic spine head sizes on CA1 pyramidal neurons following theta‐burst stimulation of Schaffer collateral axons. 2P‐SuPER further revealed nanoscopic increases in dendritic spine neck widths, a feature of synaptic plasticity that has not been thoroughly investigated due to the combined limit of resolution and penetration depth in existing imaging technologies.      

Automatic dendritic spine analysis in two-photon laser scanning microscopy images.

Dendritic spine expression plays an important role in the central nervous system. Modern fluorescence microscopy and green fluorescent protein technology have facilitated the research on spines. To quantitatively analyze the spines in fluorescence microscopy images, an automatic dendritic spine analysis method is proposed. Because of the limit of axial resolution, our method is designed to process the projection image along the z-axis and analyze the lateral spines. The method can automatically extract the dendrite centerlines and segment the spines along the dendrites according to width-based criteria. The criteria utilize a common morphological feature of the spines. It can detect some shapes of spines missed by previous methods. In addition, the proposed method is automatic once a few parameters are set. Spine numbers, lengths, and densities, which biologists are interested in, are analyzed both manually and automatically. The results of the two methods match well. The proposed method provides automatic and accurate dendritic spine analysis. It can serve as a useful tool for spine image analysis to avoid tedious manual labor.     

Dendritic spines: from structure to in vivo function

Dendritic spines arise as small protrusions from the dendritic shaft of various types of neuron and receive inputs from excitatory axons. Ever since dendritic spines were first described in the nineteenth century, questions about their function have spawned many hypotheses. In this review, we introduce understanding of the structural and biochemical properties of dendritic spines with emphasis on components studied with imaging methods. We then explore advances in in vivo imaging methods that are allowing spine activity to be studied in living tissue, from super-resolution techniques to calcium imaging. Finally, we review studies on spine structure and function in vivo. These new results shed light on the development, integration properties and plasticity of spines.     

Structured illumination imaging with quasi‐periodic patterns

Structured illumination microscopy (SIM) is a well‐established method for optical sectioning and super‐resolution. The core of structured illumination is using a periodic pattern to excite image signals. This work reports a method for estimating minor pattern distortions from the raw image data and correcting these distortions during SIM image processing. The method was tested with both simulated and experimental image data from two‐photon Bessel light‐sheet SIM. The results proves the method is effective in challenging situations, where strong scattering background exists, signal‐to‐noise ratio (SNR) is low and the sample structure is sparse. Experimental results demonstrate restoring synaptic structures in deep brain tissue, despite the presence of strong light scattering and tissue‐induced SIM pattern distortion.     

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis ¹. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.     

Two-photon imaging of dendritic spine development in the mouse cortex

Dendritic spines are the postsynaptic sites of most excitatory synapses in the mammalian brain. With the advent of two-photon microscopy and transgenic mice expressing fluorescent proteins, dendritic spines can now be imaged in the living cerebral cortex over time scales ranging from seconds to years. Recent studies with this in vivo imaging approach have begun to provide important insights into the development and plasticity of dendritic spines in the intact brain. Here, we review these studies and discuss technical requirements for image acquisition. We envision that intravital two-photon imaging at the level of individual synapses will greatly expand our current understandings of how neuronal networks are assembled and modified throughout life.     

Eph/ephrin对树突棘的调控与中枢神经系统疾病

树突棘是神经元树突上的功能性突起结构,通常作为突触后成份与投射来的轴突共同构成完整的突触连接。树突棘的形态与结构具有明显的可塑性,其变化通常会引起突触功能的改变。Eph受体酪氨酸激酶家族分子与其配体ephrin都是重要的神经导向因子,同时对树突棘结构也有直接的调控作用。Eph受体的活化可以促进树突棘的发生并影响树突棘的形态及内部结构;而Eph受体的异常也往往会损害正常的突触功能,甚至导致许多与树突棘结构异常相关的神经系统病变的发生。     

Time-lapse imaging of dendritic spines in vitro

Dendritic spines are small protrusions present postsynaptically at approximately 90% of excitatory synapses in the brain. Spines undergo rapid spontaneous changes in shape that are thought to be important for alterations in synaptic connectivity underlying learning and memory. Visualization of these dynamic changes in spine morphology are especially challenging because of the small size of spines (approximately 1 microm). Here we describe a microscope system, based on a spinning-disk confocal microscope, suitable for imaging mature dendritic spines in brain slice preparations, with a time resolution of seconds. We discuss two commonly used in vitro brain slice preparations and methods for transfecting them. Preparation and transfection require approximately 1 d, after which slices must be cultured for at least 21 d to obtain spines of mature morphology. We also describe imaging and computer analysis routines for studying spine motility. These procedures require in the order of 2 to 4 h.     

Fast extraction of neuron morphologies from large-scale SBFSEM image stacks

Neuron morphology is frequently used to classify cell-types in the mammalian cortex. Apart from the shape of the soma and the axonal projections, morphological classification is largely defined by the dendrites of a neuron and their subcellular compartments, referred to as dendritic spines. The dimensions of a neuron’s dendritic compartment, including its spines, is also a major determinant of the passive and active electrical excitability of dendrites. Furthermore, the dimensions of dendritic branches and spines change during postnatal development and, possibly, following some types of neuronal activity patterns, changes depending on the activity of a neuron. Due to their small size, accurate quantitation of spine number and structure is difficult to achieve (Larkman, J Comp Neurol 306:332, 1991). Here we follow an analysis approach using high-resolution EM techniques. Serial block-face scanning electron microscopy (SBFSEM) enables automated imaging of large specimen volumes at high resolution. The large data sets generated by this technique make manual reconstruction of neuronal structure laborious. Here we present NeuroStruct, a reconstruction environment developed for fast and automated analysis of large SBFSEM data sets containing individual stained neurons using optimized algorithms for CPU and GPU hardware. NeuroStruct is based on 3D operators and integrates image information from image stacks of individual neurons filled with biocytin and stained with osmium tetroxide. The focus of the presented work is the reconstruction of dendritic branches with detailed representation of spines. NeuroStruct delivers both a 3D surface model of the reconstructed structures and a 1D geometrical model corresponding to the skeleton of the reconstructed structures. Both representations are a prerequisite for analysis of morphological characteristics and simulation signalling within a neuron that capture the influence of spines.     

Matrix metalloproteinase-7 disrupts dendritic spines in hippocampal neurons through NMDA receptor activation

Dendritic spines are protrusions from the dendritic shaft that host most excitatory synapses in the brain. Although they first emerge during neuronal maturation, dendritic spines remain plastic through adulthood, and recent advances in the molecular mechanisms governing spine morphology have shown them to be exquisitely sensitive to changes in the micro-environment. Among the many factors affecting spine morphology are components and regulators of the extracellular matrix (ECM). Modification of the ECM is critical to the repair of injuries throughout the body, including the CNS. Matrix metalloproteinase (MMP)-7/matrilysin is a key regulator of the ECM during pathogen infection, after nerve crush and in encephalitogenic disorders. We have investigated the effects of MMP-7 on dendritic spines in hippocampal neuron cultures and found that it induces the transformation of mature, short mushroom-shaped spines into long, thin filopodia reminiscent of immature spines. These changes were accompanied by a dramatic redistribution of F-actin from spine heads into thick, rope-like structures in the dendritic shaft. Strikingly, MMP-7 effects on dendritic spines were similar to those of NMDA treatment, and both could be blocked by channel-specific antagonists. These findings are the first direct evidence that MMPs can influence the morphology of mature dendritic spines, and hence synaptic stability.     

Three-dimensional reconstruction of synapse and dendritic spines in the hippocampus of rats and ground squirrels: new paradigms of the structure and function of a synapse

The article reviews the literature data and results obtained by the authors concerning synaptic plasticity and remodeling of synaptic organelles in the central nervous system. Modern techniques of laser scanning confocal microscopy and serial thin sectioning for in vivo and in vitro studies of dendritic spines including a correlation between morphological changes and synaptic transmission efficiency are discussed, particularly, in relation to long-term potentiation. Organization of different types of dendritic spines and involvement of filopodia in spine genesis are examined. Significance of serial ultrathin sections for unbiased quantitative stereological analysis and three-dimensional reconstructions is discussed. The contact of one dendritic spine with two presynaptic boutons (multiple synapses) on both CA1 mushroom dendritic spines is discussed. The analyzed findings suggest new ideas for organization and functioning of synapses.     

Confocal scanning optical microscopy and three-dimensional imaging

Summary— Confocal scanning optical microscopy has significant advantages over conventional fluorescence microscopy: it rejects the out-of-locus light and provides a greater resolution than the wide-field microscope. In laser scanning optical microscopy, the specimen is scanned by a diffraction-limited spot of laser light and the fluorescence emission (or the reflected light) is focused onto a photodetector. The imaged point is then digitized, stored into the memory of a computer and displayed at the appropriate spatial position on a graphic device as a part of a two-dimensional image. Thus, confocal scanning optical microscopy allows accurate non-invasive optical sectioning and further three-dimensional reconstruction of biological specimens. Here we review the recent technological aspects of the principles and uses of the confocal microscope, and we introduce the different methods of three-dimensional imaging.     

High-throughput, detailed, cell-specific neuroanatomy of dendritic spines using microinjection and confocal microscopy

Morphological features such as size, shape and density of dendritic spines have been shown to reflect important synaptic functional attributes and potential for plasticity. Here we describe in detail a protocol for obtaining detailed morphometric analysis of spines using microinjection of fluorescent dyes, high-resolution confocal microscopy, deconvolution and image analysis with NeuronStudio. Recent technical advancements include better preservation of tissue, resulting in prolonged ability to microinject, and algorithmic improvements that compensate for the residual z-smear inherent in all optical imaging. Confocal imaging parameters were probed systematically to identify both optimal resolution and the highest efficiency. When combined, our methods yield size and density measurements comparable to serial section transmission electron microscopy in a fraction of the time. An experiment containing three experimental groups with eight subjects each can take as little as 1 month if optimized for speed, or approximately 4-5 months if the highest resolution and morphometric detail is sought.     

A Compartmental Model for Activity-Dependent Dendritic Spine Branching

Dendritic spines are small, mushroom-like protrusions from the arbor of a neuron in the central nervous system. Interdependent changes in the morphology, biochemistry, and activity of spines have been associated with learning and memory. Moreover, post-mortem cortices from patients with Alzheimer’s or Parkinson’s disease exhibit biochemical and physical alterations within their dendritic arbors and a reduction in the number of dendritic spines. For over a decade, experimentalists have observed perforations in postsynaptic densities on dendritic spines after induction of long-term potentiation, a sustained enhancement of response to a brief electrical or chemical stimulus, associated with learning and memory. In more recent work, some suggest that activity-dependent intraspine calcium may regulate the surface area of the spine head, and reorganization of postsynaptic densities on the surface. In this paper, we develop a model of a dendritic spine with the ability to partition its transmission and receptor zones, as well as the entire spine head. Simulations are initially performed with fixed parameters for morphology to study electrical properties and identify parameters that increase efficacy of the synaptic connection. Equations are then introduced to incorporate calcium as a second messenger in regulating continuous changes in morphology. In the model, activity affects compartmental calcium, which regulates spine head morphology. Conversely, spine head morphology affects the level of local activity, whether the spines are modeled with passive membrane properties, or excitable membrane using Hodgkin–Huxley kinetics. Results indicate that merely separating the postsynaptic receptors on the surface of the spine may add to the diversity of circuitry, but does not change the efficacy of the synapse. However, when the surface area of the spine is a dynamic variable, efficacy of the synapse may vary continuously over time.     

Two-Photon Excitation STED Microscopy in Two Colors in Acute Brain Slices

Many cellular structures and organelles are too small to be properly resolved by conventional light microscopy. This is particularly true for dendritic spines and glial processes, which are very small, dynamic, and embedded in dense tissue, making it difficult to image them under realistic experimental conditions. Two-photon microscopy is currently the method of choice for imaging in thick living tissue preparations, both in acute brain slices and in vivo. However, the spatial resolution of a two-photon microscope, which is limited to ∼350 nm by the diffraction of light, is not sufficient for resolving many important details of neural morphology, such as the width of spine necks or thin glial processes. Recently developed superresolution approaches, such as stimulated emission depletion microscopy, have set new standards of optical resolution in imaging living tissue. However, the important goal of superresolution imaging with significant subdiffraction resolution has not yet been accomplished in acute brain slices. To overcome this limitation, we have developed a new microscope based on two-photon excitation and pulsed stimulated emission depletion microscopy, which provides unprecedented spatial resolution and excellent experimental access in acute brain slices using a long-working distance objective. The new microscope improves on the spatial resolution of a regular two-photon microscope by a factor of four to six, and it is compatible with time-lapse and simultaneous two-color superresolution imaging in living cells. We demonstrate the potential of this nanoscopy approach for brain slice physiology by imaging the morphology of dendritic spines and microglial cells well below the surface of acute brain slices.     

Simultaneous imaging of morphological plasticity and calcium dynamics in dendrites

The structure and function of the nervous system are intricately connected. To investigate their relationship it is essential to image neuronal structure and function simultaneously with high spatio-temporal resolution. For this purpose, we describe here a two-step strategy. First, to visualize neurons and their entire dendritic arborization in neuronal tissue, we use ballistic delivery or single-cell electroporation of a fluorescent calcium indicator and a red fluorescent dye. Second, dual wavelength wide-field fluorescence microscopy or confocal microscopy enables imaging structural plasticity of dendrites (including filopodia and spines) and calcium dynamics together. We routinely apply this strategy to developing neurons in live tissue, but mature neurons can also be loaded and imaged as described. For labeling cells and setting up imaging equipment, approximately 2 h are required.     

Polarization of actin cytoskeleton is reduced in dendritic protrusions during early spine development in hippocampal neuron

Dendritic spines are small protrusions that receive synaptic signals in neuronal networks. The actin cytoskeleton plays a key role in regulating spine morphogenesis, as well as in the function of synapses. Here we report the first quantitative measurement of F-actin retrograde flow rate in dendritic filopodia, the precursor of dendritic spines, and in newly formed spines, using a technique based on photoactivation localization microscopy. We found a fast F-actin retrograde flow in the dendritic filopodia but not in the spine necks. The quantification of F-actin flow rates, combined with fluorescence recovery after photobleaching measurements, allowed for a full quantification of spatially resolved kinetic rates of actin turnover, which was not previously feasible. Furthermore we provide evidences that myosin II regulates the actin flow in dendritic filopodia and translocates from the base to the tip of the protrusion upon spine formation. Rac1 inhibition led to mislocalization of myosin II, as well as to disruption of the F-actin flow. These results provide advances in the quantitative understanding of F-actin remodeling during spine formation.     

Electron microscopic 3D-reconstruction of dendritic spines in cultured hippocampal neurons undergoing synaptic plasticity

Dendritic spines are assumed to constitute the locus of neuronal plasticity, and considerable effort has been focused on attempts to demonstrate that new memories are associated with the formation of new spines. However, few studies that have documented the appearance of spines after exposure to plasticity-producing paradigms could demonstrate that a new spine is touched by a bona fida presynaptic terminal. Thus, the functional significance of plastic dendritic spine changes is not clearly understood. We have used quantitative time lapse confocal imaging of cultured hippocampal neurons before and after their exposure to a conditioning medium which activates synaptic NMDA receptors. Following the experiment the cultures were prepared for 3D electron microscopic reconstruction of visually identified dendritic spines. We found that a majority of new, 1- to 2-h-old spines was touched by presynaptic terminals. Furthermore, when spines disappeared, the parent dendrites were sometime touched by a presynaptic bouton at the site where the previously identified spine had been located. We conclude that new spines are most likely to be functional and that pruned spines can be transformed into shaft synapses and thus maintain their functionality within the neuronal network.     

Super-resolution dynamic imaging of dendritic spines using a low-affinity photoconvertible actin probe

The actin cytoskeleton of dendritic spines plays a key role in morphological aspects of synaptic plasticity. The detailed analysis of the spine structure and dynamics in live neurons, however, has been hampered by the diffraction-limited resolution of conventional fluorescence microscopy. The advent of nanoscopic imaging techniques thus holds great promise for the study of these processes. We implemented a strategy for the visualization of morphological changes of dendritic spines over tens of minutes at a lateral resolution of 25 to 65 nm. We have generated a low-affinity photoconvertible probe, capable of reversibly binding to actin and thus allowing long-term photoactivated localization microscopy of the spine cytoskeleton. Using this approach, we resolve structural parameters of spines and record their long-term dynamics at a temporal resolution below one minute. Furthermore, we have determined changes in the spine morphology in response to pharmacologically induced synaptic activity and quantified the actin redistribution underlying these changes. By combining PALM imaging with quantum dot tracking, we could also simultaneously visualize the cytoskeleton and the spine membrane, allowing us to record complementary information on the morphological changes of the spines at super-resolution.     

Molecular regulation of dendritic spine shape and function

Dendritic spines are discrete membrane protrusions from dendritic shafts where the large majority of excitatory synapses are located. Their highly heterogeneous morphology is thought to be the morphological basis for synaptic plasticity. Electron microscopy and time-lapse imaging studies have suggested that the shape and number of spines can change after long-term potentiation (LTP), although there is no evidence that morphological changes are necessary for LTP induction and maintenance. An increasing number of proteins have been found to be morphogens for dendritic spines and provide new insights into the molecular mechanisms regulating spine formation and morphology.