Project description:Life exists in three dimensions, but until the turn of the century most electron microscopy methods provided only 2D image data. Recently, electron microscopy techniques capable of delving deep into the structure of cells and tissues have emerged, collectively called volume electron microscopy (vEM). Developments in vEM have been dubbed a quiet revolution as the field evolved from established transmission and scanning electron microscopy techniques, so early publications largely focused on the bioscience applications rather than the underlying technological breakthroughs. However, with an explosion in the uptake of vEM across the biosciences and fast-paced advances in volume, resolution, throughput and ease of use, it is timely to introduce the field to new audiences. In this Primer, we introduce the different vEM imaging modalities, the specialized sample processing and image analysis pipelines that accompany each modality and the types of information revealed in the data. We showcase key applications in the biosciences where vEM has helped make breakthrough discoveries and consider limitations and future directions. We aim to show new users how vEM can support discovery science in their own research fields and inspire broader uptake of the technology, finally allowing its full adoption into mainstream biological imaging.
Project description:The entorhinal cortex (EC) plays a pivotal role in memory function and spatial navigation, connecting the hippocampus with the neocortex. The EC integrates a wide range of cortical and subcortical inputs, but its synaptic organization in the human brain is largely unknown. We used volume electron microscopy to perform a 3D analysis of the microanatomical features of synapses in all layers of the medial EC (MEC) from the human brain. Using this technology, 12,974 synapses were fully 3D reconstructed at the ultrastructural level. The MEC presented a distinct set of synaptic features, differentiating this region from other human cortical areas. Furthermore, ultrastructural synaptic characteristics within the MEC was predominantly similar, although layers I and VI exhibited several synaptic characteristics that were distinct from other layers. The present study constitutes an extensive description of the synaptic characteristics of the neuropil of all layers of the EC, a crucial step to better understand the connectivity of this cortical region, in both health and disease.
Project description:Functional and structural studies investigating macroscopic connectivity in the human cerebral cortex suggest that high-order associative regions exhibit greater connectivity compared to primary ones. However, the synaptic organization of these brain regions remains unexplored. In the present work, we conducted volume electron microscopy to investigate the synaptic organization of the human brain obtained at autopsy. Specifically, we examined layer III of Brodmann areas 17, 3b, and 4, as representative areas of primary visual, somatosensorial, and motor cortex. Additionally, we conducted comparative analyses with our previous datasets of layer III from temporopolar and anterior cingulate associative cortical regions (Brodmann areas 24, 38, and 21). 9,690 synaptic junctions were 3D reconstructed, showing that certain synaptic characteristics are specific to particular regions. The number of synapses per volume, the proportion of the postsynaptic targets, and the synaptic size may distinguish one region from another, regardless of whether they are associative or primary cortex. By contrast, other synaptic characteristics were common to all analyzed regions, such as the proportion of excitatory and inhibitory synapses, their shapes, their spatial distribution, and a higher proportion of synapses located on dendritic spines. The present results provide further insights into the synaptic organization of the human cerebral cortex.
Project description:The extrageniculate visual pathway, which carries visual information from the retina through the superficial layers of the superior colliculus and the pulvinar, is poorly understood. The pulvinar is thought to modulate information flow between cortical areas, and has been implicated in cognitive tasks like directing visually guided actions. In order to better understand the underlying circuitry, we performed retrograde injections of modified rabies virus in the visual cortex and pulvinar of the Long-Evans rat. We found a relatively small population of cells projecting to primary visual cortex (V1), compared to a much larger population projecting to higher visual cortex. Reciprocal corticothalamic projections showed a similar result, implying that pulvinar does not play as big a role in directly modulating rodent V1 activity as previously thought.
Project description:Top-down, context-dependent modulation of visual processing has been a topic of wide interest, including in mouse primary visual cortex (V1). However, the organization of feedback projections to V1 is relatively unknown. Here, we investigated inputs to mouse V1 by injecting retrograde tracers. We developed a software pipeline that maps labeled cell bodies to corresponding brain areas in the Allen Reference Atlas. We identified more than 24 brain areas that provide inputs to V1 and quantified the relative strength of their projections. We also assessed the organization of the projections, based on either the organization of cell bodies in the source area (topography) or the distribution of projections across V1 (bias). Projections from most higher visual and some nonvisual areas to V1 showed both topography and bias. Such organization of feedback projections to V1 suggests that parts of the visual field are differentially modulated by context, which can be ethologically relevant for a navigating animal.
Project description:Neocortical areas communicate through extensive axonal projections, but the logic of information transfer remains poorly understood, because the projections of individual neurons have not been systematically characterized. It is not known whether individual neurons send projections only to single cortical areas or distribute signals across multiple targets. Here we determine the projection patterns of 591 individual neurons in the mouse primary visual cortex using whole-brain fluorescence-based axonal tracing and high-throughput DNA sequencing of genetically barcoded neurons (MAPseq). Projections were highly diverse and divergent, collectively targeting at least 18 cortical and subcortical areas. Most neurons targeted multiple cortical areas, often in non-random combinations, suggesting that sub-classes of intracortical projection neurons exist. Our results indicate that the dominant mode of intracortical information transfer is not based on 'one neuron-one target area' mapping. Instead, signals carried by individual cortical neurons are shared across subsets of target areas, and thus concurrently contribute to multiple functional pathways.
Project description:Unified visual perception relies on the integration of bottom-up and top-down inputs in the primary visual cortex (V1), with top-down inputs known to provide behavior-related modulation on visual processing. However, the organization of top-down inputs in V1 remains unclear. Here, using optogenetics-assisted circuit mapping, we characterized how multiple top-down inputs from higher-order cortical and thalamic areas engage excitatory and inhibitory neurons in V1. Systematic layer- and cell-type-specific profiling of the innervation properties of top-down inputs ultimately revealed that each top-down input employs a unique laminar profile when innervating V1. These profiles partially overlap in superficial layers, bypass layer 4 (L4), and clearly segregate upon reaching deep layers. Specifically, inputs from the medial secondary visual cortex (V2M) and anterior cingulate cortex (ACA) preferentially activate L6 neurons, while inputs from the ventrolateral orbitofrontal cortex (ORBvl) and lateral posterior thalamic nucleus (LP) activate L5 neurons. Having defined the inputs, we conducted independent optogenetic activation studies and discovered that ORBvl and LP inputs selectively activate two types of pyramidal neurons (Pyrs) in L5: Pyr <-- ORBvl and Pyr <-- LP neurons, each with specific electrophysiological properties and gene expression profiles. Retrograde mapping subsequently revealed that Pyr <-- ORBvl neurons preferentially innervate subcortical areas and Pyr <-- LP neurons innervate cortical areas, indicating parallel processing of ORBvl and LP inputs in Pyr-type-specific subnetworks. Further, we found mutual inhibition between these two subnetworks, as LP inputs indirectly inhibit Pyr <-- ORBvl neurons and ORBvl inputs inhibit Pyr <-- LP neurons through local inhibitory neurons. Our study thus deepens understanding of the neuronal mechanisms involved in top-down modulation of visual processing by providing a valuable resource characterizing the layer- and cell-type-specific organization of top-down inputs in V1 and by revealing that L5 Pyr-type-specific subnetworks engage in parallel processing of corticocortical and thalamocortical top-down inputs.
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.