Tissue preparation, immunohistochemistry, Imaging, and Quantification v1

Protocol for neuronal imaging used in Yoo et al 2021

Real-time Noise-suppressed Wide-Dynamic-Range Compression in Ultrahigh-Resolution Neuronal Imaging

SummaryWith a limited dynamic range of an imaging system, there are always regions with signal intensities comparable to the noise level, if the signal intensity distribution is close to or even wider than the available dynamic range. Optical brain/neuronal imaging is such a case where weak-intensity ultrafine structures, such as, nerve fibers, dendrites and dendritic spines, often coexist with ultrabright structures, such as, somas. A high fluorescence-protein concentration makes the soma order-of-magnitude brighter than the adjacent ultrafine structures resulting in an ultra-wide dynamic range. A straightforward enhancement of the weak-intensity structures often leads to saturation of the brighter ones, and might further result in amplification of high-frequency background noises. An adaptive illumination strategy to real-time-compress the dynamic range demands a dedicated hardware to operate and owing to electronic limitations, might encounter a poor effective bandwidth especially when each digitized pixel is required to be illumination optimized. Furthermore, such a method is often not immune to noise-amplification while locally enhancing a weak-intensity structure. We report a dedicated-hardware-free method for rapid noise-suppressed wide-dynamic-range compression so as to enhance visibility of such weak-intensity structures in terms of both contrast-ratio and signal-to-noise ratio while minimizing saturation of the brightest ones. With large-FOV aliasing-free two-photon fluorescence neuronal imaging, we validate its effectiveness by retrieving weak-intensity ultrafine structures amidst a strong noisy background. With compute-unified-device-architecture (CUDA)-acceleration, a time-complexity of <3 ms for a 1000×1000-sized 16-bit data-set is secured, enabling a real-time applicability of the same.

Title: Multi-Scale LM/EM Neuronal Imaging from Brain to Synapse with a Tissue Clearing Method, ScaleSF

The mammalian brain is organized over sizes that span several orders of magnitude, from synapses to the entire brain. Thus, a technique to visualize neural circuits across multiple spatial scales (multi-scale neuronal imaging) is vital for deciphering brain-wide connectivity. Here, we developed this technique by coupling successive light microscope/electron microscope (LM/EM) imaging with an ultrastructurally-preserved tissue clearing method, ScaleSF. Our multi-scale neuronal imaging incorporates 1) brain-wide macroscopic observation, 2) mesoscopic circuit mapping, 3) microscopic subcellular imaging, and 4) EM imaging of nanoscopic structures, allowing seamless integration of structural information from the brain to synapses. We applied the technique to three neural circuits of two different species, mouse striatofugal, mouse callosal, and marmoset corticostriatal projection systems, and succeeded in the simultaneous interrogation of their circuit structure and synaptic connectivity in a targeted way. Our multi-scale neuronal imaging will significantly advance the understanding of brain-wide connectivity by expanding the scales of objects.

Parallel evolution of a splicing program controlling neuronal excitability in flies and mammals

SummaryNeurons draw on alternative splicing for their increased transcriptomic complexity throughout animal phylogeny. To delve into the mechanisms controlling the assembly and evolution of this regulatory layer, we characterized the neuronal microexon program in Drosophila and compared it with that of mammals. We found that in Drosophila, this splicing program is restricted to neurons by the post-transcriptional processing of the enhancer of microexons (eMIC) domain in Srrm234 by Elav and Fne. eMIC deficiency or misexpression leads to widespread neurological alterations largely emerging from impaired neuronal activity, as revealed by a combination of neuronal imaging experiments and cell-type-specific rescues. These defects are associated with the genome-wide skipping of short neural exons, which are strongly enriched in ion channels. Remarkably, we found no overlap of eMIC-regulated exons between flies and mice, illustrating how ancient post-transcriptional programs can evolve independently in different phyla to impact distinct cellular modules while maintaining cell-type specificity.

Natural sensory context drives diverse brain-wide activity during C. elegans mating

Natural goal-directed behaviors often involve complex sequences of many stimulus-triggered components. Understanding how brain circuits organize such behaviors requires mapping the interactions between an animal, its environment, and its nervous system. Here, we use continuous brain-wide neuronal imaging to study the full performance of mating by the C. elegans male. We show that as each mating unfolds in its own sequence of component behaviors, the brain operates similarly between instances of each component, but distinctly between different components. When the full sensory and behavioral context is taken into account, unique roles emerge for each neuron. Functional correlations between neurons are not fixed, but change with behavioral dynamics. From the contribution of individual neurons to circuits, our study shows how diverse brain-wide dynamics emerge from the integration of sensory perception and motor actions within their natural context.