このプロトコルは、国家自然科学基金、中国の (#31171066 #81270464) と中国とドイツ科学センター (GZ919) からの研究補助金によって支えられました。

ここで報告した実験的なプロトコルは、動物倫理委員会の上海交通大学医学部 (# SYXK2013-0050) によって承認されているそのまま神経節と神経大腸の解剖。トランクは、この手法ではかなり経験豊富な人のため 15 分の最小値を取る。それ以降の電気生理学的記録のための組織の生存を確保するため、解剖を実行しながら深麻酔下で動物の生きているが、維持が必要です。
 1 灌流液の準備とテスト混合物 <ol>クレブス ソリューションの準備 5 L <li>: 113 mM の NaCl、KCl、1.2 mM NaH 2 PO 4、1.2 mM MgSO 4、5.9 mM NaHCO 3、25 mM 1.2 mM CaCl 2。 と 11.5 mM グルコース。95% O 2 + 5% CO 2 ガス混合ソリューションを飽和させます。冷蔵庫の中に酸素のクレブスの ~ 500 mL をあらかじめ冷たい</li>。
	必要に応じて、株式の <li>準備因数は化合物 (エタノールと 10 mM 5-ヒドロキシトリプタミン (セロトニン) 生理食塩水での 1 mM カプサイシン) をテストします。クレブス (お風呂用) 及び (腔内管理) 用生理食塩水のストックを最終濃度使用する直前に希釈します</li>。
</ol> 2。記録電極の作製 <ol><li>プル標準的なガラスから記録電極チューブ内部のフィラメント (1.5 mm 外径) 従来の電極の引き手を使用してなし。熱のための引き手設定を調整し、引き出し、引っ張られた電極のシャンクは 20 と 25 mm.</li>
</ol> 3。組織コレクション <ol><li>Anaesthetize ラット深くペントバルビ タール ナトリウム (80 mg/kg, i. p.) を使用します。</li> <li>無菌性を確保しながらメスを使用して腹壁の正中切開を実行することによって腹腔内を公開します。腸と大腸を公開する他の組織は脇をプルします</li>。
	<li>は、解剖顕微鏡の下で動物を配置します。慎重な郭清を左の PG を探し、PN とそれに参加する LSN を識別します。これらの神経の項から数ミリをカット 
	注: PG は大腸の交差点近くにあります。PG ( 図 1) を腰仙部脊髄および LSN プロジェクトから通常、3-4 PN</li>。
	<li>は、直腸を公開する恥骨の骨をカットしました。大腸; 上記組織 (すなわち 膀胱等) を削除します。PG をそのまま残すように注意してください</li>。
	<li>は、ペントバルビ タールの過量の静脈内注入によるラットを犠牲します。トランセクト コロン PG の上約 3 cm と鉗子を用いた動物から大腸を削除します</li>。
	<li>は、予冷のクレブス液でいっぱいペトリ皿に大腸を転送します。糞便を削除するには、コロンを優しく洗浄します。残尿膀胱や他の組織を項を損なうことがなく、慎重に削除</li>
</ol> 4。結腸求心性神経の解剖 <ol><li>記録室 (20 mL) にコロンを配置し、carbogenated クレブス ソリューションを継続的に組織を灌流します。15 mL/分で灌流率を設定</li>
	<li>は、口腔と肛門の両端に大腸を cannulate します。アナル方向に口頭で 10 mL/h の速度で生理食塩水で結腸腔内注入を開始します</li>。
	<li>は、解剖顕微鏡の下で主要な PG を探します。昆虫ピンを利用して、神経節を公開します。慎重な郭清を伴う神経神経節から発せられるとコロン ( 図 1) に向かって実行しているの良い枝を見つけます。神経節の近くに神経をカットします 。
	注: 図 1 に示します、PG に末梢神経の枝からの録音神経おそらく骨盤と腰椎の内臓求心性線維が混在しています。PN や、PG に近位の LSN から録音が可能また、</li>
	<li>熱浴と暖かい 34 ± 0.5 ° C でチャンバー内の温度を保つためにクレブス ソリューションをオン</li>
</ol> 5。吸引電極の作製 <ol><li>予ガラス ピペット (手順 2) を取るし、解剖顕微鏡の下で検査します。鉗子のペアで電極の先端を破る記録される神経の径と互換性のあるサイズのだ</li>。
	<li>軽くフレアの近くに配置することによって先端の面取りします</li>。
</ol> 6。電気生理学的記録 <ol><li>接続電極ホルダーに斜め電極。10 mL のシリンジを電極に正または負の圧力を適用するホルダー側ポートに接続します</li>。
	<li>ホルダーを bioamplifier のパッチアンプ用アダプターに接続し、マニピュレーターにパッチアンプ用アダプターをマウントします</li>。
	<li>は、組織お風呂に電極を移動し、ホルダーの銀線接触するまで穏やかな吸引を適用することによってクレブス ソリューションと電極を入力します。神経の断端に近い電極を置き、電極に神経を吸うため否定的な圧力を適用します。否定的な圧力を適用して神経の 〜 1 mm の電極に引かれ、タイトなシールを形成します</li>。
	<li>ターン bioamplifier とセットのフィルター 300 3,000 Hz。 オシロ スコープで信号を監視し、神経信号 (20 kHz のサンプリング レート) とスパイク データの処理にコンピューターを使用して管腔内圧力信号 (100 Hz サンプリング レート) を記録ソフトウェアします</li>。
</ol> 7。結腸求心性感受性検定 継続的に intraluminally を注入しながらコンセント カニューレの 3 ウェイ タップを閉じることによってコロンの <ol><li>適用ランプ膨満。排水のカニューレに 3 ウェイ タップ オープン時 60 mmHg に到達するまでに内圧を監視します</li>。
	<li>15 分適用薬余分な - または求心性神経の化学物質過敏症をテストする intraluminally の一定の間隔でこの手順を繰り返します</li>。
</ol>

<ol>
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著者は利益相反を宣言しません。

ここで提示されたプロトコルは、ラットの結腸求心性神経の電気生理学的特性を評価するために比較的簡単な実験です。(神経記録を設定する組織郭清) からプロトコルを完了する約 2 時間がかかります。組織コレクション (ステップ 3) 吸引電極 (手順 5) の作成は、重要なステップ。PG、LSN、PN を特定し、組織解剖時に神経節と神経を損傷しないように注意することが重要です。ガラス ピペッ?...

消化管 (GIT の)、豊かな中枢神経系に腸からの感覚信号を伝えることと、腸と脳との対話に貢献する外因性の求心性神経で支配されています。これらの外因性求心性神経の変更された興奮性および求心性入力の変更された中央処理内臓痛と機能と炎症性腸疾患1を含む GI 条件の他の症状に基づいています。大腸からの感覚情報は、主に胸腰椎/下腹と腰仙部/骨盤神経 (PN)2を通じて伝達されます。齧歯動物疾患モデルでこれらの一次求心性線維の電気生理学的性質の研究に関心の高まりがあった。しかし、齧歯動物で結腸求心性神経の電気生理学的記録体内は技術的な課題は、手術のかなりのスキルが必要です。さらに、血行動態、組織の動き、および麻酔薬も影響を与えます神経活動と体内の刺激テストする感度。したがって、近年、研究の増加する数は生体外で(前のヴィヴォ) 準備人間、モルモット、マウス、ラットなど種の結腸における感覚情報伝達のメカニズムを調べるを採用しました。求心性神経と病気の条件に変更された興奮性。3,4,5,6,7,8
前のヴィヴォ下部消化管手術の 2 種類が主に報告されている:「フラット シート」準備5,9,10 「チューブ」準備3,4。「フラット シート」マウス大腸準備のためビデオ プロトコルは、以前に発行された11をされています。このプロトコルはマウス大腸、pn) で接続、腰部の内臓神経 (LSN) は収穫と隔組織の商工会議所または。大腸は縦、切り開かれ、神経束は、パラフィン オイルが充填された記録のコンパートメントに拡張されます。モノポーラ プラチナ イリジウム電極を用いた神経活動が記録されます。プロトコルは個々 の求心性線維の受容野の同定のため公平な電気刺激によることができます。それは化学的刺激のアプリケーションだけでなく、異なる機械的刺激パラダイムのアプリケーションをローカライズする (例えば、焦点粘膜プロービングと円周方向引張)、求心性神経終末へ。神経は、組織室から別の部屋に拡張する必要があります、ので比較的長い; 添付の神経を維持する重要です。神経の正常解剖この方法に新しいものに問題が生じます。最近では、Nullensらは、マウスの腸と大腸セグメント12求心系の腸間膜の培養記録用ビデオ プロトコルを公開しました。この「チューブ」準備の添付腸間膜を持つ腸セグメントはそのままに、こうして傾斜膨満感と異なる化学物質の内、余分な内腔の管理を可能にする保持されます。組織の近くに置くことができる、吸引電極を用いた腸間膜神経が記録されますので、腸間膜神経は比較的短いにもかかわらず、求心性活動を記録できます。しかし、腸間膜神経が腸を支配する迷走神経および脊髄の求心性線維の混合集団の構成や胸腰椎下腹。腰部骨盤求心性神経は、このプロトコルで差別されることはできませんが大腸を刺激する.ここでは、そのままのページで「チューブ」大腸製剤を用いたラット結腸求心性神経の電気生理学的記録のための詳しいプロトコルを提案します。この方法は内臓腰椎の機能特性の評価ができる (下腹) と腰仙部骨盤求心性神経。

<table width="889">
	<tbody>
		<tr height="40">
			<td height="40" style="width:215px;height:40px;">Sodium Pentobarbital</td>
			<td style="width:123px;">Shanghai Westang Bio-Tech</td>
			<td style="width:344px;">B558</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Capsaicin</td>
			<td>Sigma</td>
			<td>M2028</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electrode puller</td>
			<td>MicroData Instrument Inc</td>
			<td>PMP107</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Neurolog System (Bioamplifier)</td>
			<td>Digitimer, Ltd</td>
			<td>Neurolog System</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">A/D converter</td>
			<td>Cambridge Electronic Design</td>
			<td>Micro1401</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Data processing software</td>
			<td>Cambridge Electronic Design</td>
			<td>Spike2 version 6</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Silver wire</td>
			<td>World Precision Instruments</td>
			<td>EP12</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glass tubes</td>
			<td>World Precision Instruments</td>
			<td>1B150-4</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electrode holder</td>
			<td>World Precision Instruments</td>
			<td>MEH3SBW</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Heating bath</td>
			<td>Grant</td>
			<td>GR150</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dissecting microscope</td>
			<td>Leica</td>
			<td>Zoom2000</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dissecting microscope</td>
			<td>World Precision Instruments</td>
			<td>PZMIII-BS</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cigarette lighter</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Surgical tools</td>
			<td>World Precision Instruments</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Insect pins</td>
			<td>home-made from 0.1 mm stainless steel wire</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Three way manipulator</td>
			<td>World Precision Instruments</td>
			<td>KITF-R</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Rats</td>
			<td>Any</td>
			<td>NA</td>
			<td>Any strain/sex can be used.</td>
		</tr>
	</tbody>
</table>

in vitro characterization of the electrophysiological properties of colonic afferent fibers in rats

大腸の感覚神経の機能不全は、いくつかの一般的な条件、機能と炎症性腸疾患、糖尿病などの病態に関与しています。ここでは、ラットの結腸求心性神経の電気生理学的特性の in vitro評価プロトコルについて述べる。アタッチすると、そのまま骨盤神経節 (PG) と、大腸がラット;隔 carbogenated 録音室でクレブス ソリューション膨満感を許可する口腔と肛門の端に ● キャニュレイテッド。PG から発せられる細かい神経束が識別され、吸引電極を用いたニューロン求心性神経活動が記録されます。大腸のセグメントの膨満は、ニューロンの放電の漸進的な増加を引き出します。主成分分析は、低閾値、高しきい値、広ダイナミック レンジの求心性線維を区別するために行われています。テスト混合物のバスタブまたは腔内投与による結腸求心性神経の化学物質過敏症を学ぶことができます。このプロトコルは、他の種、マウスやモルモットなどと正常で下行結腸の胸腰椎/下腹と腰仙部/骨盤求心性神経の電気生理学的特性の差異を検討するためにアプリケーションで変更できるし、病理学の条件。

図 1は、前のヴィヴォ「チューブ」大腸の準備に、PG に遠位神経から記録担当者と実験装置の模式図神経は、骨盤、腰椎の内臓求心性神経の混合物をおそらく含まれています。正常ラットから標本、結腸求心性神経は通常不規則な自発活動の低レベルを持ってください。コロンのランプを膨満感は、発火率 (図 1 b) の...

Watch this Scientific Journal Video about In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats at JoVE.com

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

感覚機能の異常は、内臓の痛みや機能および炎症性腸病気の他の症状に基づいています。に備えて、前のヴィヴォラット大腸結腸求心性神経の電気生理学的記録のためのプロトコルを次に示します。

体外ラットの結腸求心性線維の電気生理学的特性の評価

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory ...

in-vitro-characterization-electrophysiological-properties-colonic

Research

JoVE Journal

Neuroscience

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

6.8K ビュー.  Shanghai Jiaotong University School of Medicine. 感覚機能の異常は、内臓の痛みや機能および炎症性腸病気の他の症状に基づいています。に備えて、前のヴィヴォラット大腸結腸求心性神経の電気生理学的記録のためのプロトコルを次に示します。

ビデオ: In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

ラットのショ糖の渇望のインキュベーションを評価するための一般的な方法

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

神経科学

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

無傷の網膜におけるGFP発現細胞集団の電気生理学的特性

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

でProprioceptorsの可視化<em&gt;ショウジョウバエ</em&gt;幼虫および蛹

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

急性海馬スライスにおけるシナプス誘発アストログリアとニューロン活動の二重の電気生理学的記録

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling of auditory axons within the VIIIth cranial nerve would enhance the study of central auditory circuit development. This approach is challenging because multiple sensory organs of the inner ear contribute to the VIIIth nerve 1. Moreover, markers that reliably distinguish auditory versus vestibular groups of axons within the avian VIIIth nerve have yet to be identified. Auditory and vestibular pathways cannot be distinguished functionally in early embryos, as sensory-evoked responses are not present before the circuits are formed. Centrally projecting VIIIth nerve axons have been traced in some studies, but auditory axon labeling was accompanied by labeling from other VIIIth nerve components 2,3. Here, we describe a method for anterograde tracing from the acoustic ganglion to selectively label auditory axons within the developing VIIIth nerve. First, after partial dissection of the anterior cephalic region of an 8-day chick embryo immersed in oxygenated artificial cerebrospinal fluid, the cochlear duct is identified by anatomical landmarks. Next, a fine pulled glass micropipette is positioned to inject a small amount of rhodamine dextran amine into the duct and adjacent deep region where the acoustic ganglion cells are located. Within thirty minutes following the injection, auditory axons are traced centrally into the hindbrain and can later be visualized following histologic preparation. This method provides a useful tool for developmental studies of peripheral to central auditory circuit formation.

Here we describe a microdissection technique followed by fluorescent dye injection into the acoustic ganglion of early chick embryos for selective tracing of auditory axon fibers in the nerve and hindbrain.

鳥類胚性内耳神経における聴覚繊維の選択的なトレース

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

無傷アダルトゼブラフィッシュの脳における電気生理学的記録

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional afferent fibers monitor other characteristics of the muscle environment, including metabolite buildup, temperature, and nociceptive stimuli. Overall, abnormal activation of sensory neurons can lead to movement disorders or chronic pain syndromes. We describe the isolation of the extensor digitorum longus (EDL) muscle and nerve for in vitro study of stretch-evoked afferent responses in the adult mouse. Sensory activity is recorded from the nerve with a suction electrode and individual afferents can be analyzed using spike sorting software. In vitro preparations allow for well controlled studies on sensory afferents without the potential confounds of anesthesia or altered muscle perfusion. Here we describe a protocol to identify and test the response of muscle spindle afferents to stretch. Importantly, this preparation also supports the study of other subtypes of muscle afferents, response properties following drug application and the incorporation of powerful genetic approaches and disease models in mice.

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons are involved in proprioceptor signaling and also report on metabolic state and injury related events. We describe an adult mouse in vitro muscle-nerve preparation for studies on stretch-activated muscle afferents.

アン<em&gt;体外</em筋求心性神経の焼成特性を研究するための&gt;成体マウス筋肉神経の準備

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional ...

In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments

Afferent nerves not only convey information concerning normal physiology, but also signal disturbed homeostasis and pathophysiological processes of the different organ systems from the periphery towards the central nervous system. As such, the increased activity or &#39;sensitization&#39; of mesenteric afferent nerves has been allocated an important role in the pathophysiology of visceral hypersensitivity and abdominal pain syndromes.
Mesenteric afferent nerve activity can be measured in vitro in an isolated intestinal segment that is mounted in a purpose-built organ bath and from which the splanchnic nerve is isolated, allowing researchers to directly assess nerve activity adjacent to the gastrointestinal segment. Activity can be recorded at baseline in standardized conditions, during distension of the segment or following the addition of pharmacological compounds delivered intraluminally or serosally. This technique allows the researcher to easily study the effect of drugs targeting the peripheral nervous system in control specimens; besides, it provides crucial information on how neuronal activity is altered during disease. It should be noted however that measuring afferent neuronal firing activity only constitutes one relay station in the complex neuronal signaling cascade, and researchers should bear in mind not to overlook neuronal activity at other levels (e.g., dorsal root ganglia, spinal cord or central nervous system) in order to fully elucidate the complex neuronal physiology in health and disease.
Commonly used applications include the study of neuronal activity in response to the administration of lipopolysaccharide, and the study of afferent nerve activity in animal models of irritable bowel syndrome. In a more translational approach, the isolated mouse intestinal segment can be exposed to colonic supernatants from IBS patients. Furthermore, a modification of this technique has been recently shown to be applicable in human colonic specimens.

In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments

Mesenteric afferent nerves convey information from the gastrointestinal tract towards the brain regarding normal homeostasis as well as pathophysiology. Gastrointestinal afferent nerve activity can be assessed by mounting isolated intestinal segments with attached afferent nerves into an organ bath, isolating the nerve, and assessing basal as well as stimulated activity.

マウス空腸および結腸セグメントにおける腸間膜求心性神経活動のin vitro記録で

Afferent nerves not only convey information concerning normal physiology, but also signal disturbed homeostasis and pathophysiological processes of the ...

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 ...

Wireless Electrophysiological Recording of Neurons by Movable Tetrodes in Freely Swimming Fish

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain activity from freely moving fish would advance research on the neural basis of fish behavior considerably. Moreover, precise control of the recording location in the brain is critical to studying coordinated neural activity across regions in fish brain. Here, we present a technique that records wirelessly from the brain of freely swimming fish while controlling for the depth of the recording location. The system is based on a neural logger associated with a novel water-compatible implant that can adjust the recording location by microdrive-controlled tetrodes. The capabilities of the system are illustrated through recordings from the telencephalon of goldfish.

A novel wireless technique for recording extracellular neural signals from the brain of freely swimming goldfish is presented. The recording device is composed of two tetrodes, a microdrive, a neural data logger, and a waterproof case. All parts are custom-made except for the data logger and its connector.

自由に泳ぐ魚における可動テトロデスによるニューロンの無線電気生理学的記録

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain ...