Rac1活性のゆっくりとしたゆらぎはグリオーマ細胞の性質や遺伝子発現の多様性に関与している

京都大学0048新制・課程博士博士(医学)甲第18455号医博第3910号新制||医||1004(附属図書館)31333京都大学大学院医学研究科医学専攻(主査)教授 野田 亮, 教授 小川 誠司, 教授 宮本 享学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDFA

Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging

Here we describe a protocol for advanced CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis). The CUBIC protocol enables simple and efficient organ clearing, rapid imaging by light-sheet microscopy and quantitative imaging analysis of multiple samples. The organ or body is cleared by immersion for 1–14 d, with the exact time required dependent on the sample type and the experimental purposes. A single imaging set can be completed in 30–60 min. Image processing and analysis can take <1 d, but it is dependent on the number of samples in the data set. The CUBIC clearing protocol can process multiple samples simultaneously. We previously used CUBIC to image whole-brain neural activities at single-cell resolution using Arc-dVenus transgenic (Tg) mice. CUBIC informatics calculated the Venus signal subtraction, comparing different brains at a whole-organ scale. These protocols provide a platform for organism-level systems biology by comprehensively detecting cells in a whole organ or body

Dynamic modulation of pulsatile activities of oxytocin neurons in lactating wild-type mice.

Breastfeeding, which is essential for the survival of mammalian infants, is critically mediated by pulsatile secretion of the pituitary hormone oxytocin from the central oxytocin neurons located in the paraventricular and supraoptic hypothalamic nuclei of mothers. Despite its importance, the molecular and neural circuit mechanisms of the milk ejection reflex remain poorly understood, in part because a mouse model to study lactation was only recently established. In our previous study, we successfully introduced fiber photometry-based chronic imaging of the pulsatile activities of oxytocin neurons during lactation. However, the necessity of Cre recombinase-based double knock-in mice substantially compromised the use of various Cre-dependent neuroscience toolkits. To overcome this obstacle, we developed a simple Cre-free method for monitoring oxytocin neurons by an adeno-associated virus vector driving GCaMP6s under a 2.6 kb mouse oxytocin mini-promoter. Using this method, we monitored calcium ion transients of oxytocin neurons in the paraventricular nucleus in wild-type C57BL/6N and ICR mothers without genetic crossing. By combining this method with video recordings of mothers and pups, we found that the pulsatile activities of oxytocin neurons require physical mother-pup contact for the milk ejection reflex. Notably, the frequencies of photometric signals were dynamically modulated by mother-pup reunions after isolation and during natural weaning stages. Collectively, the present study illuminates the temporal dynamics of pulsatile activities of oxytocin neurons in wild-type mice and provides a tool to characterize maternal oxytocin functions

Involvement of Ca2+-dependent hyperpolarization in sleep duration in mammals

The detailed molecular mechanisms underlying the regulation of sleep duration in mammals are still elusive. To address this challenge, we constructed a simple computational model, which recapitulates the electrophysiological characteristics of the slow-wave sleep and awake states. Comprehensive bifurcation analysis predicted that a Ca2+-dependent hyperpolarization pathway may play a role in slow-wave sleep and hence in the regulation of sleep duration. To experimentally validate the prediction, we generate and analyze 21 KO mice. Here we found that impaired Ca2+-dependent K+ channels (Kcnn2 and Kcnn3), voltage-gated Ca2+ channels (Cacna1g and Cacna1h), or Ca2+/calmodulin-dependent kinases (Camk2a and Camk2b) decrease sleep duration, while impaired plasma membrane Ca2+ ATPase (Atp2b3) increases sleep duration. Pharmacological intervention and whole-brain imaging validated that impaired NMDA receptors reduce sleep duration and directly increase the excitability of cells. Based on these results, we propose a hypothesis that a Ca2+-dependent hyperpolarization pathway underlies the regulation of sleep duration in mammals

Development of an optimized backbone of FRET biosensors for kinases and GTPases

We report an optimized backbone for the rapid development of a highly sensitive intramolecular fluorescence resonance energy transfer (FRET) biosensor, which includes an optimized pair of fluorescent proteins and a long flexible linker ranging from 116 to 244 amino acids in length. With this backbone system, we developed FRET biosensors of PKA, ERK, JNK, EGFR, RSK, S6K, Akt, PKC, Ras, and Rac1

The Closely Related RNA helicases, UAP56 and URH49, Preferentially Form Distinct mRNA Export Machineries and Coordinately Regulate Mitotic Progression

UAP56 and URH49, closely related RNA helicases in humans, form different mRNA export machineries, the hTREX complex and the AREX complex, respectively. These helicases regulate different sets of genes, among which are mitotic factors. Consistent with their target genes, each helicase is required for a different step in the mitotic process