Imaging Subthreshold Voltage Oscillation With Cellular Resolution in the Inferior Olive in vitro

Abstract

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 vivo 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.

Voltage-induced fluorescence lifetime imaging of a BODIPY derivative in giant unilamellar vesicles as potential neuron membrane mimics

“…Interestingly, the resonances for the two alkene protons appeared as a pseudo-triplet with a coupling constant in the order of 17Hz rather than two doublets. A triplet peak found for the 11 B NMR spectrum at 1.03 ppm and the quartet (1 : 1 : 1 : 1) for the 19 F NMR spectrum at À143.06 ppm, with a coupling constant of around 32-34 Hz are typical for BODIPYs and further corroborate the structure. High-resolution mass spectrometry and HPLC measurements confirmed the high purity of the material (see ESI †).…”

“…Such a new method would hopefully have application in such an area as mammalian brain slice imaging which is still a challenging task. 11 The preparation of the target dye is shown in Scheme 1 starting from BD which was synthesized according to an adapted method reported elsewhere. 12 The compound aJBD was synthesised through the Knoevenagel condensation between BD and JUL-CHO as a black solid with a green metallic colour.…”

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

“…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 involvement of various voltage-gated calcium currents distributed in somatic and dendritic compartments in driving and modulating both STOs and spike width variations has been described decades ago, but the underlying mechanisms remain poorly understood. Previously, voltage imaging methodology has been used in the context of IO STOs (see Leznik and Llinás ( 2005 ); Devor and Yarom ( 2002 ); Dorgans et al ( 2020 )), but only calcium imaging would provide the necessary spatiotemporal information on these intracellular processes. On the other hand, the STOs and spike width variations are likely to be exquisitely sensitive to perturbations of calcium buffering (Rekling et al, 2012 ; Yu et al, 2014 (Iwaniuk et al, 2009 )), raising concerns that calcium probe presence would lead to aberrant or completely absent STOs or spike-width variations and therefore disqualify calcium imaging methodology entirely from behavioral use.…”