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In ecological analyses of species and community distributions there is interest in the nature of their responses to environmental gradients and in identifying the most important environmental variables, which may be used for predicting patterns of biodiversity. Methods such as random forests already exist to assess predictor importance for individual species and to indicate where along gradients abundance changes. However, there is a need to extend these methods to whole assemblages, to establish where along the range of these gradients the important compositional changes occur, and to identify any important thresholds or change points. We develop such a method, called \"gradient forest,\" which is an extension of the random forest approach. By synthesizing the cross-validated R 2 and accuracy importance measures from univariate random forest analyses across multiple species, sampling devices, and surveys, gradient forest obtains a monotonic function of each predictor that represents the compositional turnover along the gradient of the predictor. When applied to a synthetic data set, the method correctly identified the important predictors and delineated where the compositional change points occurred along these gradients. Application of gradient forest to a real data set from part of the Great Barrier Reef identified mud fraction of the sediment as the most important predictor, with highest compositional turnover occurring at mud fraction values around 25%, and provided similar information for other predictors. Such refined information allows for more accurate capturing of biodiversity patterns for the purposes of bioregionalization, delineation of protected areas, or designing of biodiversity surveys.

Glypican 4 and glypican 6 are identified as astrocyte-secreted signals that induce the formation of functional, rather than structural, synapses through the recruitment to the neuron surface of the GluA1 subunits of the AMPA glutamate receptor. Staring role for glypicans Molecular signals released by astrocytes, the dominant type of glial cell found in the brain, have previously been identified as influential regulators in the formation of new synapses in the developing central nervous system. However, these molecules mostly induce the structural synapse, with the connection itself remaining functionally silent. Here, Allen et al . biochemically isolate other astrocyte-derived signals, glypicans 4 and 6, which induce functional synapses and are sufficient to increase the frequency of excitatory synaptic events. This is achieved by enhancing the density of AMPA-sensitive glutamate receptors at the surface of the synapse. Glypican 6 defects have been observed in human disorders involving synaptic dysfunction, suggesting a role for glypicans as regulators of neuronal circuit formation in both development and disease. In the developing central nervous system (CNS), the control of synapse number and function is critical to the formation of neural circuits. We previously demonstrated that astrocyte-secreted factors powerfully induce the formation of functional excitatory synapses between CNS neurons 1 . Astrocyte-secreted thrombospondins induce the formation of structural synapses, but these synapses are postsynaptically silent 2 . Here we use biochemical fractionation of astrocyte-conditioned medium to identify glypican 4 (Gpc4) and glypican 6 (Gpc6) as astrocyte-secreted signals sufficient to induce functional synapses between purified retinal ganglion cell neurons, and show that depletion of these molecules from astrocyte-conditioned medium significantly reduces its ability to induce postsynaptic activity. Application of Gpc4 to purified neurons is sufficient to increase the frequency and amplitude of glutamatergic synaptic events. This is achieved by increasing the surface level and clustering, but not overall cellular protein level, of the GluA1 subunit of the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) glutamate receptor (AMPAR). Gpc4 and Gpc6 are expressed by astrocytes in vivo in the developing CNS, with Gpc4 expression enriched in the hippocampus and Gpc6 enriched in the cerebellum. Finally, we demonstrate that Gpc4-deficient mice have defective synapse formation, with decreased amplitude of excitatory synaptic currents in the developing hippocampus and reduced recruitment of AMPARs to synapses. These data identify glypicans as a family of novel astrocyte-derived molecules that are necessary and sufficient to promote glutamate receptor clustering and receptivity and to induce the formation of postsynaptically functioning CNS synapses.

Array tomography encompasses light and electron microscopy modalities that offer unparalleled opportunities to explore three-dimensional cellular architectures in extremely fine structural and molecular detail. Fluorescence array tomography achieves much higher resolution and molecular multiplexing than most other fluorescence microscopy methods, while electron array tomography can capture three-dimensional ultrastructure much more easily and rapidly than traditional serial-section electron microscopy methods. A correlative fluorescence/electron microscopy mode of array tomography furthermore offers a unique capacity to merge the molecular discrimination strengths of multichannel fluorescence microscopy with the ultrastructural imaging strengths of electron microscopy. This essay samples the first decade of array tomography, highlighting applications in neuroscience.

A method for 3D differentiation of human pluripotent stem cells yields brain cortical spheroids with functional neurons and astrocytes. The spheroids can be sliced for imaging and electrophysiological studies. The human cerebral cortex develops through an elaborate succession of cellular events that, when disrupted, can lead to neuropsychiatric disease. The ability to reprogram somatic cells into pluripotent cells that can be differentiated in vitro provides a unique opportunity to study normal and abnormal corticogenesis. Here, we present a simple and reproducible 3D culture approach for generating a laminated cerebral cortex–like structure, named human cortical spheroids (hCSs), from pluripotent stem cells. hCSs contain neurons from both deep and superficial cortical layers and map transcriptionally to in vivo fetal development. These neurons are electrophysiologically mature, display spontaneous activity, are surrounded by nonreactive astrocytes and form functional synapses. Experiments in acute hCS slices demonstrate that cortical neurons participate in network activity and produce complex synaptic events. These 3D cultures should allow a detailed interrogation of human cortical development, function and disease, and may prove a versatile platform for generating other neuronal and glial subtypes in vitro .

Myelin is best known for its role in increasing the conduction velocity and metabolic efficiency of long-range excitatory axons. Accordingly, the myelin observed in neocortical gray matter is thought to mostly ensheath excitatory axons connecting to subcortical regions and distant cortical areas. Using independent analyses of light and electron microscopy data from mouse neocortex, we show that a surprisingly large fraction of cortical myelin (half the myelin in layer 2/3 and a quarter in layer 4) ensheathes axons of inhibitory neurons, specifically of parvalbumin-positive basket cells. This myelin differs significantly from that of excitatory axons in distribution and protein composition. Myelin on inhibitory axons is unlikely to meaningfully hasten the arrival of spikes at their pre-synaptic terminals, due to the patchy distribution and short path-lengths observed. Our results thus highlight the need for exploring alternative roles for myelin in neocortical circuits. The brain is far away from the muscles that it controls. In humans, for example, the brain must be able to trigger the contraction of muscles that are more than a meter away. This task falls to specialized motor neurons that stretch from the brain to the spinal cord, and from the spinal cord to the muscles. Neurons transmit information (in the form of electrical nerve impulses) along their length through cable-like structures called axons. The axons of the motor neurons are so long that, if they were ‘naked’, it would take at least a second for nerve impulses to travel their entire length. Such a long delay between thoughts and actions would make rapid movement impossible. Nerve impulses are able to travel from the brain to the muscles much more quickly, because they are wrapped with a substance called myelin that acts like electrical insulation. Myelin helps the nerve impulses travel up to 100 times faster down the axon. Not surprisingly, diseases that damage myelin, such as multiple sclerosis, severely disrupt movement and sensation. Not all of the brain’s myelin is found around the long axons of motor neurons. The outer layer of the brain, known as the cerebral cortex, also contains myelin. However, most neurons within the cerebral cortex are only a few millimeters long. So what exactly is myelin doing there? Micheva et al. have now used electron microscopy and light microscopy to study the neurons in the cortex of the mouse brain. This revealed that up to half of the myelin in some layers of the cortex surrounds the axons of inhibitory neurons (which reduce the activity of the neurons they signal to). Moreover, one particular subtype of inhibitory neuron accounts for most of the myelinated inhibitory axons, namely inhibitory neurons that contain a protein called parvalbumin. Exactly why parvalbumin-containing neurons are myelinated remains a mystery. Myelin covers only short segments of the axons of these neurons, so it would speed up the transmission of signals by less than a millisecond – probably not enough to make a meaningful difference. Parvalbumin-containing neurons often signal frequently, and thus require large amounts of energy. One possibility therefore is that myelin helps to meet these energy requirements or to reduce energy consumption. Further research will be needed to test this and other ideas.

Conventional heavy metal poststaining methods on thin sections lend contrast but often cause contamination. To avoid this problem, we tested several en bloc staining techniques to contrast tissue in serial sections mounted on solid substrates for examination by field emission scanning electron microscopy (FESEM). Because FESEM section imaging requires that specimens have higher contrast and greater electrical conductivity than transmission electron microscopy (TEM) samples, our technique uses osmium impregnation (OTO) to make the samples conductive while heavily staining membranes for segmentation studies. Combining this step with other classic heavy metal en bloc stains, including uranyl acetate (UA), lead aspartate, copper sulfate and lead citrate, produced clean, highly contrasted TEM and scanning electron microscopy (SEM) samples of insect, fish and mammalian nervous systems. This protocol takes 7–15 d to prepare resin-embedded tissue, cut sections and produce serial section images.

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.

Synapse loss correlates with a cognitive decline in Alzheimer's disease (AD), but whether this is caused by fibrillar deposits known as senile plaques or soluble oligomeric forms of amyloid β (Aβ) is controversial. By using array tomography, a technique that combines ultrathin sectioning of tissue with immunofluorescence, allowing precise quantification of small structures, such as synapses, we have tested the hypothesis that oligomeric Aβ surrounding plaques contributes to synapse loss in a mouse model of AD. We find that senile plaques are surrounded by a halo of oligomeric Aβ. Analysis of >14,000 synapses (represented by PSD95-stained excitatory synapses) shows that there is a 60% loss of excitatory synapses in the halo of oligomeric Aβ surrounding plaques and that the density increases to reach almost control levels in volumes further than 50 μm from a plaque in an approximately linear fashion (linear regression, r² = 0.9; P < 0.0001). Further, in transgenic cortex, microdeposits of oligomeric Aβ associate with a subset of excitatory synapses, which are significantly smaller than those not in contact with oligomeric Aβ. The proportion of excitatory synapses associated with Aβ correlates with decreasing density (correlation, -0.588; P < 0.0001). These data show that senile plaques are a potential reservoir of oligomeric Aβ, which colocalizes with the postsynaptic density and is associated with spine collapse, reconciling the apparently competing schools of thought of \"plaque\" vs. \"oligomeric Aβ\" as the synaptotoxic species in the brain of AD patients.

Adaptive neuronal circuit function requires a continual adjustment of synaptic network parameters known as “neuromodulation”, a process prominently involving G protein-coupled receptors (GPCRs), signal transduction by heterotrimeric G proteins and regulation of ion channel gating downstream. Many of the basic molecular mechanisms of neuromodulation are now known, but the organization of neuromodulation at a network level has remained an enigma. Single-cell RNA sequencing now provides a bright light with which to illuminate this critical “dark matter” of neuroscience. Here we leverage recent advances in transcriptomics and neurotaxonomy to explore GPCR, G protein and ion channel gene expression across cell types and individual neurons in mouse hippocampus area CA1, with the goal of revealing broad outlines of that well-studied brain structure's neuromodulatory network architecture.