神经科学

Neurons are complex cells with two distinct compartments: the somatodendritic and the axonal domains. Because of their polarized morphology, it is challenging to study the differential cellular and molecular mechanisms that occur in axons and impact the soma and dendrites using conventional in vitro culture systems. Compartmentalized cultures offer a solution by physically and chemically separating the axonal from the somatodendritic domain of neurons. The microfluidic chamber model presented in this work is valuable for studying these mechanisms in primary cortical cultures derived from rat and mouse. In addition, this chamber model is compatible with various microscopy methods, such as phase contrast, and fluorescence imaging of living and fixed cells.

Key features

• Preparation and attachment of PDMS microfluidic chambers to glass coverslips.

• Primary culture of cortical neurons and plating cortical neurons in microfluidic chamber.

• Confirmation of compartmentalization using the retrograde transport of the fluorescently labeled form of cholera toxin subunit B (f-Ctb).

• Immunofluorescence and multilabeling of compartmentalized cortical neurons.

• Retrograde transport of fluorescently labeled BDNF.

The activity of numerous autophagy-related proteins depends on their phosphorylation status, which places importance on understanding the responsible kinases and phosphatases. Great progress has been made in identifying kinases regulating autophagy, but much less is known about the phosphatases counteracting their function. Genetic screens and modern proteomic approaches provide powerful tools to identify candidate phosphatases, but further experiments are required to assign direct roles for candidates. We have devised a novel protocol to test the role of purified phosphatases in dephosphorylating specific targets in situ. This approach has the potential to visualize context-specific differences in target dephosphorylation that are not easily detected by lysate-based approaches such as Western blots.

Graphical abstract:

G-protein coupled signaling pathways are organized into multi-protein complexes called signalosomes that are located within and on cellular membranes. We describe the use of silica nanoparticles coated with a unilamellar phospholipid bilayer (lipobeads) to reconstitute the activated photoreceptor G-protein α-subunit (Gtα*) with its cognate effector (phosphodiesterase-6; PDE6) for biochemical and structural studies of the activation mechanism regulating this GPCR signaling pathway. Lipobeads are prepared by resuspending dried-down phospholipid mixtures with monodisperse 70 nm silica particles, followed by extrusion through a 100 nm membrane filter. This uniform and supported liposomal preparation is easily sedimented, permitting the separation of soluble from membrane-associated proteins. Upon loading lipobeads with Gtα* and PDE6, we find that activation of PDE6 catalysis by Gtα* occurs much more efficiently than in the absence of membranes. Chemical cross-linking of membrane-confined proteins allows detection of changes in protein-protein interactions, resulting from G-protein activation of PDE6. The advantages of using lipobeads over partially purified membrane preparations or traditional liposomal preparations are generally applicable to the study of other membrane-confined signal transduction pathways.

The functional performance of a cell depends on how macromolecules, in particular proteins, come together in a precise orientation, how they assemble into protein complexes and interact with each other. In order to study protein-protein interactions at a molecular level, a variety of methods to investigate these binding processes yield affinity constants and/or the identification of binding regions. There are several well-established biophysical techniques for biomolecular interaction studies, such as fluorescence spectroscopy and surface plasmon resonance. Although these techniques have been proven to be efficient, they either need labeling or immobilization of one interaction partner. Backscattering interferometry (BSI) is a label-free detection method, which allows label- and immobilization-free interaction analysis under physiologically relevant conditions with high sensitivity and in small volumes. We used BSI to measure the interaction of the neuronal calcium sensor recoverin with its target G protein-coupled receptor kinase 1 (GRK1) as a model system. Increasing concentrations of purified recoverin were mixed with a specific concentration of a GRK1 fusion protein. In this protocol, we provide a full description of the instrumental setup, data acquisition, and evaluation. Equilibrium dissociation constants of recoverin-GRK1 interaction determined by the BSI instrumental setup are in full agreement with affinity constants obtained by different methods as described in the literature.

Calcium ions trigger many cellular events, including the release of neurotransmitters at the synaptic terminal and excitotoxic cell death. Recently, we have discovered that a transient increase in the level of cytoplasmic Ca²⁺ triggers the exposure of phosphatidylserine (PS) on the surfaces of necrotic cells in the nematode Caenorhabditis elegans. PS serves as an “eat me” signal that attracts engulfing cells to engulf and degrade necrotic cells. During the above study, we developed a microscopic imaging protocol for real-time monitoring the levels of cytoplasmic Ca²⁺ and cell surface PS in Caenorhabditis elegans touch neurons. Previously, Ca²⁺ dynamics was monitored in neurons in Caenorhabditis elegans larvae in time periods ranging from milliseconds to seconds. Methods for monitoring Ca²⁺ dynamics for a relatively long period of time during embryonic development were not available, let alone for simultaneous monitoring Ca²⁺ and PS dynamics. The protocol reported here utilizes a deconvolution imaging system with an optimized experimental setting that reduces photo-damage and allows the proper development of embryos during the real-time imaging process. This protocol enables the simultaneous measurement of cytosolic Ca²⁺ and cell surface PS levels in necrotic touch neurons during embryonic development in a period longer than six hours. Our method provides an easy and sensitive approach to perform long-time Ca²⁺ and PS recording in living animals, simultaneously or individually. This protocol can be applied to study various cellular and developmental events that involve the dynamic regulation of Ca²⁺ and/or PS.

Computational neuroscience aims to model, reproduce, and predict network dynamics for different neuronal ensembles by distilling knowledge derived from electrophysiological and morphological evidence. However, analyses and simulations often remain critically limited by the sparsity of direct experimental constraints on essential parameters, such as electron microscopy and electrophysiology pair/multiple recording evidence of connectivity statistics. Notably, available data are particularly scarce regarding quantitative information on synaptic connections among identified neuronal types. Here, we present a user-friendly data-driven pipeline to estimate connection probabilities, number of contacts per connected pair, and distances from the pre- and postsynaptic somas along the axonal and dendritic paths from commonly available two-dimensional tracings and other broadly accessible measurements. The described procedure does not require any computational background and is accessible to all neuroscientists. This protocol therefore fills the important gap from neuronal morphology to circuit organization and can be applied to many different neural systems, brain regions, animal species, and data sources.

Graphic abstract:

The processing protocol from 2D reconstructions to quantitated synaptic connections

Protein N-glycosylation plays a vital role in diverse cellular processes, and dysregulated N-glycosylation is implicated in a variety of human diseases including neurodegenerative disorders and cancer. With recent advances in high-resolution mass spectrometry-based glycoproteomics technologies enabling large-scale N-glycoproteome profiling of disease and control samples, analysis of the large datasets has become a challenge. Here, we provide a protocol for the systems-level analysis of in vivo N-glycosylation sites on N-glycosylated proteins and their changes in human disease, such as Alzheimer's disease. The protocol includes quantitation and differential analysis of N-glycopeptide abundance, in addition to integrative N-glycoproteome and proteome data analyses, to determine disease-associated changes in N-glycosylation site occupancy and identify differentially N-glycosylated proteins in human disease versus control samples. This protocol can be modified and applied to study proteome-wide N-glycosylation alterations in response to different cellular stresses or pathophysiological states in other organisms or model systems.

Over the last decade, lipids have emerged as possessing an ever-increasing number of key functions, especially in membrane trafficking. For instance, phosphatidic acid (PA) has been proposed to play a critical role in different steps along the secretory pathway or during phagocytosis. To further investigate in detail the precise nature of PA activities, we need to identify the organelles in which PA is synthesized and the PA subspecies involved in these biological functions. Indeed, PA, like all phospholipids, has a large variety based on its fatty acid composition. The recent development of PA sensors has helped us to follow intracellular PA dynamics but has failed to provide information on individual PA species. Here, we describe a method for the subcellular fractionation of RAW264.7 macrophages that allows us to obtain membrane fractions enriched in specific organelles based on their density. Lipids from these membrane fractions are precipitated and subsequently processed by advanced mass spectrometry-based lipidomics analysis to measure the levels of different PA species based on their fatty acyl chain composition. This approach revealed the presence of up to 50 different species of PA in cellular membranes, opening up the possibility that a single class of phospholipid could play multiple functions in any given organelle. This protocol can be adapted or modified and used for the evaluation of other intracellular membrane compartments or cell types of interest.

Cell-based functional assays are an important part of compound screening and drug lead optimization, and they can also play a crucial role in the determination of the residues involved in ligand binding and signaling for a particular G-protein-coupled receptor. Conventional methods used for Gα_q/15-coupled receptors rely on the use of fluorescent probes for Ca⁺⁺ sensing (such as Fura-2 and Fluo-4) or on the incorporation of [³H]-inositol into inositol 1,4,5- triphosphate (IP3). However, these methods are not suitable for screening large libraries of compounds or for screening several mutants of the same receptor. In contrast, the IP-One assay by Cisbio is a TR-FRET assay suitable for large compound library screening when using stable cell lines that express a specific 7TMR. However, when using transiently transfected mutants of a 7TMR, this assay is not ideal, as it requires a two-step protocol of cell culture. Therefore, we have optimized the IP-One assay protocol using the reverse transfection method in 384-well plates. This offers a time- and resource-efficient alternative to the two-step protocol previously used for the screening of several mutants of Gα_q/15-coupled 7TMRs.

Rats are highly social animals, and mainly communicate with one another in two ways: through ultrasonic vocalizations and pheromones. Most research on pheromones has been dedicated those regarding sexual behavior, but more recently pheromones which signal danger to conspecifics have been identified in rodents. In fact, rats are capable of communicating information regarding a specific fear to a companion with which they share a cage. If a rat is trained to associate a previously neutral odor with a foot shock and then pair housed with another rat, the companion will also display a fear response specific to the trained odor, despite never being shocked itself. This communication relies on pheromones; presenting soiled bedding from a shocked rat to an individually housed naïve rat produces the same fear response in the naïve rat. The current protocol describes how to produce this phenomenon in adult Sprague Dawley rats. It is simple and easily reproduced, requires minimal equipment, and may be completed within one week.