Abstract
IntroductionWith its preservation of cytoarchitecture and synaptic circuitry, the hippocampal slice preparation has been a critical tool for studying the electrophysiological effects of pharmacological and genetic manipulations. To analyze the maximum number of slices or readouts per dissection, long incubation times postslice preparation are commonly used. We were interested in how slice integrity is affected by incubation postslice preparation.MethodsHippocampal slices were prepared by three different methods: a chopper, a vibratome, and a rotary slicer. To test slice integrity, we compared glycogen levels and immunohistochemistry of selected proteins in rat hippocampal slices immediately after dissection and following 2 and 4 hr of incubation.ResultsWe found that immunoreactivity of the dendritic marker microtubule‐associated protein 2 (Map2) drastically decreased during this incubation period, whereas immunoreactivity of the glutamate transporter VGlut1 did not significantly change with incubation time. Astrocytic and microglial cell numbers also did not significantly change with incubation time whereas glycogen levels markedly increased during incubation.ConclusionImmunoreactivity of the dendritic marker Map2 quickly decreased after dissection with all the slicing methods. This work highlights a need for caution when using long incubation periods following slice preparation.
Highlights
With its preservation of cytoarchitecture and synaptic circuitry, the hippocampal slice preparation has been a critical tool for studying the electrophysiological effects of pharmacological and genetic manipulations
Because VGlut1 levels determine the amount of glutamate stored and released per vesicle, and variations in VGlut1 expression regulate the efficacy of glutamate synaptic transmission (Santos et al, 2009), we examined VGlu1 immunohistochemistry
We assessed the effect of postslicing incubation period on the metabolic and immunohistochemical characteristics of chopper-, vibratome-, and rotary-generated hippocampal slices
Summary
One of the most commonly studied models of mammalian brain physiology is the in vitro hippocampal slice. Even though maximal evoked electrical responses can be obtained within 30–60 min of incubation, it is common practice to incubate slices for 1–2 hr, regardless of temperature, glucose levels, or oxygen levels of incubation, to allow recovery from the trauma of preparation and the development of a stable metabolic state prior to beginning experiments (Kirov, Sorra, & Harris, 1999; Schurr, Reid, Tseng, & Edmonds, 1984; Wang & Kass, 1997; Whittingham, Lust, Christakis, & Passonneau, 1984) Both electrophysiologically and metabolically, this stable state has been shown to persist for up to 6–9 hr of incubation, at which point deterioration and electrical failure begin and proceed rapidly (Chang & Greenough, 1984; Schurr et al, 1984; Whittingham et al, 1984). In this study, we prepared hippocampal slices with three different slicers: a tissue chopper, a vibratome, and a rotary slicer
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