The classic approach to measure the spiking response of neurons involves the use of metal electrodes to record extracellular potentials. Starting over 60 years ago with a single recording site, this technology now extends to ever larger numbers and densities of sites. We argue, based on the mechanical and electrical properties of existing materials, estimates of signal-to-noise ratios, assumptions regarding extracellular space in the brain, and estimates of heat generation by the electronic interface, that it should be possible to fabricate rigid electrodes to concurrently record from essentially every neuron in the cortical mantle. This will involve fabrication with existing yet nontraditional materials and procedures. We further emphasize the need to advance materials for improved flexible electrodes as an essential advance to record from neurons in brainstem and spinal cord in moving animals.

The cyanobacterial culture HT-58-2, composed of a filamentous cyanobacterium and accompanying community bacteria, produces chlorophyll a as well as the tetrapyrrole macrocycles known as tolyporphins. Almost all known tolyporphins (A-M except K) contain a dioxobacteriochlorin chromophore and exhibit an absorption spectrum somewhat similar to that of chlorophyll a. Here, hyperspectral confocal fluorescence microscopy was employed to noninvasively probe the locale of tolyporphins within live cells under various growth conditions (media, illumination, culture age). Cultures grown in nitrate-depleted media (BG-11 vs. nitrate-rich, BG-11) are known to increase the production of tolyporphins by orders of magnitude (rivaling that of chlorophyll a) over a period of 30-45 days. Multivariate curve resolution (MCR) was applied to an image set containing images from each condition to obtain pure component spectra of the endogenous pigments. The relative abundances of these components were then calculated for individual pixels in each image in the entire set, and 3D-volume renderings were obtained. At 30 days in media with or without nitrate, the chlorophyll a and phycobilisomes (combined phycocyanin and phycobilin components) co-localize in the filament outer cytoplasmic region. Tolyporphins localize in a distinct peripheral pattern in cells grown in BG-11 versus a diffuse pattern (mimicking the chlorophyll a localization) upon growth in BG-11. In BG-11, distinct puncta of tolyporphins were commonly found at the septa between cells and at the end of filaments. This work quantifies the relative abundance and envelope localization of tolyporphins in single cells, and illustrates the ability to identify novel tetrapyrroles in the presence of chlorophyll a in a photosynthetic microorganism within a non-axenic culture.

Hox genes pattern the anterior-posterior axis of animals and are posited to drive animal body plan evolution, yet their precise role in evolution has been difficult to determine. Here, we identified evolutionary modifications in the Hox gene Abd-Bthat dramatically altered its expression along the body plan of Drosophila santomea. Abd-B is required for pigmentation in Drosophila yakuba, the sister species of D. santomea, and changes to Abd-B expression would be predicted to make large contributions to the loss of body pigmentation in D. santomea. However, manipulating Abd-B expression in current-day D. santomea does not affect pigmentation. We attribute this epistatic interaction to four other genes within the D. santomea pigmentation network, three of which have evolved expression patterns that do not respond to Abd-B. Our results demonstrate how body plans may evolve through small evolutionary steps distributed throughout Hox-regulated networks. Polygenicity and epistasis may hinder efforts to identify genes and mechanisms underlying macroevolutionary traits.

BACKGROUND: Unbiased screening studies have repeatedly identified actin-related proteins as one of the families of proteins most influenced by neurotrauma. Nevertheless, the status quo model of cytoskeletal reorganization after neurotrauma excludes actin and incorporates only changes in microtubules and intermediate filaments. Actin is excluded in part because it is difficult to image with conventional techniques. However, recent innovations in fluorescent microscopy provide an opportunity to image the actin cytoskeleton at super-resolution resolution in living cells. This study applied these innovations to an in vitro model of neurotrauma.

NEW METHOD: New methods are introduced for traumatizing neurons before imaging them with high speed structured illumination microscopy or lattice light sheet microscopy. Also, methods for analyzing structured illumination microscopy images to quantify post-traumatic neurite dystrophy are presented.

RESULTS: Human induced pluripotent stem cell-derived neurons exhibited actin organization typical of immature neurons. Neurite dystrophy increased after trauma but was not influenced by jasplakinolide treatment. The F-actin content of dystrophies varied greatly from one dystrophy to another.

COMPARISON WITH EXISTING METHODS: In contrast to fixation dependent methods, these methods capture the evolution of the actin cytoskeleton over time in a living cell. In contrast to prior methods based on counting dystrophies, this quantification scheme parameterizes the severity of a given dystrophy as it evolves from a local swelling to an almost-perfect spheroid that threatens to transect the neurite.

CONCLUSIONS: These methods can be used to investigate genetic factors and therapeutic interventions that modulate the course of neurite dystrophy after trauma.

The development of single-molecule localization microscopy (SMLM) has sparked a revolution in biological imaging, allowing 'super-resolution' fluorescence microscopy below the diffraction limit of light. The last decade has seen an explosion in not only optical hardware for SMLM but also the development or repurposing of fluorescent proteins and small-molecule fluorescent probes for this technique. In this review, written by chemists for chemists, we detail the history of single-molecule localization microscopy and collate the collection of probes with demonstrated utility in SMLM. We hope it will serve as a primer for probe choice in localization microscopy as well as an inspiration for the development of new fluorophores that enable imaging of biological samples with exquisite detail.

Cells require a constant supply of fatty acids to survive and proliferate. Fatty acids incorporate into membrane and storage glycerolipids through a series of endoplasmic reticulum (ER) enzymes, but how these enzymes are regulated is not well understood. Here, using a combination of CRISPR-based genetic screens and unbiased lipidomics, we identified calcineurin B homologous protein 1 (CHP1) as a major regulator of ER glycerolipid synthesis. Loss of CHP1 severely reduces fatty acid incorporation and storage in mammalian cells and invertebrates. Mechanistically, CHP1 binds and activates GPAT4, which catalyzes the initial rate-limiting step in glycerolipid synthesis. GPAT4 activity requires CHP1 to be N-myristoylated, forming a key molecular interface between the two proteins. Interestingly, upon CHP1 loss, the peroxisomal enzyme, GNPAT, partially compensates for the loss of ER lipid synthesis, enabling cell proliferation. Thus, our work identifies a conserved regulator of glycerolipid metabolism and reveals plasticity in lipid synthesis of proliferating cells.

The emergence of new and increasingly sophisticated behaviors after birth is accompanied by dramatic increase of newly established synaptic connections in the nervous system. Little is known, however, of how nascent connections are organized to support such new behaviors alongside existing ones. To understand this, in the larval zebrafish we examined the development of spinal pathways from hindbrain V2a neurons and the role of these pathways in the development of locomotion. We found that new projections are continually layered laterally to existing neuropil, and give rise to distinct pathways that function in parallel to existing pathways. Across these chronologically layered pathways, the connectivity patterns and biophysical properties vary systematically to support a behavioral repertoire with a wide range of kinematics and dynamics. Such layering of new parallel circuits equipped with systematically changing properties may be central to the postnatal diversification and increasing sophistication of an animal's behavioral repertoire.

We present CLADES (Cell Lineage Access Driven by an Edition Sequence), a technology for cell lineage studies based on CRISPR/Cas9. CLADES relies on a system of genetic switches to activate and inactivate reporter genes in a pre-determined order. Targeting CLADES to progenitor cells allows the progeny to inherit a sequential cascade of reporters, coupling birth order with reporter expression. This gives us temporal resolution of lineage development that can be used to deconstruct an extended cell lineage by tracking the reporters expressed in the progeny. When targeted to the germ line, the same cascade progresses across animal generations, marking each generation with the corresponding combination of reporters. CLADES thus offers an innovative strategy for making programmable cascades of genes that can be used for genetic manipulation or to record serial biological events.

Serial-section electron microscopy such as FIB-SEM (focused ion beam scanning electron microscopy) has become an important tool for neuroscientists to trace the trajectories and global architecture of neural circuits in the brain, as well as to visualize the 3D ultrastructure of cellular organelles in neurons. In this study, we examined 3D features of mitochondria in electron microscope images generated from serial sections of four regions of mouse brains: nucleus accumbens (NA), hippocampal CA1, somatosensory cortex and dorsal cochlear nucleus (DCN). We compared mitochondria in the presynaptic terminals to those in the postsynaptic/dendritic compartments, and we focused on the shape and size of mitochondria. A common feature of mitochondria among the four brain regions is that presynaptic mitochondria generally are small and short, and most of them do not extend beyond presynaptic terminals. In contrast, the majority of postsynaptic/dendritic mitochondria are large and many of them spread through significant portions of the dendrites. Comparing among the brain areas, the cerebral cortex and DCN have even larger postsynaptic/dendritic mitochondria than the NA and CA1. Our analysis reveals that mitochondria in neurons are differentially sized and arranged according to their subcellular locations, suggesting a spatial organizing principle of mitochondria at the synapse.