title: Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging.
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.
authors.affiliation: The MARCS Institute, Western Sydney University.
authors.forename: Morven A
authors.initials: MA
authors.lastname: Cameron
authors.affiliation: The MARCS Institute, Western Sydney University; School of Medicine, Western Sydney University.
authors.forename: Orsolya
authors.initials: O
authors.lastname: Kekesi
authors.affiliation: The MARCS Institute, Western Sydney University; School of Medicine, Western Sydney University.
authors.forename: John W
authors.initials: JW
authors.lastname: Morley
authors.affiliation: The MARCS Institute, Western Sydney University; School of Medicine, Western Sydney University.
authors.forename: Alba
authors.initials: A
authors.lastname: Bellot-Saez
authors.affiliation: School of Medicine, Western Sydney University.
authors.forename: Sindy
authors.initials: S
authors.lastname: Kueh
authors.affiliation: The MARCS Institute, Western Sydney University.
authors.forename: Paul
authors.initials: P
authors.lastname: Breen
authors.affiliation: The MARCS Institute, Western Sydney University.
authors.forename: André
authors.initials: A
authors.lastname: van Schaik
authors.affiliation: The MARCS Institute, Western Sydney University.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Tapson
authors.affiliation: The MARCS Institute, Western Sydney University; School of Medicine, Western Sydney University; Y.Buskila@westernsydney.edu.au.
authors.forename: Yossi
authors.initials: Y
authors.lastname: Buskila
mesh_terms: D000818:Animals; D001921:Brain / Q000502:physiology*; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D003470:Culture Media; D004594:Electrophysiology / Q000379:methods; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D009417:Nerve Tissue / Q000502:physiology*; D009474:Neurons / Q000502:physiology; D014021:Tissue Preservation / Q000379:methods*
chemical_list: D003470:Culture Media; D002118:Calcium