我々は、博士に感謝します。書かれた原稿の書式設定の支援のための初期の技術の集合まで、および博士ヴェロニカアルバレス中に彼らの援助のためのping Jun朱と進小山。この研究は、NIAAAの学内臨床と医学研究の部門によって資金を供給された。

<ol>
	<li>Zhu, P. J., Lovinger, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+potentiates+gabaergic+synaptic+transmission+in+a+postsynaptic+neuron/synaptic+bouton+preparation+from+basolateral+amygdala.">Ethanol potentiates gabaergic synaptic transmission in a postsynaptic neuron/synaptic bouton preparation from basolateral amygdala.</a> J Neurophysiol. 96, 433-441 (2006).</li><li>Zhu, P. J., Lovinger, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrograde+endocannabinoid+signaling+in+a+postsynaptic+neuron/synaptic+bouton+preparation+from+basolateral+amygdala.">Retrograde endocannabinoid signaling in a postsynaptic neuron/synaptic bouton preparation from basolateral amygdala.</a> J Neurosci. 25, 6199-6207 (2005).</li><li>Akaike, N., Moorhouse, A. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+enhances+glutamate+transmission+by+retrograde+dopamine+signaling+in+a+postsynaptic+neuron/synaptic+bouton+preparation+from+the+ventral+tegmental+area.">Ethanol enhances glutamate transmission by retrograde dopamine signaling in a postsynaptic neuron/synaptic bouton preparation from the ventral tegmental area.</a> Neuropsychopharmacology. 34, 1233-1244 (2009).</li><li>Murakami, N., Ishibashi, H., Katsurabayashi, S., Akaike, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+channel+subtypes+on+single+gabaergic+presynaptic+terminal+projecting+to+rat+hippocampal+neurons.">Calcium channel subtypes on single gabaergic presynaptic terminal projecting to rat hippocampal neurons.</a> Brain Res. 951, 121-129 (2002).</li><li>Zhu, P. J., Lovinger, D. 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A., Rothman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+secretion+and+synaptic+transmission+with+ph-sensitive+green+fluorescent+proteins.">Visualizing secretion and synaptic transmission with ph-sensitive green fluorescent proteins.</a> Nature. 394, 192-195 (1998).</li><li>Li, Z., Burrone, J., Tyler, W. J., Hartman, K. N., Albeanu, D. F., Murthy, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+vesicle+recycling+studied+in+transgenic+mice+expressing+synaptophluorin.">Synaptic vesicle recycling studied in transgenic mice expressing synaptophluorin.</a> Proc Natl Acad Sci U S A. 102, 6131-6136 (2005).</li></ol>

利害の衝突は宣言されません。

成功vibrodissociationはスライスが健康な神経細胞が含まれているし、間質の神経細胞が毒性損傷を与えることなくスライスを終了するのに十分な柔軟性であることが必要です。健全なスライスが成人の脳スライスで発見されているよりも少ないグリア/ interstiatial材料で作ることができるとき、したがって、技術は、出生後早期年齢（P1 - 21）で最適に動作します。我々の経験では、しかし、スライス自体のニューロンの生存のためのスライス標本を最適化することvibrodissociation技術のための逆効果かもしれません。我々は日常的にショ糖がvibrodissociationのために準備+、スライスが私たちの通常記録のACSFで（例えばカット）を調製することができる細胞外のNa +とCa 2の多くのために置換されたコールド変更したACSFを使用してその場の記録でのスライスを用意するのに対し。スライス自体の個々のニューロンからの記録のためにスライスを作成するときに我々はまた、Cだけのセクショニング後に35℃で通常のACSFでスライスを配置し、室温に戻す前に、この温度で​​30〜60分間のためにそれらを残す。しかし、vibrodissociationのために準備のスライスは、単にスライスした後、室温に即座に移動されます。これらの手順は、ニューロンがより容易にスライス自体から緩い動揺することができる会社間質組織の不足のため、おそらくvibrodissociation後の健康なニューロン、高収率を提供しています。我々が日常的に特定の日に私たちの記録とイメージング実験のための十分な神経細胞を得ることとして我々は徹底的に、スライスの調製条件を検討していない。それは、スライス厚やpreinc​​ubatonの手続きの変更などの追加修正は、、健康な神経細胞の収率を高めるために行われる可能性があります。非常に軽度のプロテアーゼ処理は、セルの歩留まりを向上させ、高齢動物で動作するように技術を得るための努力で試みられている。しかし、いつもにも軽度のプロテアーゼ処理は、シナプスの機能を混乱させるようだ。このように、間にプロテアーゼおよび機械的解離の組み合わせは、まだそれが日付に前脳ニューロンの信頼性が実証されていない動作するように見つけることができる。 それはvibrodissociation後の健康な神経細胞の比較的低い収率はテクニックが豊富な神経細胞サブタイプ、主に投射ニューロンを研究するために主に有用であることを意味するにも留意すべきである。小さな神経細胞サブポピュレーションを容易に識別可能なGFP発現マウスの可用性は、これらのまれな神経細胞を研究の可能性が高まります。神経細胞の収率が大幅に向上させることができる場合を除きしかし、これらのニューロン上のデータの蓄積が相対的に遅くなる可能性があります。 いくつかのステップは、カルシウム感知染料による神経細胞のロードが成功のために不可欠であることが証明されている。 AM -エステル化された色素への暴露は、37 ° Cで行われ、そして我々はより低い温度で色素負荷しようとするに成功していない。染料の濃度は、それが色素ローディングのそれより高い濃度がシナプス前boutonsでカルシウム濃度を低下させる表示されるので、ロード時間と温度の両方を考慮して最適化する必要があります。我々はより高い濃度でロードすると、周波数とsIPSCsの振幅を減少させることを観察した。小さなシナプス前boutonsの緩衝能力は相馬では異なる可能性があるとboutonsのサイズが一致していないので、カルシウム感知vibrodissociatedニューロンのシナプス前終末に染料をロードする場合、注意が必要です。ニューロンを含む料理は、​​色素ローディング、以下のHEPES緩衝外部溶液で洗浄し、洗浄後の回復時間が非常に重要ですしている。さらに、細胞全体の記録のための良好なシールを作るために、細胞は、外側の細胞膜からの非内部化色素分子を取り除くために徹底的に洗浄する必要があります。 電気生理学および生細胞イメージングのためのvibrodissociated神経細胞の使用に加えて、免疫細胞化学的手法はこれらの細胞11,12に適用することもできます。細胞は容易に固定し、抗体の多様、および他の細胞のマーカーで染色することができます。これらの細胞の使用は、GABA作動性シナプス前終末でのタンパク質発現の可視化のためのクリーンな準備を提供します。 GABA作動性ニューロンに特異的プロモーターの制御下に蛍光タンパク質を発現するマウスを使用すると、捜査官がvibrodissociated準備（図1）で個々のシナプス端末を識別することができます。このタイプのイメージング蛍光マーカーは、レーザーベースの顕微鏡とSTEDのような新しいテクニック（刺激の排出枯渇顕微鏡）を用いて端末の形態学的測定を可能にすることができる。シナプス小胞サイクリングを報告する色素で可視化は、この準備でも可能です。赤池らは、生きた細胞4の端子を視覚化するスチリル色素、FM1 - 43でGABA作動端子ラベルを貼っている。 またvibrodissociatedでプロファイルRNA発現する単一セルの逆転写酵素PCRを行うことができるはずですニューロン。このテクニックは、日常的に細胞培養で増殖させた酵素的に解離ニューロンとニューロンに適用されます。

<table border="1"><tbody><tr><td> アイテム </td><td> 会社 </td><td> カタログ＃ </td><td> コメント </td></tr><tr><td>振動組織スライサー</td><td>ライカマイクロシステムズ株式会社</td><td> VT1200S </td><td></td></tr><tr><td>細胞培養用ディッシュ（35 mm）に</td><td> BDファルコン</td><td> 353001 </td><td></td></tr><tr><td>ガラスボトムディッシュ</td><td> Willco皿</td><td> GWSB - 5040またはGWSB - 3522 </td><td>イメージングのための0.16〜0.19ミリメートルのガラスの厚さ</td></tr><tr><td>圧電マニピュレータ</td><td> EXFO -バーレイ</td><td> LSS - 3000 </td><td>また、リレーなどを使用することができます</td></tr><tr><td> SD9スクエアパルス刺激</td><td>グラステクノロジー</td><td> SD9K </td><td>圧電マニピュレータをトリガするための</td></tr><tr><td>解剖ステレオスコープ</td><td>ワイルドHeerbrugg </td><td> TYP 374590 </td><td>また、任意のステレオスコープを使用することができます</td></tr><tr><td> /ブラウンマイクロピペットプラーフレイミング</td><td>サターのインストゥルメント</td><td> P - 97 </td><td></td></tr><tr><td>薄い壁ガラス管</td><td>世界の精密機器</td><td> TW - 150F - 4 </td><td>火炎密封したガラスマイクロピペットとパッチマイクロピペット用</td></tr><tr><td> 6チャンネルの潅流バルブコントロールシステム</td><td>ワーナーインスツルメンツ</td><td> VC - 6またはVC - 6M </td><td></td></tr><tr><td>灌流ファーストステップ</td><td>ワーナーインスツルメンツ</td><td> SF - 77B </td><td></td></tr><tr><td> 20 - 63x目標と倒立顕微鏡</td><td>ニコン</td><td> TS200 Diaphot </td><td></td></tr><tr><td> EMCCDカメラ</td><td>アンドールテクノロジー</td><td> IXON EM + DU - 888 </td><td></td></tr><tr><td>エキサイト蛍光照明光源</td><td> EXFOフォトニクスソリューション（株） </td><td> X - Citeの120PC </td><td></td></tr><tr><td>カルシウム指示薬回答にみたFluo - 4、 </td><td>分子プローブ</td><td> F14201 </td><td></td></tr></tbody></table>

vibrodissociation of neurons from rodent brain slices to study synaptic transmission and image presynaptic terminals

中枢神経系から神経細胞の機械的な解離は、シナプス前boutonsは関心の分離されたニューロンに接続されたままという利点があります。これは細胞外とシナプス後細胞内環境を良好に制御することができる条件下でのシナプス伝達の検査が可能になります。 vibrodissociationとして知られているプロテアーゼの使用せずに振動ベースの技術は、機械的な分離のための最も人気のあるテクニックです。小さなボール状にファイアーポリッシュ先端とマイクロピペットは、P1 - P21齧歯類から作られる脳のスライスに置かれます。マイクロピペットは、スライス面に平行に振動し、分離されたニューロンの解放をもたらすスライスの厚さによって低下する。分離されたニューロンはvibrodissociationの数分以内に研究のための準備が整いました。細胞外環境の優れた制御隣接セルの影響から自由;適合電気生理学とイメージングの研究に適した実行可能な、比較的成熟した神経細胞の急速な生産：このテクニックは、などの一次ニューロン培養、脳スライスと酵素的に分離された神経細胞の使用上の利点があります。迅速な薬剤のアプリケーションおよび総細胞灌流を用いて十分に制御された薬理学的実験のため、およびスライスまたは細胞培養調製物中の神経細胞への相対的な細胞全体のレコーディングにおける空間クランプ改良。この製剤は、シナプス生理学、薬理学、変調と可塑性を調べるために使用することができます。生きている細胞とboutonsでプリとシナプス後要素の両方のリアルタイムイメージングは​​vibrodissociatedニューロンを用いても可能です。プリとシナプス後要素の分子成分のキャラクタリゼーションは、免疫学とイメージングベースのアプローチで達成することができる。

Watch this Scientific Journal Video about Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals at JoVE.com

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

この報告書は、添付されたシナプス前boutonsを保持する個々の実行可能な神経細胞の機械的な分離のためのテクニックを示しています。 Vibrodissociatedニューロンは、隣接セルからの影響のない急速な生産、優れた薬理学的制御と空間のクランプの改善の利点を持っている。このメソッドは、シナプスの要素のイメージングやパッ​​チクランプ記録に使用することができます。

シナプス伝達とイメージシナプス前終末を研究するために齧歯類の脳スライスからの神経細胞のVibrodissociation

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

vibrodissociation-neurons-from-rodent-brain-slices-to-study-synaptic

Research

JoVE Journal

Neuroscience

15.4K ビュー.  National Institutes of Health/National Institute on Alcohol Abuse and Alcoholism. この報告書は、添付されたシナプス前boutonsを保持する個々の実行可能な神経細胞の機械的な分離のためのテクニックを示しています。 Vibrodissociatedニューロンは、隣接セルからの影響のない急速な生産、優れた薬理学的制御と空間のクランプの改善の利点を持っている。このメソッドは、シナプスの要素のイメージングやパッ​​チクランプ記録に使用?...

ビデオ: Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &amp;#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

β-アミロイドオリゴマーの調製 1から42まで</sub海馬スライスでのシナプス可塑性の減損の>と誘導

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

神経科学

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

げっ歯類からの神経細胞とヒト以外の霊長類の脳スライスのDiOLISTIC標識

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and amphetamine), the role it plays in psychiatric illnesses (e.g. schizophrenia and Attention Deficit Hyperactivity Disorder), and its involvement in degenerative disorders like Parkinson's and Huntington's disease. Under normal physiological conditions, dopamine is known to regulate locomotor activity, cognition, learning, emotional affect, and neuroendocrine hormone secretion. One of the largest densities of dopamine neurons is within the striatum, which can be divided in two distinct neuroanatomical regions known as the nucleus accumbens and the caudate-putamen. The objective is to illustrate a general protocol for slice fast-scan cyclic voltammetry (FSCV) within the mouse striatum. FSCV is a well-defined electrochemical technique providing the opportunity to measure dopamine release and uptake in real time in discrete brain regions. Carbon fiber microelectrodes (diameter of ~ 7 &mu;m) are used in FSCV to detect dopamine oxidation. The analytical advantage of using FSCV to detect dopamine is its enhanced temporal resolution of 100 milliseconds and spatial resolution of less than ten microns, providing complementary information to in vivo microdialysis.

Using fast-scan cyclic voltammetry to measure electrically evoked presynaptic dopamine dynamics in striatal brain slices.

高速スキャンサイクリックボルタンメトリーによる線条体の脳スライスにおけるシナプス前ドーパミンのダイナミクス

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and ...

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

軸索、樹状突起および脳スライスにおける個々のニューロンの樹状突起棘から電位感受性色素記録

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals

Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca2+&#160;into the presynaptic terminal, through voltage-gated calcium channels (VGCCs) located in the release face membrane, is the trigger for vesicle fusion and neurotransmitter release. Crucial to the rapidity of synaptic transmission is the spatial and temporal synchrony between the arrival of the action potential, VGCCs and the neurotransmitter release machinery. The ability to directly record Ca2+&#160;currents from the release face membrane of individual presynaptic terminals is imperative for a precise understanding of the relationship between presynaptic Ca2+&#160;and neurotransmitter release. Access to the presynaptic release face membrane for electrophysiological recording is not available in most preparations and presynaptic Ca2+&#160;entry has been characterized using imaging techniques and macroscopic current measurements &#8211; techniques that do not have sufficient temporal resolution to visualize Ca2+ entry. The characterization of VGCCs directly at single presynaptic terminals has not been possible in central synapses and has thus far been successfully achieved only in the calyx-type synapse of the chick ciliary ganglion and in rat calyces. We have successfully addressed this problem in the giant reticulospinal synapse of the lamprey spinal cord by developing an acutely dissociated preparation of the spinal cord that yields isolated reticulospinal axons with functional presynaptic terminals devoid of postsynaptic structures. We can fluorescently label and identify individual presynaptic terminals and target them for recording. Using this preparation, we have characterized VGCCs directly at the release face of individual presynaptic terminals using immunohistochemistry and electrophysiology approaches. Ca2+ currents have been recorded directly at the release face membrane of individual presynaptic terminals, the first such recording to be carried out at central synapses.

Recording Ca2+ currents at the presynaptic release face membrane is key to a precise understanding of Ca2+ entry and neurotransmitter release. We present an acute dissociation of the lamprey spinal cord that yields functional isolated reticulospinal axons, permitting recording directly from the release face membrane of individual presynaptic terminals.

ヤツメウナギ網様体軸索の急性解離は、個別の機能シナプス前終末のリリース顔膜から録音を有効にする方法

Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca2+&#160;into the presynaptic terminal, through voltage-gated ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

胚性齧歯類の脳から単離、プライマリ脳ドーパミンニューロンの文化と長期メンテナンス

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

のシナプス変調を分析<em&gt;キイロショウジョウバエ</em&gt;長時間の光照射後の感光体

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a challenging and low-throughput technique due to its complexity and high reliance on user operation and control. This manuscript demonstrates an image-guided automatic patch clamp system for in vitro whole-cell patch clamp experiments in acute brain slices. Our system implements a computer vision-based algorithm to detect fluorescently labeled cells and to target them for fully automatic patching using a micromanipulator and internal pipette pressure control. The entire process is highly automated, with minimal requirements for human intervention. Real-time experimental information, including electrical resistance and internal pipette pressure, are documented electronically for future analysis and for optimization to different cell types. Although our system is described in the context of acute brain slice recordings, it can also be applied to the automated image-guided patch clamp of dissociated neurons, organotypic slice cultures, and other non-neuronal cell types.

This protocol describes how to conduct automatic image-guided patch-clamp experiments using a system recently developed for standard in vitro electrophysiology equipment.

脳スライスにおけるニューロン研究のための自動画像誘導パッチクランプの応用

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

海馬の齧歯動物の脳スライスにおけるシナプス密度の評価

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

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 ...