1）準備<ol><li>コンピュータ画面の前で快適な椅子で座件名、開始するには。 </li><li>画面と被写体の[後]頭骨が60 cmであるとの距離を確保するために巻き尺を使用してください。 </li><li>最後に、被験者の目の上に遮光マスクを配置します。 </li></ol> 2）V1/V2上の閃光閾値を決定する<ol><li>後でこのプロトコルでphosphenic行動を探索する際に被験者の眼内閃光閾値を知ることが重要となる。このしきい値を決定するために、最初の70％の電源にTMSマシンを設定します。 </li><li>コイルを保持するので、8の字の顔、頭蓋正中線の上。 </li><li>イニオン上記3 cm程度の時点で、シングルパルス刺激を始める。 </li><li>各パルスの後、どんなphosphenic経験を報告する対象を求める。 </li><li>この領域の周囲に小さな検索グリッド（V1/V2）でコイルを動かし始める。 </li><li>刺激は被験者から一貫して明白な閃光レポートを誘発する場所を決めます。それは、TMSは、人口の約40％で閃光を引き出すためにできなくなることが理論化されているので、被験者は何も報告しない可能性があります。このような場合は、残念ながら、この実験では動作しません。被験者は、これを退去を許可する必要があります。 </li><li> phosphenic&quot;ホットスポット&quot;が特定されると、正確に3 6連続パルスのための主題レポートの閃光まで上下TMSの電力出力を調整します。この電力レベルは、サブジェクトのV1/V2しきい値です。利用可能な場合、neuronavigationは検索およびV1/V2ホットスポットをターゲットとする場合に大きな空間的な精度を達成するために使用することができます。 </li></ol> 3）V5/MT +コンプレックスにわたってPhosphenicしきい値の決定<ol><li>同じ前述の手順とパラメータを使用して、我々は今V5/MT以上閃光閾値+複雑なを決定します。この領域を見つけるために、被験者のイニオンに横方向のポイント3センチ背が5 cmの開始。 </li><li>各パルスの後、どんなphosphenic経験を報告する対象を求める。 </li><li>あいまいで一貫性の閃光が誘発されるまで、再び、単一パルスを使用して、小規模な検索のグリッドパターンを開始します。 </li><li>最後に、正確に3 6連続パルスのための主題レポートの閃光まで上下TMSの電力出力を調整します。この電力レベルは、サブジェクトのV5/MT +複雑なしきい値です。再び、可能な場合、neuronavigationはV5/MT +ホットスポットを配置し、ターゲットとする場合に大きな空間的な精度を達成するために使用することができます。 </li></ol> 4）ベースラインの閃光の動作を決定<ol><li>閃光閾値は、両方の視覚野のために決定されると、我々はベースラインphosphenic挙動を測定する必要があります。これを行うには、第一の主題の遮光マスクを削除します。 </li><li>次に、60秒のためのコンピュータの画面の中央に表示される安定した固定のクロスを凝視されることを指示する。 </li><li>マスクを置き換えると、V1/V2のホットスポットを介して、120％で3秒間の単一パルストレインV1/V2しきい値を生成します。 </li><li>五秒間待ってから、別の列車を行う。 </li><li>再び、五秒待ってから、サード列車を行う。 </li><li>サードシングルパルストレインの後、任意の誘発閃光の場所と動機の特性を記述するために主題を求める。これはベースラインとなります。 </li><li> V5/MT +ホットスポット（V5/MTの120％にTMS電源をリセットすることを忘れないでください+しきい値）以上この同じ手順を繰り返します。 </li></ol> 5）条件ナンバーワン<ol><li> phophenicベースラインが決定されると、再び主題の遮光マスクを削除します。 </li><li> 60秒のためのコンピュータの画面の中央に表示される安定した固定のクロスを凝視されることを指示する。この時間は、むしろ空白の画面ではなく、我々はクロスの周りは、いくつかの場所で移動する一連のドットを埋め込むことになります。 <img src="/files/ftp_upload/2273/2273fig1.jpg" alt="図1" /> 図1条件いずれかに刺激を適応：単純な並進運動。すべてのドットは左または右にコヒーレントに移動。 すべてのドットが同じ方向に動いていることを確認してください。この刺激が視覚的な適応を生むのに役立つはずです。刺激に長時間暴露によって誘発されるようになる神経興奮性が変化する現象は、バイアスに続いて提示刺激の知覚を提供しています。 </li><li> 60秒後、マスクを置き換えると、V1/V2のホットスポットの上に、120％で3秒間の単一のパルストレインV1/V2しきい値を生成します。 </li><li>五秒間待ってから、さらに2回繰り返す。 </li><li> three列車の後に、任意の誘発閃光の場所と動機の特性を報告するために主題を求める。利用可能な場合は、暗い部屋で目の動きトラッキングシステムの使用は、より高い精度と閃光のelicitations中の被験者による眼球運動の定量的なモニタリングを実現することができます。 </li><li> phospまでこのthree-train/reportシーケンスを継続するHeNeレーザの動作は、ベースラインのアクティビティに戻ります。 </li><li> V5/MT +複雑なホットスポットのためにこの手順を繰り返します。 </li></ol> 6）コンディションナンバートゥー<ol><li>この二番目の条件では、条件いずれかに利用したのと同じ手順を繰り返します。 &quot;スターバースト&quot;のパターンに似て - この時間は、しかし、むしろ特異な方向に移動する一連のドットで主題を提示するよりも、それぞれが離れて中心点からの相対的な基本的な方向に移動する一連のドットで主題を提示。 <img src="/files/ftp_upload/2273/2273fig2.jpg" alt="図2" /> 図2条件2のために刺激を適応：。放射状の動き。ドットは、どちらに向かってまたは離れて中央の点から、それぞれの枢機卿の方向に移動する。 </li><li>前と同様に、60秒後、V1/V2分の3以上、120％しきい値の列車のシリーズを行って、マスクを交換し、被験者の報告を求め、そしてベースラインが戻るまで続ける。 </li><li> V5/MT +のために繰り返します。 </li></ol> 7）図<ol><li>オプションとして、各条件の後に（またはすべての条件がすべて完了した後）、グラフ上に地域とphopsphenesの各シリーズの動機の動作を描画する対象を求める。これは不可欠なステップではないが、解釈、データの別のセットをご提供します。 </li></ol> 8）代表の結果単一方向の動機の刺激への視覚的な適応は、V1/V2を介して同一のphosphenic動きを引き出す必要があります。 <img src="/files/ftp_upload/2273/2273fig3.jpg" alt="図3" /> 図3。条件最中、右と左半球（被験者の図面に基づいて）で、V1/V2から誘導される閃光の例。 V5/MT +複雑な閃光はまた、閃光が現在適応刺激の運動方向の和とベースラインの閃光のように表示されるように、影響を受ける可能性があります。 しかし、スターバーストパターンの視覚的な適応はV5/MT +複合体から同一のスターバーストphosphenicの動きを引き出す必要がありますが、V1/V2ベースラインを変更しないでください。 <img src="/files/ftp_upload/2273/2273fig4.jpg" alt="図4" /> 図4。V5/MTから誘導される閃光の例+条件2（被験者の図面に基づいて）中の右と左半球に複雑。

<ol>
	<li>Pascual-Leone, A., Davey, M., Wassermann, E. M., Rothwell, J., Puri, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Transcranial Magnetic Stimulation. , (2002).</li><li>Walsh, V., Pascual-Leone, 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> Transcranial Magnetic Stimulation: A Neurochronometrics of Mind. , (2005).</li><li>Silvanto, J., Muggleton, N. G., Cowey, A., Walsh, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+adaptation+reveals+state-dependent+effects+of+transcranial+magnetic+stimulation.">Neural adaptation reveals state-dependent effects of transcranial magnetic stimulation.</a> European Journal of Neuroscience. 25, 1874-1881 (2007).</li><li>Silvanto, J., Pascual-Leone, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=State-Dependency+of+Transcranial+Magnetic+Stimulation.">State-Dependency of Transcranial Magnetic Stimulation.</a> Brain Topography. 21, 1-10 (2008).</li><li>Silvanto, J., Cattaneo, Z., Battelli, L., Pascual-Leone, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baseline+cortical+excitablility+determines+whether+TMS+disrupts+or+facilitates+behavior.">Baseline cortical excitablility determines whether TMS disrupts or facilitates behavior.</a> Journal of Neurophysiology. 99, 2725-2730 (2008).</li><li>Silvanto, J., Muggleton, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testing+the+validity+of+the+TMS+state-dependency+approach:+targeting+functionally+distinct+motion-selective+neural+populations.">Testing the validity of the TMS state-dependency approach: targeting functionally distinct motion-selective neural populations.</a> Neuroimage. 40, 1841-1848 (2008).</li><li>Tootell, R. B., Rappas, J. B., Kwong, K. K., Malach, R., Born, R. T., Brady, T. J., Rosen, B. R., Belliveau, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+human+MT+and+related+visual+cortical+areas+using+magnetic+resonance+imaging.">Functional analysis of human MT and related visual cortical areas using magnetic resonance imaging.</a> Journal of Neuroscience. 15, 3215-3230 (1995).</li><li>Singh, K. D., Smith, A. T., Greenlee, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+frequency+and+direction+sensitivities+of+human+visual+areas+measured+using+fMRI.">Spatiotemporal frequency and direction sensitivities of human visual areas measured using fMRI.</a> Neuroimage. 12, 550-564 (2000).</li><li>Rutchmann, R. M., Schrauf, M., Greenlee, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+activation+during+dichoptic+presentation+of+optic+flow+stimuli.">Brain activation during dichoptic presentation of optic flow stimuli.</a> Exp Brain Res. 134, 533-537 (2000).</li><li>Morrone, M. C., Tosetti, M., Montanaro, D., Fiorentini, A., Cioni, G., Burr, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cortical+area+that+responds+specifically+to+optic+flow+revealed+by+fMRI.">A cortical area that responds specifically to optic flow revealed by fMRI.</a> Nat. Neuroscience. 3, 1322-1328 (2000).</li></ol>

利害の衝突は宣言されません。

この実験では、状態依存の中心で取得します。 。V1/V2でのニューロンは、単純な、方向性に対応すると考えられているため7,8、単方向動機の刺激への適応は、この動きを登録するニューロンの興奮性を増加した-などとして、彼らは、TMSに対応する最初のはずですパルス。 V5/MT +複雑なもので、この領域から誘導される閃光にも適応刺激によって影響されるか、単純な並進運動に同調?...

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<td>Light Blocking Eye Mask</td>
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<td>Ear Plugs</td>
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<td>Tape Measure</td>
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<td>Blank Graph Paper</td>
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<td>Stimuli Developed &amp; Presented on Computer using Adobe Photoshop</td>
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<td>Any Single Pulse Capable TMS Device</td>
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state-dependency effects on tms:  a look at motive phosphene behavior

経頭蓋磁気刺激（TMS）は、一過性または不変にローカライズされた磁場パルスのアプリケーションを介して皮質の興奮性を（増加またはそれを下げるのどちらか）を変調できる非侵襲的neurostimulatoryと神経調節技術です。TMSのフィールド内では1,2、長期的状態依存性は、刺激を対象とした特定の神経領域の初期、ベースライン状態を指す。推測することができるように、TMSの効果は、（観光）ターゲット皮質領域のこの主要な感受性と反応性に応じて変えることができます。3,4,5 この実験では、動機の閃光の誘発と主観的な経験を通して状態の依存関係のこの概念を検討する。閃光は、視覚的に視覚野への電磁パルスによって引き起こされる小さな光の点滅を認識されている。これらの小さなライトは、視覚野の種類が刺激されているかに応じて様々な特性を想定することができます。 V1/V2の刺激とV5/MT +複雑な視覚的な領域を介して惹起さとしてこの特定の研究では、動機の閃光を目標とする。6

Watch this Scientific Journal Video about State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior at JoVE.com

State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

この記事では、我々は、動機のphosphenicプレゼンテーションを誘導TMSで視覚的に関連する状態依存性の影響を調べる。

TMSの状態 - 依存性の効果：動機の閃光の挙動を見

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate ...

state-dependency-effects-on-tms-a-look-at-motive-phosphene-behavior

Research

JoVE Journal

Neuroscience

10.5K ビュー.  Beth Israel Deaconess Medical Center. この記事では、我々は、動機のphosphenicプレゼンテーションを誘導TMSで視覚的に関連する状態依存性の影響を調べる。

ビデオ: State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

NeuroStar TMSデバイス：うつ病の治療薬としてFDA承認されたプロトコルを実施

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

神経科学

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank's Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1&deg;C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Here, we describe a method for efficient cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. This simple protocol provides flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

高濃縮ニューロン培養の世代のための皮質組織ブロックの凍結保存

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly ...

Studying the Integration of Adult-born Neurons

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the hippocampal dentate gyrus throughout life. Previous reports have shown that adult hippocampal neurogenesis is associated with diverse brain disorders, including epilepsy, schizophrenia, depression and anxiety (1). Deciphering the process of normal and aberrant adult-born neuron integration may shed light on the etiology of these diseases and inform the development of new therapies.
SGZ adult neurogenesis mirrors embryonic and post-natal neuronal development, including stages of fate specification, migration, synaptic integration, and maturation. However, full integration occurs over a prolonged, 6-week period. Initial synaptic input to adult-born SGZ dentate granule cells (DGCs) is GABAergic, followed by glutamatergic input at 14 days (2). The specific factors which regulate circuit formation of adult-born neurons in the dentate gyrus are currently unknown.
Our laboratory uses a replication-deficient retroviral vector based on the Moloney murine leukemia virus to deliver fluorescent proteins and hypothesized regulatory genes to these proliferating cells. This viral technique provides high specificity and resolution for analysis of cell birth date, lineage, morphology, and synaptogenesis.
A typical experiment often employs two or three viruses containing unique label, transgene, and promoter elements for single-cell analysis of a desired developmental process in vivo. The following protocol describes a method for analyzing functional newborn neuron integration using a single green (GFP) or red (dTomato) fluorescent protein retrovirus and patch-clamp electrophysiology.

A way to study the integration of newborn dentate granule cells in adult animals is described. This technique uses an engineered retrovirus to label newborn neurons, followed by electrophysiological recordings to determine in vivo functional integration.

アダルト生まれのニューロンの統合を検討し

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the ...

Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological impairments 1. Two pervasive and disabling symptoms experienced by TBI survivors, memory deficits and a reduction in seizure threshold, are thought to be mediated by TBI-induced hippocampal dysfunction 2,3. In order to demonstrate how altered hippocampal circuit function adversely affects behavior after TBI in mice, we employ lateral fluid percussion injury, a commonly used animal model of TBI that recreates many features of human TBI including neuronal cell loss, gliosis, and ionic perturbation 4-6.
	Here we demonstrate a combinatorial method for investigating TBI-induced hippocampal dysfunction. Our approach incorporates multiple ex vivo physiological techniques together with animal behavior and biochemical analysis, in order to analyze post-TBI changes in the hippocampus. We begin with the experimental injury paradigm along with behavioral analysis to assess cognitive disability following TBI. Next, we feature three distinct ex vivo recording techniques: extracellular field potential recording, visualized whole-cell patch-clamping, and voltage sensitive dye recording. Finally, we demonstrate a method for regionally dissecting subregions of the hippocampus that can be useful for detailed analysis of neurochemical and metabolic alterations post-TBI.
	These methods have been used to examine the alterations in hippocampal circuitry following TBI and to probe the opposing changes in network circuit function that occur in the dentate gyrus and CA1 subregions of the hippocampus (see Figure 1). The ability to analyze the post-TBI changes in each subregion is essential to understanding the underlying mechanisms contributing to TBI-induced behavioral and cognitive deficits. 
	The multi-faceted system outlined here allows investigators to push past characterization of phenomenology induced by a disease state (in this case TBI) and determine the mechanisms responsible for the observed pathology associated with TBI.

A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.

軽度の外傷性脳損傷後の海馬回路機能の改変に関する調査

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

縦断のために脊髄商工を移植するための手順<em&gt;インビボ</emマウス脊髄の&gt;イメージング

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

網膜リボンシナプスにおける膜受容体の高分解能定量的イムノゴールド分析

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

勉強するリニアアガロースチャネルを使用します<em&gt;ショウジョウバエ</em&gt;幼虫クロールの動作

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

オプトジェネティクスを組み合わせてそのまま神経回路内でのシナプス前機能に関する遺伝子ノックダウンの効果を特徴付ける人工マイクロ Rna

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

FM Dye Cycling at the Synapse: Comparing High Potassium Depolarization, Electrical and Channelrhodopsin Stimulation

FM dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them water-soluble with the ability to reversibly enter and exit membrane lipid bilayers. These styryl dyes are relatively non-fluorescent in aqueous medium, but insertion into the outer leaflet of the plasma membrane causes a &#62;40X increase in fluorescence. In neuronal synapses, FM dyes are internalized during SV endocytosis, trafficked both within and between SV pools, and released with SV exocytosis, providing a powerful tool to visualize presynaptic stages of neurotransmission. A primary genetic model of glutamatergic synapse development and function is the Drosophila neuromuscular junction (NMJ), where FM dye imaging has been used extensively to quantify SV dynamics in a wide range of mutant conditions. The NMJ synaptic terminal is easily accessible, with a beautiful array of large synaptic boutons ideal for imaging applications. Here, we compare and contrast the three ways to stimulate the Drosophila NMJ to drive activity-dependent FM1-43 dye uptake/release: 1) bath application of high [K+] to depolarize neuromuscular tissues, 2) suction electrode motor nerve stimulation to depolarize the presynaptic nerve terminal, and 3) targeted transgenic expression of channelrhodopsin variants for light-stimulated, spatial control of depolarization. Each of these methods has benefits and disadvantages for the study of genetic mutation effects on the SV cycle at the Drosophila NMJ. We will discuss these advantages and disadvantages to assist the selection of the stimulation approach, together with the methodologies specific to each strategy. In addition to fluorescent imaging, FM dyes can be photoconverted to electron-dense signals visualized using transmission electron microscopy (TEM) to study SV cycle mechanisms at an ultrastructural level. We provide the comparisons of confocal and electron microscopy imaging from the different methods of Drosophila NMJ stimulation, to help guide the selection of future experimental paradigms.

Synaptic vesicle (SV) cycling is the core mechanism of intercellular communication at neuronal synapses. FM dye uptake and release are the primary means of quantitatively assaying SV endo- and exocytosis. Here, we compare all the stimulation methods to drive FM1-43 cycling at the Drosophila neuromuscular junction (NMJ) model synapse.

シナプスでサイクリング FM 色素: 高カリウムの脱分極、電気とチャネルロドプシン刺激の比較

FM dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

加圧齧歯動物に及ぼす影響の爆発による頭部外傷の中間の大脳動脈

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...