living neurons
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2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Aleksandra Arsić ◽  
Cathleen Hagemann ◽  
Nevena Stajković ◽  
Timm Schubert ◽  
Ivana Nikić-Spiegel

AbstractModern light microscopy, including super-resolution techniques, has brought about a demand for small labeling tags that bring the fluorophore closer to the target. This challenge can be addressed by labeling unnatural amino acids (UAAs) with bioorthogonal click chemistry. The minimal size of the UAA and the possibility to couple the fluorophores directly to the protein of interest with single-residue precision in living cells make click labeling unique. Here, we establish click labeling in living primary neurons and use it for fixed-cell, live-cell, dual-color pulse–chase, and super-resolution microscopy of neurofilament light chain (NFL). We also show that click labeling can be combined with CRISPR/Cas9 genome engineering for tagging endogenous NFL. Due to its versatile nature and compatibility with advanced multicolor microscopy techniques, we anticipate that click labeling will contribute to novel discoveries in the neurobiology field.


Author(s):  
Pietro Renna ◽  
Cristian Ripoli ◽  
Onur Dagliyan ◽  
Francesco Pastore ◽  
Marco Rinaudo ◽  
...  

2022 ◽  
Author(s):  
Wouter J Droogers ◽  
Jelmer Willems ◽  
Harold D MacGillavry ◽  
Arthur PH de Jong

Recent advances in CRISPR/Cas9-mediated knock-in methods enable labeling of individual endogenous proteins with fluorophores, to determine their spatiotemporal expression in intact biological preparations. However, multiplex knock-in methods remain limited, particularly in postmitotic cells, due to a high degree of crosstalk between genome editing events. We present Conditional Activation of Knock-in Expression (CAKE), which delivers efficient, flexible and accurate multiplex genome editing in neurons. CAKE is based on sequential gRNA expression operated by a Cre- or Flp-recombinase to control the time window for genomic integration of each donor sequence, which diminishes crosstalk between genome editing events. Importantly, CAKE is compatible with multiple CRISPR/Cas9 strategies, and we show the utilization of CAKE for co-localization of various endogenous proteins, including synaptic scaffolds, ion channels and neurotransmitter receptor subunits. Knock-in efficacy was highly sensitive to DNA vector amount, while knock-in crosstalk was dependent on the rate of donor DNA integration and timing of Cre activation. We applied CAKE to study the co-distribution of endogenous synaptic proteins using dual-color single-molecule localization microscopy, and we introduced dimerization modules to acutely control synaptic receptor dynamics in living neurons. Taken together, CAKE is a versatile method for multiplex protein labeling, enabling accurate detection, precise localization and acute manipulation of endogenous proteins in single cells.


2021 ◽  
Vol 15 ◽  
Author(s):  
Kyle R. Jenks ◽  
Katya Tsimring ◽  
Jacque Pak Kan Ip ◽  
Jose C. Zepeda ◽  
Mriganka Sur

Neurons remodel the structure and strength of their synapses during critical periods of development in order to optimize both perception and cognition. Many of these developmental synaptic changes are thought to occur through synapse-specific homosynaptic forms of experience-dependent plasticity. However, homosynaptic plasticity can also induce or contribute to the plasticity of neighboring synapses through heterosynaptic interactions. Decades of research in vitro have uncovered many of the molecular mechanisms of heterosynaptic plasticity that mediate local compensation for homosynaptic plasticity, facilitation of further bouts of plasticity in nearby synapses, and cooperative induction of plasticity by neighboring synapses acting in concert. These discoveries greatly benefited from new tools and technologies that permitted single synapse imaging and manipulation of structure, function, and protein dynamics in living neurons. With the recent advent and application of similar tools for in vivo research, it is now feasible to explore how heterosynaptic plasticity contribute to critical periods and the development of neuronal circuits. In this review, we will first define the forms heterosynaptic plasticity can take and describe our current understanding of their molecular mechanisms. Then, we will outline how heterosynaptic plasticity may lead to meaningful refinement of neuronal responses and observations that suggest such mechanisms are indeed at work in vivo. Finally, we will use a well-studied model of cortical plasticity—ocular dominance plasticity during a critical period of visual cortex development—to highlight the molecular overlap between heterosynaptic and developmental forms of plasticity, and suggest potential avenues of future research.


Author(s):  
Ilya V. Rogachevskii ◽  
Vera B. Plakhova ◽  
Valentina A. Penniyaynen ◽  
Stanislav G. Terekhin ◽  
Svetlana A. Podzorova ◽  
...  

A gamma-pyrone derivative, comenic acid, activates the opioid-like receptor-mediated signaling pathway that modulates the NaV1.8 channels in the primary sensory neuron membrane. These channels are responsible for generation of the nociceptive signal; gamma-pyrones can therefore have a great therapeutic potential as analgesics, and this effect deserves a deeper understanding. The novelty of our approach to the design of a medicinal substance is based on a combination of the data obtained on living neurons using very sensitive physiological methods and the results of quantum-chemical calculations. This approach allows to correlate the molecular structure of gamma-pyrones with their ability to evoke a physiological response of the neuron. Comenic acid can bind two calcium cations. One of them is chelated by the carbonyl and the hydroxyl functional groups, while another one forms the salt bond with the carboxylate anion. Calcium-bound gamma-pyrones are fundamentally different in electrostatic properties from the free gamma-pyrone molecules. These two calcium ions are the key elements involved in ligand-receptor binding. It is very likely ion-ionic interactions between these cations and anionic functional groups of the opioid-like receptor that activate the latter. The calculated intercationic distance of 9.5 Å is a structural criterion for effective ligand-receptor binding of calcium-bound gamma-pyrones.


Micromachines ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 925
Author(s):  
Younghoon Park ◽  
Sung-Yun Park ◽  
Kyungsik Eom

Neural interfaces, which enable the recording and stimulation of living neurons, have emerged as valuable tools in understanding the brain in health and disease, as well as serving as neural prostheses. While neural interfaces are typically based on electrical transduction, alternative energy modalities have been explored to create safe and effective approaches. Among these approaches, optical methods of linking neurons to the outside world have gained attention because light offers high spatial selectivity and decreased invasiveness. Here, we review the current state-of-art of optical neural interfaces and their clinical applications. Optical neural interfaces can be categorized into optical control and optical readout, each of which can be divided into intrinsic and extrinsic approaches. We discuss the advantages and disadvantages of each of these methods and offer a comparison of relative performance. Future directions, including their clinical opportunities, are discussed with regard to the optical properties of biological tissue.


2021 ◽  
Vol 27 (S1) ◽  
pp. 500-502
Author(s):  
Oleg Suchalko ◽  
Roman Timoshenko ◽  
Alexander Vaneev ◽  
Vasilii Kolmogorov ◽  
Nikita Savin ◽  
...  

2021 ◽  
Author(s):  
Jessica J. A. Hummel ◽  
Casper C. Hoogenraad

Molecular motors drive long-range intracellular transport of various vesicles and other cargos within a cell. Identifying which kinesin motors interact with which type of transport vesicles has been challenging, especially in complex neuronal cells. Here, we present a highly adaptable toolbox of engineered kinesin motors to control and interrogate the selectivity and regulation of cargo transport with acute chemical induction. Selectivity of cargo-motor interaction can be addressed by systematic screening of a library of kinesin tails and neuronal cargos. Additionally, our toolbox can be used to study kinesin-cargo regulatory mechanisms, and we found that cargo trafficking by KIF16B is regulated by its PX-domain. Furthermore, our toolbox enables acute manipulation of polarized trafficking in living neurons by stirring transport into axons or dendrites. Engineering kinesin motors provides a powerful tool to map the specificity of interactions between kinesin and cargos, manipulate polarized transport and investigate cargo-motor interaction modes.


Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1348
Author(s):  
Rena Kono ◽  
Yuji Ikegaya ◽  
Ryuta Koyama

Phagocytosis by glial cells has been shown to play an important role in maintaining brain homeostasis. Microglia are currently considered to be the major phagocytes in the brain parenchyma, and these cells phagocytose a variety of materials, including dead cell debris, abnormally aggregated proteins, and, interestingly, the functional synapses of living neurons. The intracellular signaling mechanisms that regulate microglial phagocytosis have been studied extensively, and several important factors, including molecules known as “find me” signals and “eat me” signals and receptors on microglia that are involved in phagocytosis, have been identified. In addition, recent studies have revealed that astrocytes, which are another major glial cell in the brain parenchyma, also have phagocytic abilities. In this review, we will discuss the roles of microglia and astrocytes in phagocytosis-mediated brain homeostasis, focusing on the characteristics and differences of their phagocytic abilities.


IBJ Plus ◽  
2021 ◽  
Author(s):  
◽  
◽  
Manuel Reyes-Sanchez ◽  
Rodrigo Amaducci ◽  
Irene Elices ◽  
...  
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