大脳視覚皮質におけるオリゴデンドロサイト前駆細胞の発達と視覚剥奪による影響

オリゴデンドロサイト前駆細胞は、感覚経験によって細胞の増殖・分化が調整される。視覚剥奪依存的オリゴデンドロサイト前駆細胞の変化は、マウスの一次視覚野においては未だ明らかとされていない。本研究では、眼優位性臨界期の開始頃、即ち生後25日目の時期特異的にオリゴデンドロサイト前駆細胞の増殖能がピークとなり、尚、開眼開始(生後14~15日目)からの両眼球剥奪によって、一次視覚野の大脳皮質下層と皮質下白質において更なる増加が観られた。これらの結果を二つの章に分けて詳細に述べた。生後25日目の健常マウスでは、細胞周期を通して分裂した娘細胞が対称的に分化へ進んだ。視覚剥奪では、対称的に細胞分裂した未分化性娘細胞が細胞周期のG1期に戻ったため増殖能が増加する一方、G0期に脱して分化へ進む細胞は減少した。これらの未分化性状態の亢進は、ソニックヘッジホッグ(Sonic hedgehog, Shh)シグナル経路の一部を媒介する可能性が挙げられた。その5日後（生後30日目）、未分化性オリゴデンドロサイト前駆細胞の殆んどは分化へ進み、僅かは更に分化が進み成熟細胞となった。視覚剥奪マウスでは、対称性モードの娘細胞が静止状態と分化状態が転換され、未分化性が維持された。やがて生後50日目の健常マウスでは、増殖細胞の殆んどが成熟オリゴデンドロサイトへ分化した。視覚剥奪マウスでは更なる成熟オリゴデンドロサイト数が見られ、それは主に対称的・非対称的に成熟細胞へ分化が増加したためであった。尚、増殖細胞に関わらず、全ての成熟細胞の分布や蛍光強度を検討すると、視覚剥奪によって更に細胞数が増加し、ミエリン形成の更なる増加も示唆された。皮質下白質においては、第一章の結果のように生後25日目に、時期特異的に細胞の増殖能がピークと増加し、視覚剥奪による更なる増加が示された。この増加は、増殖細胞の対称性・非対称性に関わらず、未分化性を促進させたためであった。視神経切断による視覚剥奪では、視神経の軸索がたどり着く視覚視床において反応性アストロサイトやミクログリア・マクロファージが増加したが、一次視覚野では影響しなかった。即ち、視神経切断による炎症反応は視床でのみ生じ、視覚野の皮質・白質では神経回路の再編成よる未分化性増加が示唆された。増殖細胞はやがて成熟細胞へ分化し、増殖5日後（生後30日目）をピークとして増加し、視覚剥奪マウスでも同様であった。皮質の結果と異なり、視覚剥奪による成熟細胞の促進は影響しなかった。本研究では、眼優位性臨界期開始頃、オリゴデンドロサイト前駆細胞の発達に重要な時期であると示唆され、やがて視覚剥奪による皮質内と白質で異なる発達・制御をもつ事が明らかとなった。視覚剥奪下、皮質下層では、生後25日目の時期特異的に増殖した細胞の未分化性維持はShhシグナル経路の一部を介して促進され、大脳視覚皮質の下層ではやがて成熟オリゴデンドロサイトへの分化を更に促進させた。白質では、生後25日目にも未分化性細胞が視覚剥奪によって増加する一方、成熟への促進は影響しなかった。従って、視覚剥奪すると神経回路の再編成が生じるが、それに伴う皮質内と白質においてオリゴデンドロサイト前駆細胞の発達時期・制御が異なる事が明かされた。この結果は、やがて神経回路再編成にオリゴデンドロサイトの発達が関与する事を示唆する。創価大

Factors Influencing Matching of Ride-Hailing Service Using Machine Learning Method

It is common to call a taxi by taxi-apps in Korea and it was believed that an app-taxi service would provide customers with more convenience. However, customers’ requests can often be denied, as taxi drivers can decide whether to take calls from customers or not. Therefore, studies on factors that determine whether taxi drivers refuse or accept calls from customers are needed. This study investigated why taxi drivers might refuse calls from customers and factors that influence the success of matching within the service. This study used origin-destination data in Seoul and Daejeon obtained from T-map Taxis, which was analyzed via a decision tree using machine learning. Cross-validation was also performed. Results showed that distance, socio-economic features, and land uses affected matching success rate. Furthermore, distance was the most important factor in both Seoul and Daejeon. The matching success rate in Seoul was lowest for trips shorter than the average at midnight. In Daejeon, the rate was lowest when the calls were made for trips either shorter or longer than the average distance. This study showed that the matching success for ride-hailing services can be differentiated particularly by the distance of the requested trip depending on the size of the city

532 nm Low-Power Laser Irradiation Facilitates the Migration of GABAergic Neural Stem/Progenitor Cells in Mouse Neocortex

<div><p>Background and Objective</p><p>Accumulating evidence has shown that low-power laser irradiation (LLI) affects cell proliferation and survival, but little is known about LLI effects on neural stem/progenitor cells (NSPCs). Here we investigate whether transcranial 532 nm LLI affects NSPCs in adult murine neocortex and in neurospheres from embryonic mice.</p><p>Study Design/Materials and Methods</p><p>We applied 532 nm LLI (Nd:YVO₄, CW, 60 mW) on neocortical surface via cranium in adult mice and on cultured cells from embryonic mouse brains in vitro to investigate the proliferation and migration of NSPCs and Akt expression using immunohistochemical assays and Western blotting techniques.</p><p>Results</p><p>In vivo experiments demonstrated that 532 nm LLI significantly facilitated the migration of GABAergic NSPCs that were induced to proliferate in layer 1 by mild ischemia. In vitro experiments using GABAergic NSPCs derived from embryonic day 14 ganglionic eminence demonstrated that 532 nm LLI for 60 min promoted the migration of GAD67-immunopositive NSPCs with a significant increase of Akt expression. Meanwhile, the LLI induced proliferation, but not migration, of NSPCs that give rise to excitatory neurons.</p><p>Conclusion</p><p>It is concluded that 532 nm LLI promoted the migration of GABAergic NSPCs into deeper layers of the neocortex in vivo by elevating Akt expression.</p></div

Trans-well migration experiments.

<p>A: Location of brain parts dissected for making neurospheres of NSPCs. Label “c” shows the cortex of E16, which includes NSPCs of excitatory and GABAergic neurons. B: Method of trans-well migration test. Bottom membrane of inserted trans-well has pores of 8 μm diameter. Neurospheres derived from “c” were placed on insert trans-well, and 532 nm laser was irradiated from above the media. Cells were fixed by 4% PFA at 48 h after LLI. C: A cell that moved through the pores stained by GAD67 (red) and Hochest (blue). Holes in the Hochest image are the pores of trans-well. Scale bar: 10 μm. Double positive cells were found only in the LLI experimental group (mean ± SD, n = 4, t-test *p < 0.05).</p

Transcranial LLI effects on pAkt and Akt expression.

<p>A: Western blot analysis of auditory cortex lysates at post-LLI and 2 days after LLI. GAPDH is a loading control. B: Quantification of blots using Image J. Band intensity was normalized to GAPDH (mean ± SD, n = 4, t-test *p < 0.05).</p

Effects of transcranial LLI on cell migration of NSPCs.

<p>A: Scheme of LLI treatment to adult mouse brain. Auditory cortical area was irradiated on the left side of the cortex through cranium. The opposite hemisphere was used as a control. B: TTC staining for detecting cell death. LLI after the mild occlusion of CCA (CCAO) did not induce cell death (left) compared with sham control (right). C: Protocol for in vivo experiments from CCAO to immunostaining. Brains were fixed at 4 h and 5 days after LLI. D: Photographs of each immunostaining at post-LLI (4 hours) and 5 days after LLI. EdU, GAD67 and Ki67 positive cells (white arrow) were found in layer 1 of post-LLI sections. EdU and GAD67 positive cells but Ki67 negative cells (yellow arrow) were found in deep layer at 5 days after LLI. GAD67 positive, EdU and Ki67 negative cell (arrowhead) is a mature inhibitory neuron. Scale bars: 10 μm. E: The laminar pattern of the cell density (mean ± SEM, n = 4) of EdU and GAD67 positive, Ki67-negative cells from layer 1 to 6 was significantly different between LLI and control groups (KS test, p<0.01). F: Cumulative fraction of total cells for non-parametric KS 2-sample test. Each accumulative curve shows higher distribution in layer 1–4 in control group, while does in layer 5–6 in LLI group. G: Total cell density of EdU and GAD67 positive and Ki67 negative (EdU+/GAD67+/Ki67-) cells did not differ between control and LLI groups.</p

LLI effects on proliferation of NSPC cells from E10 forebrain and E14 MGE.

<p>A: Location of brain parts dissected for making neurosphere of NSPCs. “a” shows cortical wall of E10 forebrain, which generates excitatory neurons, whereas “b” shows E14 MGE, which generates GABAergic neurons. B: CCK-8 assay to show biochemically cell proliferation for different duration of LLI (mean ± SD, n = 4 for each duration, t-test, *p < 0.05). The non-irradiated group was standardized as 100%. C: Cell counting of DAPI staining to clarify the increase in cell number following the dissociation from neurospheres. LLI significantly promoted proliferation (mean ± SD, n = 5, t-test, *p < 0.05).</p

LLI effects on pAkt and Akt expression of cultured cells.

<p>A: Western blot analysis of neurosphere lysates from post-LLI (4 hours) of E10 forebrain. B: Quantification of blots. Each blot intensity was normalized to GAPDH of loading control (mean ± SD, n = 3, t-test *p < 0.05).</p