Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain

Abstract

Visualizing entire neuronal networks for analysis in the intact brain has been impossible up to now. Techniques like computer tomography or magnetic resonance imaging (MRI) do not yield cellular resolution, and mechanical slicing procedures are insufficient to achieve high-resolution reconstructions in three dimensions. Here we present an approach that allows imaging of whole fixed mouse brains. We modified 'ultramicroscopy' by combining it with a special procedure to clear tissue. We show that this new technique allows optical sectioning of fixed mouse brains with cellular resolution and can be used to detect single GFP-labeled neurons in excised mouse hippocampi. We obtained three-dimensional (3D) images of dendritic trees and spines of populations of CA1 neurons in isolated hippocampi. Also in fruit flies and in mouse embryos, we were able to visualize details of the anatomy by imaging autofluorescence. Our method is ideally suited for high-throughput phenotype screening of transgenic mice and thus will benefit the investigation of disease models.

Cell type-specific Nogo-A gene ablation promotes axonal regeneration in the injured adult optic nerve

“…In order to assess the pattern of growing axons, a three-dimensional analysis of the regenerating fibers was carried out in whole-mounted optic nerves after tissue clearing using a modified protocol previously described. 37,38 In the optic nerve segments situated between 100 and 300 mm past the lesion site, branched and unbranched axons could be distinguished, as well as axons that formed U-turns within this region (Figures 2e and f). We found that the proportion of branching axons did not significantly vary between Cnp-Cre þ / À xRtn4 flox/flox and Rtn4 flox/flox control mice (Figure 2g).…”

Unleashing Optics and Optoacoustics for Developmental Biology

“…Light sheet microscopy or ultramicroscopy, originally developed in 1903 [41], reappeared in 1983 when it was applied to fixed and cleared tissue as orthogonal-plane fluorescence optical sectioning (OPFOS) [42], and as thin laser sheet imaging microscopy (TSLIM) in 2002 [43] finally being applied to whole fixed and cleared organs as ultramicroscopy [44]. However, it was not until Huisken et al in 2004 [45] presented selective plane illumination microscopy (SPIM) data applied to in vivo zebrafish imaging that light sheet microscopy really took off.…”

What is comparable in comparative cognition?

“…A recent neuroanatomical study, for example, succeeded in identifying and mapping 16 per cent of the approximately 100 000 neurons of the Drosophila brain [97], illustrating just how far we have progressed in the direction of understanding the circuitry of insects' brains. Other recent developments make the imaging of entire neural circuits, and possibly whole brains at micrometre resolution, increasingly feasible [98,99]. It is quite conceivable that as such technologies develop we will discover neural-computational solutions to cognitive tasks in non-human animals quite unlike those in humans (even where the behavioural outcomes might be similar).…”