<meta charset="utf8"></head><body>成体マウスの海馬ミクログリアの電気生理学的記録

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<table><tbody><tr><td>100 mm Petri dish</td><td>Corning</td><td>353003</td><td></td></tr><tr><td>15 mL Falcon tubes</td><td>BD</td><td>352096</td><td></td></tr><tr><td>24-well plates</td><td>BD</td><td>353047</td><td></td></tr><tr><td>35 mm Petri dish</td><td>Corning</td><td>353001</td><td>-</td></tr><tr><td>50 mL Falcon tubes</td><td>BD</td><td>352070</td><td></td></tr><tr><td>70 &amp;mu;m cell screening</td><td>Miltenyi</td><td>130-095-823</td><td>To remove cell clumps before cell sorting</td></tr><tr><td>Adult Mouse Brain Dissociation Kit</td><td>Miltenyi</td><td>130- 107-677</td><td></td></tr><tr><td>Anti-CD11b Antibody</td><td>Bio-Rad</td><td>MCA74</td><td>Goat Anti-mouse IgG also available. For blocking endogenous immunoglobulins.</td></tr><tr><td>Anti-Iba 1 Antibody</td><td>SYSY</td><td>234308</td><td>Goat Anti-guinea pig IgG) also available. For blocking endogenous immunoglobulins.</td></tr><tr><td>Axon Digidata 1440A</td><td>USA</td><td></td><td></td></tr><tr><td>Axon MultiClamp 700B Amplifier</td><td>USA</td><td></td><td></td></tr><tr><td>BSA Albumin Fraction V</td><td>BioFrox</td><td>4240GR500</td><td>Serum</td></tr><tr><td>C57BL/6 mice&amp;nbsp;</td><td>Guangdong Medical Laboratory Animal Center</td><td></td><td></td></tr><tr><td>CD11 b/c MicroBeads</td><td>Miltenyi</td><td>130-105-634</td><td></td></tr><tr><td>Chloral hydrate</td><td>Absin</td><td>abs47051394</td><td>Widely used as anesthesia in mice</td></tr><tr><td>Clampfit 10.6</td><td>USA</td><td></td><td></td></tr><tr><td>Confocal Microscope</td><td>Zeiss</td><td>&amp;nbsp;LSM 800</td><td></td></tr><tr><td>Culture medium</td><td>Fisher Scientific</td><td>C11995500BT</td><td>-</td></tr><tr><td>DAPI dye</td><td>Beyotime</td><td>C1002</td><td></td></tr><tr><td>Electrode puller</td><td>Narishige</td><td>PC-10</td><td></td></tr><tr><td>HBSS</td><td>Servicebio</td><td>G4203</td><td></td></tr><tr><td>Horizontal shaker</td><td>SCILOGEX</td><td>SLK-O3000-S</td><td></td></tr><tr><td>Image analysis software</td><td>Fiji</td><td></td><td></td></tr><tr><td>MACS Columns</td><td>Miltenyi</td><td>130-042-201</td><td></td></tr><tr><td>MACS Separators</td><td>Miltenyi</td><td>130-042-102</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Fisher Scientific</td><td>T353-500</td><td>Use fresh 4% solution in 1X PBS, pH 7.2-7.4.&amp;nbsp;</td></tr><tr><td>Poly-L-lysine</td><td>Beyotime</td><td>&amp;nbsp;C0313</td><td>Coverslip coating</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>X100</td><td></td></tr></tbody></table>

isolation and whole-cell patch-clamp recording of hippocampal microglia from adult mice

<meta charset="utf8"></head><body>成体マウスからの海馬ミクログリアの単離と全細胞パッチクランプ記録

成体マウスからの海馬ミクログリアの単離と全細胞パッチクランプ記録

Microglia are resident immune cells in the brain that interact with neurons to maintain the homeostasis of the central nervous system (CNS). Studies show ...

isolation-whole-cell-patch-clamp-recording-hippocampal-microglia-from

Nanyang Technological University

Research

JoVE Journal

Biology

Isolation and Whole-Cell Patch-Clamp Recording of Hippocampal Microglia from Adult Mice

622 ビュー.  South China Normal University. 本プロトコルは、成体マウスから初代海馬ミクログリアを単離し精製する方法を説明し、続いて、これらの急性に単離された細胞の全細胞パッチクランプ記録を実施するための指示が続きます。

ビデオ: 成体マウスからの海馬ミクログリアの単離と全細胞パッチクランプ記録

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

マウス新生児から保護免疫表現と機能皮質ミクログリアの分離

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

免疫・感染学

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

Morphologically-と神経化学的に特定し海馬介在ニューロンからホールセルパッチクランプ記録

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

神経科学

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent improvements in methodologies and applications for the study of theta oscillations. A simple characterization of the isolated hippocampal preparation is presented whereby the relationship between internal hippocampal theta oscillators is examined together with the activity of pyramidal cells, and GABAergic interneurons, of the cornu ammonis-1 (CA1) and subiculum (SUB) areas. Overall, we show that the isolated hippocampus is capable of generating intrinsic theta oscillations in vitro and that rhythmicity generated within the hippocampus can be precisely manipulated by optogenetic stimulation of parvalbumin-positive (PV) interneurons. The in vitro isolated hippocampal preparation offers a unique opportunity to use simultaneous field and intracellular patch-clamp recordings from visually-identified neurons to better understand the mechanisms underlying theta rhythm generation.

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

Here, we present a protocol for recording rhythmic neuronal network theta and gamma oscillations from an isolated whole hippocampal preparation. We describe the experimental steps from extraction of the hippocampus to details of field, unitary and whole-cell patch clamp recordings as well as optogenetic pacing of the theta rhythm.

海馬シータバンドのチューニング<em&gt;インビトロ</em&gt;：隔離されたげっ歯類胸腺腫回路からの記録方法

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent ...

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive human diseases. Considerable advances have been made to uncover the molecular signatures of homeostatic microglia as well as alterations of their gene expression in response to environmental stimuli. With the advent and maturation of single-cell genomic methodologies, it is increasingly recognized that heterogenous microglia may underlie the diverse roles they play in different developmental and pathological conditions. Further dissection of such heterogeneity can be achieved through efficient isolation of microglia from a given region of interest, followed by sensitive profiling of individual cells. Here, we provide a detailed protocol for the rapid isolation of microglia from different brain regions in a single adult mouse brain hemisphere. We also demonstrate how to use these sorted microglia for plate-based deep single-cell RNA sequencing. We discuss the adaptability of this method to other scenarios and provide guidelines for improving the system to accommodate large-scale studies.

We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease.

ディープシングルセルRNAシーケンシングのための1つの成人マウス脳半球からの領域特異的ミクログリアの分離

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol&#8217;s critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.

This is a protocol for acute slice preparation from adult and aging mouse hippocampi that takes advantage of transcardial perfusion and slice cutting with ice-cold NMDG-aCSF to reduce hypoxic damage to the tissue. The resulting slices stay healthy over many hours, and are suitable for long-term patch-clamp and field-recordings.

成虫マウスおよび老化マウスからの海馬スライスにおける低酸素症の最小化

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high ...

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 ...

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 ...

In vitroアッセイのためのMACSを用いたマウスからの視床下部ミクログリアの単離

Isolation of Hypothalamic Microglia from Freshly Perfused Adult Mouse Brain by Magnetic-Activated Cell Sorting

Microglia, as the resident macrophages of the brain, are essential for maintaining brain homeostasis. They shape neuronal circuits during development, survey their environment for debris or dead cells, as well as respond to infection and injury in the brain, among many other functions. However, their important role in neurodevelopment and synaptic plasticity and pathophysiology has not been fully defined, highlighting the need for further investigation. To gain a more comprehensive understanding of the role of microglia in these processes, we need to isolate microglia and characterize them genetically, metabolically, and functionally. However, the isolation of microglia from adult mice, especially from small brain structures, is challenging as they represent a small percentage of the total brain cells, and the yield of isolated microglia is often too low. Here, the magnetic isolation of microglia using CD11b+ microbeads allows us to sort microglial cells from the hypothalamus of a freshly perfused adult mouse brain. The current method allows us to achieve relatively high purity and yield in a short period while maintaining cell viability.

著者スポットライト:マウスの脳機能および代謝障害に対する栄養の影響の調査 

We describe a protocol for isolating microglial cells from the mouse hypothalamus (or equivalent small brain structures) using magnetically activated cell sorting (MACS), in a relatively short time. The MACS-sorted hypothalamic microglia can be used for ex vivo analysis and can be plated to perform in vitro assays.

磁気活性化細胞選別による灌流したばかりの成体マウス脳からの視床下部ミクログリアの単離  

Microglia, as the resident macrophages of the brain, are essential for maintaining brain homeostasis. They shape neuronal circuits during development, ...

成体マウスからの海馬ミクログリアの単離と全細胞パッチクランプ記録

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この動画の章

マウス新生児から保護免疫表現と機能皮質ミクログリアの分離

Morphologically-と神経化学的に特定し海馬介在ニューロンからホールセルパッチクランプ記録

海馬シータバンドのチューニング

ディープシングルセルRNAシーケンシングのための1つの成人マウス脳半球からの領域特異的ミクログリアの分離

成虫マウスおよび老化マウスからの海馬スライスにおける低酸素症の最小化

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

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

磁気活性化細胞選別による灌流したばかりの成体マウス脳からの視床下部ミクログリアの単離

磁気活性化細胞選別による灌流したばかりの成体マウス脳からの視床下部ミクログリアの単離

磁気活性化細胞選別による灌流したばかりの成体マウス脳からの視床下部ミクログリアの単離

成体マウスからの海馬ミクログリアの単離と全細胞パッチクランプ記録

要約

さらに動画を探す

この動画の章

マウス新生児から保護免疫表現と機能皮質ミクログリアの分離

Morphologically-と神経化学的に特定し海馬介在ニューロンからホールセルパッチクランプ記録

海馬シータバンドのチューニング

ディープシングルセルRNAシーケンシングのための1つの成人マウス脳半球からの領域特異的ミクログリアの分離

成虫マウスおよび老化マウスからの海馬スライスにおける低酸素症の最小化

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

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

磁気活性化細胞選別による灌流したばかりの成体マウス脳からの視床下部ミクログリアの単離

磁気活性化細胞選別による灌流したばかりの成体マウス脳からの視床下部ミクログリアの単離

磁気活性化細胞選別による灌流したばかりの成体マウス脳からの視床下部ミクログリアの単離

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