Cold-induced suspension and resetting of Ca2+ and transcriptional rhythms in the suprachiasmatic nucleus neurons.

Does the circadian clock keep running under such hypothermic states as daily torpor and hibernation? This fundamental question has been a research subject for decades but has remained unsettled. We addressed this subject by monitoring the circadian rhythm of clock gene transcription and intracellular Ca2+ in the neurons of the suprachiasmatic nucleus (SCN), master circadian clock, in vitro under a cold environment. We discovered that the transcriptional and Ca2+ rhythms are maintained at 22°C-28°C, but suspended at 15°C, accompanied by a large Ca2+ increase. Rewarming instantly resets the Ca2+ rhythms, while transcriptional rhythms reach a stable phase after the transient state and recover their phase relationship with the Ca2+ rhythm. We conclude that SCN neurons remain functional under moderate hypothermia but stop ticking in deep hypothermia and that the rhythms reset after rewarming. These data also indicate that stable Ca2+ oscillation precedes clock gene transcriptional rhythms in SCN neurons.

All-synchronized picosecond pulses and time-gated detection improve the spatial resolution of two-photon STED microscopy in brain tissue imaging.

Super-resolution in two-photon excitation (2PE) microscopy offers new approaches for visualizing the deep inside the brain functions at the nanoscale. In this study, we developed a novel 2PE stimulated-emission-depletion (STED) microscope with all-synchronized picosecond pulse light sources and time-gated fluorescence detection, namely, all-pulsed 2PE-gSTED microscopy. The implementation of time-gating is critical to excluding undesirable signals derived from brain tissues. Even in a case using subnanosecond pulses for STED, the impact of time-gating was not negligible; the spatial resolution in the image of the brain tissue was improved by approximately 1.4 times compared with non time-gated image. This finding demonstrates that time-gating is more useful than previously thought for improving spatial resolution in brain tissue imaging. This microscopy will facilitate deeper super-resolution observation of the fine structure of neuronal dendritic spines and the intracellular dynamics in brain tissue.

Single-scan volumetric imaging throughout thick tissue specimens by one-touch installable light-needle creating device.

Biological tissues and their networks frequently change dynamically across large volumes. Understanding network operations requires monitoring their activities in three dimensions (3D) with single-cell resolution. Several researchers have proposed various volumetric imaging technologies. However, most technologies require large-scale and complicated optical setups, as well as deep expertise for microscopic technologies, resulting in a high threshold for biologists. In this study, we propose an easy-to-use light-needle creating device for conventional two-photon microscopy systems. By only installing the device in one position for a filter cube that conventional fluorescent microscopes have, single scanning of the excitation laser light beam excited fluorophores throughout over 200 µm thickness specimens simultaneously. Furthermore, the developed microscopy system successfully demonstrated single-scan visualization of the 3D structure of transparent YFP-expressing brain slices. Finally, in acute mouse cortical slices with a thickness of approximately 250 µm, we detected calcium activities with 7.5 Hz temporal resolution in the neuronal population.

Lhx2 regulates the timing of ß-catenin-dependent cortical neurogenesis.

The timing of cortical neurogenesis has a major effect on the size and organization of the mature cortex. The deletion of the LIM-homeodomain transcription factor Lhx2 in cortical progenitors by Nestin-cre leads to a dramatically smaller cortex. Here we report that Lhx2 regulates the cortex size by maintaining the cortical progenitor proliferation and delaying the initiation of neurogenesis. The loss of Lhx2 in cortical progenitors results in precocious radial glia differentiation and a temporal shift of cortical neurogenesis. We further investigated the underlying mechanisms at play and demonstrated that in the absence of Lhx2, the Wnt/ß-catenin pathway failed to maintain progenitor proliferation. We developed and applied a mathematical model that reveals how precocious neurogenesis affected cortical surface and thickness. Thus, we concluded that Lhx2 is required for ß-catenin function in maintaining cortical progenitor proliferation and controls the timing of cortical neurogenesis.