Tissue collection and preparation
Rodents were anaesthetised by an intraperitoneal injection of 60 mg/kg
phenobarbital and perfused transcardially with artificial cerebrospinal
fluid (ACSF) containing sucrose. The level of anaesthesia was monitored
using pedal and tail pinch reflexes, rate, depth and pattern of
respiration through observation and colour of mucous membranes and skin.
The ACSF comprised of (in mM): 248 sucrose, 3.3 KCl, 1.4
NaH2PO4, 2.5 CaCl2, 1.2
MgCl2, 25.5 NaHCO3, and 15 glucose,
which was bubbled with 95% O2 and 5%
CO2. The animals were then decapitated and the brain
removed and coronal sections hippocampus containing the neocortex
~ 300 µm thick – were cut in ice-cold standard ACSF
using an automated vibratome (Leica, Germany). This standard ACSF
contained (in mM): 121 NaCl, 2.5 KCl, 1.3
NaH2PO4, 2 CaCl2, 1
MgCl2, 20 glucose and 26 NaHCO3,
equilibrated with 95% O2 and 5% CO2.
Slices were incubated in ACSF for one hour at room temperature (20–23
°C) prior to recording. Brain slices were placed in a submerged chamber
and superfused with ACSF at a rate of 1–2 ml min‑1for electrophysiological recordings. For neuroanatomical studies, brains
were immediately fixed after perfusion in 4% paraformaldehyde plus
0.2% picric acid in 0.1M phosphate buffer (PB) for 24 hours prior to
sectioning.