Abstract
In order to observe and quantify long-range neuronal connections in intact mouse brain by light microscopy, it is first necessary to clear the brain, thus suppressing refractive-index variations. Here we describe a method that clears the brain and preserves the signal from proteinaceous fluorophores using a pH-adjusted non-aqueous index-matching medium. Successful clearing is enabled through the use of either 1-propanol or tert-butanol during dehydration whilst maintaining a basic pH. We show that high-resolution fluorescence imaging of entire, structurally intact juvenile and adult mouse brains is possible at subcellular resolution, even following many months in clearing solution. We also show that axonal long-range projections that are EGFP-labelled by modified Rabies virus can be imaged throughout the brain using a purpose-built light-sheet fluorescence microscope. To demonstrate the viability of the technique, we determined a detailed map of the monosynaptic projections onto a target cell population in the lateral entorhinal cortex. This example demonstrates that our method permits the quantification of whole-brain connectivity patterns at the subcellular level in the uncut brain.
Highlights
Visualizing connectivity between neuronal structures by light or electron microscopy almost invariably requires cutting the preparation into a series of slices
Most of the fluorescence was still lost during the benzyl alcohol / benzyl benzoate (BABB) procedure: even when using tert-butanol for dehydration only 1.0% of the original fluorescence was retained after 5 days of clearing (Fig 1A)
Our analysis revealed monosynaptic innervation of the entorhinal cortex (EC) from Piriform Cortex (PiC), olfactory bulb (OB) mitral cells, neurons in the dorsal and lateral olfactory nucleus, the medial septum (MS) including the ventral diagonal band (VDB), the horizontal diagonal band (HDB), the magnocellular preoptic nucleus (MCPO), several thalamic nuclei, the lateral hypothalamic nuclei, the Ectorhinal cortex (ECT), the perirhinal cortex (PRh), different cortical and hippocampal regions, and the subiculum (Table 2; S4 Video)
Summary
Visualizing connectivity between neuronal structures by light or electron microscopy almost invariably requires cutting the preparation into a series of slices. Such slices must be individually imaged and aligned in a tedious and error-prone procedure before any three-dimensional structure can be successfully reconstructed. The need to slice the tissue results from the limited depth to which high-resolution imaging can be performed. For light microscopy, this depth limit can be greatly increased— removing the need to slice the tissue—by removing absorption, scattering, and optical distortion. While absorption can be reduced by draining the sample of blood and using longer wavelengths, the suppression of scattering and wave-front
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