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

Abstract

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 cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum.While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradientrefractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice.A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

Designing AAV Vectors for Monitoring the Subtle Calcium Fluctuations of Inferior Olive Network in vivo

“…Instead, experimenters have resorted to indirect methods, such as imaging IO-axon-evoked spikes in the cerebellum (Ju et al, 2019 ; Hoang et al, 2020 ; Roh et al, 2020 ; Michikawa et al, 2021 ), optogenetic modulation of IO afferents instead of IO neurons (Kim et al, 2020 ), limiting experimentation to cerebellar cortical regions where off-target transfection of axons with, e.g., the CamKII promoter is not problematic (Mathews et al, 2012 ; Gaffield et al, 2019 ) or morphological analysis (Nishiyama et al, 2007 ; Pätz et al, 2018 ; Vrieler et al, 2019 ; González-Calvo et al, 2021 ). To our knowledge, there have been no published reports of in situ, in vivo multi-cell IO network activity besides our recently-published preliminary observations (Guo et al, 2021 ).…”

“…We have previously described in detail the procedures for precise stereotactic targeting of the IO (Dorgans et al, 2020 ; Guo et al, 2021 ). In brief, postnatal (P) 35-45 C57BL/6J male mice (CLEA Japan, Shizuoka, Japan) were anesthetized with 5% isoflurane (SomnoSuite, Kent Scientific, CT, USA) and maintained on a heat pad (38° C ; TMP-5b, Supertech Instruments) attached to a stereotaxic frame (Neurostar, Tübingen, Germany) with constant 1% to 2.4% isoflurane delivery via a nose cone.…”

“…In brief, postnatal (P) 35-45 C57BL/6J male mice (CLEA Japan, Shizuoka, Japan) were anesthetized with 5% isoflurane (SomnoSuite, Kent Scientific, CT, USA) and maintained on a heat pad (38° C ; TMP-5b, Supertech Instruments) attached to a stereotaxic frame (Neurostar, Tübingen, Germany) with constant 1% to 2.4% isoflurane delivery via a nose cone. Special attention is required to correctly align the head and body for reliable targeting of deep structures such as IO (Dorgans et al, 2020 ; Guo et al, 2021 ). After shaving the scalp, skin was locally anesthetized by application of Xylocaine gel (Xylogel, gel 2%, Aspen, Japan), disinfected and incised with a sharp blade to expose the skull, and small (about 1 mm diameter) craniotomies were opened with a hand-held drill (Surgic XT Plus drill, NSK Dental, Japan).…”

“…The appropriate injectate mixes were backfilled into quartz capillary pipettes designed for targeting IO (Guo et al, 2021 ). The pipette was slowly inserted in brain tissue (~ 0.2 mm/s) to the target locations.…”

“…Even if no dramatic pathological changes was observed, overloading a brain structure with viral particles can lead to unspecific or trans-synaptic expression patterns and, thus, unreliable experimental results. Notably, while in our previous report (Guo et al, 2021 ) we were able to record IO neuron activity using a commercial CAG-promoter-driven GCaMP6s-expression vector, the expression efficacy and neuron-specificity was highly unreliable, leading to initiation of the present work.…”