Abstract
Spreading depolarization (SD) is a sudden, large, and synchronous depolarization of principal cells which also involves interneurons and astrocytes. It is followed by depression of neuronal activity, and it slowly propagates across brain regions like cortex or hippocampus. SD is considered to be mechanistically relevant to migraine, epilepsy, and traumatic brain injury (TBI), but there are many questions about its basic neurophysiology and spread. Research into SD in hippocampus using slices is often used to gain insight and SD is usually triggered by a focal stimulus with or without an altered extracellular buffer. Here, we optimize an in vitro experimental model allowing us to record SD without focal stimulation, which we call spontaneous. This method uses only an altered extracellular buffer containing 0 mM Mg2+ and 5 mM K+ and makes it possible for simultaneous patch and extracellular recording in a submerged chamber plus intrinsic optical imaging in slices of either sex. We also add methods for quantification and show the quantified optical signal is much more complex than imaging alone would suggest. In brief, acute hippocampal slices were prepared with a chamber holding a submerged slice but with flow of artificial cerebrospinal fluid (aCSF) above and below, which we call interface-like. As soon as slices were placed in the chamber, aCSF with 0 Mg2+/5 K+ was used. Most mouse slices developed SD and did so in the first hour of 0 Mg2+/5 K+ aCSF exposure. In addition, prolonged bursts we call seizure-like events (SLEs) occurred, and the interactions between SD and SLEs suggest potentially important relationships. Differences between rats and mice in different chambers are described. Regarding optical imaging, SD originated in CA3 and the pattern of spread to CA1 and the dentate gyrus was similar in some ways to prior studies but also showed interesting differences. In summary, the methods are easy to use, provide new opportunities to study SD, new insights, and are inexpensive. They support previous suggestions that SD is diverse, and also suggest that participation by the dentate gyrus merits greater attention.
Highlights
Increased neuronal excitability is a common feature underlying several neurological disorders, including but not limited to migraine, epilepsy, traumatic brain injury (TBI), and stroke (Eikermann-Haerter et al, 2013; Noseda and Burstein, 2013; Rogawski, 2013; Kim et al, 2014; Bugay et al, 2020)
We found that the likelihood of developing spreading depolarization (SD) in mouse slices significantly increased in an interface-like chamber, where 80% of mouse slices developed SD in an interface-like recording chamber when compared to 19% of slices in a classic recording chamber
When mouse and rat slices were compared in the same preparation, we found that SLEs were observed in slices from both species while SDs were exclusively in mouse slices, at least for SDs and SLEs following 0 Mg2+/5 K+ aCSF exposure
Summary
Increased neuronal excitability is a common feature underlying several neurological disorders, including but not limited to migraine, epilepsy, traumatic brain injury (TBI), and stroke (Eikermann-Haerter et al, 2013; Noseda and Burstein, 2013; Rogawski, 2013; Kim et al, 2014; Bugay et al, 2020). Seizures may be the best-known example that results from increased neuronal excitability, spreading depolarization (SD) is another example. Firing ceases, neurons appear to lose normal intrinsic electrophysiological properties such as input resistance, and there is a loss of the normal ion gradients across the membrane (Czéh et al, 1993; Somjen, 2001; Hartings et al, 2017). The activity of the subset of neurons is almost synchronous so when electrical activity is monitored extracellularly, the initial depolarization is associated with a significant negative potential shift in an extracellular recording [direct current (DC) recording]. At the peak of the depolarization, action potential firing stops and neurons are silent. This period is associated with a suppression in the extracellular recording and is followed by a slow recovery. Glia and ion pumps restore normal ionic gradients (Lian and Stringer, 2004; Dreier, 2011; Varga et al, 2020)
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