Name: investigation of spatial interaction between astrocytes and neurons in cleared brains
Uploaded: 2022-03-31T14:50:00
Description: Watch this Scientific Journal Video about Investigation of Spatial Interaction Between Astrocytes and Neurons in Cleared Brains at JoVE.com

This project has received funding from the European Research Council (ERC) under the European Union&#39;s Horizon 2020 research and innovation programme (grant agreement No 803589), the Israel Science Foundation (ISF grant No. 1815/18), and the Canada-Israel grants (CIHR-ISF, grant No. 2591/18). We thank Nechama Novick for commenting on the whole manuscript.

Experimental protocols were approved by the Hebrew University Animal Care and Use Committee and met the guidelines of the National Institute of Health Guide for the Care and Use of Laboratory Animals.
1. Viral vector transduction
NOTE: Viral vector transduction is used to express fluorophores in the brain.
<ol>
	<li>Use an atlas (e.g., Allen Brain Atlas) to locate the relevant coordination of the target area. 
	NOTE: 3D atlases can be found o...

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protoplasmic+astrocytes+in+CA1+stratum+radiatum+occupy+separate+anatomical+domains.">Protoplasmic astrocytes in CA1 stratum radiatum occupy separate anatomical domains.</a> The Journal of Neuroscience. 22 (1), 183-192 (2002).</li><li>Chai, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+circuit-specialized+astrocytes:+transcriptomic,+proteomic,+morphological,+and+functional+evidence.">Neural circuit-specialized astrocytes: transcriptomic, proteomic, morphological, and functional evidence.</a> Neuron. 95 (3), 531-549 (2017).</li><li>Taniguchi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+resource+of+Cre+driver+lines+for+genetic+targeting+of+GABAergic+neurons+in+cerebral+cortex.">A resource of Cre driver lines for genetic targeting of GABAergic neurons in cerebral cortex.</a> Neuron. 71 (6), 995-1013 (2011).</li><li>Hippenmeyer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+developmental+switch+in+the+response+of+DRG+neurons+to+ETS+transcription+factor+signaling.">A developmental switch in the response of DRG neurons to ETS transcription factor signaling.</a> PLoS Biology. 3 (5), 159 (2005).</li><li>Ye, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wiring+and+molecular+features+of+prefrontal+ensembles+representing+distinct+experiences.">Wiring and molecular features of prefrontal ensembles representing distinct experiences.</a> Cell. 165 (7), 1776-1788 (2016).</li><li>DeNardo, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+evolution+of+cortical+ensembles+promoting+remote+memory+retrieval.">Temporal evolution of cortical ensembles promoting remote memory retrieval.</a> Nature Neuroscience. 22 (3), 460-469 (2019).</li><li>Srinivasan, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+transgenic+mouse+lines+for+selectively+targeting+astrocytes+and+studying+calcium+signals+in+astrocyte+processes+in+situ+and+in+vivo.">New transgenic mouse lines for selectively targeting astrocytes and studying calcium signals in astrocyte processes in situ and in vivo.</a> Neuron. 92 (6), 1181-1195 (2016).</li><li>Chung, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+molecular+interrogation+of+intact+biological+systems.">Structural and molecular interrogation of intact biological systems.</a> Nature. 497 (7449), 332-337 (2013).</li><li>Ye, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wiring+and+molecular+features+of+prefrontal+ensembles+representing+distinct+experiences.">Wiring and molecular features of prefrontal ensembles representing distinct experiences.</a> Cell. 165 (7), 1776-1788 (2016).</li><li>Chung, K., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLARITY+for+mapping+the+nervous+system.">CLARITY for mapping the nervous system.</a> Nature Methods. 10 (6), 508-513 (2013).</li></ol>

Tissue clearing methods present a revolutionary tool in brain research, inviting questions that could not previously have been asked. From targeting the properties of a small group of cells, a single cell, or even a single synapse, CLARITY now enables the targeting of total cell populations or long-range connectivity features by using relevant fluorophores.
The outcome of the fluorophore expression and CLARITY procedure combination is not binary; many factors may interfere with the procedure l...

In recent years, the knowledge of astrocyte function and how they interact with neuronal circuits has increased dramatically. Astrocytes can influence plasticity1,2, assist in neuronal postinjury recovery3,4, and even induce de novo neuronal potentiation, with recent studies exhibiting the importance of astrocytes in memory acquisition and reward, previously regarded as purely neuronal functions5,6,7. A feature of particular interest in astrocyte research is the spatial arrangement of the cells, which maintain unique spatial organizations in the hippocampus and other brain structures8,9,10. Unlike the neuronal dendrites that intertwine between cell somata, hippocampal astrocytes inhabit visually distinguishable territories with slight overlap between their processes, creating distinct domains8,11,12,13. The evidence supporting the participation of astrocytes in neuronal circuits does not support the lack of detailed anatomical description of such populations and the neurons in their domains14.
Viral vector transduction procedures, along with transgenic animals (TG), have been popularized as a toolset to investigate brain structures, functions, and cell interactions15,16. The utilization of different promoters allows the targeting of specific cells according to their genetic properties, activation levels17,18, or projection targets. Different viruses can express different colored fluorophores in different populations. A virus can be combined with the endogenous expression of fluorophores in TG, or TG animals can be used without the need for viruses. These techniques are widely used for neuronal marking, and some labs have started using them with modifications specialized for targeting other cell types, such as astrocytes5,9,19.
The CLARITY technique, first described in 201320,21, enables the study of thick brain slices by making the entire brain transparent while leaving the microscopic structures intact. By combining the two methods-viral vector transduction and tissue clearing-the option of examining the spatial interactions between different cell type populations is now available. Most astrocyte-neuron interaction studies were performed on thin brain slices, resulting in images of incomplete astrocytes due to their large domains, thus radically restricting the number of analyzed cells. The use of the CLARITY technique allows single-cell resolution characterization of cell populations in large-scale volumes simultaneously. Imaging fluorescently tagged cell populations in clear brains does not deliver synaptic resolution but permits thorough characterization of the spatial interactions between astrocytes and a variety of neuronal cell types.
For that reason, we harnessed these state-of-the-art techniques to investigate the properties of astrocytes throughout the dorsal CA1, imaging all lamina (Stratum Radiatum, pyramidal layer, and Stratum Oriens). We measured tens of thousands of astrocytes (with viral penetrance of &#62;96%5), thereby analyzing the information of the entire astrocytic population around CA1. With efficient penetrance of the neuronal markers, we could record the interactions between the entire population of CA1 astrocytes and the four types of neuronal cells-parvalbumin (PV), somatostatin (SST), VIP inhibitory neurons, and excitatory pyramidal cells9.
Several experiments were performed using a combination of fluorescence from TG animals and differently colored viral vectors (all inhibitory cells), while others (excitatory) utilized two viral vectors expressing different fluorophores under different promoters9. This paper presents a detailed protocol, including the tagging of the desired cells in the brain, making the brain transparent using a modified CLARITY procedure, as well as imaging and analyzing complete brain structures, using various procedures and software.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AAV1-GFAP::TdTomato</td>
			<td>ELSC Vector Core Facility (EVCF)</td>
			<td></td>
			<td>viral vector used to detect astrocytes</td>
		</tr>
		<tr>
			<td>AAV5-CaMKII::eGFP</td>
			<td>ELSC Vector Core Facility (EVCF)</td>
			<td></td>
			<td>viral vector used to detect neurons</td>
		</tr>
		<tr>
			<td>AAV5-CaMKII::H2B-eGFP</td>
			<td>ELSC Vector Core Facility (EVCF)</td>
			<td></td>
			<td>viral vector used to detect neuronal nuclei</td>
		</tr>
		<tr>
			<td>AAV5-CaMKII::TdTomato</td>
			<td>ELSC Vector Core Facility (EVCF)</td>
			<td></td>
			<td>viral vector used to detect neurons</td>
		</tr>
		<tr>
			<td>Acrylamide (40%)</td>
			<td>Bio-rad</td>
			<td>#161-0140</td>
			<td></td>
		</tr>
		<tr>
			<td>Bisacrylamide (2%)</td>
			<td>Bio-rad</td>
			<td>#161-0142</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Sigma</td>
			<td>#B7901</td>
			<td>Molecular weight - 61.83 g/mol</td>
		</tr>
		<tr>
			<td>Confocal microscope, scanning, FV1000</td>
			<td>Olympus</td>
			<td></td>
			<td>4x objective (UPlanSApo, 0.16 NA)</td>
		</tr>
		<tr>
			<td>Imaris software</td>
			<td>Bitplane, UK</td>
			<td></td>
			<td>A software that allows 3D analysis of images</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>#S5881</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PFA 4%</td>
			<td>EMS</td>
			<td>#15710</td>
			<td></td>
		</tr>
		<tr>
			<td>RapiClear</td>
			<td>SunJin lab</td>
			<td>#RC147002</td>
			<td></td>
		</tr>
		<tr>
			<td>RapiClear CS</td>
			<td>SunJin lab</td>
			<td>#RCCS002</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma</td>
			<td>#L3771</td>
			<td></td>
		</tr>
		<tr>
			<td>SyGlass software</td>
			<td></td>
			<td></td>
			<td>A software that allows 3D analysis of images using virtual reality</td>
		</tr>
		<tr>
			<td>Tris base 1 M</td>
			<td>Bio-rad</td>
			<td>#002009239100</td>
			<td>Molecular weight - 121.14 g/mol</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>ChemCruz</td>
			<td>#sc-29112A</td>
			<td></td>
		</tr>
		<tr>
			<td>Two photon microscope</td>
			<td>Neurolabware</td>
			<td></td>
			<td>Ti:sapphire laser (Chameleon Discovery TPC, Coherent), GaAsP photo-multiplier tubes (Hamamatsu, H10770-40) , bandpass filter (Semrock), water immersion 16x objective (Nikon, 0.8 NA)&#160;</td>
		</tr>
		<tr>
			<td>VA-044 Initiator</td>
			<td>Wako</td>
			<td>#011-19365</td>
			<td></td>
		</tr>
	</tbody>
</table>

investigation of spatial interaction between astrocytes and neurons in cleared brains

Combining viral vector transduction and tissue clearing using the CLARITY method makes it possible to simultaneously investigate several types of brain cells and their interactions. Viral vector transduction enables the marking of diverse cell types in different fluorescence colors within the same tissue. Cells can be identified genetically by activity or projection. Using a modified CLARITY protocol, the potential sample size of astrocytes and neurons has grown by 2-3 orders of magnitude. The use of CLARITY allows the imaging of complete astrocytes, which are too large to fit in their entirety in slices, and the examination of the somata with all their processes. In addition, it provides the opportunity to investigate the spatial interaction between astrocytes and different neuronal cell types, namely, the number of pyramidal neurons in each astrocytic domain or the proximity between astrocytes and specific inhibitory neuron populations. This paper describes, in detail, how these methods are to be applied.

Successful clearing of thick brain tissue slices results in a new range of questions that can be asked regarding the properties of large cell populations as opposed to the properties of single cells or neighboring groups of cells. To achieve successful results, one should strictly adhere to the CLARITY protocol, as there is a wide range of parameters that need to be considered to reduce the variance between samples (e.g., percentage of clarity, fluorescence information, swollenness parameters).
<p class="jove_content...

Watch this Scientific Journal Video about Investigation of Spatial Interaction Between Astrocytes and Neurons in Cleared Brains at JoVE.com

Investigation of Spatial Interaction Between Astrocytes and Neurons in Cleared Brains

Combining viral vector transduction and brain clearing using the CLARITY method allows the investigation of a large number of neurons and astrocytes simultaneously.

Combining viral vector transduction and tissue clearing using the CLARITY method makes it possible to simultaneously investigate several types of brain ...

The clarity technique first described in 2013 is a tissue clearing technique that enables the samplings of larger quantities of cells, blood vessels, so even proteins such a single cell resolution in thick brain slices. This technique enables the study of intact microscopic structures by making the entire brain transparent. This protocol is a simple chip and straightforward pipeline for tissue clear.This technique can be used for clearing tissues in other systems in addition to brain tissues. To begin, seal the tube and cap using parafilm and puncture two holes in the cap. Then transfer nitrogen gas from the tank using a flexible pipe with five millimeter internal diameter and a 19 gauge needle connected at its end.Start degassing by attaching the pipe to the tube and allowing gas exchange for 30 minutes at room temperature. Detach the pipe and immediately seal the holes with modeling clay. Then transfer the degas sealed tubes to a 37 degree Celsius bath for 3 1/2 hours to polymerize the hydrogel solution.Take the brain out from the tube. Using laboratory wipes, remove the polymerized hydrogel around the brain ensuring that no residual gel remains attached to the surface of the brain. Divide the brain into thick slices that contain all areas of interest.Put the slices in the first clearing solution and incubate at 37 degrees Celsius with rotation at 70 RPM for 24 hours. In the meantime, prepare a perforated tube to place the brain. Put the perforated tube in a beaker filled with second clearing solution on a stirring device.Then place the brain in the beaker and seal it with aluminum foil to prevent bleaching. After the tissue turns transparent, transfer the brain into PBST in incubate at 37 degrees Celsius with rotation at 70 RPM for 24 hours. Replace the solution with fresh PBST and continue the incubation for another 24 hours.Then transfer the brain to PBS at room temperature for 24 hours. After 24 hours, remove the brain from PBS and transfer it to refractive index matching solution. Incubate the brain at 37 degrees Celsius overnight.First, place the sample in the middle of the slide. Using hot glue, create walls on the edges of the slide, almost as high as the tissue. Make sure to leave a small gap at one of the corners.As the hot glue layer approaches the height of the brain slice, apply one to two drops of the refractive index matching solution on the sample to moisten the upper surface and prevent bubble formation. While the hot glue is still liquid, seal the top with a cover slip, placing it as evenly as possible then fill the chamber with the refractive index matching solution. Close the gap with hot glue.If the hot glue walls extend beyond the borders of the slide, cut the extending edges. If an oil immersion objective is used for imaging, add another two to three millimeters of glue to the walls above the cover slip to retain the immersion solution. After this protocol, the brain tissue will be cleared.Clear brain slices were prepared using this protocol to visualize populations of astrocytes and neurons in the CA1 region of mouse hippocampus. All astrocytes expressed tdTomato and excitatory neurons expressed H2B-GFP in their nuclei. The chamber prepared for the clarified tissue was optimal for imaging under a two photon or confocal microscope.Using a two photon microscope, over 300 astrocytes were observed in a cleared section of the CA1 region of the mouse hippocampus. The hippocampal astrocytes in red and pyramidal cells, somata in green were visible and thick, transparent tissue representing the spatial proximity between these two cell types. Using a scanning laser confocal microscope, axonal bundles from excitatory neurons were traced across an entire hemisphere.Green bundles were traced from the dorsal hippocampus toward the super mamillary bodies. The red bundles were traced from the mamillary bodies toward their origin at the vent hippocampus. Factors, such as temperature, concentrations and incubation time influenced the outcome of the clarity procedure.Therefore, preciseness in every step of the protocol is vital for the success of achieving clear tissue. Using this technique, we measure the distances between neurons and astrocytes in several brain structures. This protocol allowed analysis of two to three order of magnitude more cells than in previous studies.

investigation-spatial-interaction-between-astrocytes-neurons-cleared

Research

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Neuroscience

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.

Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees (Apis mellifera L.)

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker bees spend the first half of their life inside the dark hive, where they are nursing the larvae or building the regular hexagonal combs for food (e.g. pollen or nectar) and brood3. The antennae are extraordinary multisensory feelers and play a pivotal role in various tactile mediated tasks4, including hive building5 and pattern recognition6. Later in life, each single bee leaves the hive to forage for food. Then a bee has to learn to discriminate profitable food sources, memorize their location, and communicate it to its nest mates7. Bees use different floral signals like colors or odors7, 8, but also tactile cues from the petal surface9 to form multisensory memories of the food source. Under laboratory conditions, bees can be trained in an appetitive learning paradigm to discriminate tactile object features, such as edges or grooves with their antennae10, 11, 12, 13. This learning paradigm is closely related to the classical olfactory conditioning of the proboscis extension response (PER) in harnessed bees14. The advantage of the tactile learning paradigm in the laboratory is the possibility of combining behavioral experiments on learning with various physiological measurements, including the analysis of the antennal movement pattern.

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees Apis mellifera L.

In this protocol we show how to condition harnessed honey bees to tactile stimuli and introduce a 2D motion capture technique for analyzing the kinematics of fine-scale antennal sampling pattern.

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker ...

An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and subsequently digests this material, commonly through lysosomal degradation. Microglia are the resident immune cells of the central nervous system (CNS) whose phagocytic function has been described in a broad range of conditions from neurodegenerative disease (e.g., beta-amyloid clearance in Alzheimer&#8217;s disease) to development of the healthy brain (e.g., synaptic pruning)1-6. The following protocol is an engulfment assay developed to visualize and quantify microglia-mediated engulfment of presynaptic inputs in the developing mouse retinogeniculate system7. While this assay was used to assess microglia function in this particular context, a similar approach may be used to assess other phagocytes throughout the brain (e.g., astrocytes) and the rest of the body (e.g., peripheral macrophages) as well as other contexts in which synaptic remodeling occurs (e.g. ,brain injury/disease).

Microglia are the resident immune cells of the central nervous system (CNS) with a high capacity to phagocytose or engulf material in their extracellular environment. Here, a broadly applicable, reliable, and highly quantitative assay for visualizing and measuring microglia-mediated engulfment of synaptic components is described.

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 &#181;m2). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta1-40 (A&#946;1-40) with or without a therapeutic&#160;intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in brain homeostasis, astrocytes supply neurons with vital metabolites and buffer extracellular water, ions, and glutamate. An integral component of the &#8220;tri-partite&#8221; synapse, astrocytes are also critical in the formation, pruning, maintenance, and modulation of synapses. To enable these highly interactive functions, astrocytes communicate among themselves and with other glial cells, neurons, the brain vasculature, and the extracellular environment through a multitude of specialized membrane proteins that include cell adhesion molecules, aquaporins, ion channels, neurotransmitter transporters, and gap junction molecules. To support this dynamic flux, astrocytes, like neurons, rely on tightly coordinated and efficient intracellular transport. Unlike neurons,&#160;where intracellular trafficking has been extensively delineated, microtubule-based transport in astrocytes has been less studied. Nonetheless, exo- and endocytic trafficking of cell membrane proteins and intracellular organelle transport orchestrates astrocytes&#8217; normal biology, and these processes are often affected in disease or in response to injury. Here we present a straightforward protocol to culture high quality murine astrocytes, to fluorescently label astrocytic proteins and organelles of interest, and to record their intracellular transport dynamics using time-lapse confocal microscopy. We also demonstrate how to extract and quantify relevant transport parameters from the acquired movies using available image analysis software (i.e., ImageJ/FIJI) plugins.

Here we describe an in vitro live-imaging method to visualize intracellular transport of organelles and trafficking of plasma membrane proteins in murine astrocytes. This protocol also presents an image analysis methodology to determine cargo transport itineraries and kinetics.

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in ...

Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) pathophysiology in vitro. Multi-electrode array (MEA) recordings are a means to record electrical field potentials from large populations of neurons and analyze network activity over time. It was previously demonstrated that the presence of hiPSC-A that are differentiated using techniques to promote a spinal cord astrocyte phenotype improved maturation and electrophysiological activity of regionally specific spinal cord hiPSC-motor neurons (MN) when compared to those cultured without hiPSC-A or in the presence of rodent astrocytes. Described here is a method to co-culture spinal cord hiPSC-A with hiPSC-MN and record electrophysiological activity using MEA recordings. While the differentiation protocols described here are particular to astrocytes and neurons that are regionally specific to the spinal cord, the co-culturing platform can be applied to astrocytes and neurons differentiated with techniques specific to other fates, including cortical hiPSC-A and hiPSC-N. These protocols aim to provide an electrophysiological assay to inform about glia-neuron interactions and provide a platform for testing drugs with therapeutic potential in ALS.

We describe a method for differentiating spinal cord human induced pluripotent-derived astrocytes and neurons and their co-culture for electrophysiological recording.

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) ...

Free-floating Immunostaining of Mouse Brains

Immunohistochemical staining of mouse brains is a routine technique commonly used in neuroscience to investigate central mechanisms underlying the regulation of energy metabolism and other neurobiological processes. However, the quality, reliability, and reproducibility of brain histology results may vary among laboratories. For each staining experiment, it is necessary to optimize the key procedures based on differences in species, tissues, targeted proteins, and the working conditions of the reagents. This paper demonstrates a reliable workflow in detail, including intra-aortic perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging, which can be followed easily by researchers in this field.
Also discussed are how to modify these procedures to satisfy the individual needs of researchers. To illustrate the reliability and efficiency of this protocol, perineuronal nets were stained with biotin-labeled Wisteria florbunda agglutinin (WFA) and arginine vasopressin (AVP) with an anti-AVP antibody in the mouse brain. Finally, the critical details for the entire procedure have been addressed, and the advantages of this protocol compared to those of other protocols. Taken together, this paper presents an optimized protocol for free-floating immunostaining of mouse brain tissue. Following this protocol makes this process easier for both junior and senior scientists to improve the quality, reliability, and reproducibility of immunostaining studies.

This protocol describes an efficient and reproducible approach for mouse brain histological studies, including perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging.