Optimized Hippocampal Slice Recordings

All experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act (1986) and the Hungarian Act of Animal Care and Experimentation (1998, XXVIII, section 243/1998), and with the guidelines of the institutional ethical code. Male Wistar rats (postnatal day 14–20; Harlan UK, Bicester, UK, or Charles River Hungary, Budapest) or CD1 mice (postnatal day 16–18; Charles River, Hungary, Budapest) were deeply anaesthetized with isoflurane and decapitated. Following decapitation, the brain was quickly removed into ice-cold cutting solution. Transverse hippocampal slices 400–450 μm in thickness were prepared using a Leica VT1000S microtome (Leica, Nussloch, Germany), and kept in an interface-type holding chamber at room temperature for at least 60 min before recording in standard or modified ACSF. The standard ACSF was composed of 126 mm NaCl, 2.5 mm KCl, 1.25 mm NaH₂PO₄, 2 mm MgCl₂, 2 mm CaCl₂, 26 mm NaHCO₃, and 10 mm glucose, prepared with ultrapure water and bubbled with 95% O₂/5% CO₂ (carbogen gas), pH 7.2–7.4. All experiments were performed using rat hippocampal slices, except the investigation of the propagation of network activities from CA3 to CA1, which was performed in slices prepared from mice. Recordings were made in either an ‘Oslo’-style interface chamber or in commercially available submerged-type slice chambers (Luigs & Neumann, Ratingen, Germany, and MED64 probes, Alpha MED Sciences, Osaka, Japan). In preliminary experiments, we found that persistent oscillations in these conventional submerged-type slice chambers were only achieved with a flow rate exceeding 10 mL/min, similar to previous observations of hippocampal network activity (Wu et al., 2005 (link)). Adding a dye to the superfusion fluid to visualize the flow, we noticed that the solution tended to flow along the edges of these chambers. The chamber design was therefore modified in either of two ways. First, in order to reduce the volume of the chamber and direct the superfusion fluid over the slice, an inert plastic insert was used (Fig. 1A and B). These plastic inserts were used in all experiments in which the effect of flow rate on generation of network oscillations was investigated. The second modification allowed a double superfusion system to be used (Supertech Ltd, Pecs, Hungary; http://www.super-tech.eu). In this design, the slices were placed on a mesh glued between two plastic rings with a thickness of 2 mm. Two separate fluid inlets allowed ACSF to flow separately above and below the slice (Fig. 1C–F). This second design was only used to study the propagation of network activity from CA3 to CA1.