Prolonged incubation of acute neuronal tissue for electrophysiology and calcium-imaging

Morven A. Cameron, Orsolya Kekesi, John W. Morley, Alba Bellot-Saez, Sindy Kueh, Paul Breen, André Van Schaik, Jonathan Tapson, Yossi Buskila

Research output: Contribution to journalArticleOtherpeer-review

7 Citations (Scopus)

Abstract

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (>24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 °C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at >24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Original languageEnglish
Article numbere55396
JournalJournal of Visualized Experiments
Volume2017
Issue number120
DOIs
Publication statusPublished - 15 Feb 2017
Externally publishedYes

Keywords

  • AM dyes
  • Brain slice
  • Calcium imaging
  • Electrophysiology
  • Issue 120
  • Neurons
  • Neuroscience
  • Papain
  • Retina

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