我々は図1の回路図を使用するための取り付けブロックの青写真とダニエラ·ペルツを描画するためのパブロTraversa、この方法論の開発にジルケSachseとベアテEisermannを認める。私たちは、原稿上のコメントのジルケSachse、パヴァーヌRamdyaとラファエルRytzに感謝しています。 R.ベルは、ベーリンガーインゲルハイム財団博士フェローシップでサポートされています。 R.ベントンの研究室での研究は、独立した研究者グラントとスイス国立科学財団を起動する欧州研究評議会によって運営されている。

<ol>
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著者らは、開示することは何もありません。

ここで説明した準備や分析方法は、対応するドライバの導入遺伝子が利用可能である触角葉（OSNs、LNとPNS）の神経細胞のいずれかの集団で臭気誘発応答を分析するために使用することができます。我々は蛍光顕微鏡を使用してアプリケーションを記述しながら、本製剤の光イメージングが均等に焦点または二光子顕微鏡22,32を用いて行うことができる。実際、これらの後者の楽器は、ニューロンExpressの場合は特に、多数のレポーター、画像の解像度を減らすことができる広視野顕微鏡の光散乱問題を軽減する。この製剤は、通常、少なくとも半時間臭気誘発応答の録音を許可しますが、この時間は、記録された測定値の数と長さに応じて各フレーム間の刺激間隔に使用される露光時間大幅に長くしたり短くすることができます。さらに、二光子励起、蛍光repの退色を減らすことができますorterと同様に、細胞の毒性は、長い期間にわたって測定22,32を許可します。 かかわらず、顕微鏡の選択の、頭部カプセル内のハエの脳の動きは時に無傷の動物のイメージング最も再発の問題です。動き補正はマイナーな問題を（ステップ5.1）修正することができますが、それは非常に安定した調製物から録音する方が良いだけです。動きのアーチファクトを低減するための戦略は、（i）適切にサボテンの棘を持つハエの口吻を抑制すること、（ii）エイコサン（前の2.6ステップ）（エイコサンがで溶融させることができるとステージに固定することにより、ハエの脚を固定する〜 37°C;）この目的のためはんだごての先端を拡張し、洗練するために銀線を使用すること、（iii）微細なピンセットを用いて食道を（触角神経の間に表示されます）ニッキング。 ここで使用されるG-CaMP1.6に加えて、G-cAMPおよび他のカルシウム指標の最適化バージョンの数は、ご利用いただけますこれは、それらの信号対雑音比は、ベースラインの蛍光および時間的ダイナミクス12,28,33が異なります。センサの正確な選択は、アカウントにこれらのすべてのプロパティは、ニューロンのタイプを分析すると生物学的な問題が対処されているを取る必要があります。電流センサは、活動電位12,34とかなりよく相関カルシウムの変化を報告し、特定の遺伝子のドライバー·ライン数の増加35と結合し、その更なる向上は、そのままその場で神経活動の光学的イメージングの可能性を強化していきます。

<table border="1"><tbody><tr><td>試薬</td><td>サプライヤー</td><td>カタログ番号</td><td>コメント</td></tr><tr><td>サボテン棘</td><td>ガーデンセンター</td><td></td><td>あまりにも柔軟性がない、〜7ミリメートル長い薄い</td></tr><tr><td>ロジン</td><td>弦楽器店</td><td></td><td></td></tr><tr><td>二成分シリコン</td><td> WPI </td><td> KWIK-SIL </td><td></td></tr><tr><td>カスタムブロックを取り付けプレキシガラスを作った</td><td>から入手可能な青写真<a href="http://neuro.uni-konstanz.de/jove_mountingblock_blueprint/" target="_blank">http://neuro.uni-konstanz.de/j ove_mountingblock_blueprint /</a> </td><td></td><td></td></tr><tr><td>銅グリッド</td><td>アテネグリッド</td><td> G220-5 </td><td>通常は電子顕微鏡の試料に使用されます。 </td></tr><tr><td>ネジ</td><td>金物店</TD&gt; <td></td><td>直径2mm </td></tr><tr><td>プラスチック製カバースリップ</td><td>プラノ-EM </td><td> L4193 </td><td> 22 X 22ミリメートル</td></tr><tr><td>はんだごてのヒント</td><td>コンラッドエレクトロニクス</td><td> 830283から62 </td><td></td></tr><tr><td>調整電源</td><td>コンラッドエレクトロニクス</td><td> 511407から62 </td><td></td></tr><tr><td>細い線</td><td> Isabellenhütte </td><td> ISA-Ω </td><td> 0.013ミリメートル</td></tr><tr><td>エイコサン</td><td>シグマ</td><td> 21,927-4 </td><td></td></tr><tr><td>顕微鏡</td><td>ツァイス</td><td> Axioexaminer D1の</td><td></td></tr><tr><td> CCDカメラ</td><td> Visitron </td><td> CoolSnap HQ </td><td></td></tr><tr><td> Metafluor </td><td> Visitron </td><td></td><td>集録ソフトウェア</td></tr><tr><td>単色光分光器</td><td> Visitron </td><td> Visichrome </td&gt; <td></td></tr><tr><td>壊れやすいブレード</td><td>ファイン科学ツール</td><td> 10050から00 </td><td></td></tr><tr><td>ブレードホルダー</td><td> WPI </td><td> 14134 </td><td></td></tr><tr><td>サファイアブレード</td><td> WPI </td><td> 500314 </td><td>ダブルブレード1mmの縁取り</td></tr><tr><td>膜のガスポンプ</td><td> KNF Neuberger AG </td><td> NMP 830 KVE </td><td></td></tr><tr><td>サファイアブレードホルダー</td><td> WPI </td><td> 500317 </td><td></td></tr><tr><td>三方弁</td><td>リー·カンパニー</td><td> LFAA1200118H </td><td>設定E </td></tr><tr><td>浮子式流量計500ミリリットル/分</td><td> Analyt-MTC </td><td> 112-08SA </td><td></td></tr><tr><td>浮子式流量計5 L / minの</td><td> Analyt-MTC </td><td> 102-08SA </td><td></td></tr><tr><td>蜜ろう</td><td> Siladent Technikで</td><td> 209212 </td><td>; </td></tr><tr><td>セルロースパッド</td><td> Kettenbach医療</td><td> 31003 </td><td></td></tr><tr><td>ピンセット</td><td>プラノ-EM </td><td> T5130 </td><td>デュモン生物学第5位</td></tr><tr><td>シリンジ</td><td> BD </td><td> 300928 </td><td> 2ミリリットル、Discardit II、2個入り</td></tr><tr><td>針</td><td>ブラウンSterican </td><td> 4665120 </td><td> 1.2ミリメートルOD </td></tr></tbody></table>

calcium imaging of odor-evoked responses in the drosophila antennal lobe

触角葉、昆虫脳内の主要な嗅覚中枢であり、脊椎動物の嗅球1-5の解剖学的および機能的に同等を表しています。外界の嗅覚情報は、それらが表現する特定の嗅覚受容体に応じて糸球体と呼ばれる神経網の異なる領域に分離する嗅細胞（OSNs）によって触角葉に送信されます。ここでは、より高い嗅覚脳領域への嗅覚情報を伝えるため、両方のローカルプロセス多くの異なった糸球体を支配することができます介在（LNS）と、投射ニューロン（PNS）とOSN軸索のシナプス。 触角葉におけるOSNs、LNSとPNの活動の光学的イメージング - 伝統的に合成カルシウム指標（例えば、カルシウム、緑、FURA-2）または電位感受性色素（例えばRH414）を使用して - 長い理解することが重要な技術であった方法嗅覚刺激が空間的および時間的パターンとして表されます。昆虫6-10の多くの種における糸球体の活動。このような蛍光カルシウム指標G-CAMP 11,12とカメレオン13と遺伝的にエンコードされた神経活動の記者の開発、生物発光カルシウム指示薬GFP-エクオリン14,15、またはシナプス伝達のレポーター、synapto-pHluorin 16が行ったショウジョウバエの嗅覚系、神経イメージング、特にアクセスキイロショウジョウバエでは 、その包括的に記述され、分子の電気生理学的および神経解剖学的特性を2,4,17を補完する 。これらの記者は、選択的に臭気誘発方法、高空間分解能と時間分解能で詳細に分析する嗅覚回路内のニューロンの定義された集団でバイナリ転写制御システム（例えばGAL4/UAS 18、LexAの/ LexAop 19,20、Qシステム21）を介して表現することができる神経活動が表現され、変調されたと22から24 &lt;形質転換/&gt;のsup。 ここでは、G-CAMP 25から27を使用して、 ショウジョウバエの触角葉で匂い誘発反応を測定するための準備と分析方法について説明します。動物の準備は低侵襲で、広い視野、蛍光共焦点顕微鏡と二光子顕微鏡を用いたイメージングに適応することができます。

Watch this Scientific Journal Video about Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe at JoVE.com

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

我々は生きているの触角葉で匂い誘発カルシウム応答を測定し、分析するための確立された技術を記述する<em&gt;キイロショウジョウバエ</em&gt;。

の臭気誘発応答のカルシウムイメージングショウジョウバエ触角葉

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

calcium-imaging-odor-evoked-responses-drosophila-antennal

Research

JoVE Journal

Neuroscience

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

25.8K ビュー.  University of Lausanne. 我々は生きているの触角葉で匂い誘発カルシウム応答を測定し、分析するための確立された技術を記述するキイロショウジョウバエ。

ビデオ: Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

Imaging Odor-Evoked Activities in the Mouse Olfactory Bulb using Optical Reflectance and Autofluorescence Signals

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. Functional optical imaging techniques are advantageous to visualize the activation of these modules in sensory cortices with high spatial resolution. In this context, endogenous optical signals that arise from molecular mechanisms linked to neuroenergetics are valuable sources of contrast to record spatial maps of sensory stimuli over wide fields in the rodent brain.
Here, we present two techniques based on changes of endogenous optical properties of the brain tissue during activation. First the intrinsic optical signals (IOS) are produced by a local alteration in red light reflectance due to: (i) absorption by changes in blood oxygenation level and blood volume (ii) photon scattering. The use of in vivo IOS to record spatial maps started in the mid 1980's with the observation of optical maps of whisker barrels in the rat and the orientation columns in the cat visual cortex1. IOS imaging of the surface of the rodent main olfactory bulb (OB) in response to odorants was later demonstrated by Larry Katz's group2. The second approach relies on flavoprotein autofluorescence signals (FAS) due to changes in the redox state of these mitochondrial metabolic intermediates. More precisely, the technique is based on the green fluorescence due to oxidized state of flavoproteins when the tissue is excited with blue light. Although such signals were probably among the first fluorescent molecules recorded for the study of brain activity by the pioneer studies of Britton Chances and colleagues3, it was not until recently that they have been used for mapping of brain activation in vivo. FAS imaging was first applied to the somatosensory cortex in rodents in response to hindpaw stimulation by Katsuei Shibuki's group4.
The olfactory system is of central importance for the survival of the vast majority of living species because it allows efficient detection and identification of chemical substances in the environment (food, predators). The OB is the first relay of olfactory information processing in the brain. It receives afferent projections from the olfactory primary sensory neurons that detect volatile odorant molecules. Each sensory neuron expresses only one type of odorant receptor and neurons carrying the same type of receptor send their nerve processes to the same well-defined microregions of &tilde;100&mu;m3 constituted of discrete neuropil, the olfactory glomerulus (Fig. 1). In the last decade, IOS imaging has fostered the functional exploration of the OB5, 6, 7 which has become one of the most studied sensory structures. The mapping of OB activity with FAS imaging has not been performed yet.
Here, we show the successive steps of an efficient protocol for IOS and FAS imaging to map odor-evoked activities in the mouse OB.

This article presents the protocols of intrinsic optical signals and flavoproteins autofluorescence signals imaging to map odor-evoked activities at the surface of the olfactory bulb in mice.

光反射率と自家蛍光信号を用いたマウス嗅球におけるイメージングの臭い誘発活動

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. ...

神経科学

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

遺伝的にコードされたカルシウムセンサーを発現する鋤鼻器のスライス標本のイメージング神経細胞応答

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

視床下部マウス脳スライスのGFPタグニューロンにおけるイメージングカルシウム応答

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe

All organisms inhabit a world full of sensory stimuli that determine their behavioral and physiological response to their environment. Olfaction is especially important in insects, which use their olfactory systems to respond to, and discriminate amongst, complex odor stimuli. These odors elicit behaviors that mediate processes such as reproduction and habitat selection1-3. Additionally, chemical sensing by insects mediates behaviors that are highly significant for agriculture and human health, including pollination4-6, herbivory of food crops7, and transmission of disease8,9. Identification of olfactory signals and their role in insect behavior is thus important for understanding both ecological processes and human food resources and well-being.
	To date, the identification of volatiles that drive insect behavior has been difficult and often tedious. Current techniques include gas chromatography-coupled electroantennogram recording (GC-EAG), and gas chromatography-coupled single sensillum recordings (GC-SSR)10-12. These techniques proved to be vital in the identification of bioactive compounds. We have developed a method that uses gas chromatography coupled to multi-channel electrophysiological recordings (termed 'GCMR') from neurons in the antennal lobe (AL; the insect's primary olfactory center)13,14. This state-of-the-art technique allows us to probe how odor information is represented in the insect brain. Moreover, because neural responses to odors at this level of olfactory processing are highly sensitive owing to the degree of convergence of the antenna's receptor neurons into AL neurons, AL recordings will allow the detection of active constituents of natural odors efficiently and with high sensitivity. Here we describe GCMR and give an example of its use.
	Several general steps are involved in the detection of bioactive volatiles and insect response. Volatiles first need to be collected from sources of interest (in this example we use flowers from the genus Mimulus (Phyrmaceae)) and characterized as needed using standard GC-MS techniques14-16. Insects are prepared for study using minimal dissection, after which a recording electrode is inserted into the antennal lobe and multi-channel neural recording begins. Post-processing of the neural data then reveals which particular odorants cause significant neural responses by the insect nervous system.
	Although the example we present here is specific to pollination studies, GCMR can be expanded to a wide range of study organisms and volatile sources. For instance, this method can be used in the identification of odorants attracting or repelling vector insects and crop pests. Moreover, GCMR can also be used to identify attractants for beneficial insects, such as pollinators. The technique may be expanded to non-insect subjects as well.

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe

Olfactory cues mediate many different behaviors in insects, and are often complex mixtures comprised of tens to hundreds of volatile compounds. Using gas chromatography with multi-channel recording in the insect antennal lobe, we describe a method for the identification of bioactive compounds.

昆虫の触角葉でガスクロマトグラフィー - マルチユニットレコーディング（GCMR）を使用して、嗅覚揮発性物質の同定

All  organisms inhabit a world full of sensory stimuli that determine their  behavioral and physiological response to their environment. Olfaction is ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

Ex Vivoショウジョウバエにおける内分泌シグナル伝達する脳の反応を可視化するためのカルシウム イメージング

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

ショウジョウバエメラメラノガスターにおける学習誘発シナプス可塑性の生体内光学カルシウムイメージング

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte function is not fully known. Pericytes have been suggested to be involved in capillary development, regulation of endothelial barrier tightness and trancytosis activity, regulation of capillary tone and to play crucial roles in certain brain pathologies.
Pericytes are challenging to investigate in the intact brain due to the difficulties in visualizing processes in the brain parenchyma, as well as the close proximity to the other cells of the NVU. The present protocol describes a method for isolation and culture of primary bovine brain capillary pericytes and their following usage in calcium imaging studies, where effects of agonists involved in brain signaling and pathologies can be investigated. Cortical capillary fragments are allowed to attach to the bottom of culture flasks and, after 6 days, endothelial cells and pericytes have grown out from the capillary fragments. The endothelial cells are removed by gentle trypsinization and pericytes are cultured for 5 additional days before passaging.
Isolated pericytes are seeded in 96-well culture plates and loaded with the calcium indicator dye (Fura-2 acetoxymethyl (AM)) to allow for measurements of intracellular calcium levels in a plate reader setup. Alternatively, pericytes are seeded on coverslips and mounted in cell chambers. Following loading with the calcium indicator (Cal-520 AM), calcium live-imaging can be performed using confocal microscopy at an excitation wavelength of 488 nm and emission wavelength of 510-520 nm.
The method described here has been used to obtain the first intracellular calcium measurements from primary brain capillary pericytes, demonstrating that pericytes are stimulated via ATP and are able to contract in vitro.

Brain capillary pericytes are essential players in the regulation of blood-brain barrier properties and blood flow. This protocol describes how brain capillary pericytes can be isolated, cultured, characterized with respect to cell type and applied for investigations of intracellular calcium signaling with fluorescent probes.

細胞体カルシウム測定とカルシウムイメージング研究のための脳毛細血管培養

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte ...

In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Responses to Taste Stimuli

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using calcium as a proxy for neuronal activity, these techniques provide a way to monitor the responses of individual cells within large neuronal ensembles to a variety of stimuli in real time. We, and others, have applied these techniques to image the responses of individual geniculate ganglion neurons to taste stimuli applied to the tongues of live anesthetized mice. The geniculate ganglion is comprised of the cell bodies of gustatory neurons innervating the anterior tongue and palate as well as some somatosensory neurons innervating the pinna of the ear. Imaging the taste-evoked responses of individual geniculate ganglion neurons with GCaMP has provided important information about the tuning profiles of these neurons in wild-type mice as well as a way to detect peripheral taste miswiring phenotypes in genetically manipulated mice. Here we demonstrate the surgical procedure to expose the geniculate ganglion, GCaMP fluorescence image acquisition, initial steps for data analysis, and troubleshooting. This technique can be used with transgenically encoded GCaMP, or with AAV-mediated GCaMP expression, and can be modified to image particular genetic subsets of interest (i.e., Cre-mediated GCaMP expression). Overall, in vivo calcium imaging of geniculate ganglion neurons is a powerful technique for monitoring the activity of peripheral gustatory neurons and provides complementary information to more traditional whole-nerve chorda tympani recordings or taste behavior assays.

Here we present how to expose the geniculate ganglion of a live, anesthetized laboratory mouse and how to use calcium imaging to measure the responses of ensembles of these neurons to taste stimuli, allowing for multiple trials with different stimulants. This allows for in depth comparisons of which neurons respond to which tastants.

マウスの神経節神経節ニューロン反応のインビボカルシウムイメージングが味覚刺激に反応する

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

インビボ マウス劣オリーブにおけるカルシウムイメージング

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

網膜神経節細胞層におけるカルシウム活性の評価

Calcium Imaging In Electrically Stimulated Flat-Mounted Retinas

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the impaired light-sensing photoreceptor cells in the degenerated retina, aiming to partially restore vision by inducing visual percepts. One common avenue of investigation involves the design and production of implantable devices with a flexible physical structure, housing a high number of electrodes. This enables the efficient and precise generation of visual percepts. However, with each technological advancement, there arises a need for a reliable and manageable ex vivo method to verify the functionality of the device before progressing to in vivo experiments, where factors beyond the device's performance come into play. This article presents a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer (GCL) following electrical stimulation. Specifically, the following steps are outlined: (1) fluorescently labeling the rat retina using genetically encoded calcium indicators, (2) capturing the fluorescence signal using an inverted fluorescence microscope while applying distinct patterns of electrical stimulation, and (3) extracting and analyzing the calcium traces from individual cells within the GCL. By following this procedure, researchers can efficiently test new stimulation protocols prior to conducting in vivo experiments.

著者スポットライト:カルシウムイメージングを使用した神経電気刺激への革新的なアプローチ 

Retinal prostheses have the ability to generate visual perceptions. To advance the development of new prostheses, ex vivo methods are needed to test devices before implantation. This article provides a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer when subjected to electrical stimulation.

電気刺激フラットマウント網膜におけるカルシウムイメージング

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the ...