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 online (e.g.,&#160;http://connectivity.brain-map.org/3d-viewer).</li>
	<li>Using stereotactic surgery, inject the viral vectors into the relevant brain structure. See9 for a detailed protocol.</li>
	<li>Wait for 3-6 weeks for fluorophore expression. 
	NOTE: A short period of time is enough if only cell bodies need to be marked. A long period of time will be needed if the axonal projections are relevant to the question, as it takes longer for the fluorophores to express in axons, which can be several millimeters long.</li>
	<li>Validate the specificity and penetrance of fluorophore expression (both of viral expression and TG animals) beforehand (Figure 1A). Before proceeding with the CLARITY procedure, designate at least one brain for thin slices and make sure the fluorophore expression is both strong and specific to the target cell feature.</li>
</ol>
2. CLARITY
NOTE: This method renders the brain transparent within 2-6 weeks.
<ol>
	<li>Perform transcranial perfusion on animals using cold Phosphate-buffered Saline (PBS) followed by 4% Paraformaldehyde (PFA) in PBS. Remove the brain and keep it in PFA overnight at 4 &#176;C in a 50 mL tube or a similar container. 
	NOTE: Before transcranial perfusion is performed, the animal must be deeply anesthetized. In the examples presented in this protocol, all animals were anesthetized using Ketamine and Xylazine (90% and 10%, respectively).</li>
	<li>Replace PFA with Hydrogel Solution (HS; see Table 1) for 48 h at 4 &#176;C. 
	NOTE: Do not allow the materials to become warmer than the refrigeration temperature (4-8 &#176;C) at this stage, or the Hydrogel will polymerize. The HS used in this protocol contains 2% acrylamide, unlike previous protocols suggesting 4%22 or 1%17. The benefit of 2% is detailed in the discussion section. When preparing the HS, work on an ice-cold surface. Store it at -20 &#176;C.</li>
	<li>Degassing 
	NOTE: The purpose of this stage is to remove all free oxygen from the tissue as O2 interferes with the polymerization process. Any nonO2 gas can be used (e.g., N2, CO2); N2 is recommended.
	<ol>
		<li>Transfer the N2 from the tank via a 5 mm (internal) flexible tubing, connected to a 19 G needle at its end (Figure 1B).</li>
		<li>Make two small holes (needle-wide) in the cap of the tube: one to introduce the gas and the other to allow the air to leave the tube (Figure 1C).</li>
		<li>Attach the pipe from the N2 gas to the tube and replace the gases for approximately 30 min at room temperature (RT).</li>
		<li>Remove the pipe and immediately seal the holes with modeling clay on every tube (Figure 1D).</li>
	</ol>
	</li>
	<li>Transfer the degassed sealed tubes to a 37 &#176;C bath for 3.5 h to polymerize the HS, which will become a gel. Be careful not to shake the tubes (Figure 1E).</li>
	<li>Extract the brain from the tube and gently remove the polymerized gel from around it using laboratory wipes. Make sure no residual gel remains attached to the surface of the tissue, as it might react with the solution in the following steps, inhibiting the tissue clearing process (Figure 1F). 
	NOTE: Because the gel contained PFA, the extraction should be done under a fume hood.</li>
	<li>Slice the brain if the question at hand requires only a part of it. Divide it in half or into very thick slices that contain all areas of interest (Figure 1G).</li>
	<li>Place the slices in the First Clearing Solution (CS1, see Table 2) in a new container and shake at 70 rpm for 24 h at 37 &#176;C.</li>
	<li>Follow the steps described below to transfer the brain from CS1 to the second Clearing Solution (CS2, see Table 2).
	<ol>
		<li>Prepare a perforated tube for the brain in advance (Figure 1H).</li>
		<li>Preheat CS2 to 40-45 &#176;C in a beaker large enough to contain the tube. Do this on a hotplate with a stirrer. Ensure the temperature does not reach 55 &#176;C to prevent the bleaching of the expressed proteins.</li>
		<li>Place the beaker filled with CS2 and the perforated tube on a stirring device (a 2 L beaker in Figure 1I). Set a moderate stirring rate that will cause the liquid to flow without distorting the tissue. 
		NOTE: Within 2-6 weeks, the tissue will become transparent (the process starts at the periphery and moves inward toward the central brain structures). The decision as to when the tissue is &#34;clear enough&#34; is up to personal judgment. The brain should be sufficiently transparent for imaging under the microscope without interference (Figure 1J not clear enough; Figure 1K clear enough). Surpassing the point at which the tissue is clear enough may cause loss of tissue rigidity.</li>
	</ol>
	</li>
	<li>Transfer from CS2 into PBST (0.5% Triton X-100 in PBS, see Table 2) in a new container at 37 &#176;C with mild shaking (70 rpm) for 24 h. 
	NOTE: In the PBST, the tissue will become whitish (Figure 1L). The clarity will return (and improve) when embedded in the Refractive Index Matching Solution (RIMS, see step 2.14).</li>
	<li>Replace the PBST with new PBST. Keep under the same conditions (37 &#176;C with mild shaking) for another 24 h.</li>
	<li>Replace PBST again with new PBST and keep at RT for 24 h.</li>
	<li>Transfer to PBS at RT for 24 h.</li>
	<li>Replace the PBS and leave at RT for an additional 24 h.</li>
	<li>Remove the brain from PBS and transfer it to RIMS at 37 &#176;C overnight. 
	NOTE: Initially, ripples in the RIMS may surround the brain. This protocol was designed and validated using two specific commercial RIMS (see Table 3). However, the protocol should not differ when using other RIMS (commercial or self-made).</li>
	<li>Keep the tissue at RT for another 24 h or until the solution reaches full equilibrium, i.e., when the tissue becomes transparent, and the solution no longer contains any visible ripples (Figure 1M).</li>
	<li>If the tissue is transparent, proceed to the chamber preparation. If at this point, the tissue becomes white instead of transparent (due to aggregates of residual SDS molecules), clean the sample again by repeating steps 2.9 and 2.10, and transfer the tissue to CS2 (step 2.8) for a few days, followed by all the steps until step 2.15.</li>
</ol>
3. Chamber preparation
NOTE: Each sample requires a slide with an imaging chamber in which the sample will be placed.
<ol>
	<li>Place the sample in the middle of the slide.</li>
	<li>Using a hot glue gun, create walls at the edges of the slide, almost as high as the tissue. Make sure to leave a small gap (approximately 5 mm) at one of the corners (Figure 2A,B).</li>
	<li>Apply 1-2 drops of the RIMS on the sample to keep the upper surface moist and prevent bubbles forming between the coverslip and the tissue.</li>
	<li>Immediately after applying the RIMS drops, add the last layer of hot glue to the walls (so that they reach the height of the brain/slice) and progress immediately (while the hot glue is still liquid) to step 3.5.</li>
	<li>Seal the top with a coverslip, placing it as evenly as possible on the top of the still-warm hot glue layer (Figure 2C).</li>
	<li>Fill the chamber with the RIMS through the gap left in the hot glue walls (Figure 2D,E).</li>
	<li>Close the gap with hot glue. Leave no air inside (Figure 2F).</li>
	<li>If the hot glue walls extend beyond the borders of the slide, cut the extending edges (Figure 2G).</li>
	<li>If the objective used for imaging is immersed, add another 2-3 mm of glue to the walls above the coverslip so that the immersion solution will last for a longer period (Figure 2H).</li>
</ol>
4. Imaging (confocal or two-photon)
<ol>
	<li>Check whether the stage can hold the chamber, as the thickness of the chamber can reach several millimeters. 
	NOTE: For example, the confocal microscope (see the Table of Materials) used in this protocol is equipped with several stages (e.g., circular, rectangular). Whichever stage is chosen to hold the chamber is irrelevant as long as its parameters fit the chamber sizes.</li>
	<li>Work with an objective with a sufficient working distance, i.e., a minimal working distance &#8805;3 mm, as the brain region of interest may be a few millimeters in depth, and the coverslip may not be completely straight as it was placed manually. 
	NOTE: As a demonstration, the results presented in Figure 1, Video 1, and Video 2 were obtained under a two-photon microscope using a water immersion 16x objective with a 3 mm working distance and magnification of 2.4 to obtain a 652 x 483 &#181;m field of view and a z-stack with 0.937 &#181;m intervals between the planes.</li>
	<li>Perform multiple-area imaging, which may take a few hours or even days. Make sure there is ample free storage in the computer as the size of each image can reach up to tens of gigabits. 
	NOTE: CLARITY imaging may result in significantly more image sections in depth. Therefore, the intensities used to capture the expression should be relatively low to prevent overexpression during the image summation.</li>
	<li>For immersion objectives, check the liquid routinely while imaging and supplement with additional liquid if needed. 
	NOTE: During the imaging of the data presented in Video 3, water was added to the surface of the chamber every 6-8 h to combat evaporation.</li>
	<li>After the image has been acquired, view and analyze it using several different software packages. 
	NOTE: Recommended software includes Imaris and SyGlass for both visualization and analysis. The precise pipeline for optimized reconstruction of astrocytes using Imaris software has been described previously9. In short, the Imaris features used in this study were Filaments to fully reconstruct cell structures and Spots to extract the position in the space of all the somata.</li>
</ol>

<ol>
	<li>Perea, G., Navarrete, M., Araque, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tripartite+synapses:+astrocytes+process+and+control+synaptic+information.">Tripartite synapses: astrocytes process and control synaptic information.</a> Trends in Neurosciences. 32 (8), 421-431 (2009).</li><li>Ciappelloni, 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=Aquaporin-4+surface+trafficking+regulates+astrocytic+process+motility+and+synaptic+activity+in+health+and+autoimmune+disease.">Aquaporin-4 surface trafficking regulates astrocytic process motility and synaptic activity in health and autoimmune disease.</a> Cell Reports. 27 (13), 3860-3872 (2019).</li><li>Sylvain, N. J., 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=The+effects+of+trifluoperazine+on+brain+edema,+aquaporin-4+expression+and+metabolic+markers+during+the+acute+phase+of+stroke+using+photothrombotic+mouse+model.">The effects of trifluoperazine on brain edema, aquaporin-4 expression and metabolic markers during the acute phase of stroke using photothrombotic mouse model.</a> Biochimica et Biophysica Acta. Biomembranes. 1863 (5), 183573 (2021).</li><li>Kitchen, P., 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=Targeting+aquaporin-4+subcellular+localization+to+treat+central+nervous+system+edema.">Targeting aquaporin-4 subcellular localization to treat central nervous system edema.</a> Cell. 181 (4), 784-799 (2020).</li><li>Adamsky, 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=Astrocytic+activation+generates+de+novo+neuronal+potentiation+and+memory+enhancement.">Astrocytic activation generates de novo neuronal potentiation and memory enhancement.</a> Cell. 174 (1), 59-71 (2018).</li><li>Adamsky, A., Goshen, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+in+memory+function:+pioneering+findings+and+future+directions.">Astrocytes in memory function: pioneering findings and future directions.</a> Neuroscience. 370, 14-26 (2018).</li><li>Nagai, J., 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=Behaviorally+consequential+astrocytic+regulation+of+neural+circuits.">Behaviorally consequential astrocytic regulation of neural circuits.</a> Neuron. 109 (4), 576-596 (2021).</li><li>Clavreul, 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=Cortical+astrocytes+develop+in+a+plastic+manner+at+both+clonal+and+cellular+levels.">Cortical astrocytes develop in a plastic manner at both clonal and cellular levels.</a> Nature Communications. 10 (1), 4884 (2019).</li><li>Refaeli, 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=Features+of+hippocampal+astrocytic+domains+and+their+spatial+relation+to+excitatory+and+inhibitory+neurons.">Features of hippocampal astrocytic domains and their spatial relation to excitatory and inhibitory neurons.</a> Glia. 69 (10), 2378-2390 (2021).</li><li>Eilam, R., Aharoni, R., Arnon, R., Malach, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+morphology+is+confined+by+cortical+functional+boundaries+in+mammals+ranging+from+mice+to+human.">Astrocyte morphology is confined by cortical functional boundaries in mammals ranging from mice to human.</a> eLife. 5, 15915 (2016).</li><li>Bushong, E. A., Marton, M. E., Ellisman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+of+astrocyte+morphology+and+the+establishment+of+astrocyte+domains+during+postnatal+hippocampal+development.">Maturation of astrocyte morphology and the establishment of astrocyte domains during postnatal hippocampal development.</a> International Journal of Developmental Neuroscience. 22 (2), 73-86 (2004).</li><li>Bushong, E. A., Martone, M. E., Ellisman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+the+relationship+between+astrocyte+morphology+and+laminar+boundaries+in+the+molecular+layer+of+adult+dentate+gyrus.">Examination of the relationship between astrocyte morphology and laminar boundaries in the molecular layer of adult dentate gyrus.</a> Journal of Comparative Neurology. 462 (2), 241-251 (2003).</li><li>Bushong, E. A., Martone, M. E., Jones, Y. Z., Ellisman, M. 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>

The authors have no conflicts of interest to disclose.

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><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)&amp;nbsp;</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 ...

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

Research

JoVE Journal

Neuroscience

2.4K Views.  The Hebrew University of Jerusalem. De helderheidstechniek die voor het eerst werd beschreven in 2013 is een weefselverwijderingstechniek die de bemonstering van grotere hoeveelheden cellen, bloedvaten, dus zelfs eiwitten zoals een enkele celresolutie in dikke hersenplakken mogelijk maakt. Deze techniek maakt de studie van intacte microscopische structuren mogelijk door het hele brein transparant te maken. Dit protocol is een eenvoudige chip en een eenvoudige pijplijn voor het wissen van weefsel.Deze techniek kan worden ...

Video: Investigation of Spatial Interaction Between Astrocytes and Neurons in Cleared Brains

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2 . Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e., axon, dendrite, or soma)3. This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell.
	In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

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 ...

Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying the human brain. One emerging technology to address this issue is the use of three dimensional (3D) neural cell cultures, termed &#39;organoids&#39; or &#39;spheroids&#39;, for long term preservation of intercellular interactions including extracellular adhesion molecules. However, these culture systems are time consuming and not systematically generated. Here, we detail a method to rapidly and consistently produce 3D cocultures of neurons and astrocytes from human pluripotent stem cells. First, pre-differentiated astrocytes and neuronal progenitors are dissociated and counted. Next, cells are combined in sphere-forming dishes with a Rho-Kinase inhibitor and at specific ratios to produce spheres of reproducible size. After several weeks of culture as floating spheres, cocultures (&#39;asteroids&#39;) are finally sectioned for immunostaining or plated upon multielectrode arrays to measure synaptic density and strength. In general, it is expected that this protocol will yield 3D neural spheres that display mature cell-type restricted markers, form functional synapses, and exhibit spontaneous synaptic network burst activity. Together, this system permits drug screening and investigations into mechanisms of disease in a more suitable model compared to monolayer cultures.

In this protocol, we aim to describe a reproducible method for combining dissociated human pluripotent stem cell derived neurons and astrocytes together into 3D sphere cocultures, maintaining these spheres in free floating conditions, and subsequently measuring synaptic circuit activity of the spheres with immunoanalysis and multielectrode array recordings.

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying ...

Developmental Biology

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Bioengineering

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

Analyzing the Size, Shape, and Directionality of Networks of Coupled Astrocytes

It has become increasingly clear that astrocytes modulate neuronal function not only at the synaptic and single-cell levels, but also at the network level. Astrocytes are strongly connected to each other through gap junctions and coupling through these junctions is dynamic and highly regulated. An emerging concept is that astrocytic functions are specialized and adapted to the functions of the neuronal circuit with which they are associated. Therefore, methods to measure various parameters of astrocytic networks are needed to better describe the rules governing their communication and coupling and to further understand their functions.
Here, using the image analysis software (e.g., ImageJFIJI), we describe a method to analyze confocal images of astrocytic networks revealed by dye-coupling. These methods allow for 1) an automated and unbiased detection of labeled cells, 2) calculation of the size of the network, 3) computation of the preferential orientation of dye spread within the network, and 4) repositioning of the network within the area of interest.
This analysis can be used to characterize astrocytic networks of a particular area, compare networks of different areas associated to different functions, or compare networks obtained under different conditions that have different effects on coupling. These observations may lead to important functional considerations. For instance, we analyze the astrocytic networks of a trigeminal nucleus, where we have previously shown that astrocytic coupling is essential for the ability of neurons to switch their firing patterns from tonic to rhythmic bursting1. By measuring the size, confinement, and preferential orientation of astrocytic networks in this nucleus, we can build hypotheses about functional domains that they circumscribe. Several studies suggest that several other brain areas, including the barrel cortex, lateral superior olive, olfactory glomeruli, and sensory nuclei in the thalamus and visual cortex, to name a few, may benefit from a similar analysis.

Here we present a protocol to assess the organization of astrocytic networks. The described method minimizes bias to provide descriptive measures of these networks such as cell count, size, area, and position within a nucleus. Anisotropy is assessed with a vectorial analysis.

It has become increasingly clear that astrocytes modulate neuronal function not only at the synaptic and single-cell levels, but also at the network ...

Visualizing Astrocyte Morphology Using Lucifer Yellow Iontophoresis

Astrocytes are essential components of neural circuits. They tile the entire central nervous system (CNS) and are involved in a variety of functions, which include neurotransmitter clearance, ion regulation, synaptic modulation, metabolic support to neurons, and blood flow regulation. Astrocytes are complex cells that have a soma, several major branches, and numerous fine processes that contact diverse cellular elements within the neuropil. In order to assess the morphology of astrocytes, it is necessary to have a reliable and reproducible method to visualize their structure. We report a reliable&#160;protocol to perform intracellular iontophoresis of astrocytes using fluorescent Lucifer yellow (LY) dye in lightly fixed brain tissue from adult mice. This method has several features that are useful to characterize astrocyte morphology. It allows for three-dimensional reconstruction of individual astrocytes, which is useful to perform morphological analyses on different aspects of their structure. Immunohistochemistry together with LY iontophoresis can also be utilized to understand the interaction of astrocytes with different components of nervous system and to evaluate the expression of proteins within the labelled astrocytes. This protocol can be implemented in a variety of mouse models of CNS disorders to rigorously examine astrocyte morphology with light microscopy. LY iontophoresis provides an experimental approach to evaluate astrocyte structure, especially in the context of injury or disease where these cells are proposed to undergo significant morphological changes.

Astrocytes are morphologically complex cells, exemplified by their multiple processes and bushy territories. To analyze their elaborate morphology, we present a reliable protocol to perform intracellular Lucifer yellow iontophoresis in lightly fixed tissue.

Astrocytes are essential components of neural circuits. They tile the entire central nervous system (CNS) and are involved in a variety of functions, ...

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 ...

Analysis of Astrocyte Territory Volume and Tiling in Thick Free-Floating Tissue Sections

Astrocytes possess an astounding degree of morphological complexity that enables them to interact with nearly every type of cell and structure within the brain. Through these interactions, astrocytes actively regulate many critical brain functions, including synapse formation, neurotransmission, and ion homeostasis. In the rodent brain, astrocytes grow in size and complexity during the first three postnatal weeks and establish distinct, non-overlapping territories to tile the brain. This protocol provides an established method for analyzing astrocyte territory volume and astrocyte tiling using free-floating tissue sections from the mouse brain. First, this protocol describes the steps for tissue collection, cryosectioning, and immunostaining of free-floating tissue sections. Second, this protocol describes image acquisition and analysis of astrocyte territory volume and territory overlap volume, using commercially available image analysis software. Lastly, this manuscript discusses the advantages, important considerations, common pitfalls, and limitations of these methods. This protocol requires brain tissue with sparse or mosaic fluorescent labeling of astrocytes, and is designed to be used with common lab equipment, confocal microscopy, and commercially available image analysis software.

This protocol describes methods for sectioning, staining, and imaging free-floating tissue sections of the mouse brain, followed by a detailed description of the analysis of astrocyte territory volume and astrocyte territory overlap or tiling.

Astrocytes possess an astounding degree of morphological complexity that enables them to interact with nearly every type of cell and structure within the ...

Investigation of Spatial Interaction Between Astrocytes and Neurons in Cleared Brains

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video