Testing neocortical slice viability in non-perfused no-magnesium artificial cerebrospinal fluid solutions

The acute in vitro brain slice model is a widely used neurophysiological research tool. When applying this method, most researchers continuously perfuse slices with carbogenated artificial cerebrospinal fluid (ACSF) to maintain pH balance and tissue oxygen delivery. Common wisdom suggests that static recordings are incompatible with submerged bath methodology because of deficiency in tissue oxygen supply. However, to our knowledge this has not been tested. In this study, we wanted to determine whether neocortical mouse slice viability could be maintained in the medium term (up to 2h) in a shallow, submerged recording bath under non-perfused, static conditions. Seizure-like events (SLEs) were generated in the slices utilizing no-magnesium ACSF and recorded for 2h under three conditions: (1) perfused ACSF condition (n=8), where slices were perfused continuously with carbogenated no-magnesium ACSF; (2) static ACSF condition (n=12), where slices were recorded in pre-carbogenated, but non-perfused (static) no-magnesium ACSF; and (3) static HEPES ACSF condition (n=12), where slices were recorded in non-perfused (static) no-magnesium ACSF with no pre-carbogenation but buffered with HEPES. SLE activity was stable for 2h across all three conditions. There was no statistically significant difference in SLE frequency, amplitude or length between static and perfused conditions. SLE frequency and amplitude were generally lower in the static HEPES buffer condition. The data indicate that robust and stable neocortical SLE activity can be generated for at least 2h in a submersion bath without ACSF perfusion if pH is adequately controlled.