doi:10.1016/j.devbrainres.2005.02.001 
Copyright © 2005 Elsevier B.V. All rights reserved.
Research report
Age dependence of pilocarpine-induced status epilepticus and inhibition of CaM kinase II activity in the rat
Michael W. Singletona, William H. Holbert IIb, c, Matthew L. Ryanb, Anh Tuyet Leeb, Jonathan E. Kurzb and Severn B. Churna, b, c, d, 
, 
aDepartment of Anatomy and Neurobiology, Virginia Commonwealth University, Richmond, VA 23298-0599, USA
bDepartment of Neurology, Virginia Commonwealth University, Richmond, VA 23298-0599, USA
cDepartment of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA 23298-0599, USA
dDepartment of Physiology, Virginia Commonwealth University, Richmond, VA 23298-0599, USA
Accepted 2 February 2005.
Available online 19 March 2005.
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Abstract
This study was conducted to characterize the post-pubertal developmental aspects on seizure susceptibility and severity as well as calcium/calmodulin protein kinase type II (CaM kinase II) activity in status epilepticus (SE). Thirty- to ninety-day-old rats, in 10-day increments, were studied. This corresponds to a developmental age group that has not received thorough attention. The pilocarpine model of SE was characterized both behaviorally and electrographically. Seven criteria were analyzed for electrographical characterization: seizure severity, SE susceptibility, the average number of discrete seizures, average time until first seizure, average time to SE, average time from first discrete seizure to SE, and death. After 1 h of SE, specific brain regions were isolated for biochemical study. Phosphate incorporation into a CaM kinase II-specific substrate, autocamtide III, was used to determine kinase activity. There was no developmental effect on the average number of discrete seizures, average time until first seizure, average time to SE, average time from first discrete seizure to SE, and death; however, there was a significant effect on SE probability and seizure severity. Once SE was expressed, all animals showed a decrease in both cortical and hippocampal CaM kinase II activities. Conversely, seizure activity in the absence of SE did not result in a decrease in CaM kinase II activity. The data suggest that there is a gradual age-dependent modulation of SE susceptibility and seizure severity within the developmental stages studied. Additionally, once status epilepticus is observed at any age, there is a corresponding SE-induced inhibition of CaM kinase II.
Keywords: Phosphorylation; Brain Development; EEG; Seizures
Neuroscience classification codes: Disorders of the nervous system; Epilepsy: basic mechanisms
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Fig. 1. Seizure progression in the Pilo–SE rat model. Sixty-day-old rats were electrographically monitored during induction of SE. (A) Typical baseline activity showed high frequency, low amplitude, and asynchronous spike wave activity. (B) After approximately 17.5 ± 1.5 min, discrete seizure activity was observed. Discrete seizures typically began with small increases in amplitude that progressed to high amplitude, low frequency activity. The ictal activity during a discrete seizure abruptly ended and baseline activity was observed. (C) Approximately 8–10 min after the first discrete seizure was observed, ictal activity initiated that progressed into a wax/wane spike frequency. The onset of this event was denoted as the onset of SE. Spike wave activity during SE typically progressed through previously described forms [22]. (D) Following the wax/wane spike frequency, the rats typically entered a fast/slow spike frequency. (E) The next stage observed and described by Handforth and Treiman is early continuous. (F) Later stages of SE include fast spiking with pauses, followed by late continuous (G). For lead placement, seeMaterials and methods.
Fig. 2. Effect of SE on substrate phosphorylation by CaM kinase II activity at various ages in the cortical brain region. Brain homogenates were prepared for basal levels (Mg) and maximally stimulated (Mx) control (Con), SE (Pilo), rats injected with pilocarpine that had discrete seizures but no SE [Pilo(–SE)], and rats that were injected with pilocarpine that did not have any discrete seizures or develop SE [Pilo(NR)], using rats aged (A) P30, (B) P50, (C) P60, (D) P70, (E) P80, and (F) P90. Cortical homogenate showed about a 30% decrease from control to SE, but no significant change between age groups. Additionally, there was a statistically significant decrease in kinase levels in the 50-day-old animals that had discrete seizures, but not SE [Pilo(–SE)]. This inhibition of the kinase was not seen at any other age group studied. Again, there was no statistically significant inhibition of CaM kinase II activity for Pilo(NR) (data not shown) when compared to control values.
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Fig. 3. Effect of SE on substrate phosphorylation by CaM kinase II activity at various ages in the hippocampal brain region. Brain homogenates were prepared for basal levels (Mg) and maximally stimulated (Mx) control (Con), SE (Pilo), rats injected with pilocarpine that had discrete seizures but no SE [Pilo(–SE)], and rats that were injected with pilocarpine that did not have any discrete seizures or develop SE [Pilo(NR)], using rats aged (A) P30, (B) P50, (C) P60, (D) P70, (E) P80, and (F) P90. In all of the age groups studied, there was a statistically significant decrease in CaM kinase II activity due to SE. Hippocampal homogenate showed about a 30% inhibition of CaM kinase II activity from control to SE animals. Similar to the cortical homogenate, there was a slight decrease in kinase levels in [Pilo(–SE)] animals, though this decrease was not statistically significant. No inhibition of CaM kinase II activity was observed for Pilo(NR) rats when compared to control values (data not shown).
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Fig. 4. Status epilepticus did not result in decreased calcium and calmodulin-dependent kinase II levels. Western blot analysis was used to determine if the SE-induced inhibition of CaM kinase II activity was due to proteolytic degradation or loss of enzyme activity. (A) Cortical samples for each age group starting with 30 days old, 50 days old, and going to 90, in 10-day increments and control vs. SE alternating in columns. As the western indicates, there was no significant difference between control and seizure rats, or between age groups. This is confirmed by statistical analysis and the bar graph in (C). There was a slight variation from control to SE animals within some ages, but these differences were not statistically significant, nor were they significantly different when compared to other ages. Hippocampal Western blots (B) were also performed and analyzed (D) using the exact same method. No statistically significant difference was observed in hippocampal Western blots between control and SE within ages or between ages. Similar results were seen for both cortical and hippocampal homogenates. This demonstrated that the SE-induced decrease in activity was not due to the loss of enzyme at any age group studied.
Table 1.
Electrographical (EEG) analysis of seizure activity
Table 2.
Characterizations of stages of SE
Abstract
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