Name: combined in vivo anatomical and functional tracing of ventral tegmental area glutamate terminals in the hippocampus
Uploaded: 2020-09-09T12:30:00.000+00:00
Duration: 9 min 36 s
Description: Watch this Scientific Journal Video about Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus at JoVE.com

この作品は、OOMに授与されたCBSブリッジンググラントによって資金提供されています。OOM、PAA、およびASは、研究を設計し、実験を行いました。ASとPAAは結果を分析した。OOMとPAAは原稿を準備しました。AAVを利用できるようにしてくれたカール・ディスセロス博士(スタンフォード大学)に感謝します。

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著者らは開示するものは何もない。

過去10年間で、AAV構造の設計は大幅に進歩しました。このように、より多くのニューロン特異的プロモーターが、改善されたトランスフェクション特異性14のためのAAV血清型の配列に組み込まれている。蛍光タンパク質、トランスポーター、受容体、イオンチャネルの遺伝子を組み合わせることで、イメージング、神経変調、シナプス活性検出用のAAVライブラリが存在するよ...

過去10年間で、ニューロン型変調の特異性を高めるための遺伝的ツールの配列が開発され、複雑なニューラルネットワーク1のマッピングが行われました。特に、神経細胞に感染し、複製する固有の能力を持つ神経刺激性ウイルスは、ニューロンサブタイプの特定のタンパク質を発現またはアブレートするために展開されています。蛍光タンパク質または遺伝的にコードされたシナプス活性指標を収容する場合、トランスフェクトされたAAVベクターは脳領域を横切るニューラルネットワークを標識および線引きする2,3。AAV構築物におけるプロモーターの選択は、ある程度の特異性(プロモーター依存性発現)を有するニューロン型におけるベクターの発現を指示する。しかし、Cre-loxの組換えを通じて、AAV構築物は、ニューロン標識4、5、6、7に対するより大きな特異性で展開される。注目すべきは、AAVベクターにパッケージ化された光活性化された微生物オプシンおよび蛍光タンパク質は、各種ニューロンサブタイプ8で発現することができ、かつ、イメージング、ニューロン型回路トレース、および光変調9,10に最適である。
AAVは、脳領域(または核)に立体的に注入され、ソーマ、樹状突起、軸索末端におけるレポータータンパク質の発現を駆動する。レポーター遺伝子(eYFP)を収容するAAVの神経発現は、ニューロン細胞体の標識化を促進し、他の脳領域11、12、13、14との間の突起の細胞細胞体および解剖学的トレースを促進する。光制御されたオプシン(例えば、hChR2)を担うAAV-eYFP構築物は、ビボ16における脳領域を標的とする神経投影の画像6、15および刺激ベースの生理学的トレース用のツールとして展開することができる。AAV血清型に応じて、ニューロン標識の方向は、前向きまたは逆行性11、12であってもよい。これまでの研究では、AAV5がニューロン12で前向きに移動することを確立しています。このように、hChR2を発現する細胞体の光刺激は、脳内の他の場所でシナプス前効果を生じる(標的)17。
ここで、AAV(血清型5)を用いて、CaMKIIαプロモーターを用いて、VTAグルタミン酸ニューロンおよび軸索突起においてeYFP(レポーター)およびhChR2(オプシン)を発現させた。この研究の結果は、CA1、CA3、およびDG海馬領域におけるVTA-グルタミン酸シナプス前末端の層依存分布を示す。また、VTAグルタミン酸ニューロンの光刺激は、ベースライン値と比較した場合に、生体内でのCA1マルチユニットおよび単一ユニットの発火率を増加させた。このプロトコルは、ニューラル回路トレース実験から得られるデータの品質を向上させることができる、手頃な価格のツールと市販のソフトウェアを利用しています。

<table><tbody><tr><td>3% Hydrogen peroxide</td><td>Fisher chemical</td><td>H312</td><td></td></tr><tr><td>AAV-CaMKII&amp;alpha;-ChR2-eGYP</td><td>Addgene</td><td>Plasmid #26969</td><td></td></tr><tr><td>BNC cable</td><td>Amazon</td><td></td><td></td></tr><tr><td>BNC Splitter</td><td>Amazon</td><td></td><td></td></tr><tr><td>Ceramic Split Mating Sleeve for &amp;Oslash;1.25mm Ferrules.</td><td>Thorlabs</td><td>ADAL1-5</td><td></td></tr><tr><td>Drill</td><td>Dremel</td><td>LR 39098</td><td></td></tr><tr><td>Gelatin coated slides</td><td>Fisher scientific</td><td>OBSLD01CS</td><td></td></tr><tr><td>Hamilton&#039;s syringe (Neuros)</td><td>WPI Inc.</td><td>06H</td><td></td></tr><tr><td>Head stage adapter</td><td>Neuronexus</td><td>Adpt-Q4-OM32</td><td></td></tr><tr><td>High impedance silicon probe</td><td>Neuronexus</td><td>Q1x1-tet-5mm-121-CQ4</td><td></td></tr><tr><td>INTAN 512ch Recording Controller</td><td>INTAN</td><td>RHD2000</td><td></td></tr><tr><td>Iodine solution</td><td>Dynarex</td><td>1425</td><td></td></tr><tr><td>Isoflurane</td><td>Piramal</td><td>NDC 66794-017-25</td><td></td></tr><tr><td>Ketamine</td><td>Spectrum</td><td>K1068</td><td></td></tr><tr><td>LED Driver</td><td>Thorlabs</td><td>LEDD1B</td><td></td></tr><tr><td>LED light source (470 nm)-blue light</td><td>Thorlabs</td><td>M470F3</td><td></td></tr><tr><td>Micromanipulator</td><td>Narishige</td><td>M0-203</td><td></td></tr><tr><td>Optic fiber</td><td>Thorlabs</td><td>CFMLC14L05</td><td></td></tr><tr><td>Pan head philips screw (M0.6 X 2mm)</td><td>Amazon</td><td>M0.6 X 2mm</td><td></td></tr><tr><td>Pre-amplifier headstage (32 Channel)</td><td>INTAN</td><td>C3314</td><td></td></tr><tr><td>Stereotaxic frame</td><td>Kopf</td><td>1530</td><td></td></tr><tr><td>TTL pulser</td><td>Prizmatix</td><td>4031</td><td></td></tr><tr><td>Urethane</td><td>Sigma</td><td>U2500</td><td></td></tr><tr><td>Xylazine</td><td>Alfa Aesar</td><td>J61430</td><td></td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Version&lt;/strong&gt;</td><td></td></tr><tr><td>Graphpad Prism</td><td>GraphPad Software</td><td>version 8</td><td></td></tr><tr><td>Intan Recording Controller</td><td>Intantech</td><td>version 2.07</td><td></td></tr><tr><td>Neuroexplorer</td><td>Nex Technologies</td><td>version 5.215</td><td></td></tr><tr><td>Plexon Offline Spike Sorter</td><td>Plexon Inc</td><td>version 4.5</td><td></td></tr><tr><td>&lt;strong&gt;ACSF Composition:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>oxygenated ACSF with 95% Oxygen/5%CO&lt;sub&gt;2&lt;/sub&gt; constantly being bubbled through the ACSF (ACSF; in mM 125 NaCl, 25 NaHCO&lt;sub&gt;3&lt;/sub&gt;, 3 KCl, 1.25 NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 1 MgCl&lt;sub&gt;2&lt;/sub&gt;, 2 CaCl&lt;sub&gt;2&lt;/sub&gt; and 25 Glucose).</td><td></td><td></td><td></td></tr></tbody></table>

combined in vivo anatomical and functional tracing of ventral tegmental area glutamate terminals in the hippocampus

脳内のニューロン亜集団の光遺伝学的調節は、研究者が生体内およびex vivoで神経回路を解剖することを可能にした。これは、神経回路内のニューロンタイプの役割と、学習に対する情報符号化におけるその重要性を決定するための前提となる。同様に、この方法は、目覚めおよび麻酔動物における2つ以上の接続された脳領域の生理学的意義を試験するために使用することができる。現在の研究は、VTAグルタミン酸ニューロンが麻酔をかけマウスのCA1(海馬)における推定ピラミッド型ニューロンの発火率をどのように調節するかを示している。このプロトコルは、海馬の層内のVTAシナプス前グルタミン酸末端のトレースのためのVTAグルタミン酸ニューロンのアデノ関連ウイルス(AAV)依存的標識を採用する。AAVベクターに収容された光制御オプシン(チャネロドプシン;hChR2)および蛍光タンパク質(eYFP)の発現は、VTAグルタミン酸末端の前向きのトレースを許可し、VTAグルタミン酸ニューロン細胞体(VTA)の光刺激を認めた。高インピーダンスの急性シリコン電極をCA1に配置し、生体内でのVTA光刺激に対するマルチユニットおよび単一ユニットの応答を検出した。この研究の結果は、海馬(CA1、CA3、およびDG)におけるシナプス前VTAグルタミン酸末端の層依存分布を示す。また、VTAグルタミン酸ニューロンの光刺激は、生体内の推定CA1錐体単位の発火およびバースト速度を増加させた。

順次トレース
AAV発現は、注射後21日目のC57BL/6マウスのVTAにおけるレポータータンパク質(eYFP)の免疫蛍光イメージング(eYFP)によって検証された(図2)。海馬におけるシナプス前VTAグルタミン酸突起の前向き標識化に成功したDG、CA3、CA1の層におけるeYFP検出によっても検証された(図6a–d;ムービー 2 と 3.
...

Watch this Scientific Journal Video about Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus at JoVE.com

Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus

現在のプロトコルは、海馬への腹側テグメンタル領域(VTA)グルタミン酸突起をトレースするための簡単な方法を示しています。VTAグルタミン酸ニューロンの光刺激をCA1記録と組み合わせて、VTAグルタミン酸末端が生体内での推定ピラミッド発火率を調節する方法を示した。

海馬の腹側テグメンタル領域グルタミン酸末端のインビボ解剖学的および機能的なトレース

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a ...

combined-vivo-anatomical-functional-tracing-ventral-tegmental-area

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

3.8K ビュー.  Louisiana State University School of Veterinary Medicine. 現在のプロトコルは、海馬への腹側テグメンタル領域(VTA)グルタミン酸突起をトレースするための簡単な方法を示しています。VTAグルタミン酸ニューロンの光刺激をCA1記録と組み合わせて、VTAグルタミン酸末端が生体内での推定ピラミッド発火率を調節する方法を示した。

ビデオ: Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus

Longitudinal Two-Photon Imaging of Dorsal Hippocampal CA1 in Live Mice

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from subcellular to network levels and at temporal scales from milliseconds to weeks. In addition, two-photon imaging can be combined with a variety of behavioral tasks to explore the causal relationships between brain function and behavior. However, in mammals, limited penetration and scattering of light have limited two-photon intravital imaging mostly to superficial brain regions, thus precluding longitudinal investigation of deep-brain areas such as the hippocampus. The hippocampus is involved in spatial navigation and episodic memory and is a long-standing model used to study cellular as well as cognitive processes important for learning and recall, both in health and disease. Here, a preparation that enables chronic optical access to the dorsal hippocampus in living mice is detailed. This preparation can be combined with two-photon optical imaging at cellular and subcellular resolution in head fixed, anesthetized live mice over several weeks. These techniques enable repeated imaging of neuronal structure or activity-evoked plasticity in tens to hundreds of neurons in the dorsal hippocampal CA1. Furthermore, this chronic preparation can be used in combination with other techniques such as micro-endoscopy, head-mounted wide field microscopy or three-photon microscopy, thus greatly expanding the toolbox to study cellular and network processes involved in learning and memory.

This method describes a chronic preparation that allows optical access to the hippocampus of living mice. This preparation can be used to perform longitudinal optical imaging of neuronal structural plasticity and activity-evoked cellular plasticity over a period of several weeks.

生きたマウスにおける後部海馬CA1の縦方向2光子イメージング

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from ...

行動学

Measurement of Glutamate Uptake using Radiolabeled L-[3H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

Glutamate removal from the extracellular space by high-affinity Na+-dependent transporters is essential to ensure that the brain&#39;s intrinsic connectivity mechanisms work properly and homeostasis is maintained. The hippocampus is a unique brain structure that manages higher cognitive functions, and is the subject of several studies regarding neurologic diseases. The investigation of physiological and pathological mechanisms in rodent models can benefit from acute hippocampal slice (AHS) preparations. AHS has the advantage of providing reliable information on cell function since the cytoarchitecture and synaptic circuits are preserved. Although AHS preparations are commonly used in neurochemistry laboratories, it is possible to find some methodological differences in the literature. Considering that distinctive slice preparation protocols might change the hippocampal regions analyzed, this current protocol proposes a standard technique for obtaining transverse AHS from resected hippocampus. This simple-to-perform protocol may be used in mice and rats&#39; experimental models and allow several ex vivo approaches investigating neurochemical dynamics (in dorsal, intermediate and ventral hippocampus) in different backgrounds (e.g., transgenic manipulations) or after in vivo manipulations (e.g., pharmacological treatments or suitable rodent models to study clinical disorders). After dissecting the hippocampus from the rodent brain, transverse slices along the septo-temporal axis (300 &#181;m thick) were obtained. These AHS contain distinct parts of the hippocampus and were subjected to an individual neurochemical investigation (as an example: neurotransmitter transporters using their respective substrates). As the hippocampus presents a high density of excitatory synapses, and glutamate is the most important neurotransmitter in the brain, the glutamatergic system is an interesting target for in vivo observed phenomena. Thus, the current protocol provides detailed steps to explore glutamate uptake in ex vivo AHS using L-[3H]-Glutamate. Using this protocol to investigate hippocampal function may help to better understand the influence of glutamate metabolism on mechanisms of neuroprotection or neurotoxicity.

Measurement of Glutamate Uptake using Radiolabeled L-[3H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

This article describes a reliable and simple way to obtain ex vivo acute hippocampal transverse slices from mice and rats using a tissue chopper. Slices obtained from resected hippocampi can be submitted for functional glutamate uptake analysis to investigate glutamatergic system homeostasis.

Glutamate removal from the extracellular space by high-affinity Na+-dependent transporters is essential to ensure that the brain&#39;s intrinsic ...

生化学

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

高解像度<em&gt;インビボ</em&gt; 3T磁気共鳴画像を用いたヒト海馬サブフィールドのためのマニュアルセグメンテーションプロトコル

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

神経科学

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

マウスの海馬領域の歯状回における樹状樹枝状分岐の評価

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat

High molecular weight biotinylated dextran amine (BDA) has been used as a highly sensitive neuroanatomical tracer for many decades. Since the quality of its labeling was affected by various factors, here, we provide a refined protocol for the application of high molecular weight BDA for studying optimal neural labeling in the central nervous system. After stereotactic injection of BDA into the ventral posteromedial nucleus (VPM) of the thalamus in the rat through a delicate glass pipette, BDA was stained with fluorescent streptavidin-Alexa (AF) 594 and counterstained with fluorescent Nissl stain AF500/525. On the background of green Nissl staining, the red BDA labeling, including neuronal cell bodies and axonal terminals, was more distinctly demonstrated in the somatosensory cortex. Furthermore, double fluorescent staining for BDA and the calcium-binding protein parvalbumin (PV) was carried out to observe the correlation of BDA labeling and PV-positive interneurons in the cortical target, providing the opportunity to study the local neural circuits and their chemical characteristics. Thus, this refined method is not only suitable for visualizing high quality neural labeling with the high molecular weight BDA through reciprocal neural pathways between the thalamus and cerebral cortex, but also will permit the simultaneous demonstration of other neural markers with fluorescent histochemistry or immunochemistry.

Here, we present a refined protocol to effectively reveal biotinylated dextran amine (BDA) labeling with a fluorescent staining method through a reciprocal neural pathway. It is suitable for analyzing the fine structure of BDA labeling and distinguishing it from other neural elements under a confocal laser scanning microscope.

トレースのラットにおける視床皮質投射のビオチン標識デキストランの高分子量アミンの運用の改善

High molecular weight biotinylated dextran amine (BDA) has been used as a highly sensitive neuroanatomical tracer for many decades. Since the quality of ...

In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional investigation of long-range connections requires targeted recordings of single neurons combined with the specific stimulation of identified distant inputs. This is often difficult to achieve with conventional and electrical stimulation techniques, because axons from converging upstream brain areas may intermingle in the target region. The stereotaxic targeting of a specific brain region for virus-mediated expression of light-sensitive ion channels allows selective stimulation of axons originating from that region with light. Intracerebral stereotaxic injections can be used in well-delimited structures, such as the anterior thalamic nuclei, in addition to other subcortical or cortical areas throughout the brain.
Described here is a set of techniques for precise stereotaxic injection of viral vectors expressing channelrhodopsin in the mouse brain, followed by photostimulation of axon terminals in the brain slice preparation. These protocols are simple and widely applicable. In combination with whole-cell patch clamp recording from a postsynaptically connected neuron, photostimulation of axons allows the detection of functional synaptic connections, pharmacological characterization, and evaluation of their strength. In addition, biocytin filling of the recorded neuron can be used for post-hoc morphological identification of the postsynaptic neuron.

This protocol describes a set of methods to identify the cell-type specific functional connectivity of long-range inputs from distant brain regions using optogenetic stimulations in ex vivo brain slices.

マウス脳スライスにおける長距離入力の光遺伝学的刺激のための生体内脳内立体注射

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional ...

Preparation of Acute Slices from Dorsal Hippocampus for Whole-Cell Recording and Neuronal Reconstruction in the Dentate Gyrus of Adult Mice

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in molecular, anatomical and functional criteria suggesting a division into different sub-circuits along its rostro-caudal extent. Owing to differential connectivity and function the most fundamental distinction is made between the dorsal and the ventral hippocampus, which are preferentially involved in spatial and emotional processing, respectively. Accordingly, in vivo work regarding spatial memory formation has focused on the dorsal hippocampus.
In contrast, electro-physiological in vitro recordings have been preferentially performed on intermediate-ventral hippocampus, largely motivated by factors like slice viability and circuit integrity. To allow for direct correlation of in vivo data on spatial processing with in vitro data we have adapted previous sectioning methods to obtain highly viable transverse brain slices from the dorsal-intermediate hippocampus for long-term recordings of principal cells and interneurons in the dentate gyrus. As spatial behavior is routinely analyzed in adult mice, we have combined this transversal slicing procedure with the use of protective solutions to enhance viability of brain tissue from mature animals. We use this approach for mice of about 3 months of age. The method offers a good alternative to the coronal preparation which is frequently used for in vitro studies on dorsal hippocampus. We compare these two preparations in terms of quality of recordings and preservation of morphological features of recorded neurons.

We present a protocol to prepare acute-slices from the dorsal-intermediate hippocampus of mice. We compare this transversal preparation with coronal slicing in terms of quality of recordings and preservation of morphological features of recorded neurons.

成体マウスのデンテート・ジラスにおける全細胞記録と神経再建のための、逆海馬からの急性スライスの作製

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in ...

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit functions in systems neuroscience. However, high absorbance and scattering of light in mammalian tissue limit intravital imaging mostly to superficial brain regions, leaving deep-brain areas, such as the hippocampus, out of reach for optical microscopy. In this video, we show the preparation and implantation of the custom-made imaging window to enable chronic in vivo imaging of the dorsal hippocampal CA1 region in head-fixed behaving mice. The custom-made window is supplemented with an infusion cannula that allows targeted delivery of viral vectors and drugs to the imaging area. By combining this preparation with wide-field imaging, we performed a long-term recording of neuronal activity using a fluorescent calcium indicator from large subsets of neurons in behaving mice over several weeks. We also demonstrated the applicability of this preparation for voltage imaging with single-spike resolution. High-performance genetically encoded indicators of neuronal activity and scientific CMOS cameras allowed the recurrent visualization of subcellular morphological details of single neurons at high temporal resolution. We also discuss the advantages and potential limitations of the described method and its compatibility with other imaging techniques.

This article demonstrates the preparation of a custom-made imaging window supplemented with infusion cannula and its implantation onto the CA1 region of the hippocampus in mice.

マウスの振る舞いの海馬における神経活動を可視化するための頭蓋骨管制法

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit ...

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

Microglia, the only immune cells resident in the brain, actively participate in neural circuit maintenance by modifying synapses and neuronal excitability. Recent studies have revealed the differential gene expression and functional heterogeneity of microglia in different brain regions. The unique functions of the hippocampal neural network in learning and memory may be associated with the active roles of microglia in synapse remodeling. However, inflammatory responses induced by surgical procedures have been problematic in the two-photon microscopic analysis of hippocampal microglia. Here, a method is presented that enables the chronic observation of microglia in all layers of the hippocampal CA1 through an imaging window. This method allows the analysis of morphological changes in microglial processes for more than 1 month. Long-term and high-resolution imaging of the resting microglia requires minimally invasive surgical procedures, appropriate objective lens selection, and optimized imaging techniques. The transient inflammatory response of hippocampal microglia may prevent imaging immediately after surgery, but the microglia restore their quiescent morphology within a few weeks. Furthermore, imaging neurons simultaneously with microglia allows us to analyze the interactions of multiple cell types in the hippocampus. This technique may provide essential information about microglial function in the hippocampus.

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

This paper describes a method for chronic in vivo observation of the resting microglia in the mouse hippocampal CA1 using precisely controlled surgery and two-photon microscopy.

インビボ マウス海馬におけるミクログリアの慢性二光子イメージング

Microglia, the only immune cells resident in the brain, actively participate in neural circuit maintenance by modifying synapses and neuronal ...

マウスの歯状回細胞におけるニューロン活動のリアルタイムイメージング 

In Vivo Calcium Imaging of Granule Cells in the Dentate Gyrus of Hippocampus in Mice

Real-time approaches are typically needed in studies of learning and memory, and in vivo calcium imaging provides the possibility to investigate neuronal activity in awake animals during behavior tasks. Since the hippocampus is closely associated with episodic and spatial memory, it has become an essential brain region in&#160;this field&#39;s research. In recent research, engram cells and place cells were studied by recording the neural activities in the hippocampal CA1 region using the miniature microscope in mice while performing behavioral tasks including open-field and linear track. Although the dentate gyrus is another important region in the hippocampus, it has rarely been studied with in vivo imaging due to its greater depth and difficulty for imaging. In this protocol, we present in detail a calcium imaging process, including how to inject the virus, implant a GRIN (Gradient-index) lens, and attach a base plate for imaging the dentate gyrus of the hippocampus. We further describe how to preprocess the calcium imaging data using MATLAB. Additionally, studies of other deep brain regions that require imaging may benefit from this method.

著者スポットライト:小型顕微鏡による歯状回顆粒細胞の神経活性の調査

The dentate gyrus of the hippocampus carries out essential and distinct functions in learning and memory. This protocol describes a set of robust and efficient procedures for in vivo calcium imaging of granule cells in the dentate gyrus in freely moving mice.

マウス海馬歯状回における顆粒細胞のin vivoカルシウムイメージング 

Real-time approaches are typically needed in studies of learning and memory, and in vivo calcium imaging provides the possibility to investigate neuronal ...

海馬の腹側テグメンタル領域グルタミン酸末端のインビボ解剖学的および機能的なトレース

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生きたマウスにおける後部海馬CA1の縦方向2光子イメージング

Measurement of Glutamate Uptake using Radiolabeled L-[³H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

高解像度

マウスの海馬領域の歯状回における樹状樹枝状分岐の評価

トレースのラットにおける視床皮質投射のビオチン標識デキストランの高分子量アミンの運用の改善

マウス脳スライスにおける長距離入力の光遺伝学的刺激のための生体内脳内立体注射

成体マウスのデンテート・ジラスにおける全細胞記録と神経再建のための、逆海馬からの急性スライスの作製

マウスの振る舞いの海馬における神経活動を可視化するための頭蓋骨管制法

インビボマウス海馬におけるミクログリアの慢性二光子イメージング

マウス海馬歯状回における顆粒細胞のin vivoカルシウムイメージング

海馬の腹側テグメンタル領域グルタミン酸末端のインビボ解剖学的および機能的なトレース

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この動画の章

生きたマウスにおける後部海馬CA1の縦方向2光子イメージング

Measurement of Glutamate Uptake using Radiolabeled L-[3H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

高解像度

マウスの海馬領域の歯状回における樹状樹枝状分岐の評価

トレースのラットにおける視床皮質投射のビオチン標識デキストランの高分子量アミンの運用の改善

マウス脳スライスにおける長距離入力の光遺伝学的刺激のための生体内脳内立体注射

成体マウスのデンテート・ジラスにおける全細胞記録と神経再建のための、逆海馬からの急性スライスの作製

マウスの振る舞いの海馬における神経活動を可視化するための頭蓋骨管制法

インビボ マウス海馬におけるミクログリアの慢性二光子イメージング

マウス海馬歯状回における顆粒細胞のin vivoカルシウムイメージング

Measurement of Glutamate Uptake using Radiolabeled L-[³H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

インビボマウス海馬におけるミクログリアの慢性二光子イメージング