我々は、この原稿の重要なレビューのために博士Sanika Chirwaに感謝します。この作品は、国立衛生研究所（DA026947、DA021471、そしてNS071122）によってサポートされていました。

1。用品<ol><li>バックグラウンドノイズを低減する防振台（TMI）の上にファラデーケージをマウントします。 </li><li>同時パッチクランプアンペロメトリー記録システムは、優れたDICの光学系と長作動距離レンズを備えた倒立顕微鏡を必要とします。車のバッテリーに灯台顕微鏡を接続します。システムのこのDC光源はさらに、電気ノイズを減少させるであろう。 </li><li>油圧マイクロマニピュレーター（Siskiyou）さらに減少ノイズ。私達の構成では、我々は、全細胞記録のために右利きのマニピュレータを使用し、電流測定のために左利き。 </li></ol> 2。記録用の電極を準備<ol><li> P-2000プラー（サター）に石英ピペットを用いてパッチ電極を引き出します。私たちのプルは、2つの熱サイクルで、約5秒続く。この熱時間は私たちの全細胞パッチピペットで一貫抵抗（MΩ3-4​​）をもたらしました。 </li><li>に電極を埋めるピペットは、2mMのドーパミンを含む溶液と右マニピュレーターにマウントします。アルミホイルでDAを含むピペット溶液を保持する容器を包む。氷上で保存する。ドーパミンは酸化可能である。光から保護し、氷の上で解決策を維持すると、ドーパミンの酸化速度が低下します。 </li><li>優しく、収納ボックスからProCFE（ダガン）低ノイズカーボンファイバーアンペロメトリー電極を取り外します（ 図1に示すように）アンペロメトリアダプタに水銀、マウントと入力し、[右maniupulatorにマウントされます。炭素繊維の遠端を保持することによって損傷から炭素繊維の先端を保護します。先端を清潔にし、無傷であることを確認するために、ラボ顕微鏡で電極を点検してください。 </li><li>外液を含有するガラスボトムシャーレに電極を入れて電流滴電極の整合性を調べます。ドーパミンの非存在下での現在のベースラインを記録します。皿に1mmのDA溶液10μlを追加します。良いアンペロメトリー電極REコー​​ド酸化電流の増加。アンペロメトリー電極が正常に動作して確実にし、各実験の開始時と終了時に、この手順を繰り返します。 </li></ol> 3。グラスボトムペトリ皿内ド​​ーパミントランスポーターを発現するように遺伝子ドーパミン神経細胞や神経細胞の初代培養を始めて<ol><li>優しく暖かい外液と細胞やドーパミンニューロン3回洗浄する。 </li><li>顕微鏡ステージ上にグラスボトムシャーレをマウントします。 </li></ol> 4。セルを視覚化し、実験を行う<ol><li>明らかに細胞を可視化するための正しい焦点を見つける。パッチ電極に正の圧力をかける。その後、静かに溶液中に両方の電極をダウンさせると、セルに近接しています。 </li><li>セル（左側）、右側にパッチ電極の隣にアンペロメトリー電極を配置します。 </li><li>パッチ電極付きセルでgigaohmシールを達成。破裂に吸引シール細胞全体の構成を実現。 </li><li>細胞内にドーパミンを含む内部液の透析のための5月8日分を許可します。 </li><li>所望の電圧ステップまたはランプ·プロトコルを使用しています。同時にパッチピペットと膜電位はパッチピペットを介して制御されながら、ドーパミントランスポーターを介して全細胞電流とドーパミンの逆輸送を測定する電流測定型電極の両方からデータを取得します。 </li></ol>

<ol>
	<li>Michael, A. C., Ikeda, M., Justice, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+contributing+to+the+recovery+of+striatal+releasable+dopamine+following+MFB+stimulation.">Mechanisms contributing to the recovery of striatal releasable dopamine following MFB stimulation.</a> Brain Res. 421, 325-335 (1987).</li><li>Gonon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+and+extrasynaptic+excitatory+action+of+dopamine+mediated+by+D1+receptors+in+the+rat+striatum+in+vivo.">Prolonged and extrasynaptic excitatory action of dopamine mediated by D1 receptors in the rat striatum in vivo.</a> J. Neurosci. 17, 5972-5978 (1997).</li><li>Sulzer, D., Pothos, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+quantal+size+by+presynaptic+mechanisms.">Regulation of quantal size by presynaptic mechanisms.</a> Rev. Neurosci. 11, 159-212 (2000).</li><li>Napolitano, A., Cesura, A. M., Da Prada, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+monoamine+oxidase+and+catechol+O-methyltransferase+in+dopaminergic+neurotransmission.">The role of monoamine oxidase and catechol O-methyltransferase in dopaminergic neurotransmission.</a> J. Neural. Transm. Suppl. 45, 35-45 (1995).</li><li>Sulzer, D., Maidment, N. T., Rayport, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine+and+other+weak+bases+act+to+promote+reverse+transport+of+dopamine+in+ventral+midbrain+neurons.">Amphetamine and other weak bases act to promote reverse transport of dopamine in ventral midbrain neurons.</a> J. Neurochem. 60, 527-535 (1993).</li><li>Salahpour, 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=Increased+amphetamine-induced+hyperactivity+and+reward+in+mice+overexpressing+the+dopamine+transporter.">Increased amphetamine-induced hyperactivity and reward in mice overexpressing the dopamine transporter.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 4405-4410 (2008).</li><li>Khoshbouei, H., Wang, H., Lechleiter, J. D., Javitch, J. A., Galli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine-induced+dopamine+efflux.+A+voltage-sensitive+and+intracellular+Na+-dependent+mechanism.">Amphetamine-induced dopamine efflux. A voltage-sensitive and intracellular Na+-dependent mechanism.</a> J. Biol. Chem. 278, 12070-12077 (2003).</li><li>Goodwin, J. S., 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=Amphetamine+and+methamphetamine+differentially+affect+dopamine+transporters+in+vitro+and+in.">Amphetamine and methamphetamine differentially affect dopamine transporters in vitro and in.</a> , 284-2978 (2009).</li><li>Kahlig, K. M., 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=Amphetamine+induces+dopamine+efflux+through+a+dopamine+transporter+channel.">Amphetamine induces dopamine efflux through a dopamine transporter channel.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 3495-3500 (2005).</li><li>Swant, J., Chirwa, S., Stanwood, G., Khoshbouei, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methamphetamine+reduces+LTP+and+increases+baseline+synaptic+transmission+in+the+CA1+region+of+mouse+hippocampus.">Methamphetamine reduces LTP and increases baseline synaptic transmission in the CA1 region of mouse hippocampus.</a> PLoS One. 5, e11382 (2010).</li><li>Gnegy, M. E., 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=Intracellular+Ca2++regulates+amphetamine-induced+dopamine+efflux+and+currents+mediated+by+the+human+dopamine+transporter.">Intracellular Ca2+ regulates amphetamine-induced dopamine efflux and currents mediated by the human dopamine transporter.</a> Mol. Pharmacol. 66, 137-143 (2004).</li></ol>

特別な利害関係は宣言されません。

同時に電圧クランプとアンペロメトリーは、次の利点があります。すべての細胞型はアクセス可能であり、記録のために使用することができます。レコーディングが行われた細胞またはニューロンの同定は、シンプルで簡単です。特に、細胞が蛍光実験者が容易に目的の細胞や神経細胞を選択することができ、目的のタンパク質に蛍光タグを追加することで、ラベルが付いていた場合。実験?...

<table border="1"><tbody><tr><td></td><td></td><td></td><td> 機器 </td></tr><tr><td>防振テーブルのw /ファラデーケージ</td><td>テクニカル·マニュファクチャリング株式会社</td><td> 63から500シリーズ</td><td>私たちはモデル63から543までを使用</td></tr><tr><td>倒立顕微鏡ニコンTE-2000 </td><td>ニコン</td><td>中止</td><td>これで、EclipseのTi </td></tr><tr><td>二つの低雑音増幅器axopatch 200B </td><td>分子デバイス</td><td></td><td> 800-635-5577 </td></tr><tr><td> 1-CV 203 BUのヘッドステージ</td><td>分子デバイス</td><td></td><td> 800-635-5578 </td></tr><tr><td> 1-HL-Uピペットホルダ</td><td>分子デバイス</td><td></td><td> 800-635-5579 </td></tr><tr><td> Digidata 1440A / Dコンバータ</td><td>分子デバイス</td><td></td><td> 800-635-5580 </td></tr><tr><td> 2つのマニピュレータSiskyou、左と右利き</td><td> Siskiyou </td><td> MX6600R MX6600L </td><td> 877-313-6418 </td></tr><tr><td>レーザーピペットプラー</td><td>サターインスツルメンツ</td><td> P-2000 </td><td> 888-883-0128 </td></tr><tr><td>低ノイズの炭素繊維電極アンペロメトリー</td><td> ProCFE </td><td></td><td> <a href="http://www.dagan.com" target="_blank">www.dagan.com</a> </td></tr><tr><td>低ノイズ石英ピペット</td><td>サターインスツルメンツ</td><td> QF100-70から7.5 </td><td> 888-883-0128 </td></tr><tr><td> 12ボルトのカーバッテリー</td><td></td><td></td><td>広く利用可能</td></tr><tr><td>車のバッテリー充電器</td><td></td><td></td><td>広く利用可能</td></tr><tr><td></td><td></td><td></td><td> 試薬 </td></tr><tr><td>塩化ナトリウム（NaCl） </td><td>シグマ</td><td> S7653 </td><td></td></tr><tr><td> HEPES </td><td>シグマ</td><td> H3375 </td><td></td></tr><tr><td>葡萄糖</td><td>シグマ</td><td> G7528 </td><td></td></tr><tr><td>硫酸マグネシウム（MgSO 4） </td><td>シグマ</td><td> M2643 </td><td></td></tr><tr><td>リン酸二水素カリウム（KH 2 PO 4） </td><td>シグマ</td><td> P5655 </td><td></td></tr><tr><td>塩化カリウム（KCl） </td><td>シグマ</td><td> P9333 </td><td></td></tr><tr><td>塩化カルシウム水和物（CaCl 2で •2H 2 0） </td><td>シグマ</td><td> 223506 </td><td></td></tr><tr><td>塩化マグネシウム六水和物（MgCl 2を •6H 2 0） </td><td>シグマ</td><td> M2670 </td><td></td></tr><tr><td> EGTA </td><td>シグマ</td><td> E0396 </td><td></td></tr></tbody></table>

single cell measurement of dopamine release with simultaneous voltage-clamp and amperometry

シナプス間隙、ドーパミンへのそのリリース後に、その前と後シナプスターゲット1を介して、その生物学的特性を発揮する。ドーパミン信号が拡散2-3、細胞外酵素4、膜輸送体5で終了します。ドーパミンニューロンの周囲のシナプス間隙に位置するドーパミントランスポーターは、内側ドーパミンフラックス（取り込み）を介して放出さアミンをクリアします。ドーパミントランスポーターはまたドーパミン5の外側への輸送または流出と呼ばれるプロセスで、内部から外部へのアミンを解放するために逆方向に働くことができます。 。フォワード（摂取）とリバース（流出）5：20年以上前スルザーらは、ドーパミントランスポーターは、次の2つのアクティビティのモードで動作することができます報告した。トランスポーターを介して流出を介して神経伝達物質放出は、細胞外空間にドーパミンを大量に移動させることができ、細胞外ドーパミンhで主要な調節的役割を果たすことが示されているomeostasis 6。ここでは、同時パッチクランプとアンペロメトリー記録は膜電位が制御されているミリ秒の時間分解能で流出機構を介してドーパミン放出を測定するために使用する方法について説明します。このため、全細胞電流と酸化（アンペロメトリー）信号はAxopatch 200Bアンプ（Molecular Devices社、全細胞電流記録、1,000 Hzのローパスベッセルフィルタが設定された）を使用して同時に測定される。アンペロメトリーは炭素繊維電極を記録するためには、第二増幅器（Axopatch 200B）に接続されており、細胞膜に隣接して配置され、700 mVに保持されます。全細胞と酸化（アンペロメトリー）電流を記録することができ、電流 - 電圧の関係は、電圧ステッププロトコルを使用して生成することができます。濃度への変換を必要とする通常のアンペロメトリックキャリブレーションとは異なり、現在は有効容積7を考慮せずに直接報告されています。従って、得られたデータいくつかの送信機はバルク溶液に失われるので、ドーパミン流出には下限を表しています。

アンペロメトリーとの複合パッチクランプは、電位依存DAT媒介DA流出。 図2Aは、細胞内環境と膜電位が全細胞パッチピペットによってクランプされるDATの媒介DA流出の代表的な実験設定や記録を示して測定することができます。アンペロメトリー電極が細胞膜（ 図2A）上に置かれている間、このテクニックを使用すると、YFP-DATのタンパク質を発現する細胞は全細胞パ?...

Watch this Scientific Journal Video about Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry at JoVE.com

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

アンペロメトリック技術は自発的ドーパミンの酸化によって生成される酸化電流を検出することにより、単一細胞からのドーパミン放出を測定します。同時電圧クランプとアンペロメトリー方法論はドーパミントランスポーターおよびドーパミンの逆輸送上のこの活動の調節の役割の全体的な &quot;活動&quot;の間の機構的な関係を明らかにする。

同時に電圧クランプとアンペロとドーパミン放出の単一細胞計測

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is ...

single-cell-measurement-dopamine-release-with-simultaneous-voltage

Research

JoVE Journal

Neuroscience

13.7K ビュー.  University of Florida. アンペロメトリック技術は自発的ドーパミンの酸化によって生成される酸化電流を検出することにより、単一細胞からのドーパミン放出を測定します。同時電圧クランプとアンペロメトリー方法論はドーパミントランスポーターおよびドーパミンの逆輸送上のこの活動の調節の役割の全体的な "活動"の間の機構的な関係を明らかにする。

ビデオ: Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

Functional Mapping with Simultaneous MEG and EEG

We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the processing of simple sensory stimuli. We will use somatosensory stimuli to locate the hand somatosensory areas, auditory stimuli to locate the auditory cortices, visual stimuli in four quadrants of the visual field to locate the early visual areas. These type of experiments are used for functional mapping in epileptic and brain tumor patients to locate eloquent cortices. In basic neuroscience similar experimental protocols are used to study the orchestration of cortical activity. The acquisition protocol includes quality assurance procedures, subject preparation for the combined MEG/EEG study, and acquisition of evoked-response data with somatosensory, auditory, and visual stimuli. We also demonstrate analysis of the data using the equivalent current dipole model and cortically-constrained minimum-norm estimates. Anatomical MRI data are employed in the analysis for visualization and for deriving boundaries of tissue boundaries for forward modeling and cortical location and orientation constraints for the minimum-norm estimates.

We use magnetoencephalography (MEG) and electroencephalography (EEG) to map brain areas involved in the processing of simple sensory stimuli.

同時MEGおよびEEGとの機能的マッピング

We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the ...

神経科学

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 ...

Single-cell Profiling of Developing and Mature Retinal Neurons

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and gene expression programs for extremely rare cell subsets has been a challenge using standard whole-tissue approaches. Gene expression profiling of individual cells allows for unprecedented access to cell types that comprise only a small percentage of the total tissue1-7. In addition, this technique can be used to examine the gene expression programs that are transiently expressed in small numbers of cells during dynamic developmental transitions8.
This issue of cellular diversity arises repeatedly in the central nervous system (CNS) where neuronal connections can occur between quite diverse cells9. The exact number of distinct cell types is not precisely known, but it has been estimated that there may be as many as 1000 different types in the cortex itself10. The function(s) of complex neural circuits may rely on some of the rare neuronal types and the genes they express. By identifying new markers and helping to molecularly classify different neurons, the single-cell approach is particularly useful in the analysis of cell types in the nervous system. It may also help to elucidate mechanisms of neural development by identifying differentially expressed genes and gene pathways during early stages of neuronal progenitor development.
As a simple, easily accessed tissue with considerable neuronal diversity, the vertebrate retina is an excellent model system for studying the processes of cellular development, neuronal differentiation and neuronal diversification. However, as in other parts of the CNS, this cellular diversity can present a problem for determining the genetic pathways that drive retinal progenitors to adopt a specific cell fate, especially given that rod photoreceptors make up the majority of the total retinal cell population11. Here we report a method for the identification of the transcripts expressed in single retinal cells (Figure 1). The single-cell profiling technique allows for the assessment of the amount of heterogeneity present within different cellular populations of the retina2,4,5,12. In addition, this method has revealed a host of new candidate genes that may play role(s) in the cell fate decision-making processes that occur in subsets of retinal progenitor cells8. With some simple adjustments to the protocol, this technique can be utilized for many different tissues and cell types.

A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.

単一セルの開発のプロファイリングと成熟網膜神経細胞

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and ...

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

単一セルと単スパイク分解能で閾値上神経活動の光記録

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

同時脳波、リアルタイム乳酸濃度の測定と齧歯類大脳皮質ニューロン活動のOptogeneticマニピュレーション

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

げっ歯類での同時のfMRIと脳深部刺激

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

単セルの精度で神経共培養の調製

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

Simultaneous Detection of c-Fos Activation from Mesolimbic and Mesocortical Dopamine Reward Sites Following Naive Sugar and Fat Ingestion in Rats

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars and fats are mediated by their innate attractions as well as learned preferences. Brain dopamine, especially meso-limbic and meso-cortical projections from the ventral tegmental area (VTA), has been implicated in both of these unlearned and learned responses. The concept of distributed brain networks, wherein several sites and transmitter/peptide systems interact, has been proposed to mediate palatable food intake, but there is limited evidence empirically demonstrating such actions. Thus, sugar intake elicits DA release and increases c-fos-like immunoreactivity (FLI) from individual VTA DA projection zones including the nucleus accumbens (NAC), amygdala (AMY) and medial prefrontal cortex (mPFC) as well as the dorsal striatum. Further, central administration of selective DA receptor antagonists into these sites differentially reduce acquisition and expression of conditioned flavor preferences elicited by sugars or fats. One approach by which to determine whether these sites interacted as a distributed brain network in response to sugar or fat intake would be to simultaneous evaluate whether the VTA and its major mesotelencephalic DA projection zones (prelimbic and infralimbic mPFC, core and shell of the NAc, basolateral and central-cortico-medial AMY) as well as the dorsal striatum would display coordinated and simultaneous FLI activation after oral, unconditioned intake of corn oil (3.5%), glucose (8%), fructose (8%) and saccharin (0.2%) solutions. This approach is a successful first step in identifying the feasibility of using cellular c-fos activation simultaneously across relevant brain sites to study reward-related learning in ingestion of palatable food in rodents.

The goal of this study is to identify reward-related distributed brain networks by delineating a reliable immunohistological technique using cellular c-fos activation to measure simultaneous changes in dopamine pathways and terminal sites after novel ingestion of fat and sugar in rats.

ラットにおけるナイーブ砂糖と脂肪の摂取後の中脳辺縁系と中間皮質ドーパミン報酬サイトからのc-Fosの活性化の同時検出

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

全細胞電圧クランプ記録のための電気生理学的方法<em&gt;ショウジョウバエ</em&gt;感光体

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ex vivo preparation of the levator auris longus (LAL) is used because it is a thin muscle that provides easy visualization of the neuromuscular junction for microelectrode impalement at the motor endplate. This method allows for the recording of spontaneous miniature endplate potentials and currents (mEPPs and mEPCs), nerve-evoked endplate potentials and currents (EPPs and EPCs), as well as the membrane properties of the motor endplate. Results obtained from this method include the quantal content (QC), number of vesicle release sites (n), probability of vesicle release (prel), synaptic facilitation and depression, as well as the muscle membrane time constant (&#964;m) and input resistance. Application of this technique to mouse models of human disease can highlight key pathologies in disease states and help identify novel treatment strategies. By fully voltage-clamping a single synapse, this method provides one of the most detailed analyses of synaptic transmission currently available.

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

The protocol described in this paper uses the mouse levator auris longus (LAL) muscle to record spontaneous and nerve-evoked postsynaptic potentials (current-clamp) and currents (voltage-clamp) at the neuromuscular junction. Use of this technique can provide key insights into mechanisms of synaptic transmission under normal and disease conditions.

オーリスの挙筋哺乳類の神経筋伝達電圧クランプ条件下での試験のための準備

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ...