Decoding the mouse spinal cord locomotor neural network using tissue clearing, tissue expansion and tiling light sheet microscopy techniques

Abstract

Decoding a biological neural network requires the structural information regarding the spatial organization, dendritic morphology, axonal projection and synaptic connection of the neurons in the network. Imaging physically sectioned nervous tissues using electron microscopy (EM) has been the only method to acquire such information. However, EM is inefficient for imaging and reconstructing large neural networks due to the low throughput and inability to target neural circuits of interest by labeling specific neuron populations genetically. Here, we present a method to image large nervous tissues from the cellular to synaptic level with high throughput using tiling light sheet microscopy combined with tissue clearing and tissue expansion techniques. We describe the method, demonstrate its capability and explore its utility for decoding large biological neural networks by studying the spinal cord locomotor neural network in genetically labeled fluorescent mice. We show our method could advance the decoding of large neural networks significantly.

Spinal premotor interneurons controlling antagonistic muscles are spatially intermingled

“…They could be either large cholinergic interneurons that are presynaptic to motor neurons, or (perhaps more likely) medial motor neurons that send recurrent axon collaterals to lateral motor neurons. In fact, while collaterals from lateral or medial motor neurons have not been traced outside their respective columns, the dendritic arborization from medial and lateral motor neuron columns extends to each other and labelling of lateral motor neurons following rabies injection of medial column innervating muscles has been observed (Goetz et al, 2015; Balaskas et al, 2019; Feng et al, 2022), indicating that some connectivity, at least from lateral to medial columns, is possible. With respect to “ectopic” labelling in other LMC pools, it is important to consider leak of the virus at the injection sites.…”

Morphology Analysis of Descending Tracts in the Mouse Spinal Cord Using Tissue Clearing, Tissue Expansion and Tiling Light Sheet Microscopy Techniques

“…1L). In the previous study, we have studied the projection patterns of three dominant descending tracts, containing the cortex, red nucleus and reticular formation 34 . In this study, we will focus on these less-studied descending pathways to analyze the projection pattern and axon features in the spinal cord using high magni cation 3D imaging microscopy.…”

“…The tissue was treated with eCubic and CMAP as previously reported 32 . Preparation of cleared spinal cord and brain(eCubic): The tissue was immersed in the delipidation solution(15 wt% urea,10% wt% Nbutyldiethanolamine,10 wt% Triton X-100 and 65 wt% ddH 2 O) at 37℃ for 3-5 days with gently shaking until the tissue became transparent.…”

“…The imaging system has been reported in previous studies 34,41,42 . The microscope used a uorescence macro zoom microscope (Olympus MVX10) with Olympus MVPLAPO 1X when the whole brain was imaged.…”

“…Thus, in order to know more details and connectome information about the axons of descending neurons in spinal cord, the hydrophilic and hydrogel-based methods were used in this study to obtain the intact 3D cleared tissue. In our previous study, we have reported optimized tissue clearing and tissue expansion methods, called eCubic and CMAP (Clearing and Magni cation Analysis of Proteome) respectively, for the large sample clearing and expansion by modifying the CUBIC and MAP methods 32 . Our previous data have shown that the tissues treated by CMAP had higher uorescence preservation and mechanical strength.…”