Large Volume Imaging of Rodent Brain Anatomy with Emphasis on Selective Plane Illumination Microscopy

Inside the human brain there are 86 Billion neurons exchanging electrical impulses to allow reaction, homoeostasis and intelligence. Their coarse organization has been described, leaving us with a fair understanding of macrocircuitry. The microcircuitry of the brain is also being explored with patch clamp recordings. But a more integrative view on mesoscale organization of the central nervous system is still lacking. Recent developments in microscopy methods have opened new windows to address this point. It is the goal of this thesis to analyze and compare anatomical features of the brain with current routine techniques as well as explore light sheet microscopy for large scale quantitative reconstructions of rodent brains. Integration efforts of the cortical column model of the Blue Brain project into more detailed cortical circuitry sparked the search of acute slice preparations suitable for long range electrophysiology. Serial section reconstruction of the rat S1 hindlimb anterograde projections was performed with special attention to the anatomical relationship between S1 hindlimb region and VPL nucleus of thalamus. It was found that a functional electrophysiological slice preparation between S1-HL and VPL is feasible. The subsequent experimental exploration proved the intact fiber path in acute slice preparations based on the morphometric measurements of the previous serial sectioning reconstruction. Another mesoscale trait investigated was the excitatory to inhibitory ratio of the juvenile rat cortex. The measured numbers for the cellular density are higher than previously published. Cellular density for S1-HL is 84535$\pm$4400 neurons/mm$^{3}$ high with an E-I ratio of 8.3. Like in other brains the cellular density is a continuously adapting function of the distance from pia and the current brain region. In the second part of this dissertation Selective Plane Illumination Microscopy(SPIM) and tissue clearing are evaluated as novel tools for whole mount rodent brain scanning, with emphasis on somata, fibers and vasculature as quantifiable brain features. Clearing methods are evaluated and a novel embedding medium for cleared tissues is presented: Thiodiethanol(TDE). Index matched brains with TDE show high degree of fluorescence conservation and transparency. Whole mount mouse and rat brains are imaged and reconstructed. The results show that whole mount microscopy is a potent tool that allows routine quantification of anatomic features on a whole brain level.


Related material