Formation of Dendritic Spines with GABAergic Synapses Induced by Whisker Stimulation in Adult Mice

AbstractDuring development, alterations in sensory experience modify the structure of cortical neurons, particularly at the level of the dendritic spine. Are similar adaptations involved in plasticity of the adult cortex? Here we show that a 24 hr period of single whisker stimulation in freely moving adult mice increases, by 36%, the total synaptic density in the corresponding cortical barrel. This is due to an increase in both excitatory and inhibitory synapses found on spines. Four days after stimulation, the inhibitory inputs to the spines remain despite total synaptic density returning to pre-stimulation levels. Functional analysis of layer IV cells demonstrated altered response properties, immediately after stimulation, as well as four days later. These results indicate activity-dependent alterations in synaptic circuitry in adulthood, modifying the flow of sensory information into the cerebral cortex

Fast Homogeneous En Bloc Staining of Large Tissue Samples for Volume Electron Microscopy

Fixation and staining of large tissue samples are critical for the acquisition of volumetric electron microscopic image datasets and the subsequent reconstruction of neuronal circuits. Efficient protocols exist for the staining of small samples, but uniform contrast is often difficult to achieve when the sample diameter exceeds a few hundred micrometers. Recently, a protocol (BROPA, brain-wide reduced-osmium staining with pyrogallol-mediated amplification) was developed that achieves homogeneous staining of the entire mouse brain but requires very long sample preparation times. By exploring modifications of this protocol we developed a substantially faster procedure, fBROPA, that allows for reliable high-quality staining of tissue blocks on the millimeter scale. Modifications of the original BROPA protocol include drastically reduced incubation times and a lead aspartate incubation to increase sample conductivity. Using this procedure, whole brains from adult zebrafish were stained within 4 days. Homogenous high-contrast staining was achieved throughout the brain. High-quality image stacks with voxel sizes of 10 × 10 × 25 nm3 were obtained by serial block-face imaging using an electron dose of ~15 e−/nm2. No obvious reduction in staining quality was observed in comparison to smaller samples stained by other state-of-the-art procedures. Furthermore, high-quality images with minimal charging artifacts were obtained from non-neural tissues with low membrane density. fBROPA is therefore likely to be a versatile and efficient sample preparation protocol for a wide range of applications in volume electron microscopy

Imaging Transient Blood Vessel Fusion Events in Zebrafish by Correlative Volume Electron Microscopy

The study of biological processes has become increasingly reliant on obtaining high-resolution spatial and temporal data through imaging techniques. As researchers demand molecular resolution of cellular events in the context of whole organisms, correlation of non-invasive live-organism imaging with electron microscopy in complex three-dimensional samples becomes critical. The developing blood vessels of vertebrates form a highly complex network which cannot be imaged at high resolution using traditional methods. Here we show that the point of fusion between growing blood vessels of transgenic zebrafish, identified in live confocal microscopy, can subsequently be traced through the structure of the organism using Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) and Serial Block Face/Scanning Electron Microscopy (SBF/SEM). The resulting data give unprecedented microanatomical detail of the zebrafish and, for the first time, allow visualization of the ultrastructure of a time-limited biological event within the context of a whole organism

Plasticity of Astrocytic Coverage and Glutamate Transporter Expression in Adult Mouse Cortex

Astrocytes play a major role in the removal of glutamate from the extracellular compartment. This clearance limits the glutamate receptor activation and affects the synaptic response. This function of the astrocyte is dependent on its positioning around the synapse, as well as on the level of expression of its high-affinity glutamate transporters, GLT1 and GLAST. Using Western blot analysis and serial section electron microscopy, we studied how a change in sensory activity affected these parameters in the adult cortex. Using mice, we found that 24 h of whisker stimulation elicited a 2-fold increase in the expression of GLT1 and GLAST in the corresponding cortical column of the barrel cortex. This returns to basal levels 4 d after the stimulation was stopped, whereas the expression of the neuronal glutamate transporter EAAC1 remained unaltered throughout. Ultrastructural analysis from the same region showed that sensory stimulation also causes a significant increase in the astrocytic envelopment of excitatory synapses on dendritic spines. We conclude that a period of modified neuronal activity and synaptic release of glutamate leads to an increased astrocytic coverage of the bouton–spine interface and an increase in glutamate transporter expression in astrocytic processes

Is EM dead?

Since electron microscopy (EM) first appeared in the 1930s, it has held centre stage as the primary tool for the exploration of biological structure. Yet, with the recent developments of light microscopy techniques that overcome the limitations imposed by the diffraction boundary, the question arises as to whether the importance of EM in on the wane. This Commentary describes some of the pioneering studies that have shaped our understanding of cell structure. These include the development of cryo-EM techniques that have given researchers the ability to capture images of native structures and at the molecular level. It also describes how a number of recent developments significantly increase the ability of EM to visualise biological systems across a range of length scales, and in 3D, ensuring that EM will remain at the forefront of biology research for the foreseeable future

Un premier pas vers l’étude du collagène du cartilage articulaire en microscopie électronique

Le cartilage articulaire est localisé à la surface des os impliqués dans une articulation et permet des mouvements avec un minimum de friction tout en agissant comme surface supportant les charges. Etant donné le nombre croissant de patients souffrant de pathologies telles que l’ostéoarthrite et l’arthrite rhumatoïde, l’étude du cartilage articulaire prend de plus en plus d’importance. Le cartilage articulaire est un sujet de choix pour être étudié par une technique d’imagerie de haute résolution comme la technique de microscopie électronique à transmission (MET), mais cette approche est complexe dû à la prépondérance de la matrice extracellulaire, composée essentiellement de protéoglycanes et de collagène et très difficile de préserver et colorer pour en extraire des informations sur l’ultrastructure. Afin de vérifier notre méthodologie pour reproduire des données déjà disponibles sur l’imagerie du cartilage par MET, et ainsi corréler les images avec le contenu en collagène, nous avons appliqué la MET à l’étude du ménisque et du plateau cartilagineux fémoral d’une articulation de la patte postérieure d’un rat adulte. Grâce à la préparation classique du tissu pour la MET focalisant sur la préservation du collagène au détriment des protéoglycanes, nous avons pu reproduire les données publiées et différencier les deux tissus en fonction de leur type de collagène prépondérant. La prochaine étape de notre travail sera d’utiliser ces images de MET pour valider les images de microscopie électronique à balayage (MEB) que nous aimerions générer à partir des mêmes tissus

Automated analysis of spine dynamics on live CA1 pyramidal cells

Dendritic spines may be tiny in volume, but are of major importance for neuroscience. They are the main receivers for excitatory synaptic connections, and their constant changes in number and in shape reflect the dynamic connectivity of the brain. Two-photon microscopy allows following the fate of individual spines in brain slice preparations and in live animals. The diffraction-limited and non-isotropic resolution of this technique, however, makes detection of such tiny structures rather challenging, especially along the optical axis (z-direction). Here we present a novel spine detection algorithm based on a statistical dendrite intensity model and a corresponding spine probability model. To quantify the fidelity of spine detection, we generated correlative datasets: Following two-photon imaging of live pyramidal cell dendrites, we used Serial Block-Face Scanning Electron Microscopy (SBEM) to reconstruct dendritic ultrastructure in 3D. Statistical models were trained on synthetic fluorescence images generated from SBEM datasets via Point Spread Function (PSF) convolution. After the training period, we tested automatic spine detection on real two-photon datasets and compared the result to ground truth (correlative SBEM data). The performance of our algorithm allowed tracking changes in spine volume automatically over several hours. Using a second fluorescent protein targeted to the endoplasmic reticulum, we could analyze the motion of this organelle inside individual spines. Furthermore, we show that it is possible to distinguish activated spines from non-stimulated neighbors by detection of fluorescently labeled presynaptic vesicle clusters. These examples illustrate how automatic segmentation in 5D (x, y, z, t, λ) allows us to investigate brain dynamics at the level of individual synaptic connections