Elucidating connectivity and functionality at the whole-brain level is one of the most challenging research goals in neuroscience. Various whole-brain optical imaging technologies with submicron lateral resolution have been developed to reveal the fine structures of brain-wide neural and vascular networks at the mesoscopic level. Among them, micro-optical sectioning tomography (MOST) is attracting increasing attention, as a variety of technological variations and solutions tailored toward different biological applications have been optimized. Here, we summarize the recent development of MOST technology in wholebrain imaging and anticipate future improvements.

Paraffin embedding is widely used in microscopic imaging for preparing biological specimens. However, owing to significant fluorescence quenching during the embedding process, it is not compatible with fluorescent-labeling techniques, such as transgenic and viral labeling using green fluorescent protein (GFP). Here, we investigate the quenching mechanism and optimize the embedding process to improve the preservation of fluorescence intensity. The results show that dehydration is the main reason for fluorescence quenching during paraffin embedding, caused by the full denaturation of GFP molecules in ethyl alcohol. To evaluate fluorescent and morphological preservation, we modified the embedding process using tertiary butanol (TBA) instead of ethyl alcohol. Fluorescence intensity following TBA dehydration increased 12.08-fold of that observed in the traditional method. We obtained uniform fluorescence maintenance throughout the whole mouse brain, while the continuous apical dendrites, spines, and axon terminals were shown evenly within the cortex, hippocampus, and the amygdala. Moreover, we embedded a whole rat brain labeled with AAV in the prelimbic cortex (Prl). With the axon terminals in different areas, such as the caudate putamen, thalamus, and pyramidal tract, the results showed a continuous tract of Prl neurons throughout the whole brain. This method was also suitable for tdTomota labeled samples. These findings indicate that this modified embedding method could be compatible with GFP and provides a potential turning point for applications in the fluorescent labeling of samples.

The glutamatergic and GABAergic neurons in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) mediated diverse brain functions. However, their whole-brain neural connectivity has not been comprehensively mapped. Here we used the virus tracers to characterize the whole-brain inputs and outputs of glutamatergic and GABAergic neurons in VTA and SNc. We found that these neurons received similar inputs from upstream brain regions, but some quantitative differences were also observed. Neocortex and dorsal striatum provided a greater share of input to VTA glutamatergic neurons. Periaqueductal gray and lateral hypothalamic area preferentially innervated VTA GABAergic neurons. Specifically, superior colliculus provided the largest input to SNc glutamatergic neurons. Compared to input patterns, the output patterns of glutamatergic and GABAergic neurons in the VTA and SNc showed significant preference to different brain regions. Our results laid the anatomical foundation for understanding the functions of cell-type-specific neurons in VTA and SNc.