Name: isolation and whole-cell patch-clamp recording of hippocampal microglia from adult mice
Uploaded: 2024-09-27T14:47:00.000+00:00
Duration: 8 min 34 s
Description: Microglia are resident immune cells in the brain that interact with neurons to maintain the homeostasis of the central nervous system (CNS). Studies show ...

This work was supported by grants from the National Natural Science Foundation of China (32170950, 32371065), the Natural Science Foundation of Guangdong Province, Nos. 2023A1515010899 and 2021A1515010804.

All experiments were approved by the Life Science Animal Care and Use Committee of South China Normal University, and appropriate standards of animal welfare were maintained (ethical approval: SCNU-SLS-2023-048). Male or female 3-5-month-old C57BL/6J mice were used for this study. The details of the reagents and equipment used are listed in the Table of Materials.
1. Preparation of solutions
<ol>
	<li>Prepare 50 mL of intracellular solution containing 160 mM of KF, 10 mM of HEPES, 10 mM of&#160;EGTA, and 2 mM of MgCl2. Adjust the pH to 7.2 with 1 M of KOH solution, and adjust the osmotic pressure to 300 mOsm with D-sucrose13,16. Store at -20 &#176;C.</li>
	<li>Prepare 50 mL of magnetic-activated cell sorting (MACS) buffer containing 0.5% bovine serum albumin (BSA) and 2 mM of ethylenediaminetetraacetic acid (EDTA) in PBS.</li>
	<li>Prepare poly-L-lysine (PLL) at a 1:1000 dilution in water. 
	NOTE: Coverslips should be coated with PLL the day before the experiment. Incubate the coated slips at 37 &#176;C overnight. Recycle PLL on the second day and dry the coated slips at 37 &#176;C.</li>
</ol>
2. Preparation of single-cell suspension from brain tissue
<ol>
	<li>Anesthetize the mice with 2.5% chloral hydrate (0.15 mL/10 g,&#160;i.p.) (following institutionally approved protocols). The disappearance of corneal reflex and pain reflex by pinching the toes or pinching the skin in mice confirm deep anesthesia. Place the animals supine on a dissecting table and pin their limbs.
	<ol>
		<li>Expose the chest and perfuse the heart17 with ice-cold sterile PBS to remove intravascular circulating blood cells. Hold the head and cut the skin along the brain midline with scissors to expose the skull.</li>
		<li>Cut the posterior of the skull bilaterally and between the eye sockets with small scissors. Cut the skull along the sagittal suture and remove the skull with tweezers. Extract the brain tissue quickly with tweezers17.</li>
	</ol>
	</li>
	<li>Place the brain tissue in 10 cm culture dishes containing ice-cold PBS. Dissect the cerebral cortex bilaterally with tweezers, isolate the hippocampal tissue, and cut it into smaller pieces. Transfer the pieces into a 15 mL centrifuge tube. 
	NOTE: Bilateral dissection of the cerebral cortex reveals the crescent of hippocampal tissue in both hemispheres of the brain.</li>
	<li>Resuspend the small pieces of hippocampal tissue in 2 mL of ice-cold Hank&#39;s balanced salt solution (HBSS) and centrifuge at 300 &#215; g for 3 min (at room temperature), then discard the HBSS.</li>
	<li>Add 1950 &#181;L of enzyme mixture 1 to the 15 mL centrifuge tube containing the hippocampal tissue pieces. Shake the centrifuge tube continuously in a 37 &#176;C water bath for 5 min. 
	NOTE: Enzyme mixture 1 is a combination of 50 &#181;L of enzyme P and 1900 &#181;L buffer Y, preheated to 37 &#176;C (present as a part of the commercially available kit, see Table of Materials).</li>
	<li>Remove the centrifuge tube from the water bath. Add 30 &#181;L of freshly prepared enzyme mixture 2 to the 15 mL centrifuge tube. Pipette the tissue fragments up and down 20 times with a 1 mL pipette tip, and incubate in a water bath for 5 min with continuous shaking at 37&#176;C. 
	NOTE: Enzyme mixture 2 is a combination of 20 &#181;L of buffer Y and 10 &#181;L of enzyme A. The above two digestion steps convert the brain tissue to a cellular suspension. The total digestion time should not exceed 15 min.</li>
	<li>Remove the centrifuge tube and pipette the suspension up and down multiple times with a 1 mL pipette until no large tissue pieces remain.</li>
	<li>Filter the cell suspension through a 70 &#181;m cell screening filter and collect the filtrate in a clean 50 mL centrifuge tube. 
	NOTE: This step removes any large pieces of undigested tissue.</li>
	<li>Transfer the filtrate to a new 15 mL centrifuge tube and centrifuge at 4 &#176;C, 300 &#215; g for 10 min. Discard the supernatant with a 1 mL pipette.</li>
	<li>Wash the cell suspension by pipetting up and down 20 times with 15 mL HBSS, and centrifuge at 4 &#176;C, 300 &#215; g for 10 min. Discard the supernatant and 
	resuspend the cell pellet in 1 mL of MACS&#160;buffer. 
	NOTE: This step provides the single-cell suspension used in subsequent experiments.</li>
</ol>
3. Acute separation of microglia
<ol>
	<li>Transfer 1 mL of the single-cell suspension into a 2 mL microcentrifuge tube and centrifuge at 4 &#176;C, 300 &#215; g for 3 min. Discard the supernatant.</li>
	<li>Resuspend the single-cell pellet in 90 &#181;L of MACS buffer. Add 10 &#181;L of CD11b/c microbeads and incubate the suspension at 4 &#176;C for 15 min on a horizontal shaker.</li>
	<li>Place the sorting column in the magnetic field of a suitable MACS separator and moisten the column twice with 500 &#181;L of MACS buffer.</li>
	<li>Wash the single-cell suspension by adding 1 mL of MACS buffer directly to the tube and centrifuge at 4 &#176;C, 300 &#215; g for 3 min. Aspirate the supernatant completely.</li>
	<li>Resuspend the cell pellet in 500 &#181;L of MACS buffer.</li>
	<li>Transfer the resuspended cell suspension to the sorting column in the magnetic field using a 1 mL pipette. Discard the flow-through. Wash the column three times with 500 &#181;L of MACS buffer while it remains in the magnetic field. 
	NOTE: The flow-through contains unlabeled cells. The column should be washed without removing it from the magnetic field.</li>
	<li>Remove the sorting column from the magnetic field. Add 1 mL MACS buffer to the column and slowly push it through with the piston. Collect the flow-through. 
	NOTE: The flow-through contains the labeled cells (i.e., the microglia). To improve the purity of the microglia, the process can be repeated with a new sorting column (optional).</li>
	<li>Seed the selected cells into a 24-well plate containing a coverslip coated with PLL. Add 200 &#181;L of culture medium and incubate for 20 min in a 37 &#176;C incubator with 95% O2, 5% CO2, and 60%-80% humidity. This step allows the microglia to attach to the coverslip for subsequent whole-cell patch-clamp and immunofluorescence experiments.</li>
</ol>
4. Immunofluorescence
<ol>
	<li>Aspirate the culture medium from the plate wells and rinse the cells three times with 0.1 M PBS on the shaker.</li>
	<li>Discard the PBS and fix the microglia on the coverslip with 4% paraformaldehyde (PFA) for 20 min at room temperature (RT).</li>
	<li>Remove the fixative and rinse the cover slip three times with 0.1 M PBS.</li>
	<li>Discard the PBS and permeabilize the cells with 200 &#181;L of blocking buffer containing 0.2% Triton X-100 and 5% BSA in PBS for 1 h at RT on the shaker.</li>
	<li>Incubate the microglia with blocking buffer plus anti-Iba1 and anti-CD11b primary antibodies18 overnight at 4 &#176;C.</li>
	<li>The next day, remove the primary antibodies and rinse the cover slip three times with PBS at RT on the shaker.</li>
	<li>Incubate the microglia with the blocking buffer containing fluorophore-coupled secondary antibodies for 2 h at RT. Add DAPI dye for the last 15 min of incubation.</li>
	<li>Remove the secondary antibodies and rinse the cover slip three times with PBS. Mount the cover slips on microscope slides for imaging.</li>
</ol>
5. Whole-cell patch-clamp recording of potassium currents in microglia
<ol>
	<li>Thaw the intracellular solution and keep it on ice.</li>
	<li>Pull patch pipettes (electrodes) from thin-walled borosilicate glass capillaries using a vertical puller17. 
	NOTE: The electrode resistance should be less than 10 M&#937;.</li>
	<li>Transfer a coverslip into the recording chamber and add the extracellular solution containing 160 mM of NaCl, 4.5 mM of KCl, 2 mM of CaCl2, 1 mM of MgCl2, and 10 mM of HEPES. Adjust the pH to 7.2 with 1 M NaOH solution before use13,16, and adjust the osmotic pressure to 300 mOsm with D-sucrose.</li>
	<li>Locate the spherical microglia under a water microscope using the 40x objective.</li>
	<li>Fill the electrode with intracellular solution, attach it to the amplifier head-stage of the electrophysiology setup, and apply positive pressure. Switch to the 4x objective and locate the electrode.
	<ol>
		<li>Lower the electrode into the bath solution and set the amplifier to voltage-clamp mode to perform the membrane test in bath mode. Switch back to the 40x objective and move the electrode tip closer to the microglia. 
		NOTE: When the electrode tip is very close to a microglial cell, the resistance should increase slightly.</li>
		<li>Apply a tiny negative pressure to hold the cell. When the resistance reaches 100 M&#937;, switch from bath to patch mode in the membrane test. When the resistance reaches 300 M&#937;, release the negative pressure.</li>
		<li>When the resistance indicates the formation of a gigaohm seal (i.e., &#62;1 G&#937;) between the electrode and the microglial membrane, switch from patch to cell mode in the membrane test.</li>
	</ol>
	</li>
	<li>Apply a small amount of suction and initiate an electric shock to rupture the membrane. The appearance of large capacitive transients indicates successful membrane rupture.</li>
	<li>Switch to voltage-clamp mode and open the recording program. Record potassium currents for 200 ms. To test potentials, apply voltage steps in 20 mV increments from -120 mV to +40 mV at 1 Hz.</li>
	<li>Analyze the obtained data offline using appropriate software.</li>
</ol>

Electrophysiological Recording of Hippocampal Microglia from Adult Mice

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The authors have nothing to disclose.

Cultured microglia from fetal or newborn mice are clearly unsuitable for studying adult microglia. Additionally, given the heterogeneity of microglia across different brain areas21, microglia isolated from the whole brain may not accurately represent the characteristics of microglia in a specific brain structure. This protocol provides a method for isolating hippocampal microglia specifically to evaluate the electrical properties of these cells. It includes methods for recording the currents gover...

Microglia, which derive from myeloid progenitors in the primitive yolk sac, are resident in the CNS and comprise about 10% to 15% of total nerve cells1,2. Functionally, in addition to surveilling the local environment, performing immune defense functions, and providing nutrition and support for neurons3,4, microglia can also directly contact neurons to regulate neuronal surface receptors and corresponding ligand binding, and indirectly interact with neurons via the secretion of cytokines5. Studies in vitro and in vivo have demonstrated that microglia express various types of potassium channels, which contribute to the maintenance of negative membrane potential6 and regulate microglial activation. Given that abnormal microglial potassium channels have been observed in various brain diseases7, it is crucial for researchers to identify potential drugs that target these potassium channels and develop strategies to regulate microglial function.
Traditional digestion and purification methods for microglia often use the whole brain, which overlooks the heterogeneity of microglia across different brain areas8. In vitro dissociation and long-term culture in serum-containing media place microglia in an active state9, which may not accurately reflect their physiological characteristics and true state.
Additionally, while outward rectifier potassium currents have been recorded in primary cultured newborn microglia and cell lines10, data from long-term cultures of fetal or newborn mouse brain tissue may differ from those of mature microglia. The present protocol aims to acutely isolate microglia and immediately perform whole-cell recordings of microglial potassium currents using adult mouse brain tissue.
Biochemical and potassium channel properties were examined using a method suitable for the isolation and purification of microglia from small tissues11,12,13,14,15. Therefore, this protocol provides guidance for researchers to elucidate the biochemical and functional properties of microglia under physiological and disease conditions. Although this protocol uses the hippocampus and potassium channels as examples, it can also be applied to the study of other brain areas and channel/cell electrophysiological properties.

<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

Microglia are resident immune cells in the brain that interact with neurons to maintain the homeostasis of the central nervous system (CNS). Studies show that the microglial surface expresses potassium channels that regulate microglial activation, while abnormalities in these potassium channels can lead to neural diseases. Currently, whole-cell patch-clamp recordings of microglia are mostly performed on cultured primary microglia from fetal or newborn mice due to difficulties in conducting electrophysiological evaluations on acutely isolated microglia. This study introduces an easy-to-follow protocol for isolating hippocampal microglia from adult mice and performing whole-cell patch-clamp recordings on the isolated cells. Briefly, the brain was removed from a mouse after decapitation, the hippocampus was dissected bilaterally, and microglia were isolated using an adult mouse brain dissociation kit. The microglia were then purified using a magnetic-activated cell sorting (MACS) method and seeded onto coverslips. Successful microglial isolation was confirmed by immunofluorescent staining with anti-CD11 and anti-Iba1 antibodies. A cover slip was placed in a recording chamber, and the whole-cell potassium currents of the acutely isolated microglia were recorded under voltage-clamp conditions.

Briefly, the process involves the isolation of hippocampal microglia from the adult mouse brain followed by whole-cell patch-clamp recording of these cells (Figure 1). The procedure begins with dissecting the hippocampus of 3 to 5-month-old C57BL/6J adult mice. Specifically, the entire brain is removed after perfusion and placed in a culture dish containing ice-cold PBS (Figure 2A). To ensure acute isolation of cells from the hippocampus, the cerebral cortex is ...

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

The present protocol describes how to isolate and purify primary hippocampal microglia from adult mice, followed by instructions for conducting whole-cell patch-clamp recordings on these acutely isolated cells.

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

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JoVE Journal

Biology

638 Views.  South China Normal University. The present protocol describes how to isolate and purify primary hippocampal microglia from adult mice, followed by instructions for conducting whole-cell patch-clamp recordings on these acutely isolated cells.

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

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).

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

Immunology and Infection

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.

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

Neuroscience

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.

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.

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.

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.

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.

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

Isolation of Hypothalamic Microglia from Mice Using MACS for In Vitro Assays

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.

Author Spotlight: Investigating the Impact of Nutrition on Mouse Brain Function and Metabolic Disorders 

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.

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

Isolation of Microglia from the Cortex of an Adult Mouse Brain

Source: Zhou, L., et al., <a href="https://app.jove.com/v/60347">Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing.</a> J. Vis. Exp. (2019)
The video demonstrates a technique for isolating microglia from the cortex of an adult mouse brain. First, the cortex undergoes segmentation and homogenization to remove neurons and other glial cells, preserving microglia. Following that, the cell suspension is exposed to anti-myelin magnetic microbeads and a magnetic field, effectively separating microglia and myeloid cells from myelin debris and cells containing myelin.

Izolacja mikrogleju z kory mózgowej dorosłej myszy

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Mechanical Tissue Dissociation
...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Primary Neuronal Culture

In Vitro Recording of Theta Oscillations in the Mouse Hippocampus

Source: Manseau, F., et.al. <a href="https://app.jove.com/v/55851">Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit.</a> J. Vis. Exp. (2017).
This video demonstrates the recording of rhythmic neuronal network activity, also known as theta oscillations, in an isolated hippocampus. Electrodes are inserted into various layers and spatial positions to detect and analyze theta oscillations. This shows the unique neuronal compositions and the connectivity of the neuronal network across different layers of the hippocampus.

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

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

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

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

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

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

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

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

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

Izolacja mikrogleju z kory mózgowej dorosłej myszy

Zapis in vitro oscylacji theta w hipokampie myszy