In summary, experiments were performed in a submerged recording chamber using horizontal hippocampal slices (thickness, 400?m) obtained from Wistar rats 7 to 9?weeks old

In summary, experiments were performed in a submerged recording chamber using horizontal hippocampal slices (thickness, 400?m) obtained from Wistar rats 7 to 9?weeks old. unexpectedly found that homozygous GHSR deletion rendered the mice less seizure susceptible than their wild-type littermates. Using multiple approaches, we found that various treatments that inactivate the GHSR lead to the attenuation of limbic seizures. This highlights the GHSR as a potential site for the development of novel anticonvulsant drugs. Methods Animals Experimental procedures were performed on male Wistar rats (260-320?g; Charles River Laboratories, France), and male GHSR+/+ and GHSR-/- littermate mice (25-35?g). All experiments were carried out in accordance with the National Rules on Animal Experiments and were approved by the Ethics Committee on Animal Experiments of the Vrije Universiteit Brussel, Belgium, and the animal Scientific Procedures Act, 1986, United Kingdom. Generation of GHSR-/- Mice GHSR-/- mice were developed by Janssen Pharmaceutica (Beerse, Belgium) in collaboration with Lexicon Genetics, Inc (The Woodlands, TX) on a C57BL/6 background, as previously described [26]. Homozygous male GHSR-/- mice Has2 and their corresponding wild-type GHSR+/+ littermates (3-5?months old) were used in this study. Genotypes were confirmed by real-time reverse transcription polymerase chain reaction using DNA isolated from mouse tail biopsy samples. Real-time reverse transcription polymerase chain reaction analysis was used to show expression or absence of the GHSR transcript. Primers sequences and additional information on DMNQ the targeted disruption of the GHSR gene can be found in the study by Verhulst et al. [26]. Mice were kept in a regulated environment (22-24? C; 50?% relative humidity; DMNQ lights on at 7:00?am and off at 9:00?pm) with free access to food (standard laboratory chow) and water (filtered). All mice were held in the animal facility for at least 1?month before being transferred to the experimentation rooms. GHSR Ligands, Chemicals, and Reagents Neuroactive substance standards, pilocarpine, and the GHSR inverse agonist [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P were supplied by Sigma-Aldrich (Bornem, Belgium). Ghrelin was supplied by PolyPeptide Laboratories (Strasbourg, France). The ghrelin mimetic capromorelin and the presumed GHSR antagonist A778193 (found in this study to be a GHSR inverse agonist) were generously provided by Dr. Luc Ver Donck and Dr. Dieder Moechars. The ghrelin fragments (1-5) amide, (1-7), (1-9), (1-11), (1-14), and (23-28) were supplied by Phoenix Pharmaceuticals (Karlsruhe, Germany). All other chemicals were analytical reagent grade or better and were supplied by Merck (Darmstadt, Germany). Aqueous solutions were made with purified water (Seralpur pro 90 CN; Belgolabo, Overijse, Belgium) and filtered through a 0.2-m membrane filter. Microdialysis in Rats Surgery Microdialysis in rat hippocampus was performed as previously described [27]. Rats received intraperitoneal injections of a mixture of ketamine and diazepam (start dose of 90.5:4.5?mg/kg) until full-body anesthesia was achieved. The animal was then mounted on a stereotaxic frame for precise intracranial steel guide cannula (CMA/Microdialysis, Solna, Sweden) implantation 3?mm above the actual membrane position in the left CA1-CA3 hippocampal DMNQ area, using the coordinates 4.6?mm lateral from the midline, 5.6?mm posterior to the bregma, and 4.6?mm ventral from the dura [27]. Ketoprofen (4?mg/kg) was administered intraperitoneally for postoperative analgesia at the end of the surgical procedure. At least 2 rats from each control and GHSR inverse agonist-treated rat groups were implanted with a sterilized radiotelemetric transmitter (F20-EET; Data Sciences International, Tilburg, The Netherlands). The measuring and reference DMNQ electrodes, both attached with a stainless steel screw at the end, were subcutaneously tunneled to the skull. The measuring electrode was stereotaxically positioned above the right hippocampus (4.6?mm lateral and 5.6?mm anterior to the bregma), whereas the reference electrode was positioned above the cerebellum (1?mm anterior according to the lambda). Microdialysis Immediately after surgery, the hippocampal guide cannula obturator was replaced by a microdialysis probe (CMA/12, 3 mm membrane length, theoretical cut-off 20?kDa; CMA/Microdialysis), which was continuously perfused with modified Ringers solution (in mM:.