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膜電位 1902年には早くも、バーンスタインは、イカの軸索の静止電位の問題を扱っていた。それは、バーンスタイン（1902）とネルンスト（1888）のこれらの初期のアイデアとの観測が、後膜の生理学の研究に影響を与えたかを考えるために興味深いです。 （MalmivuoとPlonsey、1995年までのレビューを参照してください。WWW上でも利用できる<a href="http://www.bem.fi/book/">http://www.bem.fi/book/</a> ）。イオンチャネルの機能と組織、器官およびシステムの機能に関する細胞生理を理解する上で非常に重要な生体膜の性質について行われているブレークスルーは、この日に、残されています。 外部の[K +]静止膜電位に関する実験的および理論的に派生効果の比較は、膜電位に対するイオンの影響を示している。これと同じ準備を使用して追加実験では、基本的な生理学的な質問に対処するために実行されずに残っている。いくつかは、アトウッドとパーナスによって1968年に戻って強調され、まだ完全に取り組むことにしました。この演習で得られた技術を使うと、1つは他の実験の準備にだけでなく、医療や健康に関連する生理的なアプリケーションの残りの多くの質問に答えるに進むことができます。我々はすべての動物に関連する基本的な質問に対処するためのモデルの無脊椎動物の準備の有用性を実証している。 この上記の練習中のイオンの電気化学的勾配で得た知識で、あなたは今ザリガニの神経筋の準備でシナプス伝達を調べることによって、膜の興奮性に進むことができます。 シナプス応答の測定この研究室の最初の部分、および関連する映画のために提供詳細は、膜電位を記録し、筋肉の構造を調査するための重要なステップを提供している。この研究室の第二部では、相動性および強直運動単位のNMJsで解剖し、記録シナプス伝達のデモでは、生理学の基本的な概念へのエクスポージャーを提供。無傷の動物の部分に関連した挙動を説明するために使用できることができる神経回路への暴露は、だけでなく、彼らの実験室での運動の中でも井の中の神経回路に関する今後の研究のための様々なオープンエンドの質問を調査する学生のための可能性がある確立された無脊椎動物の準備（Kennedy ら、1969;。アントンセンとエドワーズ、2003） これらの製剤はまた、シナプス促進、抑うつとの長期可塑性を（本研究室の研究で検討されていない）を調査するために使用することができます。だけでなく、その自然環境、にもザリガニのいくつかの種の中で、神経可塑性は、実験的な刺激条件（。Cooperら 、1998メルシエとアトウッド、1989）に依存します。どの程度にシナプス伝達効率と筋肉のダイナミクスを変更する機能は、動物を調査するまま提供しています。ザリガニの季節変動と脱皮周期に関連して、その動作を変更しないため、それらの神経筋システムにおいて比較的長期の活動の違いがあります。それは、爪に近い筋の相動運動神経の端末は冬の間に古典的な一過性の形態を示すが、夏季には端子の長さに沿ってより多くの静脈瘤（; Lnenickaと趙、1991 Lnenicka 1993）膨潤となることが示されている。 腹神経索内でザリガニ横の巨大な（LG）の介在で実施されたいくつかの初期の研究では、ギャップジャンクショ​​ン（;渡辺とGrundfest、1961年ジョンソン、1924）の存在を証明した。これはよくCO 2は、ギャップ結合を（ラーノら、1990）脱共役によって、電気通信に影響を及ぼすことが知られています。 Bierbowerとクーパー、、最近ではギャップジャンクションの存在を示すように、このレポートで説明されている感覚- CNS -モーター筋肉回路、2露出をCOにも敏感である、（Bierbower、2010内で神経索と通信することが示された2010） 1960同じまたは隣接するセグメント内の静的な筋肉の受容体の器官（MRO）を焼成トニックは、自発運動の駆動を占めることができればエッカート（1961）検査時なので第3 回運動根の自発的な活動が話題となっている。 （ストラウンら 、2000;ケネディと武田、1965a、B。エッカート、1961）これらの初期の研究では、アクティビティが可能性高い拠点から腹側神経索（VNC）内で駆動されたことが明らかとなった。 CO 2の存在は、自発的活動を停止して以来、人はギャップ結合またはグルタミン酸作動性興奮性ドライブがあるかもしれないどこかドライブの運動ニューロンに想定することができます。 NMJsを塞いだり、展示は、CO 2の存在下でグルタミン酸に感度を減少し、そして彼らは、ブロック可能性がありますされていますCNS内だけでなくヒツジシラミバエ（Bierbower、2010; Bierbowerとクーパー、2010;またBadre らを参照、2005）。 様々な神経修飾物質の作用も容易にNMJsの様々なタイプ（;グリフィスら、2000;。サウサードら、2000;。。ストラウンら 、2000年クーパーとクーパー、2009）で検討されている。さらに、様々な影響は中枢神経系の回路で神経修飾物質によって及ぼされている。それは5 - HTとoctopaminergicニューロンは神経回路の出力を変えることで&quot;ゲイン設定主体&quot;（Ma らとして機能する可能性が示唆されている、1992;。シュナイダーら、1996;。ホーナーら、1997;。エドワーズは、 ら 、2002）。我々は完全に個々の標的細胞に対する神経修飾物質の影響を理解する前に多くの作業が行われるままです。異なる神経修飾物質が互いに協調して動作する可能性があることを考えると、それらの混合作用の解析は今後の研究（Djokaj ら 、2001）のための領域です。さらに、いくつかの研究は、特に脊椎動物では、特定の行動を規制することができます全体の経路上の神経修飾物質の影響に対処する。この感覚- CNS -モータユニットの準備で1つは運動ニューロン（ケネディら 、1969）の活動に関する感覚入力と神経修飾物質の両方の影響を調べることができます。 （。。リビングストンら 、1980;スネッドンら 、2000）が5 - HTは、ザリガニ、ロブスター、カニの行動の状態を調節する役割を果たしていることが仮定されているので、いくつかの試みは、その濃度を決定するためになされているVNC、血リンパ、そしてロブスターの孤立神経節における（リビングストンら、1980;。ハリス、ウォリックとクラヴィッツ1984; Fadool ら、1988。）。しかし、行動のアクションを占めることができる特定の用量反応関係を排除し、記録された測定値にかなりのばらつきがあった。 上昇位置に保持されて爪を持つと、その腹部の下に隠れてテールとザリガニが支配する姿勢（リビングストンら 、1980）を示すと考えられてきた。ザリガニの腹部屈曲の状態が支配的な階層的な状態を（Listerman ら 、2000）を維持しながら、支配的なザリガニが、ペアの中で、社会的相互作用中に示すかという姿勢であることが表示されません。彼らは相手からの撤退として従順なザリガニは、さらに自分自身で自分の腹部を挟むだろう。このようなテール挟み込むも防衛態勢（Listerman ら 、2000）として見られている。これらの動作は容易に（; Bruskiとダナム、1987; Li ら、2000;。。Listermanら 、2000 Bovbjerg、1953、1956）フィールドと実験室の設定で観察されている。興味深いことに、ロブスター（リビングストンら 、1980）に記載の行動姿勢は5 - HTとオーストラリアのザリガニにおけるオクトパミン注射、Cheraxデストラクタ （マクレー、1996）用に反転されています。おそらく、全く別の応答は、オーストラリアのザリガニの浅指屈筋の準備で見られます。優位性は、一般的にザリガニの間で関連する大きさであるためさらに、1つは急速に変更の社会的条件（ストラウンら 、2000）のための非常に塑性応答システムを期待する。無脊椎動物における神経修飾物質に対する反応性の可塑性は、調査のオープンエリアです。 ウィッテンバッハ、ジョンソン、そしてホイ（1999）は、デジタルメディアや他のザリガニの準備に加えて、本レポートに記載され、同じ筋肉を含む様々なザリガニのexperimentationsための実験マニュアルを作成しました。これは、学生の演習のための優れたリソースです。

<ol><li>ザリガニ（ ザリガニ ）。アチャファラヤ生物サプライ（株）、レースランド、LA。、米国。 </li><li>標準的なザリガニ生理食塩水：ヴァンHarreveldのソリューション（1936）から変更された。 （mMで）205のNaCl、5.3 KClを、13.5のCaCl 2 2H 2 O、2.45のMgCl 2 · 6H 2 O、5 HEPES、pHを7.4に調整。セロトニン、グルタミン酸とドーパミンは、ザリガニの生理食塩水で作られています。ブアン固定液を直接使用したとメチレンブルーは、ザリガニの生理食塩水で作られています。すべての化学物質は、シグマケミカル社（セントルイス、MO）から入手されています。 </li><li>解剖ツール：ファイン＃5ピンセット、細かいはさみ、ナイフの刃ホルダー、＃26002から20虫ピン（すべてのファイン科学のツール（米国）、（株）、373 - Gビンテージパークドライブ、フォスターシティー、CA 94404から1139から取得） 。 </li><li> Sylgard底ガラス皿</li><li>ビーカー（薬液を保持する） </li><li>電気信号は、コンピュータにPowerLab 26Tインタフェース（ADInstruments、コロラドスプリングズ、コロラド州、米国）への行に記録されます。我々は、チャートまたはScopeというADInstrumentsから標準のソフトウェアを使用してください。 </li><li>細胞内だけでなく、細胞外記録のためのモデル3000 AC / DCアンプを使用することができます。 </li><li>細胞内記録用のズーム機能で顕微鏡を解剖。可視化端末上で焦点距離の記録のために直立の目標（4 ×と20倍）を持つ化合物の顕微鏡が使用されます。一つは、水銀光源を必要とします。 </li><li>細胞内記録のために我々はガラスキャピラリチューブ（FHC、ブランズウィック、ME、04011、アメリカ合衆国カタログ＃30-31-0）を使用します。細胞内電極は、20〜30オームの抵抗を持つ必要があります。細胞内電極は、3 MのKClを充填した。 </li><li>吸引電極は細胞外シグナルを記録するために使用されます。 </li><li>ファラデーケージ。 </li><li>高輝度の照明装置（光源）。 </li><li>マニピュレーター</li></ol>

membrane potentials, synaptic responses, neuronal circuitry, neuromodulation and muscle histology using the crayfish: student laboratory exercises

このレポートの目的は、生物学的膜を横切ってイオン勾配による影響の理解を深めることを支援することです。膜の外側に+ Kの（1）イオンの濃度、および特定のイオンに対する膜の（2）透過性：細胞の膜電位に影響を与えると我々はこれらの実験に取り組むその二つの側面があります。ザリガニ腹部伸筋は、それらの生化学的及び生理学的な表現型で、だけでなく、その構造にあるというトニック（遅い）と相性その他（高速）とのグループ内にあります。これらの筋肉を支配する運動ニューロンは機能的特徴に対応して異なっている。我々はこれらの筋肉だけでなく、シナプス伝達の特性を実証するために表面的、強壮腹部屈筋を使用。さらに、我々は回路の一部にはクチクラ感覚刺激の効果だけでなく、神経修飾物質の影響を実証するために感覚CNS -運動ニューロン筋の回路を紹介します。この演習で得られた技術を使うと、1つは他の実験の準備にだけでなく、医療や健康に関連する生理的なアプリケーションの残りの多くの質問に答えるために始めることができます。我々はすべての動物に関連する基本的な質問に対処するためのモデルの無脊椎動物製剤の有用性を実証している。

Watch this Scientific Journal Video about Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises at JoVE.com

Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

実験は、筋肉の構造、シナプス応答、イオン勾配や膜電位に対する透過性の影響を調べるには経験を積むために学生のための簡単​​なアプローチを示しています。また、感覚 - CNS -モーター筋回路は、神経回路上に化合物の影響をテストする手段を示すために提示されます。

膜電位、シナプス応答、神経回路、ニューロモデュレーションとザリガニを使用して筋肉の組織学：学生研究室の演習

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that ...

membrane-potentials-synaptic-responses-neuronal-circuitry

Research

JoVE Journal

Neuroscience

58.4K ビュー.  University of Kentucky. 実験は、筋肉の構造、シナプス応答、イオン勾配や膜電位に対する透過性の影響を調べるには経験を積むために学生のための簡単​​なアプローチを示しています。また、感覚 - CNS -モーター筋回路は、神経回路上に化合物の影響をテストする手段を示すために提示されます。

ビデオ: Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

Recording Behavioral Responses to Reflection in Crayfish

Social behavior depends on sensory input from the visual, mechanical and olfactory systems. One important issue concerns the relative roles of each sensory modality in guiding behavior. The role of visual inputs has been examined by isolating visual stimuli from mechanical and chemosensory stimuli. In some studies (Bruski &amp; Dunham, 1987: Delgado-Morales et al., 2004) visual inputs have been removed with blindfolds or low light intensity, and effects of remaining sensory modalities have been elucidated. An alternative approach is to study the effects of visual inputs in the absence of any appropriate mechanical and chemosensory cues. This approach aims to identify the exclusive role of visual inputs. 
We have used two methods to provide visual stimuli to crayfish without providing chemical and mechanical cues. In one method, crayfish are videotaped in an aquarium where half of the walls are covered in mirrors to provide a reflective environment, and the other half are covered in a non-reflective (matte finish) plastic. This gives the crayfish a choice between reflective and non-reflective environments. The reflective environment provides visual cues in the form of reflected images of the crayfish as it moves throughout half of the tank; these visual cues are missing from the non-reflective half of the tank. An alternative method is to videotape the behavior of crayfish in an aquarium separated by a smaller chamber at each end, with a crayfish in one small chamber providing visual cues and an inert object in the opposite small chamber providing visual input from a non-moving, non-crayfish source. 
Our published results indicate that responses of crayfish to the reflective environment depend on socialization and dominance rank. Socialized crayfish spent more time in the reflective environment and exhibited certain behaviors more frequently there than in the non-reflective environment; isolated crayfish showed no such differences. Crayfish that were housed in same-sex pairs developed a social rank of either dominant or subordinate. Responses to reflection differed between dominant and subordinate crayfish (May &amp; Mercier, 2006; May &amp; Mercier, 2007). Dominant crayfish spent more time on the reflective side, entered reflective corners more frequently and spent more time in reflective corners compared to the non-reflective side. Subordinate crayfish walked in reverse more often on the reflective side than on the non-reflective side. Preliminary data suggest similar effects from visual cues provided by a crayfish in a small adjoining chamber (May et al., 2008).

We have developed two methods for studying effects of visual cues on behavior in the absence of tactile and chemical cues. One method involves videotaping responses of crayfish to reflective walls in an aquarium; the other examines effects of visual inputs provided by a live crayfish behind a transparent partition.

ザリガニのリフレクションに行動反応の記録

Social behavior depends on sensory input from the visual, mechanical and olfactory systems.  One important issue concerns the relative roles of each ...

神経科学

Physiological Recordings of High and Low Output NMJs on the Crayfish Leg Extensor Muscle

We explain in detail how to expose and conduct electrophysiological recordings of synaptic responses for high (phasic) and low (tonic) output motor neurons innervating the extensor muscle in the walking leg of a crayfish. Distinct differences are present in the physiology and morphology of the phasic and tonic nerve terminals. The tonic axon contains many more mitochondria, enabling it to take a vital stain more intensely than the phasic axon. The tonic terminals have varicosities, and the phasic terminal is filiform. The tonic terminals are low in synaptic efficacy but show dramatic facilitated responses. In contrast, the phasic terminals are high in quantal efficacy but show synaptic depression with high frequency stimulation. The quantal output is measured with a focal macropatch electrode placed directly over the visualized nerve terminals. Both phasic and tonic terminals innervate the same muscle fibers, which suggests that inherent differences in the neurons, rather than differential retrograde feedback from the muscle, account for the morphological and physiological differentiation.

This article demonstrates how to conduct electrophysiological recordings of synaptic responses on the extensor muscle in the walking leg of a crayfish and how the nerve terminals are visualized to show the gross morphological differences of high- and low-output nerve terminals.

ザリガニの脚の伸筋に高と低出力NMJsの生理的録音

We explain in detail how to expose and conduct electrophysiological recordings of synaptic responses for high (phasic) and low (tonic) output motor ...

Muscle Receptor Organs in the Crayfish Abdomen: A Student Laboratory Exercise in Proprioception

The primary purpose of this experiment is to demonstrate primary sensory neurons conveying information of joint movements and positions as proprioceptive information for an animal. An additional objective of this experiment is to learn anatomy of the preparation by staining, dissection and viewing of neurons and sensory structures under a dissecting microscope. This is performed by using basic neurophysiological equipment to record the electrical activity from a joint receptor organ and staining techniques. The muscle receptor organ (MRO) system in the crayfish is analogous to the intrafusal muscle spindle in mammals, which aids in serving as a comparative model that is more readily accessible for electrophysiological recordings. In addition, these are identifiable sensory neurons among preparations. The preparation is viable in a minimal saline for hours which is amenable for student laboratory exercises. The MRO is also susceptible to neuromodulation which encourages intriguing questions in the sites of modulatory action and integration of dynamic signals of movements and static position along with a gain that can be changed in the system.

The primary purpose of this experiment is to understand how primary sensory neurons convey information of joint movements and positions as proprioceptive information for an animal. An additional objective of this report is present the anatomy of the preparation by dissection and viewing of neurons under a dissecting microscope.

ザリガニ腹部の筋肉の受容体の臓器：固有感覚の学生の研究室の演習

The primary purpose of this experiment is to demonstrate primary sensory neurons conveying information of joint movements and positions as proprioceptive ...

Physiological Experimentation with the Crayfish Hindgut: A Student Laboratory Exercise

The purpose of the report is to describe dissection techniques for preparing the crayfish hindgut and to demonstrate how to make physiological recordings with a force transducer to monitor the strength of contraction. In addition, we demonstrate how to visually monitor peristaltic activity, which can be used as a bioassay for various peptides, biogenic amines and neurotransmitters. This preparation is amenable to student laboratories in physiology and for demonstrating pharmacological concepts to students. This preparation has been in use for over 100 years, and it still offers much as a model for investigating the generation and regulation of peristaltic rhythms and for describing the mechanisms underlying their modulation. The pharmacological assays and receptor sub-typing that were started over 50 years ago on the hindgut still contribute to research today. This robust preparation is well suited to training students in physiology and pharmacology.

In this report we demonstrate techniques that can be used to investigate the biology of the crayfish hindgut. We show how to dissect a crayfish abdomen and study the associated anatomy, physiology and modulation of activity. The peristaltic activity and strength of contractions are measured using a force transducer.

ザリガニ後腸との生理学的実験：学生実験室実習

The purpose of the report is to describe dissection techniques for preparing the crayfish hindgut and to demonstrate how to make physiological recordings ...

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 ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

培養神経細胞における膜タンパク質の側方拡散とエキソサイトーシスは、蛍光回復と蛍光損失光退色を用いて評価

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

グルタミン酸とGABAの高速マイクロイオン導入：シナプス統合を調査するために便利なツール

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons responsible for proprioception as they are detecting joint position and movement, and muscle tension. Electrical activity from crustacean proprioceptors and tension receptors is recorded by basic neurophysiological instrumentation, and a transducer is used to simultaneously measure force that is generated by stimulating a motor nerve. In addition, we demonstrate how to stain the neurons for a quick assessment of their anatomical arrangement or for permanent fixation. Staining reveals anatomical organization that is representative of chordotonal organs in most crustaceans. Comparing the tension nerve responses to the proprioceptive responses is an effective teaching tool in determining how these sensory neurons are defined functionally and how the anatomy is correlated to the function. Three staining techniques are presented allowing researchers and instructors to choose a method that is ideal for their laboratory.

Physiological and anatomical techniques are demonstrated to address function and structure for joint proprioceptors and muscle tension receptors in crustacean walking limbs.

カニ手足の固有感覚と緊張受容体：学生実験室の練習

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

急性<em&gt;インビボ</em&gt;麻酔ラットにおけるHyperdirect経路からの局所場ポテンシャルとマルチユニット活性の電気生理学的記録

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

生体内培養-高速、シンプルで、安価な AWESAM プロトコルを使用してマウスのアストロ サイトのように

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...