Project description:The ability to acquire ever larger datasets of brain tissue using volume electron microscopy leads to an increasing demand for the automated extraction of connectomic information. We introduce SyConn2, an open-source connectome analysis toolkit, which works with both on-site high-performance compute environments and rentable cloud computing clusters. SyConn2 was tested on connectomic datasets with more than 10 million synapses, provides a web-based visualization interface and makes these data amenable to complex anatomical and neuronal connectivity queries.
Project description:In recent years, numerous studies have shown that astrocytes play an important role in neuronal processing of information. One of the most interesting findings is the existence of bidirectional interactions between neurons and astrocytes at synapses, which has given rise to the concept of "tripartite synapses" from a functional point of view. We used focused ion beam milling and scanning electron microscopy (FIB/SEM) to examine in 3D the relationship of synapses with astrocytes that were previously labeled by intracellular injections in the rat somatosensory cortex. We observed that a large number of synapses (32%) had no contact with astrocytic processes. The remaining synapses (68%) were in contact with astrocytic processes, either at the level of the synaptic cleft (44%) or with the pre- and/or post-synaptic elements (24%). Regarding synaptic morphology, larger synapses with more complex shapes were most frequently found within the population that had the synaptic cleft in contact with astrocytic processes. Furthermore, we observed that although synapses were randomly distributed in space, synapses that were free of astrocytic processes tended to form clusters. Overall, at least in the developing rat neocortex, the concept of tripartite synapse only seems to be applicable to a subset of synapses.
Project description:Knowing the proportions of asymmetric (excitatory) and symmetric (inhibitory) synapses in the neuropil is critical for understanding the design of cortical circuits. We used focused ion beam milling and scanning electron microscopy (FIB/SEM) to obtain stacks of serial sections from the six layers of the juvenile rat (postnatal day 14) somatosensory cortex (hindlimb representation). We segmented in three-dimensions 6184 synaptic junctions and determined whether they were established on dendritic spines or dendritic shafts. Of all these synapses, 87-94% were asymmetric and 6-13% were symmetric. Asymmetric synapses were preferentially located on dendritic spines in all layers (80-91%) while symmetric synapses were mainly located on dendritic shafts (62-86%). Furthermore, we found that less than 6% of the dendritic spines establish more than one synapse. The vast majority of axospinous synapses were established on the spine head. Synapses on the spine neck were scarce, although they were more common when the dendritic spine established multiple synapses. This study provides a new large quantitative dataset that may contribute not only to the knowledge of the ultrastructure of the cortex, but also towards defining the connectivity patterns through all cortical layers.
Project description:Enzymes that facilitate the local deposition of electron dense reaction products have been widely used as labels in electron microscopy (EM) for the identification of synaptic contacts in neural tissue. Peroxidases, in particular, can efficiently metabolize 3,3'-diaminobenzidine tetrahydrochloride hydrate (DAB) to produce precipitates with high contrast under EM following heavy metal staining, and can be genetically encoded to facilitate the labeling of specific cell-types or organelles. Nevertheless, the peroxidase/DAB method has so far not been reported to work in a multiplexed manner in combination with 3D volume EM techniques (e.g. Serial blockface electron microscopy, SBEM; Focused ion beam electron microscopy, FIBSEM) that are favored for the large-scale ultrastructural assessment of synaptic architecture However, a recently described peroxidase with enhanced enzymatic activity (dAPEX2) can efficienty deposit EM-visible DAB products in thick tissue without detergent treatment opening the possibility for the multiplex labeling of genetically defined cell-types in combination with volume EM methods. Here we demonstrate that multiplexed dAPEX2/DAB tagging is compatible with both FIBSEM and SBEM volume EM approaches and use them to map long-range genetically identified synaptic inputs from the anterior cingulate cortex to the periaqueductal gray in the mouse brain.
Project description:Previous studies revealed an abundance of functional Connexin43 (Cx43) hemichannels consequent to loss of plakophilin-2 (PKP2) expression in adult murine hearts. The increased Cx43-mediated membrane permeability is likely responsible for excess entry of calcium into the cells, leading to an arrhythmogenic/cardiomyopathic phenotype. The latter has translational implications to the molecular mechanisms of inheritable arrhythmogenic right ventricular cardiomyopathy (ARVC). Despite functional evidence, visualization of these "orphan" (i.e., non-paired in a gap junction configuration) Cx43 hemichannels remains lacking. Immuno-electron microscopy (IEM) remains an extremely powerful tool to localize, with nanometric resolution, a protein within its native structural landscape. Yet, challenges for IEM are to preserve the antigenicity of the molecular target and to provide access for antibodies to reach their target, while maintaining the cellular/tissue ultrastructure. Fixation is important for maintaining cell structure, but strong fixation and vigorous dehydration (as it is routine for EM) can alter protein structure, thus impairing antigen-antibody binding. Here, we implemented a method to combine pre-embedding immunolabeling (pre-embedding) with serial block-face scanning electron microscopy (SBF-SEM). We utilized a murine model of cardiomyocyte-specific, Tamoxifen (TAM) activated knockout of PKP2. Adult hearts were harvested 14 days post-TAM, at this time hearts present a phenotype of concealed ARVC (i.e., an arrhythmogenic phenotype but no overt structural disease). Thick (200 µm) vibratome slices were immunolabelled for Cx43 and treated with nanogold or FluoroNanogold, coupled with a silver enhancement. Left or right ventricular free walls were dissected and three-dimensional (3D) localization of Cx43 in cardiac muscle was performed using SBF-SEM. Reconstructed images allowed us to visualize the entire length of gap junction plaques, seen as two parallel, closely packed strings of Cx43-immunoreactive beads at the intercalated disc. In contrast, in PKP2-deficient hearts we observed bulging of the intercellular space, and entire areas where only one of the two strings could be observed, indicating the presence of orphan Cx43. We conclude that pre-embedding and SBF-SEM allowed visualization of cardiac Cx43 plaques in their native environment, providing for the first time a visual complement of functional data indicating the presence of orphan Cx43 hemichannels resulting from loss of desmosomal integrity in the heart.
Project description:A decrease in synaptic plasticity and/or a change in excitation/inhibition balance have been suggested as mechanisms underlying major depression disorder. However, given the crucial role of astrocytes in balancing synaptic function, particular attention should be given to the contribution of astrocytes in these mechanisms, especially since previous findings show that astrocytes are affected and exhibit reactive-like features in depression. Moreover, it has been shown that reactive astrocytes increase the synthesis and release of GABA, contributing significantly to tonic GABA inhibition. In this study we found decreased plasticity and increased tonic GABA inhibition in the prelimbic area in acute slices from the medial prefrontal cortex in the Flinders Sensitive Line (FSL) rat model of depression. The tonic inhibition can be reduced by either blocking astrocytic intracellular Ca2+ signaling or by reducing astrocytic GABA through inhibition of the synthesizing enzyme MAO-B with Selegiline. Blocking GABA synthesis also restores the impaired synaptic plasticity in the FSL prefrontal cortex, providing a new antidepressant mechanism of Selegiline.
Project description:In mammalian cochlea, sound-induced vibration is amplified by a three-row lattice of Y-shaped microstructures consisting of electromotile outer hair cell and supporting Deiters cell. This highly organized structure is thought to be essential for hearing of low-level sounds. Prior studies reported differences in geometry and synaptic innervation of the outer hair cells between rows, but how these fine features are achieved at subcellular level still remains unclear. Using serial block-face electron microscopy, we acquired few-hundred-micron-sized cytoarchitecture of mouse organ of Corti at nanometer resolution. Structural quantifications were performed on the Y-shapes as well as afferent and efferent projections to outer hair cells (OHCs). Several new features, which support the previously observed inter-row heterogeneity, are described. Our result provides structural bases for the gradient of mechanical properties and diverse centrifugal regulation of OHC rows.
Project description:Executive functions of the brain are believed to require tonic dopamine inputs to the prefrontal cortex (PFC). It is unclear, however, how this background dopamine activity controls synaptic plasticity in the PFC, a possible underlying mechanism of executive functions. Using PFC slices, we show that pairing of dopamine with weak tetanic stimulation, a maneuver that otherwise induces NMDA receptor-independent long-term depression (LTD), induces long-term potentiation (LTP) when "primed" with dopamine. This "priming" occurs through the combined activation of D1 and D2 receptors and requires 12-40 min to develop. Moreover, concurrent synaptic activation of NMDA receptors during priming is necessary for this novel form of LTP. We suggest that a role of background dopamine signals in the PFC is to prevent high-frequency synaptic inputs from abnormally inducing LTD and to secure the induction of LTP.