Seeking new insights into the homeostasis, modulation and plasticity of cortical synaptic networks, we have analyzed results from a single-cell RNA-seq study of 22,439 mouse neocortical neurons. Our ...analysis exposes transcriptomic evidence for dozens of molecularly distinct neuropeptidergic modulatory networks that directly interconnect all cortical neurons. This evidence begins with a discovery that transcripts of one or more neuropeptide precursor (NPP) and one or more neuropeptide-selective G-protein-coupled receptor (NP-GPCR) genes are highly abundant in all, or very nearly all, cortical neurons. Individual neurons express diverse subsets of NP signaling genes from palettes encoding 18 NPPs and 29 NP-GPCRs. These 47 genes comprise 37 cognate NPP/NP-GPCR pairs, implying the likelihood of local neuropeptide signaling. Here, we use neuron-type-specific patterns of NP gene expression to offer specific, testable predictions regarding 37 peptidergic neuromodulatory networks that may play prominent roles in cortical homeostasis and plasticity.
We assembled a semi-automated reconstruction of L2/3 mouse primary visual cortex from ∼250 × 140 × 90 μm3 of electron microscopic images, including pyramidal and non-pyramidal neurons, astrocytes, ...microglia, oligodendrocytes and precursors, pericytes, vasculature, nuclei, mitochondria, and synapses. Visual responses of a subset of pyramidal cells are included. The data are publicly available, along with tools for programmatic and three-dimensional interactive access. Brief vignettes illustrate the breadth of potential applications relating structure to function in cortical circuits and neuronal cell biology. Mitochondria and synapse organization are characterized as a function of path length from the soma. Pyramidal connectivity motif frequencies are predicted accurately using a configuration model of random graphs. Pyramidal cells receiving more connections from nearby cells exhibit stronger and more reliable visual responses. Sample code shows data access and analysis.
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•A cortical reconstruction describes neuron connectivity, function, and cell biology•Pyramidal areal synapse density appears invariant with distance from the soma•Analysis of connectivity motifs reexamines "non-randomness" in cortical networks•Pyramidal cells with more local connections give stronger and more reliable responses
Reconstruction of mouse visual cortex provides capacity for quantitative characterization of organelles, compartments, cells, circuits, and activity and their interrelations.
Neurons in the developing brain undergo extensive structural refinement as nascent circuits adopt their mature form. This physical transformation of neurons is facilitated by the engulfment and ...degradation of axonal branches and synapses by surrounding glial cells, including microglia and astrocytes. However, the small size of phagocytic organelles and the complex, highly ramified morphology of glia have made it difficult to define the contribution of these and other glial cell types to this crucial process. Here, we used large-scale, serial section transmission electron microscopy (TEM) with computational volume segmentation to reconstruct the complete 3D morphologies of distinct glial types in the mouse visual cortex, providing unprecedented resolution of their morphology and composition. Unexpectedly, we discovered that the fine processes of oligodendrocyte precursor cells (OPCs), a population of abundant, highly dynamic glial progenitors, frequently surrounded small branches of axons. Numerous phagosomes and phagolysosomes (PLs) containing fragments of axons and vesicular structures were present inside their processes, suggesting that OPCs engage in axon pruning. Single-nucleus RNA sequencing from the developing mouse cortex revealed that OPCs express key phagocytic genes at this stage, as well as neuronal transcripts, consistent with active axon engulfment. Although microglia are thought to be responsible for the majority of synaptic pruning and structural refinement, PLs were ten times more abundant in OPCs than in microglia at this stage, and these structures were markedly less abundant in newly generated oligodendrocytes, suggesting that OPCs contribute substantially to the refinement of neuronal circuits during cortical development.
A primary cilium is a membrane-bound extension from the cell surface that contains receptors for perceiving and transmitting signals that modulate cell state and activity. Primary cilia in the brain ...are less accessible than cilia on cultured cells or epithelial tissues because in the brain they protrude into a deep, dense network of glial and neuronal processes. Here, we investigated cilia frequency, internal structure, shape, and position in large, high-resolution transmission electron microscopy volumes of mouse primary visual cortex. Cilia extended from the cell bodies of nearly all excitatory and inhibitory neurons, astrocytes, and oligodendrocyte precursor cells (OPCs) but were absent from oligodendrocytes and microglia. Ultrastructural comparisons revealed that the base of the cilium and the microtubule organization differed between neurons and glia. Investigating cilia-proximal features revealed that many cilia were directly adjacent to synapses, suggesting that cilia are poised to encounter locally released signaling molecules. Our analysis indicated that synapse proximity is likely due to random encounters in the neuropil, with no evidence that cilia modulate synapse activity as would be expected in tetrapartite synapses. The observed cell class differences in proximity to synapses were largely due to differences in external cilia length. Many key structural features that differed between neuronal and glial cilia influenced both cilium placement and shape and, thus, exposure to processes and synapses outside the cilium. Together, the ultrastructure both within and around neuronal and glial cilia suggest differences in cilia formation and function across cell types in the brain.
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•Primary cilia extend from visual cortex neurons and select glia•Internal cilia ultrastructure is not stereotyped but varies between cell classes•Cilia are near synapses and surrounded by neurites and glial processes•Cilia placement and structure impact the external environment of cilia
Ott and Torres et al. identify distinguishing anatomical features of neuronal and glial primary cilia in the mouse visual cortex. Internal structural differences determine both cilium shape and the extent of cilium exposure to the extracellular neuropil, where cilia pass near, and even adjacent to, many synapses.
Morphology based analysis of cell types has been an area of great interest to the neuroscience community for several decades. Recently, high resolution electron microscopy (EM) datasets of the mouse ...brain have opened up opportunities for data analysis at a level of detail that was previously impossible. These datasets are very large in nature and thus, manual analysis is not a practical solution. Of particular interest are details to the level of post synaptic structures. This paper proposes a fully automated framework for analysis of post-synaptic structure based neuron analysis from EM data. The processing framework involves shape extraction, representation with an autoencoder, and whole cell modeling and analysis based on shape distributions. We apply our novel framework on a dataset of 1031 neurons obtained from imaging a 1mm x 1mm x 40 micrometer volume of the mouse visual cortex and show the strength of our method in clustering and classification of neuronal shapes.
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