Kainic Acid

The experiments were largely done on material from a previously published study [8 (link)]. A set of 6 week-old 24 female C57BL/6 mice (19–24 g; Charles River) were divided into 6 groups and perfused 4 hours (N = 5), 1 day (N = 5), 3 days (N = 5), 1 week (N = 5), 2.5 weeks (N = 4) and 4 weeks (N = 5) after a single intraperitoneal BrdU-injection (5-Bromo-2-deoxyuridine, 50 mg/kg BrdU in sterile 0,9% NaCl; Sigma).
Additional 18 female C57BL/6 mice (same age as above) were divided into 3 groups: seizure (N = 6), running (N = 6) and controls (N = 6). Seizure animals received a single intraperitoneal application of 30 mg/kg kainic acid (KA, Sigma) in 0.1 M phosphate buffered saline (PBS) on the day before BrdU-injections (Day 0), and only those displaying continuous convulsive seizure activity were used in these experiments. The "Runner" group was housed with 2–3 animals per cage that was equipped with a running wheel. During the first 7 days of the experiment all animals received one daily injection of BrdU. Tissue from this experiment will be used in an unrelated study. Data on dendritic morphology, etc., were exclusively generated for the present study.
To analyze the spatial relationship between DCX-positive cells and astrocytes we used 3 female transgenic mice expressing enhanced green fluorescent protein EGFP under the promoter for glial fibrillary acidic protein (GFAP). The animals were kindly provided by Helmut Kettenmann, Berlin [37 (link)] and were 7 weeks of age. For the detection of apoptosis a total of 37 female C57BL/6J mice (Charles River, Sulzfeld, Germany), 8 weeks of age, were used.
For the water maze experiment, 21 female Nestin-GFP reporter mice [69 (link)] (C57BL/6, 10 weeks of age at the beginning of the experiment) were used and randomly assigned to one of the following experimental groups: Morris water maze hidden version, Morris water maze cued version, standard laboratory conditions. On the 3 days before the experiment each animal received single injection of BrdU. Animals were subjected to water maze training at days 5 to 8 of the experiment (days 5 to 8 after the last BrdU injection). We followed the protocol devised by Wolfer and Lipp [70 (link)]. Six trials of training each maximally lasting for 2 minutes were given each day. Tissue from that experiment that has been published elsewhere [44 (link)] was analyzed for the present study. Again, the data on dendritic morphology, etc., were exclusively generated for the present study.

Seizure-induced immediate-early gene, Arc, expression has been used for labeling adult-born, circuit-incorporated neurons, primarily due to synapse and glutamate receptor formation [68 (link)]. An intraperitoneal (i.p.) kainic acid injection (10 mg/kg; Sigma, St. Louis, MO, USA. Cat. No. K0250) was given to induce mice’s tonic and clonic seizures [69 (link)]. Such kainic acid treatment may strongly stimulate dorsal DG BrdU-labeled neurons via epileptic activation onto their newly-formed synapses with other extant ones [26 (link)]. Approximately 25–35 min after the kainic acid injection, mice exhibited initial seizure activity by exhibiting head nodding, followed by forelimb clonus, and progressively to a seizure stage, characterized by repeated rearing and falling episodes. An i.p. sodium pentobarbital (50 mg/kg, SCI Pharmtech, Inc., Taoyuan, Taiwan, Cat. No. 051100) was given 30 min after the onset of the stage 5 seizure [70 (link)] to prevent further seizures. Animals were then perfused 60 min after the injection. Their brains were removed and postfixed in a 4% paraformaldehyde solution overnight at 4 °C and subsequently cryoprotected in a 30% sucrose solution for 48 h at 4 °C. Coronal sections, at 20 μm thickness, were made using a microtome (Thermo Fisher Scientific, Cleveland, OH, USA, Model: CryoStar NX50 OP). Triple staining for BrdU, NeuN, and Arc was conducted using mouse anti-BrdU (1:500, Millipore, Temecula, CA, USA, Cat. No. B5002), rabbit anti-Arc (1:1000, Synaptic Systems, Goettingen, Germany, Cat. No. 156 003), and chicken anti-NeuN (1:100, Millipore, Temecula, CA, USA, Cat. No. ABN91) primary antibodies (Supplementary Table S1) in blocking buffer, and incubated with Alexa Fluor 488-conjugated sheep anti-mouse (1:500, Jackson ImmunoResearch, West Grove, PA, USA, Cat. No. 515-545-062), Alexa Fluor 594-conjugated goat anti-rabbit (1:1000, Jackson ImmunoResearch, West Grove, PA, USA, Cat. No. 111-585-045), and CF350 conjugated donkey anti-chicken (1:100, Sigma, St. Louis, MO, USA, Cat. No. SAB4600219) secondary antibodies (Supplementary Table S1) in PBS and imaged with an Olympus fluorescent microscope (Olympus, Tokyo, Japan, Model: IX71).

Seizure-induction protocols using kainic acid (KA) were followed as described (Rojas et al., 2014 (link)). Wild type (WT) and RGS14 KO mice were individually housed in clean mouse cages without access to food and water 30 minutes prior to KA injection. KA (3 mg/mL) was prepared on the day of experimentation by dissolving KA (Tocris, 0222) in a 0.9% bacteriostatic saline solution. Mice were injected intraperitoneally (i.p.) with a single, 30 mg/kg dose of KA or 0.3 mL of saline (control). Immediately after injection, mice were monitored and scored for seizure behavior using a modified Racine scale (Racine, 1972 (link); Rojas et al., 2014 (link)). Each mouse was given a behavioral seizure score from a scale of 0–6 every 5 minutes for 90 minutes. Mice were scored based off the most severe seizure behavior that was observed during the 5 minute interval. When an mouse reached mortality, they were given a score of 7, which was not included in the mean behavioral seizure score and no further scores were given for those mice. Ninety minutes after injection, all mice were injected intraperitoneally with 10 mg/kg diazepam to terminate seizure activity. After seizure termination, mice remained singly housed, and food (dry and moistened) and water were returned to the cage, and monitored for well-being.