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.
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	<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...

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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 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...

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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.

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2.3K vistas.  The Hebrew University of Jerusalem. La técnica de claridad descrita por primera vez en 2013 es una técnica de limpieza de tejidos que permite el muestreo de grandes cantidades de células, vasos sanguíneos, por lo que incluso proteínas como una resolución de una sola célula en rodajas gruesas de cerebro. Esta técnica permite el estudio de estructuras microscópicas intactas haciendo que todo el cerebro sea transparente. Este protocolo es un chip simple y una tubería directa para la limpieza de tejidos.Esta técni...