in-vivo-calcium-imaging-in-mouse-inferior-olive

<em>In vivo</em> Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

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The inferior olive (IO) is an evolutionarily conserved brain stem structure and its output activity plays a major role in the cerebellar computation necessary for controlling the temporal accuracy of motor behavior. The precise timing and synchronization of IO network activity has been attributed to the dendro-dendritic gap junctions mediating electrical coupling within the IO nucleus. Thus, the dendritic morphology and spatial arrangement of IO neurons governs how synchronized activity emerges in this nucleus. To date, IO neuron structural properties have been characterized in few studies and with small numbers of neurons; these investigations have described IO neurons as belonging to two morphologically distinct types, "curly" and "straight". In this work we collect a large number of individual IO neuron morphologies visualized using different labeling techniques and present a thorough examination of their morphological properties and spatial arrangement within the olivary neuropil. Our results show that the extensive heterogeneity in IO neuron dendritic morphologies occupies a continuous range between the classically described "curly" and "straight" types, and that this continuum is well represented by a relatively simple measure of "straightness". Furthermore, we find that IO neuron dendritic trees are often directionally oriented. Combined with an examination of cell body density distributions and dendritic orientation of adjacent IO neurons, our results suggest that the IO network may be organized into groups of densely coupled neurons interspersed with areas of weaker coupling.

Variability and directionality of inferior olive neuron dendrites revealed by detailed 3D characterization of an extensive morphological library.

Voltage imaging with cellular resolution in mammalian brain slices is still a challenging task. Here, we describe and validate a method for delivery of the voltage-sensitive dye ANNINE-6plus (A6+) into tissue for voltage imaging that results in higher signal-to-noise ratio (SNR) than conventional bath application methods. The not fully dissolved dye was injected into the inferior olive (IO) 0, 1, or 7 days prior to acute slice preparation using stereotactic surgery. We find that the voltage imaging improves after an extended incubation period  in terms of labeled volume, homogeneous neuropil labeling with saliently labeled somata, and SNR. Preparing acute slices 7 days after the dye injection, the SNR is high enough to allow single-trial recording of IO subthreshold oscillations using wide-field (network-level) as well as high-magnification (single-cell level) voltage imaging with a CMOS camera. This method is easily adaptable to other brain regions where genetically-encoded voltage sensors are prohibitively difficult to use and where an ultrafast, pure electrochromic sensor, like A6+, is required. Due to the long-lasting staining demonstrated here, the method can be combined, for example, with deep-brain imaging using implantable GRIN lenses.

Article	Year
Variability and directionality of inferior olive neuron dendrites revealed by detailed 3D characterization of an extensive morphological library. Brain structure & function\| PubMed ID: 30929054	2019
Imaging Subthreshold Voltage Oscillation With Cellular Resolution in the Inferior Olive . Frontiers in cellular neuroscience\| PubMed ID: 33381015	2020

Marylka Yoe Uusisaari

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