fMRIによる、ヒト脳の機能的マップ

Nature Methods 7, 253 (2010) 

"A new study, pooling brain-imaging data from 35 centers across the world, shows the power of data sharing and demonstrates a universal architecture of functional connections in the human brain."

該当サイトはこちら。

Cell Stem Cell, 6:323-335,2010

MicroRNA-9 Coordinates Proliferation and Migration of Human Embryonic Stem Cell-Derived Neural Progenitors

Human pluripotent stem cells offer promise for use in cell-based therapies for brain injury and diseases. However, their cellular behavior is poorly understood. Here we show that the expression of the brain-specific microRNA-9 (miR-9) is turned on in human neural progenitor cells (hNPCs) derived from human embryonic stem cells. Loss of miR-9 suppressed proliferation but promoted migration of hNPCs cultured in vitro. hNPCs without miR-9 activity also showed enhanced migration when transplanted into mouse embryonic brains or adult brains of a mouse model of stroke. These effects were not due to precocious differentiation of hNPCs. One of the key targets directly regulated by miR-9 encodes stathmin, which increases microtubule instability and whose expression in hNPCs correlates inversely with that of miR-9. Partial inhibition of stathmin activity suppressed the effects of miR-9 loss on proliferation and migration of human or embryonic rat neural progenitors. These results identify miR-9 as a novel regulator that coordinates the proliferation and migration of hNPCs.

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ヒトESだが、neural progenitorの発生で機能するmicro RNAの論文。
loss of miR-9でmigrationが過剰になる？

Autophagy論文いくつか

Proc Natl Acad Sci U S A. 2010 Mar 15. [Epub ahead of print]
Mechanisms of acute axonal degeneration in the optic nerve in vivo.

Axonal degeneration is an initial key step in traumatic and neurodegenerative CNS disorders. We established a unique in vivo epifluorescence imaging paradigm to characterize very early events in axonal degeneration in the rat optic nerve. Single retinal ganglion cell axons were visualized by AAV-mediated expression of dsRed and this allowed the quantification of postlesional acute axonal degeneration (AAD). EM analysis revealed severe structural alterations of the cytoskeleton, cytoplasmatic vacuolization, and the appearance of autophagosomes within the first hours after lesion. Inhibition of autophagy resulted in an attenuation of acute axonal degeneration. Furthermore, a rapid increase of intraaxonal calcium levels following crush lesion could be visualized using a calcium-sensitive dye. Application of calcium channel inhibitors prevented crush-induced calcium increase and markedly attenuated axonal degeneration, whereas application of a calcium ionophore aggravated the degenerative phenotype. We finally demonstrate that increased postlesional autophagy is calcium dependent and thus mechanistically link autophagy and intraaxonal calcium levels. Both processes are proposed to be major targets for the manipulation of axonal degeneration in future therapeutic settings.

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Eur J Cancer. 2010 Mar 13. [Epub ahead of print]
Inhibition of autophagy augments 5-fluorouracil chemotherapy in human colon cancer in vitro and in vivo model.

Although 5-fluorouracil (5-FU)-based adjuvant chemotherapy is widely used in the treatment of colorectal cancer, novel therapeutic strategies need to be explored. It has been reported that autophagy is extensively implicated in cancer. However, the function of autophagy is not fully understood. In the present study, apoptosis induced by 5-FU in 3 human colon cancer cell lines (HCT116, DLD-1, and DLD-1/5-FU (a specific 5-FU-resistant sub-line)) was measured using MTT assay, DNA fragmentation assay, Hoechst 33342 staining, and caspase-3 immunoblotting. The autophagy activation induced by 5-FU treatment was revealed by microtubule-associated protein 1 light chain 3 (LC3) immunofluorescence and immunoblotting and p62 immunoblotting. Inhibition of autophagy by 3-methyladenine (3-MA) or small interference RNA targeting Atg7 (Atg7 siRNA) significantly augmented 5-FU-induced apoptosis. This synergistic effect of 5-FU and 3-MA was further confirmed in the DLD-1 xenograft tumour model. Tumour growth was suppressed more significantly with combination treatment than 5-FU treatment alone. In conclusion, autophagy was activated as a protective mechanism against 5-FU-induced apoptosis and its inhibition could be a promising strategy for adjuvant chemotherapy in colon cancer.

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BMC Cancer. 2010 Mar 16;10(1):98. [Epub ahead of print]
Genetic and epigenetic silencing of the beclin 1 gene in sporadic breast tumors.

ABSTRACT: BACKGROUND: : Beclin 1, an important autophagy-related protein in human cells, is involved in cell death and cell survival. Beclin 1 mapped to human chromosome 17q21. It is widely expressed in normal mammary epithelial cells. Although down-regulated expression with mono-allelic deletions of beclin 1 gene was frequently observed in breast tumors, whether there was other regulatory mechanism of beclin 1 was to be investigated. We studied the expression of beclin 1 and explored the possible regulatory mechanisms on its expression in breast tumors. METHOD: S: 20 pairs of tumors and adjacent normal tissues from patients with sporadic breast invasive ductal cancer (IDCs) were collected. The mRNA expression of beclin 1 was detected by real-time quantitative RT-PCR. Loss of heterozygosity (LOH) was determined by real-time quantitative PCR and microsatellite methods. The protein expression of beclin 1, p53, BRCA1 and BRCA2 was assessed by immunohistochemistry. CpG islands in 5' genomic region of beclin 1 gene were identified using MethylPrimer Program. Sodium bisulfite sequencing was used in examining the methylation status of each CpG island. RESULTS: : Decreased beclin 1 mRNA expression was detected in 70% of the breast tumors, and the protein levels were co-related to the mRNA levels. Expression of beclin 1 mRNA was demonstrated to be much higher in the BRCA1 positive tumors than that in the BRCA1 negative ones. Loss of heterozygosity was detected in more than 45% of the breast tumors, and a dense cluster of CpG islands was found from the 5' end to the intron 2 of the beclin 1 gene. Methylation analysis showed that the promoter and the intron 2 of beclin 1 were aberrantly methylated in the tumors with decreased expression. CONCLUSIONS: : These data indicated that LOH and aberrant DNA methylation might be the possible reasons of the decreased expression of beclin 1 in the breast tumors. The findings here shed some new light on the regulatory mechanisms of beclin 1 in breast cancer.

Trends in Neurosciences 30:350-356, 2007

Thalamic synchrony and dynamic regulation of global forebrain oscillations

The circuitry within the thalamus creates an intrinsic oscillatory unit whose function depends critically on reciprocal synaptic connectivity between excitatory thalamocortical relay neurons and inhibitory thalamic reticular neurons along with a robust post-inhibitory rebound mechanism in relay neurons. Feedforward and feedback connections between cortex and thalamus reinforce the thalamic oscillatory activity into larger thalamocortical networks to generate sleep spindles and spike-wave discharge of generalized absence epilepsy. The degree of synchrony within the thalamic network seems to be crucial in determining whether normal (spindle) or pathological (spike-wave) oscillations occur, and recent studies show that regulation of excitability in the reticular nucleus leads to dynamical modulation of the state of the thalamic circuit and provide a basis for explaining how a variety of unrelated genetic alterations might lead to the spike-wave phenotype. In addition, given the central role of the reticular nucleus in generating spike-wave discharge, these studies have suggested specific interventions that would prevent seizures while still allowing normal spindle generation to occur. This review is part of the INMED/TINS special issue Physiogenic and pathogenic oscillations: the beauty and the beast, based on presentations at the annual INMED/TINS symposium (http://inmednet.com).

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Neuro-circuitをどのように考えるか、という一例として。

 　→　→　→　→
↑ Core network ↓
 　←　←　←　←
     ↑
●Structure-excitatory/inhibitory/connection?
●Main molecule/neural subtype?
●Modificator?
     ↑
inhibitionで、ネットワークのoutput（synchronization）が影響するか？

PNAS 103:17949-17954, 2006

 Lipocalin-type prostaglandin D synthase produces prostaglandin D2 involved in regulation of physiological sleep

Prostaglandin (PG) D2 has been proposed to be essential for the initiation and maintenance of the physiological sleep of rats because intracerebroventricular administration of selenium tetrachloride (SeCl4), a selective inhibitor of PGD synthase (PGDS), was shown to reduce promptly and effectively the amounts of sleep during the period of infusion. However, gene knockout (KO) mice of PGDS and prostaglandin D receptor (DP1R) showed essentially the same circadian profiles and daily amounts of sleep as wild-type (WT) mice, raising questions about the involvement of PGD2 in regulating physiological sleep. Here we examined the effect of SeCl4 on the sleep of WT and KO mice for PGDS and DP1R and that of a DP1R antagonist, ONO-4127Na, on the sleep of rats. The i.p. injection of SeCl4 into WT mice decreased the PGD2 content in the brain without affecting the amounts of PGE2 and PGF2α. It inhibited sleep dose-dependently and immediately after the administration during the light period when mice normally sleep, increasing the wake time; and the treatment with this compound resulted in a distinct sleep rebound during the following dark period. The SeCl4-induced insomnia was observed in hematopoietic PGDS KO mice but not at all in lipocalin-type PGDS KO, hematopoietic and lipocalin-type PGDS double KO or DP1R KO mice. Furthermore, the DP1R antagonist ONO-4127Na reduced sleep of rats by 30% during infusion into the subarachnoid space under the rostral basal forebrain at 200 pmol/min. These results clearly show that the lipocalin-type PGDS/PGD2/DP1R system plays pivotal roles in the regulation of physiological sleep.

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●SeCl4（PGD synthaseの阻害剤）を入れると、sleep↓

●DP1R antagonistのONO-4127Naを入れると、sleep↓

●しかし、DP1R-KOマウスでは、sleepの量もcircadianも差がない

●L-PGDS-KOマウスでも、sleepの量もcircadianも差がない

  (Ref.)

↓

●PGD2以外のalternative pathwayによるcompensationの問題/阻害剤のspecificityの問題

●DP1R-KOマウスでSeCl4を入れるとどうなる？

↓

●H-PGDS、L-PGDS、DP1R-KOマウスでSeCl4を入れたら、、L-PGDS、DP1R-KOマウスではsleep↓の効果が見えなくなった。ということは、acuteの効果はやはり見える。

●sleep↓の後のリバウンドは、L&H-PGDS-DKOにすると消える。

●いずれにしても、circadianはがっつり残る→睡眠ホメオスタシスと日内変動のメカニズムがどう絡み合うのか？？

 

Current Opinion in Pharmacology 7:33-38, 2007 (Review)

Prostaglandins and adenosine in the regulation of sleep and wakefulness

Prostaglandin (PG) D2 and adenosine are potent humoral sleep-inducing factors that accumulate in the brain during prolonged wakefulness. PGD2 is produced in the brain by lipocalin-type PGD synthase, which is localized mainly in the leptomeninges, choroid plexus and oligodendrocytes, and circulates in the cerebrospinal fluid as a sleep hormone. It stimulates DP1 receptors on leptomeningeal cells of the basal forebrain to release adenosine as a paracrine signaling molecule to promote sleep. Adenosine activates adenosine A2A receptor-expressing sleep-active neurons in the basal forebrain and the ventrolateral preoptic area. Sleep-promoting neurons in the ventrolateral preoptic area send inhibitory signals to suppress the histaminergic neurons in the tuberomammillary nucleus, which contribute to arousal through histamine H1 receptors. Increased knowledge of the molecular mechanisms by which PGD2 induces sleep through activation of adenosine A2A receptors and inhibition of the histaminergic arousal system will be useful both for a better understanding of sleep/wake regulation and for the development of novel types of sleeping pills or anti-doze drugs.

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●sleep deprivationでは、PGD2もadenosineも量が上がる。

●DP1R-KOでもcircadianに差がないそう・・・
adenosineの上流であることは分かった。

●A1R→BFのCholinergic neuron？
あまり重要な役回りではなさそう

●A2AR→caffeineのターゲット

●＜重要＞A1R-KOも、A2AR-KOも、normalなcircadian profilesを示し、NREM/REMの量もWTと変わらない。reboundについてはA2AR-KOでは差が出る（A1R-KOは差なし)。

●PGD2/A2ARアゴニスト投与後のc-Fos染色で、sleep-active neurons（VLPO）が同定された。
ref.: 32, 33

●NA, Ach, A1R signal---| VLPO

（serotonineとadenosineへの反応性の違いで、Type1/2に分けられている）
●5-HT ---| VLPO Type1 neuron
   ↓
VLPO Type2 neuron
●CGS21680　→　VLPO Type2 neuron

●Figure 1がよくまとまっている。

●全体的なインプレ：
・KOマウスで差が出ない～通常のS-Wサイクルで働いているというより、S-Wサイクルのコントロールをしているニューロンの活性を著しく変動させている、という感じ？Adenosineは日内変動がある不思議。

・Thresholdの形成要因は？細胞の外部/内部因子の関わり？

●VLPOのGABA neuron　→　TMNのヒスタミン、となっているが、それだけだろうか？

Nat Neuro 13:369-378, 2010

Stimulus onset quenches neural variability: a widespread cortical phenomenon

Neural responses are typically characterized by computing the mean firing rate, but response variability can exist across trials. Many studies have examined the effect of a stimulus on the mean response, but few have examined the effect on response variability. We measured neural variability in 13 extracellularly recorded datasets and one intracellularly recorded dataset from seven areas spanning the four cortical lobes in monkeys and cats. In every case, stimulus onset caused a decline in neural variability. This occurred even when the stimulus produced little change in mean firing rate. The variability decline was observed in membrane potential recordings, in the spiking of individual neurons and in correlated spiking variability measured with implanted 96-electrode arrays. The variability decline was observed for all stimuli tested, regardless of whether the animal was awake, behaving or anaesthetized. This widespread variability decline suggests a rather general property of cortex, that its state is stabilized by an input.

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慣れない分野でタフな論文。理解が結構怪しいが。

●刺激後にneuronの活動（membrane potencial/firing rates）のvariabilityが低下することを、いくつかの指標を定義して証明。その指標を変化させるほかのfactorが影響しているわけではないことをなにやらごちゃごちゃと検討している・・・らしい・・・

●平均化することで失われてしまう情報に意味があるのではないかという問題提起

とすれば・・・

・個々のneuronのvariabilityがノイズであると考えれば、network全体として機能することでbufferingしている？

・むしろ個々のneuronのvariabilityはそれぞれの機能（処理している情報）の違いを反映している？

●Discussionより

"Mechanistically, the variance decline inplies that cortical curcuits become more stable when driven."

→こういうタイプのネットワークはいくつか考えられて、recurrent networkもその一つ。

視床下部神経核とホルモンの機能（まとめサイト）

滋賀医大のサイトでいいまとめがあったのでメモ。

サイトはこちら

Sleep. 33(1):19-28, 2010.

Genetic evidence for a role for protein kinase A in the maintenance of sleep and thalamocortical oscillations.

STUDY OBJECTIVES: Genetic manipulation of cAMP-dependent protein kinase A (PKA) in Drosophila has implicated an important role for PKA in sleeplwake state regulation. Here, we characterize the role of this signaling pathway in the regulation of sleep using electroencephalographic (EEG) and electromyographic (EMG) recordings in R(AB) transgenic mice that express a dominant negative form of the regulatory subunit of PKA in neurons within cortex and hippocampus. Previous studies have revealed that these mutant mice have reduced PKA activity that results in the impairment of hippocampus-dependent long-term memory and long-lasting forms of hippocampal synaptic plasticity. DESIGN: PKA assays, in situ hybridization, immunoblots, and sleep studies were performed in R(AB) transgenic mice and wild-type control mice. MEASUREMENTS AND RESULTS: We have found that R(AB) transgenic mice have reduced PKA activity within cortex and reduced Ser845 phosphorylation of the glutamate receptor subunit GluR1. R(AB) transgenic mice exhibit non-rapid eye movement (NREM) sleep fragmentation and increased amounts of rapid eye movement (REM) sleep relative to wild-type mice. Further, R(AB) transgenic mice have more delta power but less sigma power during NREM sleep relative to wild-type mice. After sleep deprivation, the amounts of NREM and REM sleep were comparable between wild-type and R(AB) transgenic mice. However, the homeostatic rebound of sigma power in R(AB) transgenic mice was reduced. CONCLUSIONS: Alterations in cortical synaptic receptors, impairments in sleep continuity, and alterations in sleep oscillations in R(AB) mice imply that PKA is involved not only in synaptic plasticity and memory storage but also in the regulation of sleep/wake states.

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PKA-GluRの経路について、Tgマウスによる考察。

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Neurosci Lett.469(1):1-5, 2010

Sleep fragmentation reduces hippocampal CA1 pyramidal cell excitability and response to adenosine.

Sleep fragmentation (SF) impairs the restorative/cognitive benefits of sleep via as yet unidentified alterations in neural physiology. Previously, we found that hippocampal synaptic plasticity and spatial learning are impaired in a rat model of SF which utilizes a treadmill to awaken the animals every 2 min, mimicking the frequency of awakenings observed in human sleep apnea patients. Here, we investigated the cellular mechanisms responsible for these effects, using whole-cell patch-clamp recordings. 24h of SF decreased the excitability of hippocampal CA1 pyramidal neurons via decreased input resistance, without alterations in other intrinsic membrane or action potential properties (when compared to cage controls, or to exercise controls that experienced the same total amount of treadmill movement as SF rats). Contrary to our initial prediction, the hyperpolarizing response to bath applied adenosine (30 microM) was reduced in the CA1 neurons of SF treated rats. Our initial prediction was based on the evidence that sleep loss upregulates cortical adenosine A1 receptors; however, the present findings are consistent with a very recent report that hippocampal A1 receptors are not elevated by sleep loss. Thus, increased adenosinergic inhibition is unlikely to be responsible for reduced hippocampal long-term potentiation in SF rats. Instead, the reduced excitability of CA1 pyramidal neurons observed here may contribute to the loss of hippocampal long-term potentiation and hippocampus-dependent cognitive impairments associated with sleep disruption. (c) 2009 Elsevier Ireland Ltd. All rights reserved.

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電気生理のFigが2個だけ。

New optogenetic tools

Nature Methods March, 2010のHighlightより、
新しいoptogenetic toolを見つける試みと、既存のツールをmutagenesisで改良する試みを紹介。

refs.
Chow et al. Nature 463:98-102,2010
Gunaydin et al. Nat. Neurosci. 13:387-392,2010

そのうち読む。

vGLUT3

グルタミン酸トランスポーターの一種である、vesicular glutamate transporter （vGLUT）のうち、vGLUT1,2は脳内に多く発現している（発現パターンは相補的でmutually exclusive）が、rapheにはどちらも出ていないことから、vGLUT3が発見された。rapheに高発現。ほか、内有毛細胞に高発現し、KOマウスはdeafness。また皮質と海馬のGABAergic basket cellの一部にも発現し、KOではnonconvulsive seizureが見られる（Seal et al. Neuron 57:263-275, 2008）。また、Low-threshold　C繊維にも発現し、障害時のmechanical hypersensitivityにも必要（Seal et al. Nature 462:651-655, 2009）。

行動異常は？？dataなし。

Molecular Cloning and Functional Identification of Mouse Vesicular Glutamate Transporter 3 and Its Expression in Subsets of Novel Excitatory Neurons

Martin et al. JBC 277:50734-50748,2002

ac, anterior commissure; aq, aqueduct; BST, bed nucleus of stria terminalis; cc, corpus callosum; CA1, field CA1 of Ammon's horn; CA3, field CA3 of Ammon's horn;CBL, cerebellum; CM, central medial thalamic ncl.; CPu, caudate putamen; CTX, neocortex;DG, dentate gyrus; DR, dorsal raphe ncl.;GP, globus pallidus; LHA, lateral hypothalamic area; MnR, median raphe ncl.; NTS, nucleus of the solitary tract; Sp5, spinal ncl. of the trigeminal tract; SON, supraoptic ncl.; 3V, third ventricle;PVA, anterior periventricular thalamic ncl.; Rob, ncl. raphe obscurus; VP, ventral pallidum;VPM, medial ventroposterior thalamic ncl.; Xi, xiphoid thalamic ncl.

皮膚感覚路

●後索-内側毛帯路

皮膚への接触、振動、深部感覚経路。後索を上って延髄後索核（楔束核、薄束核←E4Bヘテロでやられるところ：体内で最も長い経路の為、軸索輸送が障害され軸索末端の変性が起きやすい；上肢からの線維は楔状束核，下肢からの線維は薄束核←ソース△、要確認）。後索核から交差。

●脊髄視床路

痛覚、温度感覚。後角でシナプスを変え、前方に向かって交差し、脊髄腹側を上行。

キーワード：ヒスタミン、ブラジキニン、プロスタグランジン、K＋イオン、substance P

Aδ線維、C繊維（無髄）