Name: preparation of neuronal co-cultures with single cell precision
Uploaded: 2014-05-20T13:00:00
Description: Watch this Scientific Journal Video about Preparation of Neuronal Co-cultures with Single Cell Precision at JoVE.com

著者らは、SEMイメージングのためのSU-8の作製とマリア·ベッカー（ISAS）のためのウルリッヒマルクグラーフ（ISAS）に感謝しています。研究は財政的にドイツ学術振興（DFG WE3737/3-1）、BundesministeriumエリーゼBildungウントForschung助成金（BMBF 0101-31P6541）でかつMinisteriumエリーゼイノベーション、WissenschaftウントForschung DESランデスノルトライン·ウェストファーレンによってサポートされていました。平家Hardelaufおかげインターナショナルライプニッツ研究科「システム生物学ラボオンチップ」の財政支援のために。

プロトコルは、神経科学者を対象としており、 図2にまとめた。そのためには、商業や機関の施設に委託されたPDMS複製に使用SU-8マスターズの微細加工をお勧めします。 2レイヤーマスクのデザインは、化学のウェブサイト12の王立協会の補足説明（ZIP形式）として自由にご利用いただけます。重要なのは、最初のSU-8層は、2.5〜3.0ミクロンの深さまで製造され、25〜30ミク?...

<ol>
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マイクロ流体アレイ化技術は、最小限の文化を確立するための単精度ニューロンの取り扱いが可能になり、その種の最初のものです。マイクロ流体構造を用いて細胞のパターンを揃えるその場生体材料のパターニング方法において 、強力なアプローチと相まって、これらのミニマルな文化が減少した地元の絡み合い高intercompartment接続レベルを有する。これらの特徴は、区画間?...

<html>神経組織は非常に複雑である。空間的に細胞接触を通じて、特に軸索と樹状突起増生によって定義層や区画内とプラスチックの接続と一緒に注文された異種の細胞の混合物である。新技術は、より深い洞察力や病気、開発、健康的な機能の中心UNRAVELメカニズムを獲得する大きな実験的な自由を与えることが必要である。 Campenot室1,2、最近微細加工の実施例3,4は、選択的に異なる体細胞集団と、それらの神経突起増生を撹乱する能力を備えたネットワーク接続された神経細胞の共培養のex vivo製造に使用することができる。これらのマイクロ流体デバイスは、たとえば、化学、5,6またはレーザー軸索切断6-8、タウオパチー9、ウイルス伝播10,11、および軸索4におけるmRNAの局在後の軸索の変性と再生を研究するために使用されてきた。 ENT」&gt; 神経生物学者の範囲を拡大するために、技術開発を最小限の神経細胞の共培養を準備する必要があります。これは、単一の細胞と細胞内の精度でシステムの調査のための神経回路網の解きほぐしを可能にします。最小限の細胞数の要件は、パーキンソン病、耳から螺旋神経節、末梢ニューロンに関連するドーパミン作動性黒質細胞を含む希少細胞タイプを分析し、幹細胞の可能性を開く。これ以外に、携帯経済は3Rのイニシアティブに関連しています。これらのマイクロ流体プラットフォームは、大規模な毒性スクリーニングまたは他のハイスループットを用いて、動物のニューロンを必要とするデータに富む一連の実験を考えることができる。 本論文では、マイクロ流体デバイスの製造および使用のためのプロトコルが記載されている。 その場 biomateri 内と組み合わせたマイクロ流体アレイ化他のパターニング方法は、最小限の細胞数を用いて高度に相互接続神経細胞の共培養の登録に使用することができる。マイクロ流体アレイ化は、差動フロー· ​​アプローチの微細構造トラップは流体回路（ 図1のSEM像と一緒に示されている）内に配置されることにより、12月15日 、に基づいています。神経突起伸長チャネルへの入口-パス0→1は、微細構造化アパーチャの直線状のアレイにニューロンを搬送するためのより低い流動抵抗（R 2&gt; R 1）を有する。単一のセルによるトラップの占有率が局部的に近隣のトラップに、その後の細胞を捕捉するための流線をそらすために流れを妨げる。アレイ内のトラップの完全な占有は、過剰なニューロンの除去のための操作のバイパスモードを生成するために蛇行路（0→2）に流線をそらすために、流体の比（R 1&gt; R 2）を切り替える。 図1。マイクロ流体回路。マイクロピラーメニスカス固定有する二重層区画化ニューロン共培養アレイのA）神経突起伸長チャネルによって相互接続された培養チャンバに隣接有する単一ニューロン整列のために微分抵抗流体回路を、B、C）SEM画像である。この設計では、三叉状ニューロントラッピング構造は、神経突起増殖物の束形成を促進するために使用した。図や王立化学協会（RSC）の許可を得て複製伝説12。 <a href="https://www.jove.com/files/ftp_upload/51389/51389fig1highres.jpg" target="_blank">拡大画像を表示するにはここをクリックしてください。</a> 平面基板上のマイクロパターン神経回路網の製造は容易にexamplのために（達成することができ<html>我々のグループからのESは、Frimat ら 16と平家ら 17）を参照してください。しかし、PDMSデバイス内およびマイクロ流体チャネルへのこれらのマイクロメートルスケール整列の要件を、生理活性物質パターンをカプセル化することは、主要な技術的課題を提起する。セクション3.1にするためのプロトコルは、チップ内、またはその場で 、生体材料のパターンの製造は、提示されている。これらのパターンは、長い培養時間スケールの間にニューロンの登録を有効にして、区画間の増生を促進する。メニスカスピニング微細構造ニューロン部位および神経突起伸長のチャンネルを配列して、所謂水マスクを整列させるために使用される。露出表面は生体材料のパターンを定義するために解砕されるのに対し、水マスクは、プラズマ処理の間、接着分子コーティングを保護する。また、プロトコルは、細胞培養のために、異なる共培養区画の選択的治療のために必要な流体の単離のために設けられている。 ve_content &quot;&gt;プロトコルは同様に、 その場での生体材料のパターニングに蒸発し、表面張力現象を利用し、簡単です。ポリレプリケーション（ジメチルシロキサン）（PDMS）マイクロ流体デバイス18のためのソフトリソグラフィーのユーザーフレンドリーな原則を活用するように設計されており、唯一の安価なハンドヘルド型プラズマ源を必要とする。マイクロ流体回路を効果的にプログラム正しい底部ポートに材料を分配し、上方から吸引するだけで問題これらの操作を行うセル負荷と区画特定の治療法。この方法では、神経生物学者を与えることを意図している自分の研究室でマイクロ流体デバイスを準備し、使用する自由。

<table border="0" cellpadding="0" cellspacing="0" width="857">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">PDMS Sylgard 184</td>
			<td style="width:163px;">Dow Corning</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PDMS Elastosil RT 601</td>
			<td style="width:163px;">Wacker</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Coverslips</td>
			<td style="width:163px;">VWR</td>
			<td>630-1590</td>
			<td>130-160 mm thick</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">3 mm Biopsy Punches</td>
			<td style="width:163px;">Kai Medical</td>
			<td style="width:143px;"></td>
			<td>Handle with care - extremely sharp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tygon Tubing</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:143px;">S-50-HL</td>
			<td>1.65 mm ID; 3.35 mm OD</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 mL Syringe (Inkjekt and Omnifix)</td>
			<td style="width:163px;">Braun</td>
			<td style="width:143px;">6064204</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">4-Way Tubing Connector</td>
			<td style="width:163px;">VWR or Fisher Scientific</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Flow Regulator</td>
			<td style="width:163px;">Harvard Apparatus</td>
			<td style="width:143px;">722645</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">0.5 mm Pins</td>
			<td style="width:163px;">Dressmaking Departments</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">PLL-g-PEG</td>
			<td style="width:163px;">SuSoS</td>
			<td style="width:143px;">PLL(20)-g[3.5]-PEG(5)</td>
			<td>Stability in storage can be an issue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">poly-D-lysine</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:143px;">P6407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">poly-lysine-FITC</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:143px;">P3069</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">poly-ornithine</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:143px;">P4957</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">fibronectin</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:143px;">F2006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">laminin</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:143px;">L2020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">PBS</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:143px;">P4417</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Inverted Fluorescent Microscope</td>
			<td style="width:163px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">Example Aspiration Pump</td>
			<td style="width:163px;">KNF Neuberger, Laboport</td>
			<td style="width:143px;">N811KVP</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">Hand Held Corona Discharge Device</td>
			<td style="width:163px;">Leybold-Heraeus, USA</td>
			<td style="width:143px;">VP23</td>
			<td>May not comply with your country&#39;s safety standards</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Femto Plasma Oven</td>
			<td style="width:163px;">Diener Electronic</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Vacuum Dessicator or Centrifuge</td>
			<td style="width:163px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of neuronal co-cultures with single cell precision

Campenot室のマイクロ流体の実施形態は、神経科学のコミュニティから大きな関心を集めている。これらの相互接続された共培養プラットフォームは、感染および疾患の伝播に発達および機能的神経生物学に及ぶ、様々な問題を調査するために使用することができる。しかし、従来のシステムは、重要な細胞の一次ドーパミン作動性黒質、スパイラル神経節、およびショウジョウバエキイロニューロンなどの少量の細胞を研究するための不適切な入力（コンパートメントごとに何千）、および高スループット実験のための非現実的が必要です。緻密培養物はまた、非常に局所的に二つの文化を相互接続するいくつかの増殖物（&lt;10％）で、交絡している。 （I）、マイクロ流体、単一のニューロンが方法を配列し、プラズマパターニング生体材料コーティングはREGIするための（II）水マスキング法：本論文では簡単なマイクロ流体とパターニングプロトコルはこれらの課題に対処が記載されているSTERニューロンとコンパートメント間の伸長を促進する。最小限のニューロン共培養物は、高レベル（&gt; 85％）intercompartment接続を用いて調製した単一細胞精度の高スループット神経生物学実験のために使用することができる。

水マスキングエクスプロイトは、表面張力。気液界面での圧力耐性が[曲率半径に反比例<img fo:content-width="1in" src="files/ftp_upload/51389/51389eq1.jpg" width="113" />マイクロ流体寸法で非常に安定インターフェイスを生成する。マイクロピラーを効果的な場所に水マスクを固定。水マスク形成は、加熱によって増加率は、マイクロ流体ポートからの蒸発によって駆動される。低倍率（4X - 10X）からの熱を?...

Watch this Scientific Journal Video about Preparation of Neuronal Co-cultures with Single Cell Precision at JoVE.com

Preparation of Neuronal Co-cultures with Single Cell Precision

生体材料コーティングのチップ内のプラズマパターニングのための単一ニューロンマイクロ流体アレイ化と水のマスキングのためのプロトコルが記載されている。高度に相互接続された共培養細胞は、最小限の入力を用いて調製することができる。

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 ...

preparation-of-neuronal-co-cultures-with-single-cell-precision

Research

JoVE Journal

Neuroscience

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

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is terminated by diffusion2-3, extracellular enzymes4, and membrane transporters5. The dopamine transporter, located in the peri-synaptic cleft of dopamine neurons clears the released amines through an inward dopamine flux (uptake). The dopamine transporter can also work in reverse direction to release amines from inside to outside in a process called outward transport or efflux of dopamine5. More than 20 years ago Sulzer et al. reported the dopamine transporter can operate in two modes of activity: forward (uptake) and reverse (efflux)5. The neurotransmitter released via efflux through the transporter can move a large amount of dopamine to the extracellular space, and has been shown to play a major regulatory role in extracellular dopamine homeostasis6. Here we describe how simultaneous patch clamp and amperometry recording can be used to measure released dopamine via the efflux mechanism with millisecond time resolution when the membrane potential is controlled. For this, whole-cell current and oxidative (amperometric) signals are measured simultaneously using an Axopatch 200B amplifier (Molecular Devices, with a low-pass Bessel filter set at 1,000 Hz for whole-cell current recording). For amperometry recording a carbon fiber electrode is connected to a second amplifier (Axopatch 200B) and is placed adjacent to the plasma membrane and held at +700 mV. The whole-cell and oxidative (amperometric) currents can be recorded and the current-voltage relationship can be generated using a voltage step protocol. Unlike the usual amperometric calibration, which requires conversion to concentration, the current is reported directly without considering the effective volume7. Thus, the resulting data represent a lower limit to dopamine efflux because some transmitter is lost to the bulk solution.

The amperometric technique measures dopamine release from a single cell by detecting the oxidative current produced by spontaneous dopamine oxidization. Simultaneous voltage clamp and amperometry methodology reveal the mechanistic relationship between the overall "activity" of dopamine transporter and the regulatory role of this activity on the reverse transport of dopamine.

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 ...

神経科学

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 Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Much information about the coupling of presynaptic ionic currents with the release of neurotransmitter has been obtained from invertebrate preparations, most notably the squid giant synapse1. However, except for the preparation described here, few vertebrate preparations exist in which it is possible to make simultaneous measurements of neurotransmitter release and presynaptic ionic currents. Embryonic Xenopus motoneurons and muscle cells can be grown together in simple culture medium at room temperature; they will form functional synapses within twelve to twenty-four hours, and can be used to study nerve and muscle cell development and synaptic interactions for several days (until overgrowth occurs). Some advantages of these co-cultures over other vertebrate preparations include the simplicity of preparation, the ability to maintain the cultures and work at room temperature, and the ready accessibility of the synapses formed2-4. The preparation has been used widely to study the biophysical properties of presynaptic ion channels and the regulation of transmitter release5-8. In addition, the preparation has lent itself to other uses including the study of neurite outgrowth and synaptogenesis9-12, molecular mechanisms of neurotransmitter release13-15, the role of diffusible messengers in neuromodulation16,17, and in vitro synaptic plasticity18-19.

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings.

からの同時プリとポストシナプスの電気生理学的記録<em&gt;アフリカツメガエル</em&gt;神経筋共培養

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges.&#160;Methods for performing this technique and the equipment necessary are described.&#160;Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

動物を振る舞う、覚醒中のシングルユニット記録のためにマイクロワイヤの組織化された配列を移植するための手順

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

神経発火率を操作することによってアストロサイト受容体の可塑性を誘導すること

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. 
Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Here we present a protocol that is based on spinal cord slices cultured on multi-electrode arrays to study functional regeneration of propriospinal connections in vitro.

マルチ電極アレイ上に成長させた器官脊髄共培養における機能的再生の調査

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

覚醒マウスにおける神経活性物質のMicroiontophoreticアプリケーションと組み合わせることで神経活動の細胞外記録

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

子宮内エレクトロポレーションは、神経部分集団の興奮とシングルセルの接続を研究するためのアプローチ

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity

There is strong evidence that macrophages can participate in the regeneration or repair of injured nervous system. Here, we describe a protocol in which macrophages are induced to produce conditioned medium (CM) that promotes neurite outgrowth. Adult dorsal root ganglion (DRG) neurons are acutely dissociated and plated. After the neurons are stably attached, peritoneal macrophages are co-cultured on a cell culture insert overlaid on the same well. Dibutyryl cyclic AMP (db-cAMP) is applied to the co-cultures for 24 h, after which the cell culture insert containing the macrophages is moved to another well to collect CM for 72 h. The CM from the co-cultures treated with db-cAMP, when applied to a separate adult DRG neuron culture, exhibits robust neurite outgrowth activity. The CM obtained from the db-cAMP-treated cultures consisting of single cell type alone, either DRG neuron or peritoneal macrophage, did not exhibit neurite outgrowth activity. This indicates that the interaction between neurons and macrophages is indispensable for the activation of macrophages secreting molecular factors with neurite outgrowth activity into CM. Thus, our co-culture paradigm will also be useful to study intercellular signaling in the neuron-macrophage interaction to stimulate the macrophages to be endowed with a pro-regenerative phenotype.

The current protocol presents experimental procedures to stimulate cultured macrophages to be endowed with capacity to release molecular factors that promote neurite outgrowth. Treatment of cAMP to the neuron-macrophage co-cultures induces the macrophages to produce conditioned medium that possesses strong neurite outgrowth activity.

アクティブに大食細胞の神経突起伸展作用と分子因子を分泌する神経細胞・ マクロファージ共培養

There is strong evidence that macrophages can participate in the regeneration or repair of injured nervous system. Here, we describe a protocol in which ...

GM-Free Generation of Blood-Derived Neuronal Cells

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other therapeutic strategies, there is currently no cure available for the injured or diseased brain. Cell replacement appears as a promising therapeutic strategy for neurodegenerative conditions. To this day, neural stem cells (NSCs) have been successfully generated from fetal tissues, human embryonic cells (ES) or induced pluripotent stem cells (iPSC). A process of dedifferentiation was initiated by activation of the novel human GPI-linked glycoprotein, which leads to generation of pluripotent stem cells. These blood-derived pluripotent stem cells (BD-PSCs) differentiate in vitro into cells with a neural phenotype as shown by brightfield and immunofluorescence microscopy. Ultrastructural analysis of these cells by means of electron microscopy confirms their primitive structure as well as neuronal-like morphology and subcellular characteristics.

We present a genetically modified-free (GM-free) method to obtain cells with a neuronal phenotype from reprogrammed peripheral blood cells. Activation of a signaling pathway linked to novel human GPI-linked protein reveals an efficient GM-free method for obtaining human pluripotent stem cells.

血液由来神経細胞のGMフリー生成

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other ...