Lateralization, maturation, and anteroposterior topography in the lateral habenula revealed by ZIF268/EGR1 immunoreactivity and labeling history of neuronal activity

We report habenular lateralization in a simple transgenic mouse model used for labeling a facet of neuronal activity history. A transgenic construct comprised of a zif268/egr1 immediate-early gene promoter and a gene for normal Venus fluorescent protein with a membrane tag converted promoter activity into long-life fluorescent proteins, which was thought to describe a facet of neuronal activity history by summing neuronal activity. In addition to mapping the immediate-early gene-immunopositive cells, this method helped demonstrate the functionality of the lateral habenular nucleus (LHb). During postnatal development, the LHb was activated between postnatal days 10 and 16. The water-immersion restraint stress also activated the LHb over a similar period. LHb activation was functionally lateralized, but had no directional bias at the population level. Moreover, the posterior LHb was activated in the early stage after the stress, while the anterior LHb was activated in the later stage. Our results indicate lateralization, maturation, and anteroposterior topography of the LHb during postnatal development and the stress response

Red fluorescent cAMP indicator with increased affinity and expanded dynamic range

cAMP is one of the most important second messengers in biological processes. Cellular dynamics of cAMP have been investigated using a series of fluorescent indicators; however, their sensitivity was sub-optimal for detecting cAMP dynamics at a low concentration range, due to a low ligand affinity and/or poor dynamic range. Seeking an indicator with improved detection sensitivity, we performed insertion screening of circularly permuted mApple, a red fluorescent protein, into the cAMP-binding motif of PKA regulatory subunit Iα and developed an improved cAMP indicator named R-FlincA (Red Fluorescent indicator for cAMP). Its increased affinity (Kd = 0.3 μM) and expanded dynamic range (860% at pH 7.2) allowed the detection of subtle changes in the cellular cAMP dynamics at sub-μM concentrations, which could not be easily observed with existing indicators. Increased detection sensitivity also strengthened the advantages of using R-FlincA as a red fluorescent indicator, as it permits a series of applications, including multi-channel/function imaging of multiple second messengers and combinatorial imaging with photo-manipulation. These results strongly suggest that R-FlincA is a promising tool that accelerates cAMP research by revealing unobserved cAMP dynamics at a low concentration range

Auto-Luminescent Genetically-Encoded Ratiometric Indicator for Real-Time Ca2+ Imaging at the Single Cell Level

Background: Efficient bioluminescence resonance energy transfer (BRET) from a bioluminescent protein to a fluorescent protein with high fluorescent quantum yield has been utilized to enhance luminescence intensity, allowing single-cell imaging in near real time without external light illumination. Methodology/Principal Findings: We applied BRET to develop an autoluminescent Ca2+ indicator, BRAC, which is composed of Ca^[2+]-binding protein, calmodulin, and its target peptide, M13, sandwiched between a yellow fluorescent protein variant, Venus, and an enhanced Renilla luciferase, RLuc8. Adjusting the relative dipole orientation of the luminescent protein's chromophores improved the dynamic range of BRET signal change in BRAC up to 60%, which is the largest dynamic range among BRET-based indicators reported so far. Using BRAC, we demonstrated successful visualization of Ca2+ dynamics at the single-cell level with temporal resolution at 1 Hz. Moreover, BRAC signals were acquired by ratiometric imaging capable of canceling out Ca^[2+]-independent signal drifts due to change in cell shape, focus shift, etc. Conclusions/Significance: The brightness and large dynamic range of BRAC should facilitate high-sensitive Ca2+ imaging not only in single live cells but also in small living subjects

Visualization of spatiotemporal activation of Notch signaling: Live monitoring and significance in neural development

AbstractNotch signaling plays various key roles in cell fate determination during CNS development in a context-dependent fashion. However, its precise physiological role and the localization of its target cells remain unclear. To address this issue, we developed a new reporter system for assessing the RBP-J-mediated activation of Notch signaling target genes in living cells and tissues using a fluorescent protein Venus. Our reporter system revealed that Notch signaling is selectively activated in neurosphere-initiating multipotent neural stem cells in vitro and in radial glia in the embryonic forebrain in vivo. Furthermore, the activation of Notch signaling occurs during gliogenesis and is required in the early stage of astroglial development. Consistent with these findings, the persistent activation of Notch signaling inhibits the differentiation of GFAP-positive astrocytes. Thus, the development of our RBP-J-dependent live reporter system, which is activated upon Notch activation, together with a stage-dependent gain-of-function analysis allowed us to gain further insight into the complexity of Notch signaling in mammalian CNS development

Optical Recording of Neuronal Activity with a Genetically-Encoded Calcium Indicator in Anesthetized and Freely Moving Mice

Fluorescent calcium (Ca2+) indicator proteins (FCIPs) are promising tools for functional imaging of cellular activity in living animals. However, they have still not reached their full potential for in vivo imaging of neuronal activity due to limitations in expression levels, dynamic range, and sensitivity for reporting action potentials. Here, we report that viral expression of the ratiometric Ca2+ sensor yellow cameleon 3.60 (YC3.60) in pyramidal neurons of mouse barrel cortex enables in vivo measurement of neuronal activity with high dynamic range and sensitivity across multiple spatial scales. By combining juxtacellular recordings and two-photon imaging in vitro and in vivo, we demonstrate that YC3.60 can resolve single action potential (AP)-evoked Ca2+ transients and reliably reports bursts of APs with negligible saturation. Spontaneous and whisker-evoked Ca2+ transients were detected in individual apical dendrites and somata as well as in local neuronal populations. Moreover, bulk measurements using wide-field imaging or fiber-optics revealed sensory-evoked YC3.60 signals in large areas of the barrel field. Fiber-optic recordings in particular enabled measurements in awake, freely moving mice and revealed complex Ca2+ dynamics, possibly reflecting different behavior-related brain states. Viral expression of YC3.60 – in combination with various optical techniques – thus opens a multitude of opportunities for functional studies of the neural basis of animal behavior, from dendrites to the levels of local and large-scale neuronal populations

Flexible and dynamic nucleosome fiber in living mammalian cells

© Landes Bioscience, 2013. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Nucleus 4 (2013): 349-356, doi:10.4161/nucl.26053.Genomic DNA is organized three dimensionally within cells as chromatin and is searched and read by various proteins by an unknown mechanism; this mediates diverse cell functions. Recently, several pieces of evidence, including our cryomicroscopy and synchrotron X-ray scattering analyses, have demonstrated that chromatin consists of irregularly folded nucleosome fibers without a 30-nm chromatin fiber (i.e., a polymer melt-like structure). This melt-like structure implies a less physically constrained and locally more dynamic state, which may be crucial for protein factors to scan genomic DNA. Using a combined approach of fluorescence correlation spectroscopy, Monte Carlo computer simulations, and single nucleosome imaging, we demonstrated the flexible and dynamic nature of the nucleosome fiber in living mammalian cells. We observed local nucleosome fluctuation (~50 nm movement per 30 ms) caused by Brownian motion. Our in vivo-in silico results suggest that local nucleosome dynamics facilitate chromatin accessibility and play a critical role in the scanning of genome information.This work was supported by a grant-in-aid for a MEXT grant, JST CREST, Yamada Science Foundation and Takeda Science Foundation. Nozaki T and Hihara A are JSPS fellows

Intracellular Calcium Spikes in Rat Suprachiasmatic Nucleus Neurons Induced by BAPTA-Based Calcium Dyes

Background: Circadian rhythms in spontaneous action potential (AP) firing frequencies and in cytosolic free calcium concentrations have been reported for mammalian circadian pacemaker neurons located within the hypothalamic suprachiasmatic nucleus (SCN). Also reported is the existence of "Ca2+ spikes" (i.e., [Ca2+]c transients having a bandwidth of 10～100 seconds) in SCN neurons, but it is unclear if these SCN Ca2+ spikes are related to the slow circadian rhythms. Methodology/Principal Findings: We addressed this issue based on a Ca2+ indicator dye (fluo-4) and a protein Ca2+ sensor (yellow cameleon). Using fluo-4 AM dye, we found spontaneous Ca2+ spikes in 18% of rat SCN cells in acute brain slices, but the Ca2+ spiking frequencies showed no day/night variation. We repeated the same experiments with rat (and mouse) SCN slice cultures that expressed yellow cameleon genes for a number of different circadian phases and, surprisingly, spontaneous Ca2+ spike was barely observed (<3%). When fluo-4 AM or BAPTA-AM was loaded in addition to the cameleon-expressing SCN cultures, however, the number of cells exhibiting Ca2+ spikes was increased to 13～14%. Conclusions/Significance: Despite our extensive set of experiments, no evidence of a circadian rhythm was found in the spontaneous Ca2+ spiking activity of SCN. Furthermore, our study strongly suggests that the spontaneous Ca2+ spiking activity is caused by the Ca2+ chelating effect of the BAPTA-based fluo-4 dye. Therefore, this induced activity seems irrelevant to the intrinsic circadian rhythm of [Ca2+]c in SCN neurons. The problems with BAPTA based dyes are widely known and our study provides a clear case for concern, in particular, for SCN Ca2+ spikes. On the other hand, our study neither invalidates the use of these dyes as a whole, nor undermines the potential role of SCN Ca2+ spikes in the function of SCN

SuperNova, a monomeric photosensitizing fluorescent protein for chromophore-assisted light inactivation

Takemoto, K., Matsuda, T., Sakai, N. et al. SuperNova, a monomeric photosensitizing fluorescent protein for chromophore-assisted light inactivation. Sci Rep 3, 2629 (2013). https://doi.org/10.1038/srep02629

A Transient Rise in Free Mg 2+ Ions Released from ATP-Mg Hydrolysis Contributes to Mitotic Chromosome Condensation

細胞分裂期の染色体凝縮はマグネシウムイオンの増加によって起こる --生細胞イメージングにより新たなメカニズムを検証--. 京都大学プレスリリース. 2018-01-19.For cell division, negatively charged chromatin, in which nucleosome fibers (10 nm fibers) are irregularly folded [ 1–5 ], must be condensed into chromosomes and segregated. While condensin and other proteins are critical for organizing chromatin into the appropriate chromosome shape [ 6–17 ], free divalent cations such as Mg2+ and Ca2+, which condense chromatin or chromosomes in vitro [ 18–28 ], have long been considered important, especially for local condensation, because the nucleosome fiber has a net negative charge and is by itself stretched like “beads on a string” by electrostatic repulsion. For further folding, other positively charged factors are required to decrease the charge and repulsion [ 29 ]. However, technical limitations to measure intracellular free divalent cations, but not total cations [ 30 ], especially Mg2+, have prevented us from elucidating their function. Here, we developed a Förster resonance energy transfer (FRET)-based Mg2+ indicator that monitors free Mg2+ dynamics throughout the cell cycle. By combining this indicator with Ca2+ [ 31 ] and adenosine triphosphate (ATP) [ 32 ] indicators, we demonstrate that the levels of free Mg2+, but not Ca2+, increase during mitosis. The Mg2+ increase is coupled with a decrease in ATP, which is normally bound to Mg2+ in the cell [ 33 ]. ATP inhibited Mg2+-dependent chromatin condensation in vitro. Chelating Mg2+ induced mitotic cell arrest and chromosome decondensation, while ATP reduction had the opposite effect. Our results suggest that ATP-bound Mg2+ is released by ATP hydrolysis and contributes to mitotic chromosome condensation with increased rigidity, suggesting a novel regulatory mechanism for higher-order chromatin organization by the intracellular Mg2+-ATP balance