We thank Souvik Maiti for help in designing the antagomirs. We also acknowledge Rangeetha J. Naik, Rakesh Dey, and Bijay Pattnaik for their help with experimental methods. This work was funded by the Council of Scientific and Industrial Research (BSC0123). HS, MV and RR acknowledge fellowship from the Council of Scientific and Industrial Research, India. MAS acknowledge fellowship from the University Grants Commission, India.

注意：動物被験体を含むすべての手順は、ゲノミクス研究所と統合生物学における制度的動物倫理委員会（IAEC）、ニューデリー（IGIB / AEC / 2013分の10）によって承認されています。このプロトコルは、具体的のmiR-29の脳とノックダウンでアンタゴ-29の標的化送達のために調整されています。 1.アンタゴデザイン戦略<ol><li> miRBase 11から成熟miRNA配列を取得する<a href="http://www.mirbase.org/">（http://www.mirbase.org/）。</a> </li><li> miRBaseレコードに「遺伝子ファミリー」リンクを使用して、関連するmiRNAファミリーメンバーの配列を取得します。 <a href="http://www.mirbase.org/cgi-bin/mirna_summary.pl?fam=MIPF0000009">（http://www.mirbase.org/cgi-bin/mirna_summary.pl?fam=MIPF0000009）。</a> </li><li>逆シーケンスを補完<a href="http://www.bioinformatics.org/sms/rev_comp.html">：http://www.bioinformatics.org/sms/rev_comp.htmlは</a>に便利なツールを提供します相補配列を逆にします。 </li><li>次のガイドラインを使用して、LNAによって変更される位置をマーク： <ol><li> LNAのピリミジンがプリン12よりも安定であるため、LNA修飾のために離れて3-4拠点を置いチミン残基を選択してください。 </li><li>前の手順が5つ未満の変更を生成する場合3-4残基によって分離されたシトシン残基を選択してください。 </li><li>これはシード配列を回避するため、アンタゴの5 &#39;末端に5変更に優先順位を付けます。 </li></ol></li></ol> 2.アンタゴ-神経ペプチド複合体の準備注：これは、プロトコルの中で最も重要なステップであり、それは標準化が必要です。プロトコルを標準化するために、任意の蛍光標識されたオリゴヌクレオチド（FLO）は、アンタゴmir 9の代わりに使用することができます。ペプチドは、高純度であるべきである（HPLCグレード、98％純度）.Sequencesペプチドおよびアンタゴの電荷を表1に示します。 <ol><li> MOの数を決定しますレアンタゴのマウスのアンタゴ、その毒性と重量のモル電荷に基づいて注入されます。ペプチド：アンタゴmirのモル電荷比に基づいて注入されるペプチドのモル数を計算します。 注：アンタゴのモル電荷比を標準化：毒性およびマウス脳における効果的な送達のためのペプチド。標準化のためのペプチド：蛍光標識オリゴヌクレオチドの異なるモル電荷比を使用してください。私たちは、アンタゴmir-29とコントロールがマウスの4microgram /グラムの体重では毒性の両方であることがわかりました。アンタゴmir-29および制御2microgram /グラム体重で使用したが、この効果的な濃度は、各マイクロRNAについて変化する可能性があります。 </li><li>ステップ1で計算されるような所望の最終濃度に滅菌10％のD-グルコースとストック溶液とは別のマイクロチューブ中のペプチドとアンタゴを希釈します。 </li><li>適度なボルテックス速度で渦のペプチド溶液をマイクロチューブにしてください。 </li><li> Aの1/3を追加します。ペプチド溶液チューブにntagomirソリューション、ゆっくりと、それはボルテックスミキサーで完全に混合されている間、滴下。 </li><li>次の1分間ボルテックスで溶液の混合を続行した後、混合物を1分間放置します。 </li><li>同じマイクロチューブ中のペプチド溶液と残りのアンタゴ溶液を混合するために、ステップ4と5をさらに2回繰り返します。ゆっくりとした添加は、トランスフェクションに有効である単分散複合体の形成に重要です。急速な添加は、複合体とその降水量の凝集につながることができます。 </li><li>ボルテックスすることなく、RTで30分間、複合体をインキュベートします。インキュベーションのこの時間の間に、注射が行われる研究室にマウスを慣らします。 </li></ol>アンタゴ-神経ペプチド複合体の3尾静脈注射<ol><li>動物を拘束するために、適切なサイズの拘束またはdecapiconeでマウスを置きます。 </li><li> PR綿棒を使用して、尾部の表面を清掃してください温水（約40℃）で電子浸しました。これは、血管拡張を促進し、静脈の可視性を向上します。 </li><li> 15〜20°の角度で - （1.0 CC 0.5）のインスリン注射器、小容量で尾アプローチ。シリンジ内に空気を導入しないように注意してください。尾の先端部で開始します。複合体を注入します。針を外して、任意の出血を止めるために注射部位（約5〜10秒）に直接消毒綿棒を適用します。 </li><li>濃縮としてトウモロコシの穂軸やティッシュペーパーで、3-5のグループ内の個別換気ケージにマウスを交換してください。同じケージに注射していないし、注射したマウスを保管しないでください。 </li></ol> 4.行動アッセイ注：注射後の回復を可能にするために注入し、行動アッセイの間に3〜4時間の時間間隔をおいてください。静かな部屋にマウスを持参してください。順化のこの期間中に、動物を邪魔しないでください。 <ol><li>にマウスを順応新しい部屋で30分間、その後、次の行動アッセイを開始します。それぞれの行動分析の間に10分の間隔をおいてください。 </li><li>後肢留め： 注：このテストは、運動機能障害および神経学的障害を示しています。 <ol><li>クリアなバックグラウンドで尾の近くにベースを保持して静かにマウスを持ち上げます。 </li><li> 10〜15秒間後肢位置を確認してください。運動失調マウス腹部に向かって時間の50％以上が13を停止し 、一方または両方の後肢を引っ込める傾向にある一方で、野生型マウスは、腹部から離れた後肢をsplays。 </li><li>ケージにマウスを返します</li></ol></li><li>レッジ試験： <ol><li>尾を保持することによって静かにマウスを持ち上げて、ケージの棚に保管してください。 </li><li>ケージの隅で特異的にマウスを観察します。野生型マウスは、恐怖とバランスを失うことなく、容易にコーナーを渡すことができます。ケージ棚に沿ってコーナーに歩きながら運動失調マウスは、そのバランス、フリーズや揺れを失います。 </li></ol></li><lI&gt;マウスフットプリントアッセイ： 注：このアッセイのために（〜5センチの高さの壁で〜5センチ幅の、〜70 cmの長。）狭い滑走路の床に、準備ができて吸収シートを保ちます<ol><li>片手で静かにマウスを持って、ブラシで後肢にインクを適用します。 </li><li>狭い滑走路の床に吸収シートを配置します。 </li><li>マウスはもう一方の端から滑走路に直線的に吸収シートを歩いたり、上で実行できるようにします。 </li><li>新鮮な吸収シートを使用して、それぞれのマウスを使用してプロセスをさらに2回繰り返します。 </li><li>前進中の2つの連続した​​ステップ間の距離を測定します。動物はちょうどそれぞれ、その実行を開始し、仕上げされた最初と最後の数足跡を含めないでください。 </li></ol></li></ol>

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The authors have nothing to disclose.

Here we demonstrate a widely accessible methodology to study the effects of miRNA modulation. Currently, most attempts at in vivo characterization of miRNA functions involve the creation of knockout mice or a transgenic that expresses a miRNA sponge. Most miRNAs, even the cell type specific ones are expressed in more than one organ. For instance, miRNAs initially thought to be specific to the hematopoietic system are also expressed in the brain, due to the presence of microglia. Thus even a cell type specifi...

マイクロRNAは、遺伝子発現と疾患への関与のための直接的な証拠の調節におけるそれらの普遍的役割に起因する新規治療ターゲットとして浮上しています。薬は1,2をターゲットとして、miRNAは、積極的に自分の可能性について検討されています。さらに、miRNA発現の変化は、いくつかの疾患および3 miRNA発現の人工的な摂動により、これらの変化のシミュレーションが疾患症状に関与する細胞経路を研究するために使用することが可能に関連付けられています。 miRNAを標的とする薬剤の組織特異的送達は、現在のmiRNAベースの薬剤開発のための大きな課題です。アンタゴとのmiRNA模倣体は、miRNAレベル4-6を摂動するための有望な薬剤です。しかし、それらの特異性および有効性を高める特殊な特徴は、それらがmiRNA発現のインビボでの摂動のために使用することができる前に、アンタゴの設計に組み込まれなければなりません。 マイクロRNAは、特に関連しています<html>現在不治の神経変性および神経発達疾患における標的として。血液脳関門は、脳内のアンタゴの配信に制限を課します。定位注射は広く脳7内の特定の位置に分子を送達するためのげっ歯類モデルで使用されています。それは、スキル、計測および時間の大規模な投資を必要とします。定位注射は侵襲的であり、手術を伴う、少なくとも軽傷を引き起こし、ローカル配信に制限されます。ニューロンを標的化するための嗜好を有するペプチドを貫通する細胞の使用は、それらがトランス血管経路を介して送達することができるので、これらの制限に対抗するが、血液脳関門を破ることができます。狂犬病ウイルス糖タンパク質（RVG）に由来するようなペプチドは、以前にマウス8で日本脳炎ウイルスに対するsiRNAを送達するために使用されました。私たちは、アンタゴmir配信のためにペプチドを使用して、miRNAが効果的にマウス脳9にノックダウンできることを見出しました。 ontent &quot;&gt; miRNAの第二の主要課題は、ノックダウンは、miRNAの小さなサイズから生じると密接に関連した配列の存在は、私たちは3の密接に関連したアイソフォームで構成されてMMU-のmiR-29ファミリー、のmiR-29aの例を取る。アイソフォームa、bおよびc。アンタゴはまた、一般的にその安定性を向上させ、ヌクレアーゼによる攻撃にそれらが耐性をレンダリングするために骨格に沿って変更されます。ロックされた核酸（LNAのは）彼らは熱安定性を向上させるというさらなる利点を提供し、さらにオーバー以降劣化をターゲットにつながります立体障害10。すべての骨格に沿って修正を導入することは有効であるが高価になることができます。私たちは、以前の最適な数を超えた修正は、さらに有効性を増強ない可能性があることを見てきました。アンタゴの設計は、したがって、アンタゴの最適な変更を伴います。 向神経ペプチドと非共有結合的に複雑なアンタゴには、ノナアルギニン内線にヘプタ充電に使用されます。 D-アルギニン彼らは、プロテアーゼによる切断を受けないよう、より高い安定性を付与するため、残基が使用されています。ノナアルギニンストレッチにヘプタは、それらが細胞型特異性を付与しませんが、効率的細胞透過剤として作用します。共有結合ノナアルギニンリンカーにRVGペプチドをリンクすることにより、神経向性、細胞透過性ペプチドを生成しました。ペプチドの正に荷電した残基は、複合体を形成するために、負に帯電した核酸骨格と相互作用します。これらの複合体は、効果的に培養された細胞内および組織内へのインビボでの DNAまたはRNAをトランスフェクトするために使用することができます。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Vortex</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Restrainer or Decapicone</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Narrow runway</td>
			<td></td>
			<td></td>
			<td>~70-cm-long, ~5-cm-wide with ~5-cm-high walls.</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescently labelled oligonucleotides (siGLO)</td>
			<td>GE Healthcare Dharmacon INC</td>
			<td>D0016300120</td>
			<td></td>
		</tr>
		<tr>
			<td>10% sterile D-glucose</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antagomir-29</td>
			<td>Exiqon</td>
			<td>custom synthesis</td>
			<td></td>
		</tr>
		<tr>
			<td>Antagomir-control</td>
			<td>Exiqon</td>
			<td>custom synthesis</td>
			<td></td>
		</tr>
		<tr>
			<td>Neuropeptide RVG</td>
			<td>G.L.Biochem (Shanghai) Ltd.</td>
			<td>custom synthesis</td>
			<td>&#62;98% purity</td>
		</tr>
		<tr>
			<td>Neuropeptide RVM</td>
			<td>G.L.Biochem (Shanghai) Ltd.</td>
			<td>custom synthesis</td>
			<td>&#62;98% purity</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Other</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Warm water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Absorbent sheets</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ink</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Brush</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antiseptic</td>
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a simple alternative to stereotactic injection for brain specific knockdown of mirna

MicroRNAs (miRNAs) are key regulators of gene expression. In the brain, vital processes like neurodevelopment and neuronal functions depend on the correct expression of microRNAs. Perturbation of microRNAs in the brain can be used to model neurodegenerative diseases by modulating neuronal cell death. Currently, stereotactic injection is used to deliver miRNA knockdown agents to specific location in the brain. Here, we discuss strategies to design antagomirs against miRNA with locked nucleotide modifications (LNA). Subsequently describe a method for brain specific delivery of antagomirs, uniformly across different regions of the brain. This method is simple and widely applicable since it overcomes the surgery, associated injury and limitation of local delivery in stereotactic injections. We prepared a complex of neurotropic, cell-penetrating peptide Rabies Virus Glycoprotein (RVG) with antagomir against miRNA-29 and injected through tail vein, to specifically deliver in the brain. The antagomir design incorporated features that allow specific targeting of the miRNA and formation of non-covalent complexes with the peptide. The knock-down of the miRNA in neuronal cells, resulted in apoptotic cell death and associated behavioural defects. Thus, the method can be used for acute models of neuro-degeneration through the perturbation of miRNAs.

ここに提示された手順を用いて、蛍光標識されたオリゴヌクレオチド50microgram（FLO）と〜1：15のモル電荷比の850microgramのRVGペプチドの複合体（FLO：ペプチド）を調製し、尾静脈を介して一回だけ注入しました。非神経向性狂犬病ウイルスのマトリックス（RVM）ペプチドおよびFLOの複合体は、配信対照として使用しました。翌日、マウスの脳および肝臓を単離し、単一細胞懸濁液を調製しまし?...

Watch this Scientific Journal Video about A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA at JoVE.com

A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

miRNAの脳特異的ノックダウンのための定位注射への単純な代替

MicroRNAs (miRNAs) are key regulators of gene expression.  In the brain, vital processes like neurodevelopment and neuronal functions depend on the ...

a-simple-alternative-to-stereotactic-injection-for-brain-specific

Research

JoVE Journal

Neuroscience

12.6K ビュー.  CSIR-Institute of Genomics and Integrative Biology, New Delhi, India. MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

ビデオ: A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

春雨とカッペリーニの処理試験：ラットとマウスの器用な前足の機能の簡単な定量的指標

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

神経科学

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

非心室プラスミド注入し、エレクトロポレーションにより産後のラット脳への遺伝子デリバリー

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

大人のラットの特定の脳領域の興奮毒性病変に対する定位手術

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to study the molecular composition of membranes produced a significant decline in its use. Recently, there has been a major revival in freeze-fracture electron microscopy thanks to the development of effective ways to reveal integral membrane proteins by immunogold labeling. One of these methods is known as detergent-solubilized Freeze-fracture Replica Immunolabeling (FRIL).
The combination of the FRIL technique with optogenetics allows a correlated analysis of the structural and functional properties of central synapses. Using this approach it is possible to identify and characterize both pre- and postsynaptic neurons by their respective expression of a tagged channelrhodopsin and specific molecular markers. The distinctive appearance of the postsynaptic membrane specialization of glutamatergic synapses further allows, upon labeling of ionotropic glutamate receptors, to quantify and analyze the intrasynaptic distribution of these receptors. Here, we give a step-by-step description of the procedures required to prepare paired replicas and how to immunolabel them. We will also discuss the caveats and limitations of the FRIL technique, in particular those associated with potential sampling biases. The high reproducibility and versatility of the FRIL technique, when combined with optogenetics, offers a very powerful approach for the characterization of different aspects of synaptic transmission at identified neuronal microcircuits in the brain.
Here, we provide an example how this approach was used to gain insights into structure-function relationships of excitatory synapses at neurons of the intercalated cell masses of the mouse amygdala. In particular, we have investigated the expression of ionotropic glutamate receptors at identified inputs originated from the thalamic posterior intralaminar and medial geniculate nuclei. These synapses were shown to relay sensory information relevant for fear learning and to undergo plastic changes upon fear conditioning.

This article illustrates how the expression of neurotransmitter receptors can be quantified and the pattern analyzed at synapses with identified pre and postsynaptic elements using a combination of viral transduction of optogenetic tools and the freeze-fracture replica immunolabeling technique.

複合光遺伝学および凍結破砕レプリカ免疫標識をマウス扁桃体におけるグルタミン酸受容体の入力固有の配置を検討すること

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to ...

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to the interphotoreceptor/subretinal compartment that has direct exposure to photoreceptors and the retinal pigment epithelium (RPE). Subretinal injections of plasminogen as well as recent preclinical and clinical trials have demonstrated safety and/or efficacy of delivering viral vectors and stem cells to individuals with advanced retinal disease. Mouse models of retinal disease, particularly hereditary retinal dystrophies, are essential for testing these therapies. The most common injection procedure in rodents is to use small transcorneal or transcleral incisions with an anterior approach to the retina. With this approach, the injection needle penetrates the neurosensory retina disrupting the underlying RPE and on insertion can easily nick the lens, causing lens opacification and impairment of noninvasive imaging. Accessing the subretinal space via a transcleral, posterior approach avoids these problems: the needle crosses the sclera approximately 0.5 mm from the optic nerve, without retinal penetration and avoids disrupting the vitreous. Collateral damage is limited to that associated with the focal sclerotomy and the effects of a transient, serous retinal detachment. The simplicity of the method minimizes ocular injury, ensures rapid retinal reattachment and recovery, and has a low failure rate. The minimal damage to the retina and RPE allows for clear assessment of the efficacy and direct effects of the therapeutic agents themselves. This manuscript describes a novel subretinal injection technique that can be used to target viral vectors, pharmacological agents, stem cells or induced pluripotent stem (iPS) cells to the subretinal space in mice with high efficacy, minimal damage, and fast recovery.

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections are the most common technique for delivering large therapeutic agents such as proteins and viral vectors to photoreceptors and the retinal pigment epithelium. An alternative method in mice that successfully targets the subretinal space with minimal collateral damage and fast recovery times is described here.

網膜下腔にアクセスするための代替と検証済みの注入方法<i&gt;経由で</i&gt;経強後方アプローチ

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

使用して報酬を求める行動の変化を測定するための簡単な方法<em&gt;キイロショウジョウバエ</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

外傷によって影響を受ける脳のレーザーキャプチャーマイクロダイ セクション定位アトラス誘導レーザー

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

ひと成人皮膚線維芽細胞から誘導される神経細胞の高収率文化の生成の単純です

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

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 ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Ex Vivoラット脳切片を用いた神経変性疾患の主要なイベントを勉強する方法

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...