著者らは、ハートウィグ・ヴィーボルトの優れた技術支援と、ハンブルク・エッペンドルフ大学医療センターのUKE顕微鏡イメージング施設(Umif)に、顕微鏡とサポートを提供してくれたことに感謝したいと考えています。この研究は、DZHK(ドイツ心臓血管研究センター)[FKZ 81Z4710141]によって資金提供されました。

著者らは開示するものは何もない。

突然の心停止が世界的に最も一般的な死因であり続けるため、VAの発症に先立つ交感神経系のニューロンおよびグリア細胞における細胞および分子プロセスの理解は高い関心を持つ。そこで、現在の原稿では、このネットワーク内のマウスエレメントであるマウスSGを同定し、RNA、タンパク質、細胞レベルでのその後の分析を行う方法の基本的なレパートリーを提供していま...

心臓自律神経系は、交感神経、副交感神経、感覚成分の緊密に調整された平衡であり、心臓が適切な生理学的応答を有する環境変化に適応することを可能にする1,2。この平衡における障害は、例えば、交感神経活動の増加、心室性不整脈(VA)3、4の維持と同様に発症の重要な促進要因として確立されている。従って、自律的変調は、β遮断薬による交感神経活性の薬理学的減少を通じて達成され、数十年5、6年のVA患者の治療の基礎となっている。しかし、薬理学的およびカテーテルベースの介入にもかかわらず、関連する数の患者はまだ再発VA7に苦しんでいる。
心臓への交感神経入力は、主に星状神経節(SG)の神経細胞体を介して媒介され、交感神経鎖の両側の星形構造は、脳幹から心臓8、9、10に多数の胸腔内神経を介して情報を中継する。傷害後にSGから発芽した神経はVAと突然心死11、12に関連し、自律変調13、14の標的としてSGを強調する。心臓への交感神経入力の減少は、局所麻酔薬の経皮的注入を介して一時的に、またはビデオ支援胸腔鏡15、16を介してSGの部分的除去によって永久に達成することができる。心臓交感神経脱用は、有望な結果を有する治療難治性VA患者のためのオプションを提示する14,16,17.我々は、神経および神経化学的リモデリング、神経炎症およびグリア活性化が自律神経機能障害18、18を寄与または悪化させる可能性のある交感神経リモデリングの特徴であることを、これらの患者の植え付けられたSGから学んだ。それでも、これらのニューロンにおける基礎となる細胞および分子プロセスは、現在まで不明瞭なままであり、例えば、コリン作動性表現型20,21へのニューロントランス分化の役割。実験研究は、VAを治療するための新しいアプローチを提示し、例えば、光遺伝学22を介した交感神経活性の減少であるが、SGの詳細な特徴付けは、VAと手をつないで行く多くの心臓病理に依然として欠けている。これらの病理を模倣するマウスモデルは、潜在的に不整脈12、23の発症に先行する神経リモデリングを研究することを可能にする。これらは、心臓および神経系の自律神経特性解析のための更なる形態学的および機能的分析によって完了することができる。本プロトコルでは、VAの理解を深めるためにマウスSGを解剖し特徴付けできる方法の基本的なレパートリーを提供する。

<table><tbody><tr><td>96-well plate</td><td>TPP</td><td>92097</td><td>RNAscope</td></tr><tr><td>Adhesion Slides SuperFrost plus&amp;nbsp; 25 x 75 x 1 mm</td><td>R. Langenbrinck</td><td>03-0060</td><td>Microscopy</td></tr><tr><td>Albumin bovine Fraction V receptor grade lyophil.</td><td>Serva</td><td>11924.03</td><td>Whole mount staining</td></tr><tr><td>bisBenzimide H33342 trihydrochloride (Hoechst)</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>B2261</td><td>Whole mount staining</td></tr><tr><td>Chicken anti neurofilament</td><td>EMD Millipore</td><td>AB5539</td><td>Whole mount staining</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Merck, KGA, Darmstadt, Germany</td><td>D8418</td><td>Whole mount staining</td></tr><tr><td>Donkey anti chicken IgY Alexa 647&amp;nbsp;</td><td>Merck, KGA, Darmstadt, Germany</td><td>AP194SA6</td><td>Whole mount staining</td></tr><tr><td>Donkey anti goat IgG Alexa 568&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A11057</td><td>Whole mount staining</td></tr><tr><td>Donkey anti rabbit IgG Alexa 488&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A21206</td><td>Whole mount staining</td></tr><tr><td>Drying block 37-100 mm</td><td>Whatman (Sigma Aldrich)</td><td>WHA10310992&amp;nbsp;</td><td>Whole mount staining</td></tr><tr><td>Eosin Y</td><td>Sigma Aldrich</td><td>E4009</td><td>Whole mount staining</td></tr><tr><td>Ethanol 99 % denatured with MEK, IPA and Bitrex (min. 99,8 %)</td><td>Th.Geyer</td><td>2212.5000</td><td>Whole mount staining</td></tr><tr><td>Eukitt mounting medium</td><td>AppliChem</td><td>253681.0008</td><td>Whole mount staining</td></tr><tr><td>Fluoromount-G</td><td>Southern Biotech</td><td>0100-01</td><td>Whole mount staining</td></tr><tr><td>Fluoromount-G + DAPI</td><td>Southern Biotech</td><td>0100-20</td><td>Whole mount staining</td></tr><tr><td>Goat anti choline acetyltransferase</td><td>EMD Millipore</td><td>AP144P</td><td>Whole mount staining</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; 30% (w/w)</td><td>Merck, KGA, Darmstadt, Germany</td><td>H1009</td><td>Whole mount staining</td></tr><tr><td>Heparin Sodium 25.000 UI / 5ml</td><td>Rotexmedica</td><td>PZN: 3862340</td><td>Preparation SG</td></tr><tr><td>High-capacity cDNA reverse transctiption kit</td><td>Life technologies</td><td>&amp;nbsp;4368813</td><td>RNA isolation</td></tr><tr><td>Isoflurane (Forene)</td><td>Abbott Laboratories</td><td>2594.00.00</td><td>Preparation SG</td></tr><tr><td>Mayer&#039;s hemalum solution</td><td>Merck</td><td>1.09249.0500</td><td>Whole mount staining</td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>34860</td><td>Whole mount staining</td></tr><tr><td>Microscope cover glasses 20x20 mm or smaller</td><td>Marienfeld</td><td>0101040</td><td>Whole mount staining</td></tr><tr><td>miRNeasy Mini Kit</td><td>Qiagen</td><td>217004</td><td>RNA isolation</td></tr><tr><td>NanoDrop 2000c</td><td>Thermo Fisher Scientific</td><td>ND-2000C</td><td>RNA isolation</td></tr><tr><td>Opal 570 Reagent Pack</td><td>Akoya Bioscience</td><td>FP1488001KT</td><td>RNAscope</td></tr><tr><td>Paraformaldehyde, 16% w/v aq. soln., methanol free&amp;nbsp;</td><td>Alfa Aesar</td><td>43368</td><td>Whole mount staining</td></tr><tr><td>Pasteur pipettes, LDPE, unsterile, 3 ml, 154 mm</td><td>Th.Geyer</td><td>7691202</td><td>Whole mount staining</td></tr><tr><td>Phosphate-buffered saline tablets</td><td>Gibco</td><td>18912-014</td><td>Whole mount staining</td></tr><tr><td>Pinzette Dumont SS Forceps</td><td>FineScienceTools</td><td>11203-25</td><td>Preparation SG</td></tr><tr><td>QIAzol Lysis Reagent</td><td>Qiagen&amp;nbsp;</td><td>79306</td><td>RNA isolation</td></tr><tr><td>Rabbit anti tyrosine hydroxylase</td><td>EMD Millipore</td><td>AB152</td><td>Whole mount staining</td></tr><tr><td>RNAlater</td><td>Merck</td><td>R0901-100ML</td><td>RNA isolation (optional)</td></tr><tr><td>RNAscope Multiplex Fluorescent Reagent Kit v2</td><td>biotechne (ACD)</td><td>323100</td><td>RNAscope</td></tr><tr><td>RNAscope Probe-Mm-S100b-C2</td><td>biotechne (ACD)</td><td>431738-C2</td><td>RNAscope</td></tr><tr><td>RNAscope Probe-Mm-Tubb3</td><td>biotechne (ACD)</td><td>423391</td><td>RNAscope</td></tr><tr><td>Stainless steel beads 7 mm&amp;nbsp;</td><td>Qiagen&amp;nbsp;</td><td>69990</td><td>RNA isolation</td></tr><tr><td>Sudan black B</td><td>Roth</td><td>0292.2</td><td>Whole mount staining</td></tr><tr><td>TaqMan Gene Expression Assay Cdkn1b (Mm00438168_m1)</td><td>Thermo Fisher Scientific</td><td>4331182</td><td>Gene expression analysis</td></tr><tr><td>TaqMan Gene Expression Assay Choline acetyltransferase (Mm01221880_m1)</td><td>Thermo Fisher Scientific</td><td>4331182</td><td>Gene expression analysis</td></tr><tr><td>TaqMan Gene Expression Assay MKi67 (Mm01278617_m1)</td><td>Thermo Fisher Scientific</td><td>4331182</td><td>Gene expression analysis</td></tr><tr><td>TaqMan Gene Expression Assay PTPCR (Mm01293577_m1)</td><td>Thermo Fisher Scientific</td><td>4331182</td><td>Gene expression analysis</td></tr><tr><td>TaqMan Gene Expression Assay S100b (Mm00485897_m1)</td><td>Thermo Fisher Scientific</td><td>4331182</td><td>Gene expression analysis</td></tr><tr><td>TaqMan Gene Expression Assay Tyrosin Hydroxylase (Mm00447557_m1)</td><td>Thermo Fisher Scientific</td><td>4331182</td><td>Gene expression analysis</td></tr><tr><td>TaqMan mastermix</td><td>Applied biosystems</td><td>4370074</td><td>Gene Expression analysis&amp;nbsp;</td></tr><tr><td>Tissue Lyser II</td><td>Qiagen</td><td>85300</td><td>RNA isolation</td></tr><tr><td>Triton X-100 10% solution</td><td>Sigma-Aldrich</td><td>93443-100ml</td><td>Whole mount staining</td></tr><tr><td>Tween-20</td><td>Sigma-Aldrich</td><td>P9416-100ML</td><td>RNAscope</td></tr><tr><td>Wacom bamboo pen</td><td>Wacom</td><td>CTL-460/K</td><td>Cell size measurements</td></tr><tr><td>Whatman prepleated qualitative filter paper, Grade 595 1/2</td><td>Sigma-Aldrich</td><td>WHA10311647</td><td>Whole mount staining</td></tr><tr><td>Wheat Germ Agglutinin, Alexa Fluor 633 Conjugate</td><td>Thermo Fisher Scientific</td><td>W21404</td><td>RNAscope</td></tr></tbody></table>

location, dissection, and analysis of the murine stellate ganglion

自律神経系は心臓電気生理学の実質的な原動力である。特にその交感神経枝の役割は、心室性不整脈(VA)の病態生理学における継続的な調査の問題である。星状神経節(SG)のニューロン   は、交感神経鎖の両側の星形構造であり   、交感神経系の重要な要素です。SGは、治療難治性VA患者における心交感神経変性による治療のための認識された標的である。SGにおける神経細胞リモデリングおよびグリア活性化はVA患者に記載されているが、不整脈の発症に先立つ可能性のある基礎となる細胞および分子プロセスは十分に理解されておらず、自律神経変調を改善するために解明されるべきである。マウスモデルは交感神経リモデリングを研究することができますが、マウスSGの同定は経験の浅い研究者にとって困難です。したがって、マウスSGの詳細な細胞および分子生物学的研究は、多くの一般的な心疾患に欠けている。ここでは、RNAレベル(遺伝子発現解析のためのRNA単離、その際のハイブリダイゼーション)、タンパク質レベル(免疫蛍光全体実装染色)、および細胞レベル(基本的形態、細胞サイズ測定)での分析のために成体マウスのSGを解剖および研究するための基本的なレパートリーについて説明します。調製技術の課題を克服するための潜在的な解決策と、自己蛍光の消光による染色の改善方法を提示します。これにより、細胞組成およびリモデリングプロセスを決定するために、確立されたマーカーを介して神経細胞とグリア細胞を可視化することができます。ここで示す方法は、VAになりやすいマウスの自律神経機能不全に関するさらなる情報を得るためにSGを特徴付けることを可能にし、心臓の自律神経系の神経およびグリア成分を調査する追加の技術によって補完することができる。

図 1は、SG を識別して解剖する方法を視覚化します。 左右の長角のコリ筋肉はSGとリブケージから内側に入る方向の重要なランドマークです。解剖は、筋肉と最初の肋骨の間の点線に沿って行われる。SGと交感神経鎖は白い構造として見えるようになります(図1C)。図1Dは、左の長い...

Watch this Scientific Journal Video about Location, Dissection, and Analysis of the Murine Stellate Ganglion at JoVE.com

Location, Dissection, and Analysis of the Murine Stellate Ganglion

心臓自律神経系、特に交感神経の神経系における病態生理学的変化は、心室性不整脈の発症および維持に寄与する。本プロトコルでは、マウスの星状神経節を特徴付け、基礎となる分子および細胞プロセスの理解を深める方法を示す。

ネズミ星状神経節の位置、解剖、分析

The autonomic nervous system is a substantial driver of cardiac electrophysiology. Especially&#160;the role of its sympathetic branch is an ongoing matter ...

location-dissection-and-analysis-of-the-murine-stellate-ganglion

Research

JoVE Journal

Medicine

9.9K ビュー.  EVK Düsseldorf, cNEP, cardiac Neuro- and Electrophysiology Research Consortium. 心臓自律神経系、特に交感神経の神経系における病態生理学的変化は、心室性不整脈の発症および維持に寄与する。本プロトコルでは、マウスの星状神経節を特徴付け、基礎となる分子および細胞プロセスの理解を深める方法を示す。

ビデオ: Location, Dissection, and Analysis of the Murine Stellate Ganglion

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous activity of participating neurons leads to the emergence of the integral functionality of the network. Here we present the methodology of application of this technique to identified pattern generating neurons in the crab stomatogastric ganglion. We demonstrate the loading of these neurons with the fluorescent voltage-sensitive dye Di-8-ANEPPQ and we show how to image the activity of dye loaded neurons using the MiCAM02 high speed and high resolution CCD camera imaging system. We demonstrate the analysis of the recorded imaging data using the BVAna imaging software associated with the MiCAM02 imaging system. The simultaneous voltage-sensitive dye imaging of the detailed activity of multiple neurons in the crab stomatogastric ganglion applied together with traditional electrophysiology techniques (intracellular and extracellular recordings) opens radically new opportunities for the understanding of how central pattern generator neural networks work.

Here we present the methodology for fast and high resolution fluorescent voltage-sensitive dye imaging of detailed activity of neurons in the crab stomatogastric ganglion.

電位感受性色素を持つカニ口と胃の神経節における神経細胞の光学的イメージング

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

神経科学

Homarus Americanus Stomatogastric Nervous System Dissection

With the goal of understanding how nervous systems produce activity and respond to the environment, neuroscientists turn to model systems that exhibit the activity of interest and are accessible and amenable to experimental methods. The stomatogastric nervous system (STNS) of the American lobster (Homarus americanus; also know was the Atlantic or Maine lobster) has been established as a model system for studying rhythm generating networks and neuromodulation of networks. The STNS consists of 3 anterior ganglia (2 commissural ganglia and an oesophageal ganglion), containing modulatory neurons that project centrally to the stomatogastric ganglion (STG). The STG contains approximately 30 neurons that comprise two central pattern generating networks, the pyloric and gastric networks that underlie feeding behaviors in crustaceans1,2. While it is possible to study this system in vivo3, the STNS continues to produce its rhythmic activity when isolated in vitro. Physical isolation of the STNS in a dish allows for easy access to the somata in the ganglia for intracellular electrophysiological recordings and to the nerves of the STNS for extracellular recordings. Isolating the STNS is a two-part process. The first part, dissecting the stomach from the animal, is described in an accompanying video article4. In this video article, fine dissection techniques are used to isolate the STNS from the stomach. This procedure results in a nervous system preparation that is available for electrophysiological recordings.

Homarus Americanus Stomatogastric Nervous System Dissection

We describe the fine dissection of the stomatogastric nervous system from the stomach of the American lobster (Homarus americanus).

ロブスター属Americanus口胃神経系の解剖

With the goal of understanding how nervous systems produce activity and respond to the environment, neuroscientists turn to model systems that exhibit the ...

生物学

Cancer Borealis Stomatogastric Nervous System Dissection

The stomatogastric ganglion (STG) is an excellent model for studying cellular and network interactions because it contains a relatively small number of cells (approximately 25 in C. borealis) which are well characterized. The cells in the STG exhibit a broad range of outputs and are responsible for the motor actions of the stomach. The stomach contains the gastric mill which breaks down food with three internal teeth, and the pylorus which filters the food before it reaches the midgut. The STG produces two rhythmic outputs to control the gastric mill and pylorus known as central pattern generators (CPGs). Each cell in the STG can participate in one or both of these rhythms. These CPGs allow for the study of neuromodulation, homeostasis, cellular and network variability, network development, and network recovery.
The dissection of the stomatogastric nervous system (STNS) from the Jonah crab (Cancer borealis) is done in two parts; the gross and fine dissection. In the gross dissection the entire stomach is dissected from the crab. During the fine dissection the STNS is extracted from the stomach using a dissection microscope and micro-dissection tools (see figure 1). The STNS includes the STG, the oesophageal ganglion (OG), and the commissural ganglia (CoG) as well as the nerves that innervate the stomach muscles. Here, we show how to perform a complete dissection of the STNS in preparation for an electrophysiology experiment where the cells in the STG would be recorded from intracellularly and the peripheral nerves would be used for extracellular recordings. The proper technique for finding the desired nerves is shown as well as our technique of desheathing the ganglion to reveal the somata and neuropil.

Cancer Borealis Stomatogastric Nervous System Dissection

The stomatogastric nervous system (STNS) of the Jonah crab (C. borealis) can be used for electrophysiology, immunohistochemistry, and cell culture studies. The STNS extraction is done in two parts: the gross and fine dissection.

癌ボレアリス口胃神経系の解剖

The stomatogastric ganglion (STG) is an excellent model for studying cellular and network interactions because it contains a relatively small number of ...

Analyzing Murine Schwann Cell Development Along Growing Axons

The development of peripheral nerves is an intriguing process. Neurons send out axons to innervate specific targets, which in humans are often more than 100 cm away from the soma of the neuron. Neuronal survival during development depends on target-derived growth factors but also on the support of Schwann cells (SCs). To this end SC ensheath axons from the region of the neuronal soma (or the transition from central to peripheral nervous system) to the synapse or neuromuscular junction. Schwann cells are derivatives of the neural crest and migrate as precursors along emerging axons until the entire axon is covered with SCs. This shows the importance of SC migration for the development of the peripheral nervous system and underlines the necessity to investigate this process. In order to analyze SC development, a setup is needed which next to the SCs also includes their physiological substrate for migration, the axon. Due to intrauterine development in vivo time-lapse imaging, however, is not feasible in placental vertebrates like mouse (mus musculus). To circumvent this, we adapted the superior cervical ganglion (SCG) explant technique. Upon treatment with nerve growth factor (NGF) SCG explants extend axons, followed by SC precursors migrating along the axons from the ganglion to the periphery. The beauty of this system is that the SC are derived from a pool of endogenous SC and that they migrate along their own physiological axons which are growing at the same time. This system is especially intriguing, because the SC development along axons can be analyzed by time-lapse imaging, opening further possibilities to gain insights into SC migration.

Here we describe a Schwann cell (SC) migration assay in which SCs are able to develop along extending axons.

成長中の軸索に沿ってマウスのシュワン細胞の発達を分析する

The  development of peripheral nerves is an intriguing process. Neurons send out  axons to innervate specific targets, which in humans are often more than ...

Surgical Technique for Superior Cervical Ganglionectomy in a Murine Model

Growing evidence suggests that the sympathetic nervous system plays an important role in cancer progression. Adrenergic innervation regulates salivary gland secretion, circadian rhythm, macular degeneration, immune function, and cardiac physiology. Murine surgical sympathectomy is a method for studying the effects of adrenergic innervation by allowing for complete, unilateral adrenergic ablation while avoiding the need for repeated pharmacologic intervention and the associated side effects. However, surgical sympathectomy in mice is technically challenging because of the small size of the superior cervical ganglion. This study describes a surgical technique for reliably identifying and resecting the superior cervical ganglion to ablate the sympathetic nervous system. The successful identification and removal of the ganglion are validated by imaging the fluorescent sympathetic ganglia using a transgenic mouse, identifying post-resection Horner's syndrome, staining for adrenergic markers in the resected ganglia, and observing diminished adrenergic immunofluorescence in the target organs following sympathectomy. This model enables future studies of cancer progression as well as other physiological processes regulated by the sympathetic nervous system.

The present protocol describes a mouse model of the ablation of adrenergic innervation by identifying and resecting the superior cervical ganglion.

マウスモデルにおける上頚部神経節摘出術の手術手技

Growing evidence suggests that the sympathetic nervous system plays an important role in cancer progression. Adrenergic innervation regulates salivary ...

がん研究

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 ...

Dissection of Pelvic Autonomic Ganglia and Associated Nerves in Male and Female Rats

The bilateral major pelvic ganglia (MPG; synonym, pelvic ganglia) are the primary source of postganglionic sympathetic and parasympathetic neurons innervating pelvic organs of rodents; the functionally equivalent structure in humans is the inferior hypogastric plexus. The major pelvic ganglia also provide the route by which lumbar and sacral sensory axons reach the pelvic organs. These complex, mixed ganglia can prove challenging to identify and dissect for further experimental study of normal autonomic mechanisms or to establish preclinical models of disease, injury or visceral pain. Here we describe a protocol to access and visualize these ganglia and their associated nerve tracts. We provide this protocol with schematics for both male and female rats, as the ganglion size and landmarks for identification differ between sexes. The protocol describes removal of the ganglion for in vitro studies, but this method can be integrated into a surgical recovery protocol for experimental interventions (e.g., nerve crush, nerve resection) or for mapping neuronal circuits (e.g., by microinjection of neural tracers). We also demonstrate the primary structures of the ganglion and its associated nerves immediately following dissection and following immunohistochemical staining.

The major pelvic ganglia contain parasympathetic and sympathetic neurons that innervate pelvic organs. Here we describe a dissection method and provide schematics for identification of these ganglia and their associated nerves. These methods can be applied to experimental manipulation of these ganglia in vivo or removal post-mortem for further study.

男性および女性ラットにおける骨神経神経と関連神経の解剖

The bilateral major pelvic ganglia (MPG; synonym, pelvic ganglia) are the primary source of postganglionic sympathetic and parasympathetic neurons ...

Whole Central and Peripheral Nervous System Mice Dissection

Animal models represent the workhorse of the neuroscience field. Despite this, today, there is still no step-by-step protocol to dissect a complete rodent nervous system, nor is there a complete schematic representing it that is freely available. Only methods to harvest the brain, the spinal cord, a specific dorsal root ganglion, and the sciatic nerve (separately) are available. Here, we provide detailed pictures and a schematic of the central and peripheral murine nervous system. More importantly, we outline a robust procedure to perform its dissection. The 30 min pre-dissection step allows isolating the intact nervous system within the vertebra with muscles free of viscera and skin. A 2-4 h dissection follows it under a micro-dissection microscope to expose the spinal cord and the thoracic nerves, and finally peel the whole central and peripheral nervous system off the carcass. This protocol represents a significant step forward in studying the anatomy and pathophysiology of the nervous system globally. For example, the dissected dorsal root ganglions from a neurofibromatosis type I mice model can be further processed for histology to unravel changes in tumor progression.

Here, we present a protocol to complete the dissection of central and peripheral nervous systems of mice. The dissected tissues are further analyzed in downstream applications, such as taking gross pictures or histology.

Animal models represent the workhorse of the neuroscience field. Despite this, today, there is still no step-by-step protocol to dissect a complete rodent ...

Low-input Nucleus Isolation and Multiplexing with Barcoded Antibodies of Mouse Sympathetic Ganglia for Single-nucleus RNA Sequencing

The cardiac autonomic nervous system is crucial in controlling cardiac function, such as heart rate and cardiac contractility, and is divided into sympathetic and parasympathetic branches. Normally, there is a balance between these two branches to maintain homeostasis. However, cardiac disease states such as myocardial infarction, heart failure, and hypertension can induce the remodeling of cells involved in cardiac innervation, which is associated with an adverse clinical outcome.
Although there are vast amounts of data for the histological structure and function of the cardiac autonomic nervous system, its molecular biological architecture in health and disease is still enigmatic in many aspects. Novel technologies such as single-cell RNA sequencing (scRNA-seq) hold promise for the genetic characterization of tissues at single-cell resolution. However, the relatively large size of neurons may impede the standardized use of these techniques. Here, this protocol exploits droplet-based single-nucleus RNA sequencing (snRNA-seq), a method to characterize the biological architecture of cardiac sympathetic neurons in health and disease. A stepwise approach is demonstrated to perform snRNA-seq of the bilateral superior cervical (SCG) and stellate ganglia (StG) dissected from adult mice.
This method enables long-term sample preservation, maintaining an adequate RNA quality when samples from multiple individuals/experiments cannot be collected all at once within a short period of time. Barcoding the nuclei with hashtag oligos (HTOs) enables demultiplexing and the trace-back of distinct ganglionic samples post sequencing. Subsequent analyses revealed successful nuclei capture of neuronal, satellite glial, and endothelial cells of the sympathetic ganglia, as validated by snRNA-seq. In summary, this protocol provides a stepwise approach for snRNA-seq of sympathetic extrinsic cardiac ganglia, a method that has the potential for broader application in studies of the innervation of other organs and tissues.

This protocol describes the detailed, low-input sample preparation for single-nucleus sequencing, including the dissection of mouse superior cervical and stellate ganglia, cell dissociation, cryopreservation, nucleus isolation, and hashtag barcoding.

単一核RNAシーケンシングのためのマウス交感神経節のバーコード化抗体による低入力核単離および多重化

The cardiac autonomic nervous system is crucial in controlling cardiac function, such as heart rate and cardiac contractility, and is divided into ...

医学

マウス交感神経ニューロンの標的化:腰椎交感神経切除術アプローチ

Surgical Lumbar Sympathectomy in Mice

Peripheral nerve injuries are common, and full functional recovery after injury is achieved in only 10% of patients. The sympathetic nervous system plays many critical roles in maintaining bodily homeostasis, but it has rarely been studied in the context of peripheral nerve injury. The extent of postganglionic sympathetic neuronal functions in distal targets in the periphery is currently unclear. To better explore the role of sympathetic innervation of peripheral targets, a surgical &#34;knock-out&#34; model provides an alternative approach. Although this can be achieved chemically, chemical destruction of postganglionic sympathetic neurons can be nonspecific and dose-dependent. The use of a surgical lumbar sympathectomy in mice, once thought to be &#34;virtually not practicable&#34; in small animals, allows for specific targeting of postganglionic sympathetic neurons that innervate the hind limbs. This manuscript describes how to surgically remove the L2-L5 lumbar sympathetic ganglia from a mouse as a survival surgery, which reliably decreases the hind paw sweat response and the number of sympathetic axons in the sciatic nerve.

著者スポットライト:交感神経ニューロンの可塑性の探索

This manuscript presents a protocol for surgically removing the postganglionic lumbar sympathetic neurons from a mouse. This procedure will facilitate a multitude of studies aimed at investigating the role of sympathetic innervation in distal tissue targets.

マウスの外科的腰椎交感神経切除術

Peripheral nerve injuries are common, and full functional recovery after injury is achieved in only 10% of patients. The sympathetic nervous system plays ...

ネズミ星状神経節の位置、解剖、分析

要約

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この動画の章

電位感受性色素を持つカニ口と胃の神経節における神経細胞の光学的イメージング

ロブスター属Americanus口胃神経系の解剖

癌ボレアリス口胃神経系の解剖

成長中の軸索に沿ってマウスのシュワン細胞の発達を分析する

マウスモデルにおける上頚部神経節摘出術の手術手技

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

男性および女性ラットにおける骨神経神経と関連神経の解剖

Whole Central and Peripheral Nervous System Mice Dissection

単一核RNAシーケンシングのためのマウス交感神経節のバーコード化抗体による低入力核単離および多重化

マウスの外科的腰椎交感神経切除術

ネズミ星状神経節の位置、解剖、分析

要約

さらに動画を探す

この動画の章

電位感受性色素を持つカニ口と胃の神経節における神経細胞の光学的イメージング

ロブスター属Americanus口胃神経系の解剖

癌ボレアリス口胃神経系の解剖

成長中の軸索に沿ってマウスのシュワン細胞の発達を分析する

マウスモデルにおける上頚部神経節摘出術の手術手技

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

男性および女性ラットにおける骨神経神経と関連神経の解剖

Whole Central and Peripheral Nervous System Mice Dissection

単一核RNAシーケンシングのためのマウス交感神経節のバーコード化抗体による低入力核単離および多重化

マウスの外科的腰椎交感神経切除術

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