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Review
. 2020 Oct 23:14:569517.
doi: 10.3389/fnins.2020.569517. eCollection 2020.

Dissecting Neuronal Activation on a Brain-Wide Scale With Immediate Early Genes

Alessandra Franceschini  1 , Irene Costantini  1   2 , Francesco S Pavone  1   2   3 , Ludovico Silvestri  1   2   3
Affiliations
Review

Dissecting Neuronal Activation on a Brain-Wide Scale With Immediate Early Genes

Alessandra Franceschini et al. Front Neurosci. .

Abstract

Visualizing neuronal activation on a brain-wide scale yet with cellular resolution is a fundamental technical challenge for neuroscience. This would enable analyzing how different neuronal circuits are disrupted in pathology and how they could be rescued by pharmacological treatments. Although this goal would have appeared visionary a decade ago, recent technological advances make it eventually feasible. Here, we review the latest developments in the fields of genetics, sample preparation, imaging, and image analysis that could be combined to afford whole-brain cell-resolution activation mapping. We show how the different biochemical and optical methods have been coupled to study neuronal circuits at different spatial and temporal scales, and with cell-type specificity. The inventory of techniques presented here could be useful to find the tools best suited for a specific experiment. We envision that in the next years, mapping of neuronal activation could become routine in many laboratories, allowing dissecting the neuronal counterpart of behavior.

Keywords: high-throughput microscopy; image analysis; immediate early genes; light-sheet microscopy; tissue clearing; whole-brain mapping.

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Figures

FIGURE 1
FIGURE 1
Scheme representing the different steps to perform whole-brain neuronal activation mapping. (A) Behavioral testing. (B) In vivo neuronal activation tagging with endogenous fluorescence proteins. (C) Sample preparation protocol: clearing and staining (optional, when the tagging is not performed). (D) Imaging with advanced fluorescence microscopy. (E) Data analysis with different algorithms. Created with BioRender.com.
FIGURE 2
FIGURE 2
Diagram of immediate early gene (IEG)-based approaches for neuron tagging. For each method, information about the characteristic of the final effect is described. In particular: the duration time (transient or not), the location (whole-brain or not), and if the method can be coupled with optogenetic and chemogenetic tools (manipulation). TRE, tetracycline responsive element; Dox, doxycycline; tTA, artificial transcription factor; TXM, 4-hydroxytamoxifen; CreER, tamoxifen-inducible recombinase; rtTA, reverse tetracycline transactivator; TeTb, bidirectional Tet promoter. Created with BioRender.com.
FIGURE 3
FIGURE 3
Physical principle underlying tissue clearing. In normal tissue (left) cellular components have a refractive index n2 significantly larger than that of the surrounding medium (water, n1). This inhomogeneity results in scattering of light and thus opaqueness of the sample. Clearing methods try to match refractive indices (right), so that tissue appears as a homogeneous optical medium where light can travel unhindered. Created with BioRender.com.
FIGURE 4
FIGURE 4
Representation of the three steps necessary to obtain neuronal quantification across the entire murine brain. (A) Scheme of advanced imaging approaches for whole-brain reconstruction: serial two-photon tomography (STP), block-face serial microscopy tomography (FAST), and light-sheet microscopy (LSM). (B) The spatial position of individual cells (left) can be automatically detected (right) using various algorithms, including machine learning ones (see text). (C) Finally, brain volumes can be spatially aligned to reference atlas using different computational approaches (see text).
FIGURE 5
FIGURE 5
Schematic of experimental pipelines that could be used for activation mapping.
See this image and copyright information in PMC

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