著者は、優れた魚の飼育と技術サポートのため西夏羅に感謝したいと思います。この作品は、健康補助R21EY019401（RT）とP30EY04068（RT）の国立研究所によって資金を供給し、ウェイン州立大学、眼科部門に失明を防止するための研究から無制限の助成金を含め、RTに資金を立ち上げました。 JTは、ウェイン州立大学大学院で提供されるトマスC.ランブルフェローシップによってサポートされていました。

このプロトコルで説明されているすべての手順は、医学のウェイン州立大学で動物使用委員会によって承認された。 1。暗順応<ol><li>転送〜ダークエンクロージャに通常の住宅システムから10成体アルビノまたは着色された魚。可能であれば、タンクを通じて通常の水の流れを可能にゼブラフィッシュ住宅モジュールに組み込まれている暗い筐体を使用しています。 （そのようなシステムが利用できない場合には、酸素と魚を通気することを確認し、完全に暗いエンクロージャ内に水槽を配置）。 </li><li> 10日間暗闇の中で魚を保管してください。動物を餌または暗箱に新しい魚を追加する場合は、光の長い期間に魚をさらす避けるためにできるだけ早く移動してください。 </li></ol> 2。 UV露光<ol><li> UV源の電源がOFFになっていることを確認してください。蛍光実体顕微鏡からの紫外線フィラメントを取り外します。 <ol><li>反転を使用してくださいUVフィラメント用のスタンドとして直径15cmのガラスシャーレ（または類似した2センチメートルの高さのようなもの）。実験台にシャーレテープ。 </li><li>反転したペトリ皿の上部にテープ紫外線フィラメント。フィラメント張り出し月末〜2ミリメートルペトリ皿になるよう配置します。 </li></ol></li><li> 250mlのガラスビーカーを取得します。背面アルミホイルでビーカーのカバー下部の½、側面、そして、箔の &quot;光沢のある&quot;側がビーカーの内側に面していることを確認する。ビーカーを卒業されている場合は、露出したビーカーの明確な半分を残して、ホイルでビーカーの文言の半分をカバーしています。 </li><li>魚施設システムから100mlの水で250mlのビーカーを埋める。 </li><li> 4リットルのビーカーに250mlのビーカーを置きます。水位が250mlのビーカーに100mlの水ラインと偶数​​になるまで水を4リットルのビーカーを埋める。 </li><li> 250mlのビーカーに10暗処置動物の最大値を追加します。アルミニウムの小片で250mlのビーカーをカバー箔。 </li><li> UVフィラメントに隣接全体ビーカー装置を置きます。フィラメントは、4 Lのビーカーの外側に触れると250ミリリットルのビーカーの露出部分に直面しなければならない。 250mlのビーカーを4 Lのビーカーの底の中心にいることを確認してください。 </li><li>露出する動物のためにできるように、しかし、UVフィラメントの先端を見てから、任意のラボ要員を防止、4Lビーカー背後に大規模な、不透明な画面を配置します。警告：この障壁はUV源の電源を入れる前に所定の位置にあることを確認してください。 </li><li> UV電源をオンにします。 30分のタイマーを設定します。どんな必要な警告ラベルは、その無防備なラボの担当者が誤って紫外線に自分自身を公開しないように置きます。 </li><li> 30分後、UV電源を遮断する。魚と250mlのビーカーを取り外します。注：UV暴露の複数回が必要な場合、露出プロトコルを繰り返す前（〜20分）電源がクールダウンしましょう​​。 Tを交換することを確認してください彼は各露光のために新鮮なシステム水で100mlの水。 4リットルのビーカー中の水を交換する必要はない。 </li></ol> 3。ハロゲンランプ露光<ol><li> 1.8 L明確なアクリルの魚飼育用の水槽に250mlのビーカーから魚を移す。オーバーフローマークに給油して下さい。 </li><li>ハロゲンランプ光処理領域を設定する。地元の金物屋からの4つの250 Wのハロゲンランプユーティリティを入手します。同じ方向に2つのランプに面しており、中央には別に29センチメートルを手配。同様の方法で、他の2つのランプを配置します。 <ol><li>彼らは、ランプの2つのセットの間の内〜73センチメートルを残して、ランプの第1のセットを対向されるようにランプの第2のセットを配置する。これは29×73センチメートルの長方形状の光治療領域を作成します。 </li></ol></li><li>地元の金物屋から小さなファンを得る。ちょうど光処理領域外、ランプの2つのセットの間の等距離のファンを配置します。 </li><li>の中心に水の完全な2 1.8 Lタンクを置きランプの2つのセットの間の等距離光処理領域。ランプ、タンクの外壁の間の距離は〜29 cmであるべきである。注：のみ1 1.8 Lタンクに10魚を治療する場合でも、このような構成を使用しています。両方のタンクは適切な範囲内で、各タンクの水の温度を維持するために必要とされる。 </li><li>各タンク内の酸素エアレーターを置きます。 </li><li>ファンに最も近い端で1〜2センチのギャップを残し、透明アクリル蓋と各タンクをカバーしています。それがこの隙間に空気を爆破するようにファンを配置します。タンクの一方または両方に温度計を配置する。 </li><li>ライト、ファン、エアレーターの電源をオンにします。最大4日間の光治療を維持する。これは動物に多くのストレスの水質と結果に影響を与えるような魚は、光治療中に供給されるべきではありません。 </li><li>日常的に温度と水位を監視します。 30-33で温度を維持℃に必要に応じて、ファンの回転速度および/またはタンク及びリチウムの間の距離を調整するghts。 <ol><li>必要に応じて、通常の毎日のシステム水で各タンクトップオフ。 </li><li>常に100％近くの生存率を維持するために、健康な成人ゼブラフィッシュ（通常生後6-9ヶ月）を使用します。魚は水槽で死体で発見された場合しかし、それをすぐに削除し、全体のタンク内の水を交換してください。 </li></ol></li></ol> 4。組織採取<ol><li>ハロゲンライト治療の発症後48時間は、治療エリアから魚を取り外す。 <ol><li>新鮮なエタノールホルムアルデヒド固定液（1部37％ホルムアルデヒド、9部100％エタノール）を準備。 </li><li> 2 - フェノキシエタノールの麻酔過剰投与（0.4 mg / Lで）とゼブラフィッシュを安楽死させる。 </li><li>ペーパータオルに安楽死させたゼブラフィッシュを転送します。湾曲したピンセットを使って目を摘出。 </li><li> 4℃で固定し、店O / Nで除核の目を置き</li></ol></li><li>目をCryoprotect。 <ol><li>室温で30分間、5％スクロースを1×PBSで目を洗い、その後に置き換える2時間のために新鮮な5％蔗糖1X PBS。次に、4℃で30％のショ糖1X PBS O / Nで目を洗う4℃で組織凍結培地、30％ショ糖1X PBS O / Nの1:1（等分）で目を洗う</li></ol></li><li> -80℃で100％の組織凍結培地や店舗で目を埋め込む℃にオリエント組織の凍結切片を腹/背軸上で実行されるように、目。 </li><li>スライドガラス上の凍結切片組織と場所。 55℃で2時間のためのウォームスライド-80°Cまたは直後にストアスライドは、標準的な免疫組織化学を行う。 </li><li>蛍光顕微鏡40,51と切片の組織および画像上の標準的な免疫組織化学を実行します。 </li></ol>

著者らは、開示することは何もありません。

ここでは、広範な光受容体の損失や堅牢な再生レスポンスの継続明るい光露光結果と短い紫外線照射を組み合わせることを示している。個人LIRDの方法と比較して、この結合方法は、また、網膜の両方の半分にロッドとコーンの両方を損傷するための最も効果的なプロトコルです。重要なことに、この処置は、着色された動物と同様に、 アルビノ動物において効果的である。 <p class=...

光誘起網膜変性（LIRD）は、一般的にげっ歯類および損傷ロッドと錐体光受容体にゼブラフィッシュの両方で使用されています。成体ゼブラフィッシュでは、光受容体の変性は、細胞周期を再入力し、過渡増幅前駆細胞を生成するためにミュラーグリア細胞をトリガします。これらの前駆細胞は、彼らが最終的に新たな光受容体を生じさせる損傷領域への移行として増殖し続けています。現在、二広く使用されLIRDパラダイム、各々の再生応じて感光体の損失と対応する差異の程度の差が生じる。がある多くの遺伝的および薬理学的ツールは、再生時のその他の個々の遺伝子の役割をテストすることが可能であるので、堅牢なLIRDパラダイムを開発する必要がある。ここで我々は2以前に確立LIRD技術の使用を組み合わせているロッドとコーン光受容体の両方の広範かつ一貫性のある損失の結果そのLIRDプロトコルを記述します。さらにこのプロトコルはLIRD研究のためのアルビノ背景に関心のあるトランスジェニック系統を維持する必要がなくなり着色された動物における使用のために拡張することができる。

<table border="0" cellpadding="0" cellspacing="0" width="586">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UV light source</td>
			<td style="width:215px;">Leica</td>
			<td style="width:155px;">EL600</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass Petri dish (150 x 20 mm)</td>
			<td>Sigma-Aldrich/Pyrex</td>
			<td>CLS3160152BO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">250 ml glass beaker</td>
			<td>Sigma-Aldrich/Pyrex</td>
			<td>CLS1000250</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4 L glass beaker</td>
			<td>Sigma-Aldrich/Pyrex</td>
			<td>CLS10004L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluminum foil</td>
			<td>Fisher</td>
			<td>01-213-105</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">250 W halogen lamps</td>
			<td>Workforce</td>
			<td>265-669</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.8 L clear acrylic tanks</td>
			<td>Aquaneering</td>
			<td>ZT180T</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.8 L clear acrylic tank lids</td>
			<td>Aquaneering</td>
			<td>ZT180LCL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fan</td>
			<td>Honeywell</td>
			<td>HT-900</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aerator</td>
			<td>Tetra</td>
			<td>77853-900</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermometer</td>
			<td>Cole-Parmer</td>
			<td>YO-08008-58</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bent forceps (5/45)</td>
			<td>World Precision Instruments</td>
			<td>504155</td>
		</tr>
	</tbody>
</table>

a novel light damage paradigm for use in retinal regeneration studies in adult zebrafish

光誘起網膜変性（LIRD）は、一般的にげっ歯類および損傷ロッドと錐体光受容体にゼブラフィッシュの両方で使用されています。成体ゼブラフィッシュでは、光受容体の変性は、細胞周期を再入力し、過渡増幅前駆細胞を生成するためにミュラーグリア細胞をトリガします。これらの前駆細胞は、彼らが最終的に新たな光受容体を生じさせる損傷領域への移行として増殖し続けています。現在、二広く使用されLIRDパラダイム、各々の再生応じて感光体の損失と対応する差異の程度の差が生じる。がある多くの遺伝的および薬理学的ツールは、再生時のその他の個々の遺伝子の役割をテストすることが可能であるので、堅牢なLIRDパラダイムを開発する必要がある。ここで我々は2以前に確立LIRD技術の使用を組み合わせているロッドとコーン光受容体の両方の広範かつ一貫性のある損失の結果そのLIRDプロトコルを記述します。さらにこのプロトコルはLIRD研究のためのアルビノ背景に関心のあるトランスジェニック系統を維持する必要がなくなり着色された動物における使用のために拡張することができる。

上述光治療プロトコルはLIRDの個々の方法と比較した。暗い処置成体動物のアルビノ （ 図3-5）において、個々の光治療が重要なロッドの損失（ 図3）とコーン（ 図4）感光体が得られた。ただし、両方の個々の治療は、主に比較的軽い治療（ 図3及び図4）から保護腹側網膜を残して、網膜の背側半分に感光体が損傷してい?...

Watch this Scientific Journal Video about A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish at JoVE.com

A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish

多重光損傷プロトコルが損傷光受容体に記述され、その結果、成体ゼブラフィッシュ網膜再生の応答を誘導しています。このプロトコルは、着色された動物は、その損害賠償の全体にわたる網膜桿体と錐体光受容体の大部分を使用することができる改良された方法を説明します。

大人のゼブラフィッシュにおける網膜再生研究で使用するための新規な光ダメージパラダイム

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, ...

a-novel-light-damage-paradigm-for-use-retinal-regeneration-studies

Research

JoVE Journal

Neuroscience

10.1K ビュー.  Wayne State University School of Medicine. 多重光損傷プロトコルが損傷光受容体に記述され、その結果、成体ゼブラフィッシュ網膜再生の応答を誘導しています。このプロトコルは、着色された動物は、その損害賠償の全体にわたる網膜桿体と錐体光受容体の大部分を使用することができる改良された方法を説明します。 

ビデオ: A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

げっ歯類におけるランニングホイール活動の概日リズムの記録および分析

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

神経科学

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

蛍光色素で大人のゼブラフィッシュの網膜神経節細胞の逆行性標識

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.
In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

ゼブラフィッシュ成体終脳の刺し傷傷害：脊椎動物の脳神経発生·再生を調査する方法

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

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 ...

Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to synaptic degeneration, inflammation, neuronal death, and learning deficits. Humans cannot regenerate lost neurons in the case of AD in part due to impaired proliferative capacity of the neural stem/progenitor cells (NSPCs) and reduced neurogenesis. Therefore, efficient regenerative therapies should also enhance the proliferation and neurogenic capacity of NSPCs. Zebrafish (Danio rerio) is a regenerative organism, and we can learn the basic molecular programs with which we could design therapeutic approaches to tackle AD. For this reason, the generation of an AD-like model in zebrafish was necessary. Using our methodology, we can introduce synthetic derivatives of A&#946;42 peptide with tissue penetrating capability into the adult zebrafish brain, and analyze the disease pathology and the regenerative response. The advantage over the existing methods or animal models is that zebrafish can teach us how a vertebrate brain can naturally regenerate, and thus help us to treat human neurodegenerative diseases better by targeting endogenous NSPCs. Therefore, the amyloid-toxicity model established in the adult zebrafish brain may open new avenues for research in the field of neuroscience and clinical medicine. Additionally, the simple execution of this method allows for cost-effective and efficient experimental assessment. This manuscript describes the synthesis and injection of A&#946;42 peptides into zebrafish brain.

Modeling Amyloid-&amp;#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

This protocol describes the synthesis, characterization, and injection of monomeric amyloid-&#946;42 peptides for generating amyloid toxicity in adult zebrafish to establish an Alzheimer&#39;s disease model, followed by histological analyses and detection of aggregations.

Β42 アミロイド毒性と大人のゼブラフィッシュ脳の神経変性をモデリング

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to ...

M&#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after severe damage. Investigating the regeneration pathways in zebrafish might bring new insights to develop innovative strategies for the treatment of retinal degenerative diseases in mammals. Herein, we focused on the induction of a focal lesion to the outer retina in adult zebrafish by means of a 532 nm diode laser. A localized injury allows investigating biological processes that take place during retinal degeneration and regeneration directly at the area of damage. Using non-invasive optical coherence tomography (OCT), we were able to define the location of the damaged area and monitor subsequent regeneration in vivo. Indeed, OCT imaging produces high-resolution, cross-sectional images of the zebrafish retina, providing information which was previously only available with histological analyses. In order to confirm the data from real-time OCT, histological sections were performed and regenerative response after the induction of the retinal injury was investigated by immunohistochemistry.

M&amp;#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

The zebrafish is a popular animal model to study mechanisms of retinal degeneration/regeneration in vertebrates. This protocol describes a method to induce localized injury disrupting the outer retina with minimal damage to the inner retina. Subsequently, we monitor in vivo the retinal morphology and the M&#252;ller glia response throughout retinal regeneration.

ゼブラフィッシュの再生モデルと網膜変性のレーザー誘起のミュラー グリア細胞の活性化

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after ...

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding diseases, and future therapies hinge on fundamental understandings about how different retinal cells function normally. Gaining such information with biochemical methods has proven difficult because contributions of particular cell types are diminished in the retinal cell milieu. Live retinal imaging can provide a view of numerous biological processes on a subcellular level, thanks to a growing number of genetically encoded fluorescent biosensors. However, this technique has thus far been limited to tadpoles and zebrafish larvae, the outermost retinal layers of isolated retinas, or lower resolution imaging of retinas in live animals. Here we present a method for generating live ex vivo retinal slices from adult zebrafish for live imaging via confocal microscopy. This preparation yields transverse slices with all retinal layers and most cell types visible for performing confocal imaging experiments using perfusion. Transgenic zebrafish expressing fluorescent proteins or biosensors in specific retinal cell types or organelles are used to extract single-cell information from an intact retina. Additionally, retinal slices can be loaded with fluorescent indicator dyes, adding to the method&#39;s versatility. This protocol was developed for imaging Ca2+ within zebrafish cone photoreceptors, but with proper markers it could be adapted to measure Ca2+ or metabolites in M&#252;ller cells, bipolar and horizontal cells, microglia, amacrine cells, or retinal ganglion cells. The retinal pigment epithelium is removed from slices so this method is not suitable for studying that cell type. With practice, it is possible to generate serial slices from one animal for multiple experiments. This adaptable technique provides a powerful tool for answering many questions about retinal cell biology, Ca2+, and energy homeostasis.

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

Imaging retinal tissue can provide single-cell information that cannot be gathered from traditional biochemical methods. This protocol describes preparation of retinal slices from zebrafish for confocal imaging. Fluorescent genetically encoded sensors or indicator dyes allow visualization of numerous biological processes in distinct retinal cell types.

Ex Vivoイメージング実験アダルト ゼブラフィッシュから準備新鮮な網膜スライス

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding ...

Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ablation of neurons is an effective method for this purpose when neurons are selectively labeled with fluorescent probes. In the present study, protocols for laser ablating a subpopulation of neurons using a two-photon microscope and testing of its functional and behavioral consequences are described. In this study, prey capture behavior in zebrafish larvae is used as a study model. The pretecto-hypothalamic circuit is known to underlie this visually-driven prey catching behavior. Zebrafish pretectum were laser-ablated, and neuronal activity in the inferior lobe of the hypothalamus (ILH; the target of the pretectal projection) was examined. Prey capture behavior after pretectal ablation was also tested.

Here, we present a protocol to ablate a genetically labeled subpopulation of neurons by a two-photon laser from Zebrafish larvae.

二光子励起レーザーとゼブラフィッシュ幼虫におけるカルシウム イメージング法と行動記録を用いた評価を用いたニューロン集団のアブレーション

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ...

A Novel In Vitro Model of Blast Traumatic Brain Injury

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the detonation of explosive devices, however, the mechanisms that underlie the brain damage resulting from blast overpressure exposure are not entirely understood and are believed to be unique to this type of brain injury. Preclinical models are crucial tools that contribute to better understand blast-induced brain injury. A novel in vitro blast TBI model was developed using an open-ended shock tube to simulate real-life open-field blast waves modelled by the Friedlander waveform. C57BL/6N mouse organotypic hippocampal slice cultures were exposed to single shock waves and the development of injury was characterized up to 72 h using propidium iodide, a well-established fluorescent marker of cell damage that only penetrates cells with compromised cellular membranes. Propidium iodide fluorescence was significantly higher in the slices exposed to a blast wave when compared with sham slices throughout the duration of the protocol. The brain tissue injury is very reproducible and proportional to the peak overpressure of the shock wave applied.

A Novel In Vitro Model of Blast Traumatic Brain Injury

This paper describes a novel model of primary blast traumatic brain injury. A compressed-air driven shock tube is used to expose in vitro mouse hippocampal slice cultures to a single shock wave. This is a simple and rapid protocol generating a reproducible brain tissue injury with a high throughput.

爆発外傷性脳損傷の新規In Vitroモデル

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the ...

An Emerging Target Paradigm to Evoke Fast Visuomotor Responses on Human Upper Limb Muscles

To reach towards a seen object, visual information has to be transformed into motor commands. Visual information such as the object&#8217;s color, shape, and size are processed and integrated within numerous brain areas, then ultimately relayed to the motor periphery. In some instances, a reaction is needed as fast as possible. These fast visuomotor transformations, and their underlying neurological substrates, are poorly understood in humans as they have lacked a reliable biomarker. Stimulus-locked responses (SLRs) are short latency (&#60;100 ms) bursts of electromyographic (EMG) activity representing the first wave of muscle recruitment influenced by visual stimulus presentation. SLRs provide a quantifiable output of rapid visuomotor transformations, but SLRs have not been consistently observed in all subjects in past studies. Here we describe a new, behavioral paradigm featuring the sudden emergence of a moving target below an obstacle that consistently evokes robust SLRs. Human participants generated visually guided reaches toward or away from the emerging target using a robotic manipulandum while surface electrodes recorded EMG activity from the pectoralis major muscle. In comparison to previous studies that investigated SLRs using static stimuli, the SLRs evoked with this emerging target paradigm were larger, evolved earlier, and were present in all participants. Reach reaction times (RTs) were also expedited in the emerging target paradigm. This paradigm affords numerous opportunities for modification that could permit systematic study of the impact of various sensory, cognitive, and motor manipulations on fast visuomotor responses. Overall, our results demonstrate that an emerging target paradigm is capable of consistently and robustly evoking activity within a fast visuomotor system.

Presented here is a behavioral paradigm that elicits robust fast visuomotor responses on human upper limb muscles during visually guided reaches.

人間の上肢筋肉に対する高速ビスオモター応答を呼び起こす新たな標的パラダイム

To reach towards a seen object, visual information has to be transformed into motor commands. Visual information such as the object&#8217;s color, shape, ...