私たちは作業に参加の仲間たちに感謝ここにまとめました。この作品は、ドイツ学術振興（SFB 894）、フォルクスワーゲン財団（TLZ）によるDFG​​ Schwerpunktprogramm 1392 &#39;嗅覚の統合解析 &quot;からの補助金によって支えられている。 TLZは、フォルクスワーゲン財団のリヒテンベルグ教授です。

<ol>
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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feedback+loops+link+odor+and+pheromone+signaling+with+reproduction.">Feedback loops link odor and pheromone signaling with reproduction.</a> Cell. 123, 683-695 (2005).</li><li>Wilson, J. M., Dombeck, D. A., Diaz-Rios, M., Harris-Warrick, R. M., Brownstone, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+calcium+imaging+of+network+activity+in+XFP-expressing+neurons+in+the+mouse.">Two-photon calcium imaging of network activity in XFP-expressing neurons in the mouse.</a> J. Neurophysiol. 97, 3118-3125 (2007).</li><li>Hu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+near-atmospheric+concentrations+of+CO2+by+an+olfactory+subsystem+in+the+mouse.">Detection of near-atmospheric concentrations of CO2 by an olfactory subsystem in the mouse.</a> Science. 317, 953-957 (2007).</li><li>Perez, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transient+receptor+potential+channel+expressed+in+taste+receptor+cells.">A transient receptor potential channel expressed in taste receptor cells.</a> Nat. Neurosci. 5, 1169-1176 (2002).</li><li>Trollinger, D. R., Cascio, W. E., Lemasters, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+loading+of+Rhod+2+into+mitochondria+shows+mitochondrial+Ca2++transients+during+the+contractile+cycle+in+adult+rabbit+cardiac+myocytes.">Selective loading of Rhod 2 into mitochondria shows mitochondrial Ca2+ transients during the contractile cycle in adult rabbit cardiac myocytes.</a> Biochem Biophys. Res. Commun. 236, 738-742 (1997).</li><li>Meshik, X. A., Hyrc, K. L., Goldberg, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+Asante+Calcium+Red+-+a+novel+ratiometric+indicator+with+long+excitation+wavelength.">Properties of Asante Calcium Red - a novel ratiometric indicator with long excitation wavelength.</a> , (2010).</li></ol>

特別な利害関係は宣言されません。

神経科学の主要な問題は、脳が社会的な情報の処理方法を理解することです。社会的認識のために必要な情報の主なソースは、嗅覚やフェロモンの信号により符号化されている。神経鼻の集団、特に脳、視床下部における信号の認識によって、これらの信号の検出は、多くの社会的プロセスと影響ホルモンおよび他の神経内分泌因子13-16の重要な役割を果たしています。複数のニューロンと多機能の核を含む視床下部のように脳の領域における神経応答を解析するのに不可欠な障害は、関心のある特定のニューロンの同定である。 多くのトランスジェニックマウス系統は、主にGFPは神経細胞を識別するのに役立ちますている、開発されている。残念なことに、GFPの蛍光特性は、このようなのFluo-3、Fluo-4を17としてのCa 2 +指示薬色素を用いた測定を複雑にします。そこで我々はinvesti始めゲートFURA-赤3,4のように、赤方偏移のCa 2 +指示薬色素を用いた神経細胞をGFPタグ付き。のFura-赤基づくのCa 2 +イメージングは、以前膵β細胞とHEK細胞に発現し5,6 GFPタグ受容体GFPタグと組み合わせた。 FURA-2と同様に、フラ·赤とGFPの間にいくつかのクロストークは2が報告されています。しかし、特定の帯域幅と二色ビームスプリッタフィルタで高品質の発光フィルタセットを使用すると、/ある程度染料間のブリードスルークロストークを制限することができます。それは、GFPタグニューロンと（1）脳スライスを使用して、同時に両方のチャネルを（緑色と赤色蛍光）を測定することにより、共焦点顕微鏡の設定を確認するのが賢明ですが、Ca 2 +指示薬色素でロードされ、同様にしないGFPタグニューロンなし（2）脳スライスとしてではなく、赤方偏移のCa 2 +指示薬がロードされます。する場合には、正規の実験と同様に共焦点顕微鏡で同じ設定を使用して、治験責任医師は、次に注意することができますyの蛍光は避けるべきである異種のチャネルで検出されています。 一部の研究者は、2 rhodo-の異形を使用するか、2,18,19 GFPタグ細胞と組み合わせて赤方偏移し代替染料としてX rhod1の使用をお勧めします。しかし、rhodは色素がミトコンドリア20に集中する傾向を持っているように見えるし、多くのニューロンに低い標識効率17を持っているAM。改良された赤方偏移のCa 2 +指標の必要性のために、研究者や企業が現在うまくいけば、優れた性能を持つ21新しいプローブの開発を行っています。

There was a typo in the abstract of <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/4213">Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices</a>.

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

<table border="1"><tbody><tr><td> 名前 </td><td> 会社 </td><td> 猫。 N° </td></tr><tr><td>寒天</td><td>シグマ</td><td> A1296 </td></tr><tr><td> Fura-red/AM </td><td>インビトロジェン</td><td> F-3021 </td></tr><tr><td>プルロニックF-127 </td><td>シグマ</td><td> P2443 </td></tr><tr><td>ジメチルスルホキシド</td><td>フィッシャー·サイエンティフィック</td><td> BP231 </td></tr><tr><td>振動刃ミクロトームハイラックスV 50 </td><td>ツァイス</td><td> 9770170 </td></tr><tr><td>ミクロトームハイラックスV 50の冷却装置CU 65 </td><td>ツァイス</td><td> 9920120 </td></tr><tr><td> O 2 / CO 2インキュベーター、CB210-UL </td><td>バインダー</td><td> 0019389 </td></tr><tr><td>スーパー接着剤、ロックタイト406TM </td><td>ヘンケル</td><td> 142580 </td></tr><tr><td>ダブルへら、スプーン形状</td><td>Bochem </td><td> 3182 </td></tr><tr><td> Microspoonのへら、スプーン形状</td><td> Bochem </td><td> 3344 </td></tr><tr><td>春はさみ、モリア-Vannas·ウォルフ - 7ミリメートルブレード</td><td>ファイン科学ツール</td><td> 15370から52 </td></tr><tr><td>春のはさみ、Vannas - 3ミリメートルブレード</td><td>ファイン科学ツール</td><td> 15000から00 </td></tr><tr><td>ワーグナーはさみ</td><td>ファイン科学ツール</td><td> 14071から12 </td></tr><tr><td>医療鉗子、デュモン7bは</td><td>ファイン科学ツール</td><td> 11270から20 </td></tr><tr><td>大きな長方形の露天風呂商工会議所（RC-27） </td><td>ワーナー·インスツルメンツ</td><td> 64から0238 </td></tr><tr><td>共焦点顕微鏡ラッドラディアンス2100 </td><td>ツァイス</td><td> NA </td></tr></tbody></table>

imaging calcium responses in gfp-tagged neurons of hypothalamic mouse brain slices

脳内の情報の符号化、正確な分子手順並びに視床下部が残っているとしての脳領域の多機能の核内の特定のニューロンの活動に関わる中心的な問題の根底にあるメカニズムについての我々の知識の巨大な増加にもかかわらず。この問題は、様々な神経ホルモンのシグナル伝達カスケードの調節に関与する分子成分の識別を含む。細胞内Ca 2 +の上昇は両方のシグナル伝達のレベルとシナプス部位で、神経細胞の感度を調節するのに重要な役割を果たしている。 新しいツールは、特定のプロモーターの制御下に緑色蛍光タンパク質（GFP）を発現させることにより、脳の神経細胞の無数のニューロンを特定するために浮上している。空間的にも時間的刺激によって誘発されるCa 2 +の GFPタグニューロン、非緑色蛍光Ca 2 +指示薬色素nの応答を監視するには使用するeeds。さらに、共焦点顕微鏡は、組織内の深さの異なる平面内のニューロンを可視化するとアウトフォーカスの蛍光を制限するために、その能力のために組織切片におけるイメージング個々のニューロンの好きな方法である。レシオメトリックのCa 2 +指示薬FURA-2は、GFPタグニューロン1と組み合わせて使用されています。しかし、染料は紫外線（UV）光によって励起される。レーザのコストおよびUV光の限られた光の浸透深さは、多くの研究室での使用を妨げた。また、GFP蛍光はFURA-2信号2と干渉する場合があります。したがって、私たちは赤い蛍光Ca 2 +指示薬色素を使用することを決めた。 FURA-赤の巨大なストロークシフトは単一の励起波長を用いて、GFPとの組み合わせで赤色蛍光の多色分析を可能にします。我々はGFPタグ嗅覚ニューロン3と組み合わせてFURA-赤を使用して以前に良好な結果を得た。嗅覚組織切片のためのプロトコルは、電子を動作するように見えたquallyよく視床下部ニューロン4インチのFura-赤基づくのCa 2 +イメージングにも成功し、膵β細胞とHEK細胞に発現し5,6 GFPタグ受容体GFPタグと組み合わせた。 FURA-赤の少し癖は、インジケータがカルシウム7をバインドした後650nmでの蛍光強度が減少することである。したがって、低Ca 2 +濃度と休息ニューロンの蛍光は、比較的高い強度を有している。それは注意すべきで、そのほかの赤のCa 2 +指示薬色素が存在しないか、現在開発されて、それはまた別のニューロンと脳領域におけるより良いまたは改善された結果を与えるかもしれない。

Watch this Scientific Journal Video about Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices at JoVE.com

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

このプロトコルでは、カルシウムイメージングの最近の進捗状況を更新<sup&gt; 2 +</sup赤色蛍光Caを用いた脳組織切片におけるGFPタグニューロンの&gt;信号<sup&gt; 2 +</sup&gt;指示薬色素。

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 ...

imaging-calcium-responses-gfp-tagged-neurons-hypothalamic-mouse-brain

Nanyang Technological University

Research

JoVE Journal

Neuroscience

17.8K ビュー.  University of Saarland, Homburg, Germany. このプロトコルでは、カルシウムイメージングの最近の進捗状況を更新 2 +信号 2 +指示薬色素。

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

Dual Somatic Recordings from Gonadotropin-Releasing Hormone (GnRH) Neurons Identified by Green Fluorescent Protein (GFP) in Hypothalamic Slices

Gonadotropin-Releasing Hormone (GnRH) is a small neuropeptide that regulates pituitary release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). These gonadotropins are essential for the regulation of reproductive function. The GnRH-containing neurons are distributed diffusely throughout the hypothalamus and project to the median eminence where they release GnRH from their axon terminals into the hypophysiotropic portal system (1). In the portal capillaries, GnRH travels to the anterior pituitary gland to stimulate release of gonadotropins into systemic circulation. GnRH release is not continuous but rather occurs in episodic pulses. It is well established that the intermittent manner of GnRH release is essential for reproduction (2, 3).
Coordination of activity of multiple GnRH neurons probably underlies GnRH pulses. Total peptide content in GnRH neurons is approximately 1.0 pg/cell (4), of which 30% likely comprises the releasable pool. Levels of GnRH during a pulse (5, 6), suggest multiple GnRH neurons are probably involved in neurosecretion. Likewise, single unit activity extracted from hypothalamic multi-unit recordings during LH release indicates changes in activity of multiple neurons (7). The electrodes with recorded activity during LH pulses are associated with either GnRH somata or fibers (8). Therefore, at least some of this activity arises from GnRH neurons.
The mechanisms that result in synchronized firing in hypothalamic GnRH neurons are unknown. Elucidating the mechanisms that coordinate firing in GnRH neurons is a complex problem. First, the GnRH neurons are relatively few in number. In rodents, there are 800-2500 GnRH neurons. It is not clear that all GnRH neurons are involved in episodic GnRH release. Moreover, GnRH neurons are diffusely distributed (1). This has complicated our understanding of coordination of firing and has made many technical approaches intractable. We have optimized loose cell-attached recordings in current-clamp mode for the direct detection of action potentials and developed a recording approach that allows for simultaneous recordings from pairs of GnRH neurons.

Dual Somatic Recordings from Gonadotropin-Releasing Hormone GnRH Neurons Identified by Green Fluorescent Protein GFP in Hypothalamic Slices

Activity in neuronal systems often requires synchronous action potential discharges from neurons within a specific population. For example, pulses of gonadotropin-releasing hormone (GnRH) likely require coordinated activity between GnRH neurons. We present our methodological approach for reliably obtaining simultaneous electrophysiological recordings from the diffusely distributed GnRH neurons.

視床下部スライスに緑色蛍光タンパク質（GFP）によって識別されるゴナドトロピン放出ホルモン（GnRH）ニューロンから二重の体細胞の録音

Gonadotropin-Releasing Hormone (GnRH) is a small neuropeptide that regulates pituitary release of luteinizing hormone (LH) and follicle-stimulating ...

神経科学

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

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

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

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

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 ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

カルシウムイメージングの急性脳室下帯スライスの調製

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

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

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

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

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

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

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

GLUT4 タンパク質デコンボリューション顕微鏡を用いたマウス一次視床下部ニューロンにおける人身売買のライブ映像

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

脳スライスにおける神経樹状突起における 2 光子カルシウム イメージング

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

線虫の行動で個々 のニューロンのレシオ メトリック カルシウム イメージング

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

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 ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

脳スライスと一次細胞培養における錐体ニューロンの弾道標識

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

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 ...