NIHのR01 DK55619、NIHのR21をCA139063

<ol>
	<li>Routh, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose-sensing+neurons:+are+they+physiologically+relevant?.">Glucose-sensing neurons: are they physiologically relevant?.</a> Physiol. Behav. 76, 403-413 (2002).</li><li>Canabal, D. D., Potian, J. G., Duran, R. G., McArdle, J. J., Routh, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperglycemia+impairs+glucose+and+insulin+regulation+of+nitric+oxide+production+in+glucose-inhibited+neurons+in+the+ventromedial+hypothalamus.">Hyperglycemia impairs glucose and insulin regulation of nitric oxide production in glucose-inhibited neurons in the ventromedial hypothalamus.</a> Am. J. Physiol. 293, 592-600 (2007).</li><li>Canabal, D. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose,+insulin,+and+leptin+signaling+pathways+modulate+nitric+oxide+synthesis+in+glucose-inhibited+neurons+in+the+ventromedial+hypothalamus.">Glucose, insulin, and leptin signaling pathways modulate nitric oxide synthesis in glucose-inhibited neurons in the ventromedial hypothalamus.</a> American journal of physiology. Reg. Integr. Comp. Physiol. 292, 1418-1428 (2007).</li><li>Murphy, B. A., Fakira, K. A., Song, Z., Beuve, A., Routh, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMP-activated+protein+kinase+and+nitric+oxide+regulate+the+glucose+sensitivity+of+ventromedial+hypothalamic+glucose-inhibited+neurons.">AMP-activated protein kinase and nitric oxide regulate the glucose sensitivity of ventromedial hypothalamic glucose-inhibited neurons.</a> Am. J. Physiol. Cell Physiol. 297, C750-C758 (2009).</li><li>Murphy, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fasting+enhances+the+response+of+arcuate+neuropeptide+Y-glucose-inhibited+neurons+to+decreased+extracellular+glucose.">Fasting enhances the response of arcuate neuropeptide Y-glucose-inhibited neurons to decreased extracellular glucose.</a> Am. J. Physiol. Cell Physiol. 296, C746-C756 (2009).</li><li>Kang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucokinase+is+a+critical+regulator+of+ventromedial+hypothalamic+neuronal+glucosensing.">Glucokinase is a critical regulator of ventromedial hypothalamic neuronal glucosensing.</a> Diabetes. 55, 412-420 (2006).</li><li>Kang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prior+hypoglycemia+enhances+glucose+responsiveness+in+some+ventromedial+hypothalamic+glucosensing+neurons.">Prior hypoglycemia enhances glucose responsiveness in some ventromedial hypothalamic glucosensing neurons.</a> Reg. Integr. Comp. Physiol. 294, R784-R792 (2008).</li><li>Paxinos, G., Franklin, K. B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (2004).</li><li>Paxinos, G., Watson, 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> The Rat Brain in Stereotaxic Coordinates. , (1998).</li><li>Song, Z., Levin, B. E., McArdle, J. J., Bakhos, N., Routh, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convergence+of+pre-+and+postsynaptic+influences+on+glucosensing+neurons+in+the+ventromedial+hypothalamic+nucleus.">Convergence of pre- and postsynaptic influences on glucosensing neurons in the ventromedial hypothalamic nucleus.</a> Diabetes. 50, 2673-2681 (2001).</li><li>Song, Z., Routh, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+glucose+and+lactate+on+glucosensing+neurons+in+the+ventromedial+hypothalamic+nucleus.">Differential effects of glucose and lactate on glucosensing neurons in the ventromedial hypothalamic nucleus.</a> Diabetes. 54, 15-22 (2005).</li></ol>

我々は、開示することは何もありません。

成体マウスの神経細胞の活動を研究することができることへの鍵は、健康的な神経細胞を解離させる能力である。成体マウスの視床下部ニューロンの解離は幼若マウス由来のニューロンと比較して、プロトコルのいくつかの重要なステップでより困難である。我々は、多くの方法でこの問題を克服している。厚手の500ミクロンの脳スライスを作ることは、若いマウスからの脳組織のために使?...

脳は神経内分泌および自律神経系を通じてエネルギー恒常性を調節する。視床下部腹内側部（VMH）は、腹内側核（VMN）と弓状核（ARC）で構成され、エネルギー恒常性の中央制御に重要である。専門的なグルコース感知ニューロンは、VMH内で、神経活動と周辺グルコース恒常1をリンクします。励起されたグルコース（GE）グルコース（GI）ニューロンは、細胞外グルコースが増加するにつれて、それらの活性を低下させる阻害したニューロンが増加;ニューロンを感知するグルコースの2種類がある。 VMHのグルコース感知ニューロンは、一般的に電気生理学またはカルシウム/膜電位感受性色素イメージングを用いて研究している。 電気生理学的パッチクランプ法は、ex vivoで神経活動の研究にゴールドスタンダードであると考えられている。この技術では、ガラスマイクロピペット電極は、高抵抗を介して細胞膜に結合している（GΩ）シール。パッチクランプ電極は、活動電位の周波数（電流クランプ）をリアルタイムで記録またはイオンコンダクタンス（電圧クランプ）は、単一のニューロン内での変更を可能にする。パッチクランプ法は、特定のイオンチャネルコンダクタンスの変化に関する詳細な情報を提供するが、主な欠点は、1つのニューロンが同時に観察され得ることである。それも、具体的な実験的治療を開始する前に、グルコース感知ニューロンからその1が記録されていることを確認するために記録の約30〜45分かかります。また、GI及びGEニューロンは&lt;総VMHニューロン集団の20％を含む。この問題を配合すると、これらのニューロンの識別の細胞マーカーの多くの場合、不足です。従って、明らかである他の技術は、パッチクランプ分析は面倒であることができない貴重な電気的情報、時間がかかり、低い収率を与えるにもかかわらず。 解離VMHニューロンの蛍光イメージングの使用は、HUの研究を可能にする同時にニューロンのndreds。カルシウム感受性色素は、間接的に神経活動の変化に相関する細胞内カルシウムの変化を測定するために使用することができる。膜電位感受性色素、膜電位の変化をモニターするために使用される。細胞膜電位を測定することは、細胞内カルシウムレベルの変化と比較して、ニューロンの活動のより直接的な指標である。さらに、膜電位色素（MPD）イメージングは​​、潜在的に活動電位発火が変更されておらず、細胞内カルシウムレベルは変化しない可能性があり、膜電位の小さい変化を検出する。これらの蛍光イメージング技術の両方が、幼若マウス2-7からVMHグルコース感知ニューロンを研究するために使用されてきた。結果は、パッチクランプ電気生理学を用いて得られたものよりも詳細であるが、イメージング実験の強度は、それらが同時に必然的にグルコース感知ニューロンの有意な数を含む細胞の大きな集団を評価することである。 MPDイメージングIより均一に全体VMH全体局在している消化管の神経細胞を研究するために特に有用だ、したがって解離しVMH（〜15％GI）で勉強するための十分な集団を提供する。 GEのニューロンが密腹-VMNとARCとVMN間のセルが悪い領域に局在している間は対照的に、彼らはVMH内のニューロン（&lt;1％、GE）のかなりの数を表すものではありません。さらに、単離された神経細胞を研究することによって、星状細胞とシナプス前効果が除去される。生理的接続およびプロセスが失われるので、これは一次ニューロン効果を研究する利点、ならびに不利であることができる。 パッチクランプ電気生理学およびMPD /カルシウム色素イメージングの両方を制限する要因は、若い動物（ 例えば 。マウスやラット&lt;8週齢）を使用する必要がある。これは、加齢に伴って起こる減少し、神経細胞の生存能力と組み合わせて、増加した結合組織に主にある。脳スライスの電気生理学スタッド中新設、結合組織の増加は、それがより困難にニューロンを可視化することができる。増大した結合組織はまた、硬いイメージング研究のための健康なニューロンの大多数を解離することができる。さらに、若い動物からのニューロンは、パッチクランプ記録や撮影のいずれかの間長く生き残る。しかし、若いマウスの使用が主な制限することができます。神経活動および/または神経伝達物質または循環の栄養素に対する応答は、年齢とともに変化する。エネルギーバランスは密接生殖状態に固定されているので、たとえば、エネルギーバランスを調節する視床下部ニューロンは、事前VS思春期後の動物で異なる反応を示すことがある。さらに、多くの疾患は、長期治療を必要とするか、または成人期まで現れていない。このような疾患の主な例は、食事性肥満や2型糖尿病である。グルコース感知ニューロンは、これらの疾患において役割を果たしていると考えられているので、我々が正常MPD撮像expのに使用するために、健康な成人VMHニューロンを培養するための方法論を開発eriments。

<table border="0" cellpadding="0" cellspacing="0" width="947">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Neurobasal-A Medium (Custom)</td>
			<td style="width:115px;">Invitrogen</td>
			<td style="width:180px;">0050128DJ</td>
			<td style="width:427px;">custom made glucose free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Hibernate-A Medium (Custom)</td>
			<td style="width:115px;">BrainBits</td>
			<td style="width:180px;"></td>
			<td>custom made glucose free</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">Penicillin streptomycin (20,000 U/ml)</td>
			<td style="width:115px;">Invitrogen</td>
			<td style="width:180px;">15140</td>
			<td>other vendors acceptable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">Stericup vacuum filter units (0.22 &#956;m)</td>
			<td style="width:115px;">Millipore</td>
			<td style="width:180px;"></td>
			<td>other vendors acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">25 mm Glass coverslips</td>
			<td style="width:115px;">Warner</td>
			<td style="width:180px;">#1 25mm round</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">18 mm Glass coverslips</td>
			<td style="width:115px;">Warner</td>
			<td style="width:180px;">#1 18mm round</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">GlutaMAX</td>
			<td style="width:115px;">Invitrogen</td>
			<td style="width:180px;">35050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">B27 minus insulin (50x)</td>
			<td style="width:115px;">Invitrogen</td>
			<td style="width:180px;">0050129SA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Razor blade</td>
			<td style="width:115px;">VWR</td>
			<td style="width:180px;">55411</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">Vibratome &#38; cooling chamber</td>
			<td style="width:115px;">Vibratome</td>
			<td style="width:180px;">Series 1000 Sectioning system</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Vibratome blades</td>
			<td style="width:115px;">Polysciences</td>
			<td style="width:180px;">22370</td>
			<td>injector or double edge blades from other vendors acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Papain, suspension</td>
			<td style="width:115px;">Worthington</td>
			<td style="width:180px;">LS003124</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">BSA, suitable for cell culture</td>
			<td style="width:115px;">Sigma</td>
			<td style="width:180px;"></td>
			<td>other vendor acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">DNAse, for cell culture</td>
			<td style="width:115px;">Invitrogen</td>
			<td style="width:180px;"></td>
			<td>other vendor acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">cloning cylinders, 6 mm x 8 mm</td>
			<td style="width:115px;">Bellco Glass</td>
			<td style="width:180px;">2090-00608</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">Membrane Potential Dye (blue)</td>
			<td style="width:115px;">Molecular Devices</td>
			<td style="width:180px;">R8042</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">In-line heater</td>
			<td style="width:115px;">Warner</td>
			<td style="width:180px;">SF-28</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Syringe pumps</td>
			<td style="width:115px;">WPI</td>
			<td style="width:180px;">sp100i</td>
			<td>other vendor acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Closed chamber</td>
			<td style="width:115px;">Warner</td>
			<td style="width:180px;">RC-43C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Polyethylene tubing</td>
			<td style="width:115px;">Warner</td>
			<td style="width:180px;">PE-90</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">Metamorph</td>
			<td style="width:115px;">Molecular Devices</td>
			<td style="width:180px;"></td>
			<td>alternate image analysis software acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Microscope</td>
			<td style="width:115px;">Olympus</td>
			<td style="width:180px;">BX61 WI</td>
			<td>
			used with 10X&#160;objective
			</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Camera</td>
			<td style="width:115px;">Photometrics</td>
			<td style="width:180px;">Cool Snap HQ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Narrow Cy3 Filter Set</td>
			<td style="width:115px;">Chroma</td>
			<td style="width:180px;">41007a</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">Illumination System</td>
			<td style="width:115px;">Sutter Instruments</td>
			<td style="width:180px;">Lambda DG-4</td>
			<td></td>
		</tr>
	</tbody>
</table>

membrane potential dye imaging of ventromedial hypothalamus neurons from adult mice to study glucose sensing

神経活動の研究はしばしば増加結合組織に関連する技術的な問題へのげっ歯類歳未満2ヶ月から神経細胞を使用して実行し、年齢とともに起こる神経細胞の生存率を減少している。ここでは、成人老齢マウスから健康的な視床下部ニューロンの解離のための方法論を説明します。成人老齢マウスからニューロンを研究する能力は、後の年齢で現れる疾患モデルを使用することができ、より正確な発生的に、特定の研究のためかもしれません。単一ニューロンを研究するために電気を使用するのではなく、解離したニューロンの蛍光撮像は、ニューロンの集団の活性を研究するために使用することができる。希望の神経細胞のタイプは、視床下部グルコース感知ニューロンの希である異種のニューロン集団を研究するときに特に便利です。私たちは、茶への回答を研究する大人の腹内側視床下部ニューロンの膜電位色素イメージングを利用細胞外のグルコースのnges。グルコース感知ニューロンは、エネルギーバランスの調節において中心的な役割を果たしていると考えられている。大人のげっ歯類でグルコース感知を研究する能力は機能不全のエネルギーバランスに関連する疾患の優位ので、特に有用である（ 例えば 。肥満）年齢とともに増加。

離れて他の視床下部のエリアからVMHの正確な解剖は一貫性のある結果を得ることが重要である。他のエリアを含めることは、計算された脱分極ニューロンの％を変え、VMHニューロン集団が希薄化する可能性があり。さらに、神経細胞を検出するグルコースは、VMHのグルコース感知ニューロンから機能的にも機構的に異なる可能性が外側視床下部などの他の視床下部の地域で確認されている。 ...

Watch this Scientific Journal Video about Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing at JoVE.com

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

成人老化マウスからの単一ニューロンの活性は、特定の脳領域のニューロンを解離し、蛍光膜電位色素イメージングを用いて研究することができる。グルコースの変化に対する応答を試験することによって、この手法は、成体腹内側視床下部ニューロンのグルコース感受性を研究するために使用することができる。

グルコース感知を勉強する成体マウス由来の膜視床下部腹内側部のニューロンの電位色素イメージング

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

membrane-potential-dye-imaging-ventromedial-hypothalamus-neurons-from

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Neuroscience

13.6K ビュー.  Rutgers New Jersey Medical School. 成人老化マウスからの単一ニューロンの活性は、特定の脳領域のニューロンを解離し、蛍光膜電位色素イメージングを用いて研究することができる。グルコースの変化に対する応答を試験することによって、この手法は、成体腹内側視床下部ニューロンのグルコース感受性を研究するために使用することができる。 

ビデオ: Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

のレーザーキャプチャーマイクロダイセクションショウジョウバエ末梢ニューロン

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

神経科学

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of &Delta;&Psi;m renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine &Delta;&Psi;m and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. 
Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine &Delta;&psi;m in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess &Delta;&Psi;m or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of &Delta;&Psi;m or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of &Delta;&Psi;m or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in &#x2206;&#x03A8;m and ROS in different cell types and in neurons isolated from other brain regions.

We demonstrate application of the fluorescence indicator, TMRM, in cortical neurons to determine the relative changes in TMRM fluorescence intensity before and after application of a specific stimulus. We also show application of the fluorescence probe H2DCF-DA to assess the relative level of reactive oxygen species in cortical neurons.

ライブラット皮質ニューロンのミトコンドリア膜電位と活性酸素種の定量

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

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

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

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 ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

FM染料を用いて培養小脳顆粒神経細胞のシナプス小胞プールの補充の定量分析

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

胎児マウスから海馬ニューロンの単離および培養

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

培養大人の背根神経節ニューロンの軸索再生の遺伝学的研究

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

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 ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

イメージング初代培養マウス神経細胞における形質膜に受容体の挿入をpHluorinタグ付き

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

脳スライスビオチン：<em&gt;生体外</emアダルトニューロンにおいて地域固有の細胞膜のタンパク質輸送を測定するために&gt;の取り組み

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with enhanced [Ca2+]i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca2+]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+]i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+]i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+]i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+]i signals in the striatal microcircuitry.

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

The properties and functions of astrocyte intracellular Ca2+ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca2+ signals within striatal astrocytes in situ.