この研究は、国立衛生研究所（グラント1UM1HL112856）によってサポートされていました。

全ての手順およびプロトコルはイリノイ大学シカゴ校の制度動物実験委員会によって承認された。ここに記載の実験は、急性非生存実験であり、眼軟膏の無駄がありませんでした。外科器具はDIH 2 O中70％エタノールで洗浄する際に無菌状態の維持にのみ発生実験の終了時にラットの犠牲は、IVケタミン/キシラジンの過剰摂取を経由して発生した。 楽器と化学物質の作製<ol><li> PBS中の5-HTのHClの0.05 Mのストック溶液を準備します。その後、0.203 mMの最終5-HT濃度にPBSで株式を希釈。 </li><li>を20μg/μlの濃度でごま油でドロナビノールを希釈します。 </li><li> （PE）-50チューブ（ID 0.58ミリメートル、外径0.965ミリメートル）ポリエチレン20cmの長さをカット。チューブの一端には、はさみを使ってベベルの先端を切断し、他の正方形の終わりに、23グラムの針を挿入します。この23グラムの針が1ミリリットルに接続します注射器、および50 U / mlのヘパリンの0.3ミリリットルでそれを埋める。 </li><li> 4-0編み絹糸の4枚をカットし、サイドに設定。 </li><li>のdiH 2 O中70％エタノールで2湾曲グレーフェ鉗子、及び3マイクロクランプを外科用ハサミを殺菌</li></ol>大腿静脈の2カテーテルカテーテル挿入のためのプロトコルは、イェスペルセンらから変更されている。21 <ol><li>腹腔内（IP）ケタミン/キシラジン（10 mg / kgを100ミリグラム/ kg）でのSprague-Dawleyラットを麻酔。ラットのつま先をつまんで麻酔の適切なレベルを確認するためにあらゆる動きを観察します。左大腿の腹側面、そして首の腹側面を剃る。地元の施設内動物管理使用委員会によって必要に応じて、乾燥を防ぐために、眼軟膏を適用します。 </li><li>外科ボード上の仰臥位でラットを固定します。左後肢大腿部に皮膚を切り取るために外科用ハサミを使用してください。 </li><li>鉗子を使用して、行動鈍いdissectio表在筋のnは大腿静脈を露出させる。大腿動脈から静脈を分離し、そして大腿静脈を中心に2つのスレッドを配置するために鉗子を使用してください。 </li><li>プルアップ静脈の血流を停止するように湾曲したグレーフェ鉗子を使用してください。 22 G注射針を使用して、大腿静脈を穿刺した後、静脈にPE-50チューブの斜角の端を挿入します。 </li><li> PE-50チューブは、注射器のプランジャーを後退させることによって、正しく挿入されたかどうかを確認してください。血液はPE-50チューブに入っ見られるべきである。 2つのスレッドを使用して静脈とPE-50チュービング約2ノットを結ぶ。 </li></ol> IV注入を介して3. 5-HT誘発性無呼吸<ol><li>呼吸を測定するためにラットを中心に、圧電ひずみゲージを配置します。 </li><li>コンピュータ上のアンプソフトウェアを介して電子増幅器の増幅レベル用の設定を変更します：（100倍）を増幅し、バンドパスフィルタ（1-10 Hz）のひずみゲージから得呼吸信号、＆＃で「highpasフィルタ」を設定する8220; AC @ 1 HZ」と「10ヘルツ」の「ローパスフィルタ」と入力して増幅を &quot;トータルゲイン」を「初期ゲイン」と「100」は「10」。 </li><li>コンピューター上の記録ソフトウェアを介して「アナログ入力」を開くことで、サンプリングレートの設定を変更します。 、アナログ - デジタル変換器を使用して（500 Hzのサンプリングレート）をデジタル化する「1000Hzの」の「サンプル」と「1」（有効サンプリングレートは500Hz）によって他のすべての記録点を「スキップ」を選択し、信号を記録する録音ソフトウェアを使用して。 </li><li>カテーテルから1ミリリットルの注射器を取り外し、カテーテルに5-HTの0.203 mMので満たされた500μlの精密ガラスシリンジを挿入します。 </li><li>輸液ポンプに500μlの精密ガラス注射器を置きます。 63ミリリットル/時の速度で350μL/ 5-HT液の1kgあたり12.5μgの/ kgで注入する。見られているラットにおける複数回の注入を行い、観察する無呼吸、コンピューターのモニターに記録された呼吸信号に（≥2.5秒）、呼吸の一時停止SAの</li><li>首の手術を進める前に、呼吸パターンを監視し、ラットにおける足指のピンチから痛みの反射をチェックする。呼吸が不規則であるか、またはつま先ピンチ痛み反射がある場合、IPケタミン/キシラジン管理する場合（100mg / kgの5ミリグラム/ kg）をした後、麻酔の適切なレベルを再確認する。 </li></ol> 4.頸部外科は節状神経節を露出させるために<ol><li>外科ボード上の仰臥位でラットを固定します。首の外科用ハサミを使用して正中縦カットを行います。 </li><li> （手術部位の明確なこの筋肉を維持するために2マイクロクランプを使用して）広頸筋筋に鈍的切開を使用して、sternohyoideusとomohyoideus筋肉を公開します。内頸動脈とその分岐の1つ、翼口蓋動脈を露出させるためにこれらの筋肉を分離する。 </li><li>それは内頸動脈に沿って実行され、目に沿ってように迷走神経を観察<html> Eの翼口蓋動脈。どのように迷走神経と翼口蓋動脈を観察し、舌咽神経（脳神経IX）および脊髄副神経（脳神経XI）と一緒に、頭蓋骨の基部に後部裂傷孔を入力してください。 </li><li>節状神経節は、それが後部引き裂か孔に入る直前に迷走神経の腫脹として表示されていることに注意してください。また、咽頭と喉頭神経枝は節状神経節の、それぞれ、前方および後方の側面を外れていることに注意してください。5 </li><li>グレーフェの鉗子を使用して、動脈から迷走神経を分離し、迷走神経の周りに糸の作品を配置。迷走神経に若干の緊張を適用するために、スレッド上のクランプを置き、注入少ない抵抗を提供するために、任意の結合組織の節状神経節をきれいに。 </li><li>損傷が節状神経節に行われなかったことを確認するために、セクション3で述べたように、5-HT誘発性無呼吸を繰り返します。 </li></ol>ドロナビノールのitle &quot;&gt; 5。Intranodose神経節インジェクション<ol><li>ごま油でドロナビノールの5μlの先端を斜めに35°のカスタムメイドの28グラム1/2インチ注射針を貼付10μlの気密精密ガラスシリンジを埋める。 </li><li>迷走神経に若干の緊張を適用するために、スレッド上にマイクロクランプを置き、それを通して穿刺しないように注意しながら節状神経節を穿刺。注射器の内容全体を注入するために、ゆっくりと注射器（≥60秒）を押します。注射器の内容の一部は神経節の外に漏れ出すことに注意してください。 </li><li> 3章で述べたように、5-HT誘発性無呼吸を繰り返します。 </li></ol>

<ol>
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The authors declare that they have no competing financial interests.

節状神経節への神経化学物質を正常に注入するための重要なステップは、1）特定し、節状神経節から結合組織を清掃する。 2）注入前の節状神経節の整合性を確認した。 3）と微妙に注入するための小さなゲージの針を用いて、しかし完全に、結節神経節を経て穿刺しない。 迷走神経は、首、腹部の多くの器官を神経支配、ならびにCNSへの心拍数、血圧、気管支肺の炎症...

Afferent signaling via the vagus nerve (cranial nerve X) transmits important general visceral information to the central nervous system (CNS) from baro-, chemo-, hepatic osmo-, cardiac, pulmonary, and gastric receptors located in the organs of the abdomen and thorax. The vagus nerve communicates information from stimuli such as heart rate, blood pressure, bronchopulmonary irritation, and gastrointestinal distension to the nucleus of solitary tract (NTS) of the medulla. The cell bodies of the pseudounipolar neurons of the vagus nerve are located in the nodose and petrosal ganglia, of which the majority is found in the former. Nodose ganglion cells contain a wealth of receptors for amino acids, monoamines, neuropeptides, and other neurochemicals that when activated, can modify afferent vagus nerve activity.1 Numerous innervations of the afferent vagus nerves coupled with the diversity of receptors located on the nodose ganglia illustrate the biological importance of this cranial nerve, and systemic drugs that do not cross into the CNS targeted at receptors on nodose ganglia can be used to treat various diseases, such as sleep apnea, gastroesophageal reflux disease, or chronic cough.2-4
The ease of access to the nodose ganglia lends itself to experimental manipulation by midline longitudinal incision made at the neck. The vagus nerve emerges from the posterior lacerated foramen at the base of the skull, and immediately displays a swelling of the nerve that is the nodose ganglion. The nodose ganglion is easily recognizable due to two nerve branches that arise from it: anteriorly the pharyngeal branch; and posteriorly superior laryngeal branch.5 Previous experimental manipulations of the nodose ganglia involved electrophysiological recordings,6 injections of immunohistochemical or immunofluorescent compounds for nerve tracings,7-10 superfusion or injections of neuroexcitotoxins,11-13 injections of adeno-associated virus to knockdown receptors,14,15 and injections of receptor-specific neurochemicals to change the activity of the vagus nerve.16,17
Systemic injections of neurochemicals are problematic in that systemic treatment affects both peripheral and central nervous systems. Thus, systemic treatment does not isolate the effect of neurochemicals on afferent vagal nerve activity. This protocol describes a method using readily available equipment of intranodose injections in the Sprague-Dawley rat that modulates vagus nerve activity without affecting the central nervous system. Stimulation of serotonin type 3 (5-HT3) receptors on nodose ganglia by intravenous (IV) infusion of serotonin (5-HT) induces the Bezold-Jarisch reflex, a vagal response trifecta of bradycardia, hypotension, and apnea, which can be abolished by supranodose vagatomy.11,17-19 Apnea is easily measured by placing a respiratory transducer around the abdomen of the rat.17,18 Cannabinoids decrease 5-HT-induced current in nodose ganglia cells,20 and intranodose ganglia injections of dronabinol attenuate 5-HT-induced apnea.

<table border="0" cellpadding="0" cellspacing="0" width="660">
	<tbody>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Name of Material/ Equipment</td>
			<td style="width:155px;">Company</td>
			<td style="width:141px;">Catalog Number</td>
			<td style="width:175px;">Comments/Description</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">5-HT HCl</td>
			<td style="width:155px;">MP Biomedicals</td>
			<td style="width:141px;">215376591</td>
			<td style="width:175px;">12.5 &#181;g/kg per 350 &#181;l/kg</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Dronabinol (Marinol) 10 mg Capsules (80 &#181;g/&#181;l)</td>
			<td style="width:155px;">AbbVie</td>
			<td style="width:141px;">NDC 0051-0023-21</td>
			<td style="width:175px;">Dilute with sesame oil to 20 &#181;g/&#181;l</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Sesame Oil</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:141px;">S3547</td>
			<td style="width:175px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Intramedic Polyethylene-50</td>
			<td style="width:155px;">BD</td>
			<td style="width:141px;">427411</td>
			<td style="width:175px;">Ordered from VWR (Cat. # 63019-047)</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Graefe Forceps</td>
			<td style="width:155px;">Roboz</td>
			<td style="width:141px;">RS-5138</td>
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a method of nodose ganglia injection in sprague-dawley rat

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located in the organs of the abdomen and thorax. The vagus nerve communicates information from stimuli such as heart rate, blood pressure, bronchopulmonary irritation, and gastrointestinal distension to the nucleus of solitary tract of the medulla. The cell bodies of the vagus nerve are located in the nodose and petrosal ganglia, of which the majority are located in the former. The nodose ganglia contain a wealth of receptors for amino acids, monoamines, neuropeptides, and other neurochemicals that can modify afferent vagus nerve activity. Modifying vagal afferents through systemic peripheral drug treatments targeted at the receptors on nodose ganglia has the potential of treating diseases such as sleep apnea, gastroesophageal reflux disease, or chronic cough. The protocol here describes a method of injection neurochemicals directly into the nodose ganglion. Injecting neurochemicals directly into the nodose ganglia allows study of effects solely on cell bodies that modulate afferent nerve activity, and prevents the complication of involving the central nervous system as seen in systemic neurochemical treatment. Using readily available and inexpensive equipment, intranodose ganglia injections are easily done in anesthetized Sprague-Dawley rats.

図1は、ドロナビノールのintranodose節注射の前と後の無呼吸を誘導するために5-HTの注入を有していたラットのサンプル呼吸記録を表す。 5-HTは、ドロナビノールの11,17-19 Intranodose神経節の注射は抑制性のCB受容体を活性化する。徐脈、低血圧、及び無呼吸のベツォルト-ヤーリッシュ反射に貢献節状神経節に対する5-HT 3受容体を活性化し、またはアロステリックに5-HT...

Watch this Scientific Journal Video about A Method of Nodose Ganglia Injection in Sprague-Dawley Rat at JoVE.com

A Method of Nodose Ganglia Injection in Sprague-Dawley Rat

Afferent vagal signaling transmits important information to central nervous system from receptors located in organs of the abdomen and thorax. The nodose ganglia of vagus nerves contain many types of receptors that modulate vagal activity. This protocol describes a method of local injections of neurochemicals into the nodose ganglia.

スプラーグ - ドーリーラットのメソッド節状神経節の注入

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located ...

a-method-of-nodose-ganglia-injection-in-sprague-dawley-rat

Research

JoVE Journal

Neuroscience

18.5K ビュー.  University of Illinois at Chicago. Afferent vagal signaling transmits important information to central nervous system from receptors located in organs of the abdomen and thorax. The nodose ganglia of vagus nerves contain many types of receptors that modulate vagal activity. This protocol describes a method of local injections of neurochemicals into the nodose ganglia.

ビデオ: A Method of Nodose Ganglia Injection in Sprague-Dawley Rat

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

非心室プラスミド注入し、エレクトロポレーションにより産後のラット脳への遺伝子デリバリー

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

神経科学

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 ...

Minimally-invasive Technique for Injection into Rat Optic Nerve

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central nervous system in a minimally-invasive surgery without bone removal. This technique describes an approach to visualization and direct injection of the optic nerve following minor fascial dissection from the orbital ridge, using a conjunctival traction suture to gently pull the eye down and out. Representative examples of an injected optic nerve show successful injection of dyed beads.

Direct injection into the rat optic nerve is useful for regenerative research. We demonstrate a minimally-invasive technique for direct injection into a rat optic nerve that does not involve opening the skull. Using this method, surgical complications are minimized and recovery is more rapid.

ラット視神経への注入のための低侵襲テクニック

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central ...

A Rat Model of Central Fatigue Using a Modified Multiple Platform Method

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed as a water tank with narrow platforms on the bottom. The model rats were put into the tank and stood on the platforms for 14 h (18:00 - 8:00) per day for a consecutive 21 days, with a blank control group set for contrast. At the end of modeling, rats in the model group showed an obvious fatigued appearance. To assess the model, we performed several behavioral tests, including the open field test (OFT), the elevated plus maze (EPM) test, and the exhaustive swimming (ES) test. The results showed that anxiety, spatial cognition impairment, poor muscle performance, and declined voluntary activity presented in model rats confirm the diagnosis of&#160;central fatigue. Changes of the central neurotransmitters also verified the result. In conclusion, the model successfully simulated central fatigue, and future study with the model may help reveal the pathological mechanism of the disease.

Here, we present a protocol to introduce a rat model of central fatigue using the modified multiple platform method (MMPM).

ラット モデル改良を使用して中枢性疲労の複数プラットフォーム メソッド

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed ...

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

新鮮凍結ひと組織から腸管神経節の隔離のためのレーザー キャプチャ レーザーマイクロダイ セクションの最適化

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications.

レンチ ウイルスのベクトルの直噴ハイライト ラット脊髄における複数の運動経路

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

E14-E16スプラーグドーリーラット胚から分離された混合原発海馬および皮質ニューロンからの神経球の生成

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

Culturing Rat Sympathetic Neurons from Embryonic Superior Cervical Ganglia for Morphological and Proteomic Analysis

Sympathetic neurons from the embryonic rat superior cervical ganglia (SCG) have been used as an in vitro model system for peripheral neurons to study axonal growth, axonal trafficking, synaptogenesis, dendritic growth, dendritic plasticity and nerve-target interactions in co-culture systems. This protocol describes the isolation and dissociation of neurons from the superior cervical ganglia of E21 rat embryos, followed by the preparation and maintenance of pure neuronal cultures in serum-free medium. Since neurons do not adhere to uncoated plastic, neurons will be cultured on either 12 mm glass coverslips or 6-well plates coated with poly-D-lysine. Following treatment with an antimitotic agent (Ara-C, cytosine &#946;-D-arabinofuranoside), this protocol generates healthy neuronal cultures with less than 5% non-neuronal cells, which can be maintained for over a month in vitro. Although embryonic rat SCG neurons are multipolar with 5-8 dendrites in vivo; under serum-free conditions, these neurons extend only a single axon in culture and continue to be unipolar for the duration of the culture. However, these neurons can be induced to extend dendrites in the presence of basement membrane extract, bone morphogenetic proteins (BMPs), or 10% fetal calf serum. These homogenous neuronal cultures can be used for immunocytochemical staining and for biochemical studies. This paper also describes optimized protocol for immunocytochemical staining for microtubule associated protein-2 (MAP-2) in these neurons and for the preparation of neuronal extracts for mass spectrometry.

This paper describes the isolation and culturing of embryonic rat sympathetic neurons from the superior cervical ganglia. It also provides detailed protocols for immunocytochemical staining and for preparing neuronal extracts for mass spectrometric analysis.

形態学およびプロテオーム解析のための胚性子宮頸部神経節からのラット交感神経の培養

Sympathetic neurons from the embryonic rat superior cervical ganglia (SCG) have been used as an in vitro model system for peripheral neurons to study ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered tissue samples. Traditional immunolabeling procedures require cutting the sample into thin sections, which restricts the ability to label and examine intact structures. However, if brain tissue can remain intact during processing, structures and circuits can remain intact for the analysis. Previously established clearing methods take significant time to completely clear the tissue, and the harsh chemicals can often damage sensitive antibodies. The iDISCO method quickly and completely clears tissue, is compatible with many antibodies, and requires no special lab equipment. This technique was initially validated for the use in mice tissue, but the current protocol adapts this method to image hemispheres of control and transgenic rat brains. In addition to this, the present protocol also makes several adjustments to preexisting protocol to provide clearer images with less background staining. Antibodies for Iba-1 and tyrosine hydroxylase were validated in the HIV-1 transgenic rat and in F344/N control rats using the present hydrophobic tissue clearing method. The brain is an interwoven network, where structures work together more often than separately of one another. Analyzing the brain as a whole system as opposed to a combination of individual pieces is the greatest benefit of this whole brain clearing method.

Here we present a hydrophobic tissue clearing method that allows for the viewing of target molecules as part of intact brain structures. This technique has now been validated for F344/N control and HIV-1 transgenic rats of both sexes.

ラット脳組織の疎水性組織除去法

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...

Establishment of a Rat Model of Superior Sagittal-Sinus Occlusion via a Thread-Embolism Method

The mechanisms contributing to the natural onset of cerebral venous sinus thrombosis (CVST) are mostly unknown, and a variety of uncontrollable factors are involved in the course of the disease, resulting in great limitations in clinical research. Therefore, the establishment of stable CVST animal models that can standardize a variety of uncontrollable confounding factors have helped to circumvent shortcomings in clinical research. In recent decades, a variety of CVST animal models have been constructed, but the results based on these models have been inconsistent and incomplete. Hence, in order to further explore the pathophysiological mechanisms of CVST, it is necessary to establish a novel and highly compatible animal model, which has important practical value and scientific significance for the diagnosis and treatment of CVST. In the present study, a novel Sprague-Dawley (SD) rat model of superior sagittal sinus (SSS) thrombosis was established via a thread-embolization method, and the stability and reliability of the model were verified. Additionally, we evaluated changes in cerebral venous blood flow in rats after the formation of CVST. Collectively, the SD-rat SSS-thrombosis model represents a novel CVST animal model that is easily established, minimizes trauma, yields good stability, and allows for accurately controlling ischemic timing and location.

Here, we establish a novel Sprague-Dawley (SD) rat model of superior sagittal sinus (SSS) thrombosis via a thread-embolization method, and the stability and reliability of the model were verified.

スレッド塞栓法による上矢状・中症のラットモデルの確立

The mechanisms contributing to the natural onset of cerebral venous sinus thrombosis (CVST) are mostly unknown, and a variety of uncontrollable factors ...