Optogenetic control of early embryos labeling using photoactivatable Cre recombinase 3.0.

Establishing a highly efficient photoactivatable Cre recombinase PA-Cre3.0 can allow spatiotemporal control of Cre recombinase activity. This technique may help to elucidate cell lineages, as well as facilitate gene and cell function analysis during development.

New Alzheimer's disease model mouse specialized for analyzing the function and toxicity of intraneuronal Amyloid β oligomers.

Oligomers of intracellular amyloid β protein (Aβ) are strongly cytotoxic and play crucial roles in synaptic transmission and cognitive function in Alzheimer's disease (AD). However, there is currently no AD model mouse in which to specifically analyze the function of Aβ oligomers only. We have now developed a novel AD model mouse, an Aβ-GFP transgenic mouse (Aβ-GFP Tg), that expresses the GFP-fused human Aβ protein, which forms only Aβ oligomers within neurons throughout their life.

Primary cultured neuronal networks and type 2 diabetes model mouse fatty liver tissues in aqueous liquid observed by atmospheric SEM (ASEM): Staining preferences of metal solutions.

Neural networking, including axon targeting and synapse formation, is the basis of various brain functions, including memory and learning. Diabetes-mellitus affects peripheral nerves and is known to cause fatty liver disease. Electron microscopy (EM) provides the resolution required to observe changes in fine subcellular structures caused by such physiological and pathological processes, but samples are observed in vacuum.

Development of new fusion proteins for visualizing amyloid-β oligomers in vivo.

The intracellular accumulation of amyloid-β (Aβ) oligomers critically contributes to disease progression in Alzheimer's disease (AD) and can be the potential target of AD therapy. Direct observation of molecular dynamics of Aβ oligomers in vivo is key for drug discovery research, however, it has been challenging because Aβ aggregation inhibits the fluorescence from fusion proteins. Here, we developed Aβ1-42-GFP fusion proteins that are oligomerized and visualize their dynamics inside cells even when aggregated.

Observation of tissues in open aqueous solution by atmospheric scanning electron microscopy: applicability to intraoperative cancer diagnosis.

In the atmospheric scanning electron microscope (ASEM), a 2- to 3-µm layer of the sample resting on a silicon nitride-film window in the base of an open sample dish is imaged, in liquid, at atmospheric pressure, from below by an inverted SEM. Thus, the time-consuming pretreatments generally required for biological samples to withstand the vacuum of a standard electron microscope are avoided. In the present study, various mouse tissues (brain, spinal cord, muscle, heart, lung, liver, kidney, spleen and stomach) were fixed, stained with heavy metals, and visualized in radical scavenger D-glucose solution using the ASEM.

Immuno-electron microscopy of primary cell cultures from genetically modified animals in liquid by atmospheric scanning electron microscopy.

High-throughput immuno-electron microscopy is required to capture the protein-protein interactions realizing physiological functions. Atmospheric scanning electron microscopy (ASEM) allows in situ correlative light and electron microscopy of samples in liquid in an open atmospheric environment. Cells are cultured in a few milliliters of medium directly in the ASEM dish, which can be coated and transferred to an incubator as required.

Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM.

Correlative light-electron microscopy of cells in a natural environment of aqueous liquid facilitates high-throughput observation of protein complex formation. ASEM allows the inverted SEM to observe the wet sample from below, while an optical microscope observes it from above quasi-simultaneously. The disposable ASEM dish with a silicon nitride (SiN) film window can be coated variously to realize the primary-culture of substrate-sensitive cells in a few milliliters of culture medium in a stable incubator environment.

Immuno EM-OM correlative microscopy in solution by atmospheric scanning electron microscopy (ASEM).

In the atmospheric scanning electron microscope (ASEM), an inverted SEM observes the wet sample from beneath an open dish while an optical microscope (OM) observes it from above. The disposable dish with a silicon nitride (SiN) film window can hold a few milliliters of culture medium, and allows various types of cells to be cultured in a stable environment. The use of this system for in situ correlative OM/SEM immuno-microscopy is explored, the efficiency of the required dual-tagged labeling assessed and the imaging capabilities of the ASEM documented.

Direct observation of protein microcrystals in crystallization buffer by atmospheric scanning electron microscopy.

X-ray crystallography requires high quality crystals above a given size. This requirement not only limits the proteins to be analyzed, but also reduces the speed of the structure determination. Indeed, the tertiary structures of many physiologically important proteins remain elusive because of the so-called "crystallization bottleneck".

Inhibitory synaptic modulation of renshaw cell activity in the lumbar spinal cord of neonatal mice.

In the mammalian spinal cord, Renshaw cells (RCs) are excited by axon collaterals of motoneurons (MNs), and in turn, provide recurrent inhibition of MNs. They are considered an important element in controlling the motor output. However, how RCs are modulated by spinal circuits during motor behaviors remains unclear.

Cre complementation with variable dimerizers for inducible expression in neurons.

Cre complementation is a process of reconstitution of the activity of DNA recombinase by noncovalent association of multiple segments of Cre recombinase, which are enzymatically inactive by themselves. Cre complementation is potentially useful in restriction of Cre activity in a specific subset of cells, with temporal regulation, by limiting overlap in expression of Cre fragments. We analyzed the efficiency of Cre complementation using three different dimerizing modules in the context of non-neuronal cells and found differential Cre complementation efficiency.

Muscarinic acetylcholine receptors stimulate Ca2+ influx in PC12D cells predominantly via activation of Ca2+ store-operated channels.

Activation of muscarinic acetylcholine receptors (mAChRs) causes the rapid release of Ca2+ from intracellular stores and a sustained influx of external Ca2+ in PC12D cells, a subline of the widely studied cell line PC12. Release of Ca2+ from intracellular stores and a sustained influx of Ca2+ are also observed following exposure to thapsigargin, a sesquiterpene lactone that depletes intracellular Ca2+ pools by irreversibly inhibiting the Ca2+ pump of the endoplasmic reticulum. In this study, we show that carbachol and thapsigargin empty the same intracellular Ca2+ stores, and that these stores are a subset of intracellular stores depleted by the Ca2+ ionophore ionomycin.

Simultaneous observation of stably associated presynaptic varicosities and postsynaptic spines: morphological alterations of CA3-CA1 synapses in hippocampal slice cultures.

Dendritic spines are highly motile structures, but the extent and mode of coordination in motility between spines and presynaptic varicosities with synaptic contacts is not clear. To analyze movements of dendritic spines and axonal varicosities simultaneously, we labeled CA1 pyramidal cells with green fluorescent protein and CA3 pyramidal cells with rhodamine-dextran in hippocampal slice cultures. Varicosities and spines were visualized using two-photon microscopy to detect close association of two components.

DHP-insensitive L-type-like Ca channel of ascidian acquires sensitivity to DHP with single amino acid change in domain III P-region.

TuCa1, an ascidian homolog of L-type Ca channel alpha(1)-subunit, has many critical sites required for binding 1,4-dihydropyridines (DHPs), but is insensitive to DHPs and methyl 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylate (FPL-64176). We have substituted Ser for Ala(1016) at the P-region of domain III in TuCa1 (TuCa1/A1016S) and functionally expressed the channel in Xenopus oocyte along with rabbit alpha(2)/delta and beta(2b). TuCa1/A1016S has gained DHP sensitivity as high as that of a mammalian neuronal L-type Ca channel (rbCII), but remained resistant to FPL-64176.

The ascidian dihydropyridine-resistant calcium channel as the prototype of chordate L-type calcium channel.

This review describes recent findings on voltage-gated Ca channel (Cav channel) cloned from ascidians, the most primitive chordates. Ascidian L-type like Cav channel has several unusual features: (1). it is closely related to the prototype of chordate L-type Cav channels by sequence alignment; (2).

Synchronized formation and remodeling of postsynaptic densities: long-term visualization of hippocampal neurons expressing postsynaptic density proteins tagged with green fluorescent protein.

To explore mechanisms governing the formation and remodeling of postsynaptic density (PSD), we used dissociated cultures of hippocampal neurons isolated from transgenic embryos expressing green fluorescent protein (GFP)-tagged PSD proteins PSD-Zip45 (Homer 1c) and PSD-95. Expression of GFP-tagged PSD molecules was stable, and the remodeling process of PSDs could be followed for >1 week. A higher expression level of GFP-PSD-Zip45 enabled us to quantitatively analyze the amount of PSD-Zip45 clusters during development.

Coexpression of a Ca(v)1.2 protein lacking an N-terminus and the first domain specifically suppresses L-type calcium channel activity.

L-type Ca(2) channels play a critical role in many types of cells, including nerve, muscle and endocrine cells. The most popular and effective tools for analyzing the roles of L-type calcium channels (L-channels) are specific antagonists such as dihydropyrigines. With these drugs however, it is difficult to target specific cells.