博士課程の訓練助成金T90 DA 022763（MA）;とNIHグラントR01 - EY017699とNSFの助成BCS - 0633281（SK）の著者は、国際脳研究財団特別研究員（DC）でサポートされています。著者は、有益なコメントをランドルフブレイクに感謝しています。

<ol>
	<li>Blake, R., Logothetis, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+competition.">Visual competition.</a> Nat. Rev. Neurosci. 3, 13-21 (2002).</li><li>Rees, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroimaging+of+visual+awareness+in+patients+and+normal+subjects.">Neuroimaging of visual awareness in patients and normal subjects.</a> Curr. Op. Neurobiol. 11, 150-156 (2001).</li><li>Bahrami, B., Carmel, D., Walsh, V., Rees, G., Lavie, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unconscious+orientation+processing+depends+on+perceptual+load.">Unconscious orientation processing depends on perceptual load.</a> Journal of Vision. 8, 1-10 (2008).</li><li>Bahrami, B., Carmel, D., Walsh, V., Rees, G., Lavie, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+attention+can+modulate+unconscious+orientation+processing.">Spatial attention can modulate unconscious orientation processing.</a> Perception. 37, 1520-1528 (2008).</li><li>Carmel, D., Walsh, V., Lavie, N., Rees, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Right+parietal+TMS+shortens+dominance+durations+in+binocular+rivalry.">Right parietal TMS shortens dominance durations in binocular rivalry.</a> Current Biology. , .</li><li>Schurger, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+very+inexpensive+MRI-compatible+method+for+dichoptic+visual+stimulation.">A very inexpensive MRI-compatible method for dichoptic visual stimulation.</a> J. Neurosci. Methods. 177, 199-202 (2009).</li><li>Tong, F., Nakayama, K., Vaughn, J. T., Kanwisher, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binocular+rivalry+and+visual+awareness+in+human+extrastriate+cortex.">Binocular rivalry and visual awareness in human extrastriate cortex.</a> Neuron. 21, 753-759 (1998).</li><li>Kovacs, I., Papathomas, T. V., Ming, Y., Feher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=When+the+brain+changes+its+mind:+interocular+grouping+during+binocular+rivalry.">When the brain changes its mind: interocular grouping during binocular rivalry.</a> Proc. Natl. Acad. Sci. USA. 93, 15508-15511 (1996).</li><li>Blake, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+primer+on+binocular+rivalry,+including+current+controversies.">A primer on binocular rivalry, including current controversies.</a> Brain and Mind. 2, 5-38 (2001).</li><li>Cowey, A., Stoerig, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurobiology+of+blindsight.">The neurobiology of blindsight.</a> Trends Neurosci. 14, 140-145 (1991).</li><li>Nasrallah, M., Carmel, D., Lavie, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murder+she+wrote:+enhanced+sensitivity+to+negative+word+valence.">Murder she wrote: enhanced sensitivity to negative word valence.</a> Emotion. 9, 609-618 (2009).</li><li>Persaud, N., McLeod, P., Cowey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-decision+wagering+objectively+measures+awareness.">Post-decision wagering objectively measures awareness.</a> Nat Neurosci. 10, 257-261 (2007).</li><li>Tsuchiya, N., Koch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+flash+suppression+reduces+negative+afterimages.">Continuous flash suppression reduces negative afterimages.</a> Nat. Neurosci. 8, 1096-1101 (2005).</li><li>Bahrami, B., Lavie, N., Rees, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attentional+Load+Modulates+Responses+of+Human+Primary+Visual+Cortex+to+Invisible+Stimuli.">Attentional Load Modulates Responses of Human Primary Visual Cortex to Invisible Stimuli.</a> Curr. Biol. 17, 509-513 (2007).</li><li>Wilson, H. R., Blake, R., Lee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+travelling+waves+in+visual+perception.">Dynamics of travelling waves in visual perception.</a> Nature. 412, 907-910 (2001).</li><li>Meng, M., Tong, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+attention+selectively+bias+bistable+perception?+Differences+between+binocular+rivalry+and+ambiguous+figures.">Can attention selectively bias bistable perception? Differences between binocular rivalry and ambiguous figures.</a> Journal of Vision. 4, 539-551 (2004).</li></ol>

利害の衝突は宣言されません。

我々はそれを作成するための視野闘争の性質、いくつかの方法を記載している、そしてそれが使用されている場合どのような考慮事項が考慮する必要があります。はじめにで説明するように、視野闘争の適切な使用は、実験的に意識の軌跡（または遺伝子座）、知覚的選択と無意識の処理についての質問に対処することが可能になります。適切にこのような調査を実施するため、しかし、人はそのような安定した収束、と、無意識の処理を調査する場合、完全な抑制をもたらす可能性のあるディスプレイを使用しての必要性を維持することの重要性などの問題を認識する必要があります。 ライバルを作成するためにどの方法を使用するかを選択するときに、それは考慮に入れ、各方法の長所と短所を取ることが重要です。そこにはポイントは1つが、多色刺激を使うことに興味を持っている場合、赤青メガネを使用して、例えば、ではない、しかし一方で、このメソッドはおそらくMRIスキャナで使用するのが最も簡単です。同様に、ミラーステレオスコープは、別々の画像が対応する網膜の場所に落ちることを保証するのは非常に信頼性の高い方法です。しかし、各観察者に必要な個々の調整やMRIスキャナでステレオスコープを入れるに関わる技術的な問題はいくつかのためのこの方法はあまり魅力的にするかもしれないの研究。 最後に、適切な依存措置を採用するために、視野闘争の経験の特性を認識することが重要です。研究の質問は、無意識の処理に関係するときに、参加者が抑制画像の実際に気づかずに、独立した検証が不可欠です。いずれかの実験的操作が支配と抑圧の期間をどのように影響するかに興味を持っているときは、そのような平均値や中央値などの期間だけではなく、中心傾向の測度の（γ-形）の分布を調べるために多くの照明かもしれない、そしてそれが重要になることがありますオブザーバーは、支配の鋭いまたは徐々に（断片的または波のような）の遷移を経験したかどうかを確認する。 我々は、この魅惑的な現象を採用するに興味がある研究者は、この導入が役に立つことを願っています。

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<td>Mirror stereoscope</td>
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how to create and use binocular rivalry

私たちの目のそれぞれは、通常、私たちの周りの世界のわずかに異なる画像を見ている。脳は、単一の一貫した表現に、これら二つの画像を組み合わせることができます。しかし、目が互いに十分に異なる画像が表示されるとき、面白いことが起きます。むしろ複合意識知覚に融合する二つの画像よりも、何transpiresは、1つのイメージが意識を支配する知覚交替のパターンです、もう一方抑制され、二つの画像、通常は数秒ごとに間の支配交互に。この知覚現象は、視野闘争として知られています。それぞれの目には不変の視覚入力が視覚意識と知覚の交替につながるため、視野闘争が、知覚的選択と人間と動物の両方のモデルで意識を研究するために有用と考えられている。視野闘争刺激を作成するには、必要なことは、同じ知覚の場所で別のイメージでそれぞれの目を提示することである。これをするいくつかの方法がありますが、フィールドへの新規参入者は、最善の、特定のニーズに合うどの方法がよくわからない。この記事の目的は、視野闘争を作成し、使用する安価で簡単ないくつかの方法を説明することです。我々は詳細高価な特殊機器を必要とし、各方法の長所と短所を記述しないメソッドを。記載されている方法は、赤青メガネ、ミラーステレオスコープとプリズムのゴーグルの使用を含む。

Watch this Scientific Journal Video about How to Create and Use Binocular Rivalry at JoVE.com

How to Create and Use Binocular Rivalry

定期的に他のが抑制され、一方のイメージが支配し、優位性の交互：目が同じ位置に異なる画像が表示されているときに視野闘争が発生します。ライバルは、知覚的選択と視覚的な認知度を調査するのに便利です。ここでは、視野闘争刺激を作成して使用するためのいくつかの簡単な方法を説明します。

どのように視野闘争を作成および使用方法

Each of our eyes normally sees a slightly different image of the world around us. The brain can combine these two images into a single coherent ...

Research

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Neuroscience

75.0K ビュー.  New York University. 定期的に他のが抑制され、一方のイメージが支配し、優位性の交互：目が同じ位置に異なる画像が表示されているときに視野闘争が発生します。ライバルは、知覚的選択と視覚的な認知度を調査するのに便利です。ここでは、視野闘争刺激を作成して使用するためのいくつかの簡単な方法を説明します。

ビデオ: How to Create and Use Binocular Rivalry

How to Build a Laser Speckle Contrast Imaging (LSCI) System to Monitor Blood Flow

Laser Speckle Contrast Imaging (LSCI) is a simple yet powerful technique that is used for full-field imaging of blood flow. The technique analyzes fluctuations in a dynamic speckle pattern to detect the movement of particles similar to how laser Doppler analyzes frequency shifts to determine particle speed. Because it can be used to monitor the movement of red blood cells, LSCI has become a popular tool for measuring blood flow in tissues such as the retina, skin, and brain. It has become especially useful in neuroscience where blood flow changes during physiological events like functional activation, stroke, and spreading depolarization can be quantified. LSCI is also attractive because it provides excellent spatial and temporal resolution while using inexpensive instrumentation that can easily be combined with other imaging modalities. Here we show how to build a LSCI setup and demonstrate its ability to monitor blood flow changes in the brain during an animal experiment.

How to Build a Laser Speckle Contrast Imaging LSCI System to Monitor Blood Flow

This video demonstrates how to build a Laser Speckle Contrast Imaging (LSCI) system that can easily be used to monitor blood flow.

どのようにレーザーを構築するためには、血流を監視するためにコントラストイメージング（LSCI）システムのスペック

Laser Speckle Contrast Imaging (LSCI) is a simple yet powerful technique that is used for full-field imaging of blood flow.  The technique analyzes ...

神経科学

Isolation of Cerebrospinal Fluid from Rodent Embryos for use with Dissected Cerebral Cortical Explants

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF differentiates from the amniotic fluid upon closure of the anterior neural tube. CSF volume then increases over subsequent days as the neuroepithelial progenitor cells lining the ventricles and the choroid plexus generate CSF. The embryonic CSF contacts the apical, ventricular surface of the neural stem cells of the developing brain and spinal cord. CSF provides crucial fluid pressure for the expansion of the developing brain and distributes important growth promoting factors to neural progenitor cells in a temporally-specific manner. To investigate the function of the CSF, it is important to isolate pure samples of embryonic CSF without contamination from blood or the developing telencephalic tissue. Here, we describe a technique to isolate relatively pure samples of ventricular embryonic CSF that can be used for a wide range of experimental assays including mass spectrometry, protein electrophoresis, and cell and primary explant culture. We demonstrate how to dissect and culture cortical explants on porous polycarbonate membranes in order to grow developing cortical tissue with reduced volumes of media or CSF. With this method, experiments can be performed using CSF from varying ages or conditions to investigate the biological activity of the CSF proteome on target cells.

The ventricular cerebrospinal fluid (CSF) bathes the neuroepithelial and cerebral cortical progenitor cells during early brain development in the embryo. Here we describe the method developed to isolate ventricular CSF from rodent embryos of different ages in order to investigate its biological function. In addition, we demonstrate our cerebral cortical explant dissection and culture technique that allows for explant growth with minimal volumes of culture medium or CSF.

解剖大脳皮質植で使用するための齧歯類の胚から脳脊髄液の分離

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF ...

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.

We describe here the use of a pH-sensitive green fluorescent protein variant, pHluorin, to study the spatio-temporal dynamics of axon guidance receptors trafficking at the cell surface.&#160;The pHluorin-tagged receptor is expressed both in cell culture and in vivo, using electroporation of the chick embryo.

細胞培養およびひよこ胚における軸索誘導受容体のダイナミクス評価におけるpHluorinの使用

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and decreases waste in costs and reagents. Here we describe optimization of a multiplex assay to assess apoptosis following a palmitic acid (PA) challenge in an in vitro hypothalamic model, using both fluorescent and luminescent based assays to measure viable cell counts and caspase-3/7 activity in a 96-well microtiter plate format. Following PA challenge, viable cells were determined by a resazurin-based fluorescent assay. Caspase-3/7 activity was then determined using a luminogenic substrate, DEVD, and normalized to cell number. This multiplexing assay is a useful technique for determining change in caspase activity following an apoptotic stimulus, such as saturated fatty acid challenge. The saturated fatty acid PA can increase hypothalamic oxidative stress and apoptosis, indicating the potential importance of assays such as that described here in studying the relationship between saturated fatty acids and neuronal function.

Multiplex assays can provide beneficial information for basic cellular mechanisms&#160;and eliminate waste of reagents and unnecessary repetitive experiments. We describe here a multiplex caspase-3/7 activity assay, using fluorescent- and luminescent-based methods, to determine cell viability in an in vitro hypothalamic model following oxidative challenge with palmitic acid.

視床下部細胞モデルでアポトーシスを決定するために、カスパーゼの多重化アッセイの使用

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

胎児アルコール スペクトルの無秩序のモデルラットにおける海馬機能障害を評価するためにトレース瞬目反射条件づけの使用

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

How to Find Effects of Stimulus Processing on Event Related Brain Potentials of Close Others when Hyperscanning Partners

The partners of each pair must be able to pass the McGill Friendship Questionnaire without communicating. Each partner is then seated in front of a screen in one of two adjacent rooms. These rooms are separated by a glass window through which participants communicate to maintain feelings of togetherness while being fitted with the EEG cap. After checking for adequate EEG signals, the glass is covered by a curtain to prevent visual communication. Then, partners must be silent but are instructed to try to feel in the presence of their partner during the entire experiment. Just before it starts, participants are told that each of them will be presented with one image at a time and that these images will occur at the same time for both of them on their own screen. They are also instructed that, for each trial, the simultaneous images will always be different. However, unbeknownst to them, trials are randomized: only half of them are consistent with this instruction and actually include two different images. These trials form the DSC, that is, the different-stimuli condition. The other half of the trials are inconsistent with the instruction. They include two identical images and form the ISC (identical-stimuli condition). After the experiment, participants are sorted into two groups: those who reported that they felt in the presence of their partner during the majority of the trials and those who reported they did not. The impact of the stimulus processing of the partner is found by subtracting the mean voltages of the ERPs of the ISC (inconsistent with the instructions) from the ERPs of the DSC (consistent with the instructions) in at least two time windows (TWs): firstly, in the 75 to 150 ms TW, where the absolute values of these subtractions are greater, especially at right frontal sites, in those who felt in the presence of their partner than in those who did not; secondly, in the LPP time window (i.e., from 650 to 950 ms post onset), where ERPs are significantly less positive in the DSC than in the ISC in those in whom the raw results of the early (75-150ms) subtractions are negative.

This protocol describes key steps involved in assessing the sensitivity of the brain of one person to the stimulus processing of a close other by selecting pairs of partners, recording their electroencephalogram (EEG) simultaneously and computing their event-related brain potentials (ERPs).

検索効果の刺激の処理にイベント関連脳電位の近くに他の方法と Hyperscanning パートナー

The partners of each pair must be able to pass the McGill Friendship Questionnaire without communicating. Each partner is then seated in front of a screen ...

Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions in the immature and adult brain. Here we describe a zinc histochemical staining procedure that reveals differences in staining patterns among different layers and brain regions. Others have utilized this procedure not only to reveal the distribution of zinc-containing neurons and circuits in the brain, but also to successfully delineate areal and laminar boundaries in the developing and adult brain in several species. Here we illustrate this staining procedure with images from developing and adult ferret brains. We reveal a zinc-staining pattern that serves as an anatomical marker of areas and layers, and can be reliably used to distinguish visual cortical areas in the developing and adult visual cortex. The main goal of this protocol is to present a histochemical method that allows the accurate identification of layers and regions in the developing and adult brain where other methods fail to do so. Secondarily, in conjunction with densitometric image analysis, this method allows one to assess the distribution of synaptic zinc to reveal potential changes throughout development. This protocol describes in detail the reagents, tools, and steps necessary to successively stain frozen brain sections. Although this protocol is described using ferret brain tissue, it can easily be adapted for use in rodents, cats, or monkeys as well as in other brain regions.

We describe a histochemical procedure that reveals characteristic laminar and areal zinc staining patterns in different brain regions. The zinc-staining pattern may be used in conjunction with other anatomical markers to reliably distinguish layers and regions in the developing and adult brain.

さまざまな地域と発展途上と大人の脳のラミナを明らかにするシナプス亜鉛化学の使用

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions ...

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for nerve fibers in the central and peripheral nervous systems. This method is particularly applicable to dorsal root ganglia (DRG), which are primary sensory neurons that exhibit a pseudo-unipolar structure of nervous processes. The patterns and features of the action potentials passed along axons are recordable in these neurons. The present study uses in vivo single-fiber recordings to observe the conduction failure of sciatic nerves in complete Freund&#8217;s adjuvant (CFA)-treated rats. As the underlying mechanism cannot be studied using in vivo single-fiber recordings, patch-clamp-recordings of DRG neurons are performed on preparations of intact DRG with the attached sciatic nerve. These recordings reveal a positive correlation between conduction failure and the rising slope of the after-hyperpolarization potential (AHP) of DRG neurons in CFA-treated animals. The protocol for in vivo single fiber-recordings allows the classification of nerve fibers via the measurement of conduction velocity and monitoring of abnormal conditions in nerve fibers in certain diseases. Intact DRG with attached peripheral nerve allows observation of the activity of DRG neurons in most physiological conditions. Conclusively, single-fiber recording combined with electrophysiological recording of intact DRGs is an effective method to examine the role of conduction failure during the analgesic process.

Single-fiber recording is an effective electrophysiological technique that is applicable to the central and peripheral nervous systems. Along with the preparation of intact DRG with the attached sciatic nerve, the mechanism of conduction failure is examined. Both protocols improve the understanding of the peripheral nervous system&#39;s relationship with pain.

インビボシングルファイバー記録と無傷の後部根神経を用いて伝導障害のメカニズムを調べる

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for ...

Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model Parkinson&#39;s disease synucleinopathy and nigrostriatal degeneration. The standardization of &#945;-syn PFF generation and in vivo application is critical in order to ensure consistent, robust &#945;-syn pathology. Here, we present a detailed protocol for the generation of fibrils from monomeric &#945;-syn, post-fibrilization quality control steps, and suggested parameters for successful neurosurgical injection of &#945;-syn PFFs into rats or mice. Starting with monomeric &#945;-syn, fibrilization occurs over a 7-day incubation period while shaking at optimal buffer conditions, concentration, and temperature. Post-fibrilization quality control is assessed by the presence of pelletable fibrils via sedimentation assay, the formation of amyloid conformation in the fibrils with a thioflavin T assay, and electron microscopic visualization of the fibrils. Whereas successful validation using these assays is necessary for success, they are not sufficient to guarantee PFFs will seed &#945;-syn inclusions in neurons, as such aggregation activity of each PFF batch should be tested in cell culture or in pilot animal cohorts. Prior to use, PFFs must be sonicated under precisely standardized conditions, followed by examination using electron microscopy or dynamic light scattering to confirm fibril lengths are within optimal size range, with an average length of 50 nm. PFFs can then be added to cell culture media or used in animals. Pathology detectable by immunostaining for phosphorylated &#945;-syn (psyn; serine 129) is apparent days or weeks later in cell culture and rodent models, respectively.&#160;

The goal of this article is to outline the steps required for the generation of fibrils from monomeric alpha-synuclein, subsequent quality control, and use of the preformed fibrils in vivo.

モノマーからのαシヌクレイン予備型フィブリルの生成と生体内での使用

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model ...

How to Obtain Reliable Visual Event-related Potentials in Newborns

The present study discusses the characteristics of visual event-related potentials (VEPs) and outlines methodological steps for obtaining reliable measurements in newborns. Obtaining high-quality, reliable VEPs is crucial for the early detection of abnormal development of the central nervous system in at-risk newborns, and for implementing successful early interventions. Recommendations are based on a previous study which showed that when post-conceptional age, polysomnography-identified sleep stages, and light-emitting diodes (LEDs) googles as the luminous source are controlled, no more than 4 repetitions of VEP averages are required to obtain replicable recordings, variability decreases, and reliable VEPs can be obtained. By controlling for these sources of variability and using statistical analyses, we were able to clearly and reliably identify the amplitude and latency of three main components (NII, PII and NIII) present in 100% of newborns (n = 20) during active sleep. Recording VEPs during awake states, quiet sleep and transitional sleep is not recommended because VEP morphology may differ significantly from one average to the next, leading to the risk of misleading clinical prognoses. Moreover, it is easier to obtain VEPs during active sleep because this state can be clearly and reliably identified at this stage of development, sleep cycles are short enough to allow measurements to be taken in a reasonable time, and the method does not require new o expensive equipment.

Several important points for obtaining high-quality reliable visual evoked potentials (VEPs) in newborns while minimizing variability and the risk of misleading prognoses are presented.