AP = 22.0 mm, L = +0.5 mm, DV = 21.5 mm). All electrodes were affixed to the skull using dental cement (Kemdent, Wiltshire, UK). Following surgery, animals were placed in an incubator (Harvard Apparatus, MA, USA) to recover from anaesthesia before being returned to their home cage. Twenty- four hr post-surgery, experimental animals were connected to a digital EEG monitoring apparatus (AS40 amplifier system; Grass Technologies, RI, USA) and a 30 min baseline cortical/ intrahippocampal EEG was recorded before administration of SKF 81297 (5.0 mg/kg) or vehicle. A subgroup of animals were pretreated with CP 55,940 (0.25 mg/kg) or vehicle, 30 min prior to SKF 81297. EEGs were recorded for 1 hr post-injection and subsequently analysed by a trained observer unaware of genotype or treatment for each animal. Electrographic seizures were defined as high frequency, high amplitude spiking (.1 Hz, more than twice baseline amplitude) with corresponding behavioural seizure activity. D1R-Mediated Activation of ERK in the Hippocampus Tissue preparation and immunofluorescence Procedures were as described previously [11,12]. In brief, mice were rapidly anaesthetized by i.p. injection of pentobarbital (500 mg/kg, Sanofi-Aventis, France) prior to intracardiac perfu- sion of 4% (w/v) paraformaldehyde (PFA) in 0.1 M Na2HPO4/ NaH2PO4 buffer, pH 7.5, delivered with a peristaltic pump at 20 ml/min over 5 min. Brains were post-fixed overnight in the same solution and stored at 4uC. Thirty mm-thick sections were cut with a vibratome (Leica, France) and stored at 220uC in a solution containing 30% (v/v) ethylene glycol, 30% (v/v) glycerol and 0.1 M sodium phosphate buffer, until they were processed for immunofluorescence. After permeabilization, free-floating sections were incubated with rabbit polyclonal antibodies for diphospho- ERK (phospho-Thr202/Tyr204-ERK1/2, 1:500; Cell Signaling Technology, Beverly, MA, USA), phospho-Ser10-acetyl-Lys14-H3 (1:500, Millipore, Stockholm, Sweden), phospho-Ser235/236- ribosomal protein S6 (rpS6; 1:500, Cell Signaling Technology, Figure 2. SKF 81297 induces electrophysiological seizures. (A) Representative trace showing hippocampal electrographic seizure events during 60 min following administration of SKF 81297 (5.0 mg/kg, i.p.). Lower panel shows in detail the first and fourth electrographic events. (B) Quantification of seizure incidence and mean seizure duration determined over a 60 min period of observation. Mean 6 SEM (n = 5 animals). Seizure incidence and duration peaked between 30–45 min. Electrographic events between 0–15 min and 45–60 min were observed in only one animal each (data not shown). (C) Representative hippocampal traces over 60 min recording period showing seizures following SKF 81297 administration. Pretreatment with CP 55,940 (0.25 mg/kg, i.p.) abolishes all SKF 81297-induced seizures. doi:10.1371/journal.pone.0019415.g002 PLoS ONE | www.plosone.org 3 May 2011 | Volume 6 | Issue 5 | e19415