Investigation of Spatial Interaction Between Astrocytes and Neurons in Cleared Brains

Summary

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

Abstract

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.

Introduction

In recent years, the knowledge of astrocyte function and how they interact with neuronal circuits has increased dramatically. Astrocytes can influence plasticity^1,2, assist in neuronal postinjury recovery^3,4, 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 functions^5,6,7. A feature of particular interest in astrocyte rese....

Protocol

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.

1. 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., http://connectivity.brain-map.org/3d-viewer).
2. Using stereotactic surgery, inject the viral vectors into the relevant brain....

Results

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).

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
AAV1-GFAP::TdTomato	ELSC Vector Core Facility (EVCF)		viral vector used to detect astrocytes
AAV5-CaMKII::eGFP	ELSC Vector Core Facility (EVCF)		viral vector used to detect neurons
AAV5-CaMKII::H2B-eGFP	ELSC Vector Core Facility (EVCF)		viral vector used to detect neuronal nuclei
AAV5-CaMKII::TdTomato	ELSC Vector Core Facility (EVCF)		viral vector used to detect neurons
Acrylamide (40%)	Bio-rad	#161-0140
Bisacrylamide (2%)	Bio-rad	#161-0142
Boric acid	Sigma	#B7901	Molecular weight - 61.83 g/mol
Confocal microscope, scanning, FV1000	Olympus		4x objective (UPlanSApo, 0.16 NA)
Imaris software	Bitplane, UK		A software that allows 3D analysis of images
NaOH	Sigma	#S5881
PBS
PFA 4%	EMS	#15710
RapiClear	SunJin lab	#RC147002
RapiClear CS	SunJin lab	#RCCS002
SDS	Sigma	#L3771
SyGlass software			A software that allows 3D analysis of images using virtual reality
Tris base 1 M	Bio-rad	#002009239100	Molecular weight - 121.14 g/mol
Triton X-100	ChemCruz	#sc-29112A
Two photon microscope	Neurolabware		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)
VA-044 Initiator	Wako	#011-19365