High-throughput microcontact printing of proteins in microwell cell culture plates.

Microcontact printing (MCP) is used to pattern a surface with a specific compound, allowing the spatially restricted response of cells to be assayed as they encounter a molecule of interest. MCP is a relatively low-cost and accessible technique that uses commercially available reagents and common cell culture equipment. However, it can be technically challenging, slow, and incompatible with microwell cell culture plates that are widely used for screening and other applications. Here, we describe a novel protocol using medical biopsy punches to transfer patterns into standard 96-well plates via polydimethylsiloxane (PDMS) cutouts. We demonstrate that this method can be used to deposit patterns of poly-D-lysine (PDL) into the microwells of glass-bottom plates. As a proof-of-concept, we show that cultured rodent glial cells preferentially grow and extend processes on the pattern. This method will allow larger scale MCP experiments in which different patterns, proteins, or other factors can be assayed in parallel.•Biopsy punches enable both cutting out small circular stamps and plunging them into tissue culture microwells to transfer proteins.•Compared to standard MCP, this method offers a more rapid workflow to pattern proteins onto substrates, and allows use of microwell plates that permits larger-scale experiments.

Hypomyelination, hypodontia and craniofacial abnormalities in a Polr3b mouse model of leukodystrophy.

RNA polymerase III (Pol III)-related hypomyelinating leukodystrophy (POLR3-HLD), also known as 4H leukodystrophy, is a severe neurodegenerative disease characterized by the cardinal features of hypomyelination, hypodontia and hypogonadotropic hypogonadism. POLR3-HLD is caused by biallelic pathogenic variants in genes encoding Pol III subunits. While approximately half of all patients carry mutations in POLR3B encoding the RNA polymerase III subunit B, there is no in vivo model of leukodystrophy based on mutation of this Pol III subunit. Here, we determined the impact of POLR3BΔ10 (Δ10) on Pol III in human cells and developed and characterized an inducible/conditional mouse model of leukodystrophy using the orthologous Δ10 mutation in mice. The molecular mechanism of Pol III dysfunction was determined in human cells by affinity purification-mass spectrometry and western blot. Postnatal induction with tamoxifen induced expression of the orthologous Δ10 hypomorph in triple transgenic Pdgfrα-Cre/ERT; R26-Stopfl-EYFP; Polr3bfl mice. CNS and non-CNS features were characterized using a variety of techniques including microCT, ex vivo MRI, immunofluorescence, immunohistochemistry, spectral confocal reflectance microscopy and western blot. Lineage tracing and time series analysis of oligodendrocyte subpopulation dynamics based on co-labelling with lineage-specific and/or proliferation markers were performed. Proteomics suggested that Δ10 causes a Pol III assembly defect, while western blots demonstrated reduced POLR3BΔ10 expression in the cytoplasm and nucleus in human cells. In mice, postnatal Pdgfrα-dependent expression of the orthologous murine mutant protein resulted in recessive phenotypes including severe hypomyelination leading to ataxia, tremor, seizures and limited survival, as well as hypodontia and craniofacial abnormalities. Hypomyelination was confirmed and characterized using classic methods to quantify myelin components such as myelin basic protein and lipids, results which agreed with those produced using modern methods to quantify myelin based on the physical properties of myelin membranes. Lineage tracing uncovered the underlying mechanism for the hypomyelinating phenotype: defective oligodendrocyte precursor proliferation and differentiation resulted in a failure to produce an adequate number of mature oligodendrocytes during postnatal myelinogenesis. In summary, we characterized the Polr3bΔ10 mutation and developed an animal model that recapitulates features of POLR3-HLD caused by POLR3B mutations, shedding light on disease pathogenesis, and opening the door to the development of therapeutic interventions.

Gel nail polish as an alternative to traditional coverslip sealants: A quick solution to a sticky situation.

A widespread protocol to seal coverslips on a microscope slide for histological analysis utilizes air-drying nail polish. Nail polish is applied to glue the coverslip in place and prevent the leakage of mounting media. Air drying takes time, typically overnight, and generates an unpleasant smell. Equally familiar is the waiting game, lightly touching the polish to check its dryness, while being careful not to disrupt the coverslip, often leaving sticky spots on one's fingertips. An advantageous solution to these drawbacks is to use gel nail polish, which rapidly hardens and dries by being cured under a LED/UV lamp. We show that UV-cured gel nail polish provides a rapid, stable, scentless, nontoxic, and cost-effective solution for coverslip sealing. Cured in 10 s, with no impact on fluorescent labels, gel polish hardens completely and the slide is ready to be imaged. Furthermore, we show that gel nail polish can be used to generate 3D ridges and structures to support coverslipping thicker samples. Gel nail polish is purposefully unscented, and the brands used in our study employ environmentally conscious, vegan, and cruelty-free ingredients. UV-cured gel nail polish is a cost-effective alternative that presents an easy, accessible, and inexpensive solution to traditional coverslip sealing methods.•Inexpensive method to rapidly seal coverslips onto a microscope slide to immediately image samples for Histological analyses.•Utilizes LED/UV light to cure gel nail polish in 10 s without bleaching fluorophores.•Can be used to generate 3D ridges and structures to support coverslipping thicker samples.

An optimized and validated protocol for the purification of PDGFRα+ oligodendrocyte precursor cells from mouse brain tissue via immunopanning.

Immunopanning is an efficient and reliable method for isolating primary cells from rodent brain tissue, making it a valuable tool for researchers interested in in vitro glial models. Here, we present an immunopanning protocol optimized for the isolation of Platelet-Derived Growth Factor Receptor Alpha positive (PDGFRα+) oligodendrocyte precursor cells (OPCs) from mouse brain tissue that results in a high yield of pure OPCs from minimal quantities of starting tissue.•The protocol presented here is optimized for a PDGFRα-dependent selection of mouse OPCs using a commercial antibody, accounting for the relatively weaker adhesion of OPCs to the anti-PDGFRα plate as compared to other oligodendrocyte lineage markers (e.g., MOG).•A modified papain digestion step, with 95% O2/5% CO2 gas that is humidified prior to perfusion, significantly enhances the yield of dissociated cells and final yield of OPCs.•Isolating OPCs at the PDGFRα+ stage permits the expansion of cells in culture, facilitating studies using transgenic mice, and enables studies on the development of the oligodendrocyte lineage without the spatial and temporal complexity of in vivo studies.

High-Throughput Strategy for Glycine Oxidase Biosensor Development Reveals Glycine Release from Cultured Cells.

Glycine is an important biomarker in clinical analysis due to its involvement in multiple physiological processes. As such, the need for low-cost analytical tools for glycine detection is growing. As a neurotransmitter, glycine is involved in inhibitory and excitatory neurochemical transmission in the central nervous system. In this work, we present a 10 µM Pt-based electrochemical enzymatic biosensor based on the flavoenzyme glycine oxidase (GO) for localized real-time measurements of glycine. Among GO variants at position 244, the H244K variant with increased glycine turnover was selected to develop a functional biosensor. This biosensor relies on amperometric readouts and does not require additional redox mediators. The biosensor was characterized and applied for glycine detection from cells, mainly HEK 293 cells and primary rat astrocytes. We have identified an enzyme, GO H244K, with increased glycine turnover using mutagenesis but which can be developed into a functional biosensor. Noteworthy, a glycine release of 395.7 ± 123 µM from primary astrocytes was measured, which is ∼fivefold higher than glycine release from HEK 293 cells (75.4 ± 3.91 µM) using the GO H244K biosensor.

AANAT1 functions in astrocytes to regulate sleep homeostasis.

How the brain controls the need and acquisition of recovery sleep after prolonged wakefulness is an important issue in sleep research. The monoamines serotonin and dopamine are key regulators of sleep in mammals and in Drosophila. We found that the enzyme arylalkylamine N-acetyltransferase 1 (AANAT1) is expressed by Drosophila astrocytes and specific subsets of neurons in the adult brain. AANAT1 acetylates monoamines and inactivates them, and we found that AANAT1 limited the accumulation of serotonin and dopamine in the brain upon sleep deprivation (SD). Loss of AANAT1 from astrocytes, but not from neurons, caused flies to increase their daytime recovery sleep following overnight SD. Together, these findings demonstrate a crucial role for AANAT1 and astrocytes in the regulation of monoamine bioavailability and homeostatic sleep.

Sleep is essential for our physical and mental health. A lack of sleep can affect our energy and concentration levels and is often linked to chronic illnesses and mood disorders. Sleep is controlled by an internal clock in our brain that operates on a 24-hour cycle, telling our bodies when we are tired and ready for bed, or fresh and alert to start a new day. In addition, the brain tracks the need for sleep and drives the recovery of sleep after periods of prolonged wakefulness ­ a process known as sleep-wake homeostasis. Chemical messengers in the brain such as dopamine and serotonin also play an important part in regulating our sleep drive. While dopamine keeps us awake, serotonin can both prevent us from and help us falling asleep, depending on the part of the brain in which it is released. Most research has focused on the role of different brain circuits on sleep, but it has been shown that a certain type of brain cell, known as astrocyte, may also be important for sleep regulation. So far, it has been unclear if astrocytes could be involved in regulating the need for recovery sleep after a sleep-deprived night ­ also known as rebound sleep. Now, Davla, Artiushin et al. used sleep-deprived fruit flies to investigate this further. The flies were kept awake over 12 hours (from 6pm to 6am), using intermittent physical agitation. The researchers found that astrocytes in the brains of fruit flies express a molecule called AANAT1, which peaked at the beginning of the night, declined as the night went on and recovered by morning. In sleep deprived flies, it inactivated the chemical messengers and so lowered the amount of dopamine and serotonin in the brain. However, in mutant flies that lacked AANAT1, both dopamine and serotonin levels increased in the brain after sleep deprivation. When AANAT1 was selectively removed from astrocytes only, sleep-deprived flies needed more rebound sleep during the day to make up for lost sleep at night. This shows that both astrocytes and AANAT1 play a crucial role in sleep homeostasis. Molecules belonging to the AANAT family exist in both flies and humans, and these results could have important implications for the science of sleep. The study of Davla, Artiushin et al. paves the way for understanding the mechanisms of sleep homeostasis that are similar in both organisms, and may in the future, help to identify sleep drugs that target astrocytes and the molecules they express.

Deep learning for high-throughput quantification of oligodendrocyte ensheathment at single-cell resolution.

High-throughput quantification of oligodendrocyte myelination is a challenge that, if addressed, would facilitate the development of therapeutics to promote myelin protection and repair. Here, we established a high-throughput method to assess oligodendrocyte ensheathment in-vitro, combining nanofiber culture devices and automated imaging with a heuristic approach that informed the development of a deep learning analytic algorithm. The heuristic approach was developed by modeling general characteristics of oligodendrocyte ensheathments, while the deep learning neural network employed a UNet architecture and a single-cell training method to associate ensheathed segments with individual oligodendrocytes. Reliable extraction of multiple morphological parameters from individual cells, without heuristic approximations, allowed the UNet to match the accuracy of expert-human measurements. The capacity of this technology to perform multi-parametric analyses at the level of individual cells, while reducing manual labor and eliminating human variability, permits the detection of nuanced cellular differences to accelerate the discovery of new insights into oligodendrocyte physiology.

Spatial memory formation requires netrin-1 expression by neurons in the adult mammalian brain.

Netrin-1 was initially characterized as an axon guidance molecule that is essential for normal embryonic neural development; however, many types of neurons continue to express netrin-1 in the postnatal and adult mammalian brain. Netrin-1 and the netrin receptor DCC are both enriched at synapses. In the adult hippocampus, activity-dependent secretion of netrin-1 by neurons potentiates glutamatergic synapse function, and is critical for long-term potentiation, an experimental cellular model of learning and memory. Here, we assessed the impact of neuronal expression of netrin-1 in the adult brain on behavior using tests of learning and memory. We show that adult mice exhibit impaired spatial memory following conditional deletion of netrin-1 from glutamatergic neurons in the hippocampus and neocortex. Further, we provide evidence that mice with conditional deletion of netrin-1 do not display aberrant anxiety-like phenotypes and show a reduction in self-grooming behavior. These findings reveal a critical role for netrin-1 expressed by neurons in the regulation of spatial memory formation.

An Automated Strategy for Unbiased Morphometric Analyses and Classifications of Growth Cones In Vitro.

During neural circuit development, attractive or repulsive guidance cue molecules direct growth cones (GCs) to their targets by eliciting cytoskeletal remodeling, which is reflected in their morphology. The experimental power of in vitro neuronal cultures to assay this process and its molecular mechanisms is well established, however, a method to rapidly find and quantify multiple morphological aspects of GCs is lacking. To this end, we have developed a free, easy to use, and fully automated Fiji macro, Conographer, which accurately identifies and measures many morphological parameters of GCs in 2D explant culture images. These measurements are then subjected to principle component analysis and k-means clustering to mathematically classify the GCs as "collapsed" or "extended". The morphological parameters measured for each GC are found to be significantly different between collapsed and extended GCs, and are sufficient to classify GCs as such with the same level of accuracy as human observers. Application of a known collapse-inducing ligand results in significant changes in all parameters, resulting in an increase in 'collapsed' GCs determined by k-means clustering, as expected. Our strategy provides a powerful tool for exploring the relationship between GC morphology and guidance cue signaling, which in particular will greatly facilitate high-throughput studies of the effects of drugs, gene silencing or overexpression, or any other experimental manipulation in the context of an in vitro axon guidance assay.