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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

We introduce a reproducible and stable optical recording method for brain slices using voltage-sensitive dye. The article describes voltage-sensitive dye staining and recording of optical signals using conventional hippocampal slice preparations.

Abstract

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural circuits. Due to the fractional change in the light signal, it has been difficult to use this method as a quantitative assay. This article describes special optics and slice handling systems, which render this technique stable and reliable. The present article demonstrates the slice handling, staining, and recording of the VSD-stained hippocampal slices in detail. The system maintains physiological conditions for a long time, with good staining, and prevents mechanical movements of the slice during the recordings. Moreover, it enables staining of slices with a small amount of the dye. The optics achieve high numerical aperture at low magnification, which allows recording of the VSD signal at the maximum frame rate of 10 kHz, with 100 pixel x 100-pixel spatial resolution. Due to the high frame rate and spatial resolution, this technique allows application of the post-recording filters that provide sufficient signal-to-noise ratio to assess the changes in neural circuits.

Introduction

Wide-field single photon voltage-sensitive dye (VSD) imaging of bulk-stained brain slice preparations has become a useful quantitative tool to assess the dynamics of neural circuits1,2,3,4. After the analysis of the changes in optical properties due to membrane excitation5,6,7, VSD imaging was first described in the early 1970s by Cohen and others6,8,9.....

Protocol

All animal experiments were performed according to protocols approved by the Animal Care and Use Committee of Tokushima Bunri University. The following protocol for slice preparation is almost a standard procedure27 , but the modifications have been the protocols of staining and recording with VSD.

1. Preparation Before the day of Experiment

  1. Prepare the stock A (Table 1), stock B (Table 2), and stock C (Table 3

Representative Results

Figure 5 shows the representative optical signal upon electrical stimulation of the Schaffer collateral in area CA1 of a mouse hippocampal slice. The consecutive images in Figure 5A show the optical signal before any spatial and temporal filters were applied, while Figure 5B shows the same data after applying a 5 x 5 x 5 cubic filter (a Gaussian Kernel convolution, 5 x 5 spatial- and 5 to tempor.......

Discussion

The slice physiology is vital for collecting the right signal. The use of the ring-membrane filter system in this protocol ensures that the slice remains healthy and un-distorted throughout the procedure2,16,17. Other systems can be used to retain slice physiology during the recording, but the slice should not get deformed at any time as the imaging needs every part of the slice to be healthy. The ring-membrane filter system is .......

Acknowledgements

TT received the JSPS KAKENHI Grant (JP16H06532, JP16K21743, JP16H06524, JP16K0038, and JP15K00413) from MEXT and grants from the Ministry of Health, Labour and Welfare (MHLW-kagaku-ippan-H27 [15570760] and H30 [18062156]). We would like to thank Editage (www.editage.jp) for English language editing.

....

Materials

NameCompanyCatalog NumberComments
High speed image acquisition systemBrainvision co. Ltd.MiCAM - UltimaImaging system
High speed image acquisition systemBrainvision co. Ltd.MiCAM 02Imaging system
Macroscepe for wide field imagingBrainvision co. Ltd.THT macroscopemacroscope
High powere LED illumination system with photo-diodode stablilizerBrainvision co. Ltd.LEX-2GLED illumination
Image acquisition softwareBrainvision co. Ltd.BV-anaimage acquisition software
Multifunctional electric stimulatorBrainvision co. Ltd.ESTM-8Stimulus isolator+AD/DA converter
SlicerLeicaVT-1200Sslicer
SlicerLeicaVT-1000slicer
Blade for slicerFeather Safety Razor Co., Ltd.#99027carbon steel razor blade
Membrane filter for slice supportMerk Millipore Ltd., MA, USAOmnipore, JHWP01300, 0.45 µm pores,membrane filter/ 0.45 13
Numerical analysis softwareWavemetrics Inc., OR, USAIgorProanalysing software
Stimulation isolatorWPI Inc.A395Stimulus isolator
AD/DA converterInstrutechITC-18AD/DA converter
Voltage sensitive dye Di-4-ANEPPSInvitrogen, Thermo-Fisher Scientific, Waltham, MA, USAcatalog number: D-1199VSD: Di-4-ANEPPS
poloxamerInvitrogen, Thermo-Fisher Scientific, Waltham, MA, USAPluronic F-127 P30000MPpoloxamer / Pluronic F-127 (20% solution in DMSO)
polyethoxylated castor oilSigma-AldrichCremophor EL C5135polyethoxylated castor oil

References

  1. Tominaga, Y., Taketoshi, M., Tominaga, T. Overall Assay of Neuronal Signal Propagation Pattern With Long-Term Potentiation (LTP) in Hippocampal Slices From the CA1 Area With Fast Voltage-Sensitive Dye Imaging. Frontiers in Cellular Neuroscience. 12, 389 (2018).
  2. Tominaga, T., Kajiwara, R., Tominaga, Y.

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