A detailed description of the methods used can be found in the online
supplementary materials.
Male Wistar-Han rats (n = 20) were implanted with cannulae and electrodes, and a second set of rats (n = 16) were implanted with cannulae. Experimental protocols conformed to NIH guidelines, and were approved by INSERM and by the IACUC of the University of California-Irvine. Continuous video/EEG monitoring was performed.
To induce status epilepticus (SE), kainic acid (KA) was given by intraperitoneal injection once per hour (5 mg/kg), and pilocarpine hydrochloride (310 mg / kg) was injected 30 minutes after a preliminary scopolamine injection (1mg/kg).
To assess molecular changes and
in vitro physiology, rats were infused with ordered or scrambled oligonucleotides (ODNs) on days 1 (10 nmol), and 2 (5nmol) after the SE. Electrophysiological and biochemical studies were performed on the day following the 2
nd infusion. For long-term effects of the ODN
in vivo, repeated infusions alternated full dose (10 nmol/hemisphere) and half dose the following post-SE days: day 1 (full dose), day 2 (half dose), day 3 (half dose), day 6 (full dose), day 8 (half dose), day 10 (full dose). Recordings were discontinued on day 13 post-SE.
Analysis of seizures, interictal activities and theta rhythm were performed as described previously
25 (link).
Organotypic hippocampal slice cultures were prepared as described before
26 (link),27 (link) using P8 rats. Phosphorothioate oligodeoxynucleotides, were added in the culture medium (1 μM) 3 hours after treatment with kainic acid that provoked seizure-like events
28 (link). Cultures were assessed for HCN expression 2-days after KA, and for NRSF at 4h-7d.
Animals used for NRSF, HCN1, HCN2 and Kv4.2 measurements were decapitated and the hippocampi were rapidly dissected. Organotypic slice culture tissues were harvested directly from the membranes. All tissues were processed for Western Blot analyses as described in the
supplementary methods.
Chromatin immunoprecipitation was performed, as described in the
supplementary methods, to detect the physical binding of transcription factors and histones to DNA.
Hippocampal slices were prepared from the dorsal hippocampus and recordings performed as previously described
10 (link).
SPSS software was used for statistical analysis. We used non parametric Mann-Whitney test for samples lower than 6 in size, and t-test otherwise. Results are expressed as mean ± s.e.m, with p<0.05 considered significant.
McClelland S., Flynn C., Dubé C., Richichi C., Zha Q., Ghestem A., Esclapez M., Bernard C, & Baram T.Z. (2011). Neuron-restrictive silencer factor-mediated cyclic nucleotide gated channelopathy in experimental temporal lobe epilepsy. Annals of neurology, 70(3), 454-465.
