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NEUROPHARMACOLOGY

Altered Calcium/Calmodulin Kinase II Activity Changes Calcium Homeostasis That Underlies Epileptiform Activity in Hippocampal Neurons in Culture

Department of Anatomy and Neurobiology (D.S.C., S.N.H.), Department of Pharmacology and Toxicology (R.J.D.), Department of Neurology (R.E.B., S.S., L.S.D., R.J.D.), and Department of Biochemistry and Molecular Biophysics (R.J.D.), Virginia Commonwealth University, Richmond, Virginia

Received July 5, 2006; accepted September 11, 2006.

	Abstract

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Epilepsy is characterized by the occurrence of spontaneous recurrent epileptiform discharges (SREDs) in neurons. A decrease in calcium/calmodulin-dependent protein kinase II (CaMK-II) activity has been shown to occur with the development of SREDs in a hippocampal neuronal culture model of acquired epilepsy, and altered calcium (Ca²⁺) homeostasis has been implicated in the development of SREDs. Using antisense oligonucleotides, this study was conducted to determine whether selective suppression of CaMK-II activity, with subsequent induction of SREDs, was associated with altered Ca²⁺ homeostasis in hippocampal neurons in culture. Antisense knockdown resulted in the development of SREDs and a decrease in both immunocytochemical staining and enzyme activity of CaMK-II. Evaluation of [Ca²⁺]_i using Fura indicators revealed that antisense-treated neurons manifested increased basal [Ca²⁺]_i, whereas missense-treated neurons showed no change in basal [Ca²⁺]_i. Antisense suppression of CaMK-II was also associated with an inability of neurons to restore a Ca²⁺ load. Upon removal of oligonucleotide treatment, CaMK-II suppression and Ca²⁺ homeostasis recovered to control levels and SREDs were abolished. To our knowledge, the results demonstrate the first evidence that selective suppression of CaMK-II activity results in alterations in Ca²⁺ homeostasis and the development of SREDs in hippocampal neurons and suggest that CaMK-II suppression may be causing epileptogenesis by altering Ca²⁺ homeostatic mechanisms.

It has been suggested that chronic elevations in intracellular Ca²⁺ ([Ca²⁺]_i) and alterations in Ca²⁺ homeostatic mechanisms in epileptic neurons play a role in the induction and maintenance of the epileptic phenotype (DeLorenzo et al., 1998, 2005; Raza et al., 2001, 2004). Calcium is an important second messenger involved in diverse cellular events, such as membrane excitability and synaptic activity (Delorenzo et al., 2005). Elevated [Ca²⁺]_i can lead to excitotoxicity, which precedes epileptogenesis and epilepsy (DeLorenzo et al., 1998, 2005; Raza et al., 2004; Sun et al., 2004). In normal cells, [Ca²⁺]_i is regulated by several Ca²⁺ homeostatic mechanisms (Carafoli et al., 1997). Studies in epileptic neurons have shown that disruption of Ca²⁺ extrusion and sequestration mechanisms can lead to elevated basal [Ca²⁺]_i with a decreased ability to restore resting [Ca²⁺]_i levels following an external Ca²⁺ load (Pal et al., 2001; Raza et al., 2001; Sun et al., 2004). Although many studies have linked increases in [Ca²⁺]_i and altered Ca²⁺ homeostatic mechanisms to epilepsy (Delorenzo et al., 2005), it remains unclear what molecular events during epileptogenesis cause these changes in Ca²⁺ homeostasis.

Calcium-calmodulin-dependent protein kinase II (CaMK-II) plays a major role in modulating neuronal excitability and function (Kelly et al., 1984), with alterations in CaMK-II levels linked to neuronal hyperexcitability. Decreases in CaM kinase II have been reported in numerous in vivo and in vitro models of epilepsy (Bronstein et al., 1993), including kindling (Wasterlain and Farber, 1984; Taft et al., 1987), electrical stimulation SE (Perlin et al., 1992), pilocarpine (Churn et al., 2000a), and low Mg²⁺ in cultured neurons (Blair et al., 1999). Furthermore, CaMK-II knockout mice demonstrated the epileptic phenotype and developed spontaneous seizures (Butler et al., 1995). It has been demonstrated that knocking down CaMK-II activity in cultured hippocampal neurons with an antisense oligonucleotide resulted in epileptiform activity as evidenced by the presence of SREDs (Churn et al., 2000b). It was also shown in cultured hippocampal neurons that CaMK-II activity was decreased in association with the development of SREDs (Blair et al., 1999). These studies implicate a role for altered CaMK-II function toward the induction and maintenance of the epileptic phenotype and suggest that alterations in CaMK-II activity may play a role in the induction of epileptogenesis by altering Ca²⁺ homeostatic mechanisms.

This study was initiated to evaluate whether the decrease in CaMK-II activity during epileptogenesis could represent a molecular mechanism that underlies alterations in Ca²⁺ homeostasis observed in the epileptic phenotype. Antisense oligonucleotides were used to experimentally suppress CaMK-II function in hippocampal neurons in culture. Following knockdown of CaMK-II, electrophysiological studies were initiated to determine the presence or absence of SREDs, and Ca²⁺-imaging studies were employed using the fluorescent calcium indicators Fura-2 and Fura-FF to evaluate [Ca²⁺]_i dynamics. Similar experiments were performed on neurons that were allowed to recover from antisense knockdown of CaMK-II. Antisense oligonucleotide suppression of -CaMK-II in neurons resulted in the development of SREDs, higher basal [Ca²⁺]_i, and a decreased ability to restore resting [Ca²⁺]_i after a Ca²⁺ load compared with control and antisense-recovered neurons. The results provide the first direct evidence that CaMK-II activity is involved in maintaining Ca²⁺ homeostasis in neurons and that the alterations in Ca²⁺ homeostasis resulting from suppression of CaMK-II may underlie the development of SREDs. Because of the pathophysiological similarities to the glutamate injury and low Mg²⁺ in vitro models of AE, knocking down CaMK-II levels in cultured hippocampal neurons provide a novel model for studying epileptiform activity.

	Materials and Methods

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Hippocampal Neuronal Culture. Primary hippocampal cultures were prepared as described previously by our laboratory (Sombati and Delorenzo, 1995). In brief, hippocampal cells were isolated from 2-day postnatal Sprague-Dawley rats (Harlan, Indianapolis, IN) and plated at a density of 2.4 x 10⁴ cells/cm² onto glial support layers previously plated onto poly-L-lysine (0.05 mg/ml)-coated Lab-Tek two-well cover glass chambers (Nalge-Nunc International, Naperville, IL). Cultures were maintained at 37°C in a 5% CO₂/95% O₂ air atmosphere and fed twice weekly with Neurabasal A medium (Invitrogen, Carlsbad, CA) enriched with B-27 serum-free supplement (Invitrogen) and 0.5 mM L-glutamine. Cultures were exposed to 5 µM cytosine arabinoside 2 days after plating to inhibit non-neuronal growth. Experimental studies and [Ca²⁺]_i measurements were performed on neurons maintained for 15 to 18 days in vitro (DIV) to ensure adequate neuronal development.

CaMK-II Antisense Knockdown Analysis. Antisense and missense oligonucleotide probes (Operon Biotechnologies, Huntsville, AL) were constructed as described previously (Churn et al., 2000b). For the -CaMK-II knockdown, an antisense oligonucleotide complimentary to the +1 to +18 nucleotides was constructed with the sequence 5'-GGT AGC CAT CCT GGC ACT-3'; the missense sequence for -CaMK-II was 5'-GGT AGC CAT AAG GGC ACT-3'. Knockdown treatment for CaMK-II involved treating hippocampal cultures for 3 days with a 3.0 µM concentration of either antisense or missense oligonucleotides every 24 h, as determined by measuring CaMK-II activity (Churn et al., 2000b). Oligonucleotide treatment was initiated at 13 DIV to allow for analysis to occur by 17 DIV. Untreated neurons were used as controls. During the treatment regimen, hippocampal cultures were maintained in Neurobasal A medium supplemented with B-27. At the end of the 3-day treatment protocol, cultures were utilized for Fura-2 [5-oxazolecarboxylic acid, 2-(6-(bis(2-((acetyloxy)methoxy)-2-oxoethyl)amino)-5-(2-(bis(2-((acetoxy(methoxy)-2-oxoethyl)amino-5-methylphenoxy)ethoxy)-2-benzofuanyl)-,(acetoxy)methyl ester] and Fura-FF [Ca²⁺]_i measurements. To assess the recovery and restoration of CaMK-II activity, cultures were treated with antisense or missense oligonucleotide every 24 h for 3 days. After the 3rd day of treatment, oligonucleotide treatments were discontinued and the neurons were allowed to recover in oligonucleotide-free medium. After full recovery of CaMK-II activity, cultures were utilized for [Ca²⁺]_i measurements.

Measurement of CaMK-II Activity. CaMK-II activity was determined measuring CaMK-II-dependent substrate phosphorylation of the synthetic peptide Syntide II (Sigma, St. Louis, MO) following the methods of Churn (Churn, 1995). Phosphorylation reaction solutions contained 41 µg of protein, 60 µM Syntide II, 10 mM MgCl₂, 7 µM [-³²P]ATP, 10 mM PIPES, pH 7.4, ± 5 µM CaCl₂, and ± 1 µg of calmodulin. Standard reactions were performed in a shaking water bath at 30°C. The phosphorylation reactions were initiated by adding Ca²⁺, continued for 1 min, and stopped by adding 20 µM EDTA. Ten-microliter aliquots of assay solution were blotted onto P-81 phosphocellulose filter paper (Whatman Inc., Florham Park, NJ). Each reaction was quantitated in triplicate. The filter paper was then washed three times in 50 mM phosphoric acid, rinsed with acetone, and allowed to air-dry. Radioactive phosphate was then quantitated by scintillation counting (Churn, 1995).

Immunocytochemical Staining of -CaMK-II. After briefly washing in phosphate-buffered saline (PBS), neuronal cultures were fixed in 4% paraformaldehyde in PBS for 10 min followed by a 3 x 5-min wash in PBS. Fixed cultures were blocked and permeabilized in SuperBlock blocking buffer (Pierce, Rockford, IL) containing 0.2% Triton X-100 for 60 min at room temperature. Cells were then incubated with a mouse monoclonal antibody to the CaMK-II -subunit (10 µg/ml, clone 6G9; Biomol, Plymouth Meeting, PA) in SuperBlock blocking buffer containing 0.1% Triton X-100 overnight at 4°C. Cells were washed 4 x 5 min in PBS containing 0.1% Triton X-100. After the wash, cells were incubated with Texas Red (Vector Laboratories, Burlingame, CA) conjugated anti-mouse IgG (20 µg/ml) in Super-Block blocking buffer for 60 min at room temperature. Cells were washed 4 x 5 min in PBS containing 0.1% Triton X-100 followed by one wash in PBS alone. Labeled cells were coated with Vectashield (Vector Laboratories) and coverslipped. Control staining was carried out in an identical manner, with the exception of removal of the primary antibody. Fluorescence microscopy was carried out on an Olympus inverted microscope (Olympus America) fitted with a 20x objective and a Texas Red filter cube allowing for excitation/emission of 595/615 nm. Fluorescent images were captured with a Q-fire digital camera and evaluated with Olympus MicroSuite imaging software (Soft Imaging System, Lakewood, CO).

Whole-Cell Current-Clamp Analysis of Hippocampal Neuronal Cultures. Electrophysiological analysis was performed using previously established procedures in our laboratory (Sombati and Delorenzo, 1995). In brief, cell culture media were replaced with physiological bath recording solution (pBRS) containing 145 mM NaCl, 2.5 mM KCl, 10 mM HEPES, 10 mM glucose, 2 mM CaCl₂, 1 mM MgCl₂, and 2 µM glycine, pH 7.3; osmolarity was adjusted to 325 mOsm with sucrose. Cell culture plates were mounted on the stage of an inverted microscope (Nikon Diaphot, Japan), continuously perfused with pBRS, and then studied using the whole-cell currentclamp recording procedure. Patch electrodes with a resistance of 2 to 4 M were pulled on a Brown-Flaming P-80C electrode puller (Sutter Instruments, Novato, CA) and then fire-polished. For whole-cell current-clamp analysis, the electrode was filled with a solution containing 140 mM K⁺ gluconate, 1 mM MgCl₂, and 10 mM Na-HEPES, pH 7.2; osmolarity was adjusted to 310 ± 5 mOsm with sucrose. Data were digitized and transferred to videotape using a pulse code modulator device (Neurodata, New York, NY) and then played back on a DC-500 Hz chart recorder (Astro-Med Dash II, Warwick, RI). Intracellular recordings were carried out using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA) in whole-cell current-clamp mode.

Intracellular Free Calcium Measurements. Intracellular free Ca²⁺ measurements were carried out using previously established procedures (Pal et al., 1999, 2001; Raza et al., 2001; Sun et al., 2004). Hippocampal neurons were loaded with acetoxymethyl form of the membrane-permeable ratiometric fluorescent Ca²⁺ indicators Fura-2 or Fura-FF (1 µM; Invitrogen) in pBRS for 30 min at 37°C. Loaded cells were washed three times with pBRS and incubated for an additional 15 min to allow for cellular esterase cleavage of the acetoxymethyl moiety and intracellular trapping of the free acid Fura-2 or Fura-FF indicators. Culture dishes were mounted onto a heated stage at 37°C on an Olympus IX-70 inverted microscope fitted with a 20x fluorite water immersion objective and coupled to an ultra high-speed fluorescence imaging system (Olympus America/PerkinElmer Life and Analytical Sciences, Boston, MA). The fluorescence excitation source was a 75-W Xenon arc lamp (Olympus America). Neutral density filters of variable opacities were used to attenuate unwanted excitation. Alternating excitation wavelengths of 340 and 380 nm were generated using a Lambda 10-2 filter wheel (Sutter Instruments Co., Novato CA), and 510-nm emissions were acquired using a Fura filter cube (Olympus America) with a dichroic at 400 nm. Alternating emissions from 340- and 380-nm excitation were captured with an ORCA-ER high-speed digital CCD camera (Hamamatsu Photonics K.K., Hamamatsu City, Japan). Image acquisition and processing were computer-controlled using the Ultra-VIEW Imaging system software version 5.2 (PerkinElmer Elmer Life and Analytical Sciences). Using the image analysis software, a region of interest was selected for each pyramidal-shaped neuron in the field, and intracellular free Ca²⁺ levels were presented as an absolute ratio value of 340/380-nm excitation-emissions. To calculate 340/380 ratio values, image pairs at each wavelength were captured and digitized at varying intervals, and the images at each wave-length were averaged over four frames.

Background autofluorescence values for both 340- and 380-nm excitations were obtained by imaging a field of neurons lacking the fluorescent indicator and were subtracted from experimental 340/380-nm excitation-emissions values from indicator-loaded cells. Absolute ratio values of 340/380-nm excitation-emissions of intracellular free Ca²⁺ levels were used in this study as a quantitative measure of [Ca²⁺]_i levels, employing established techniques (Pal et al., 1999, 2000; Raza et al., 2001; Sun et al., 2004).

Experimental studies were carried out on oligonucleotide-treated hippocampal cultures to evaluate [Ca²⁺]_i dynamics. In some experiments, recording solution during Ca²⁺ imaging contained 400 nM tetrodotoxin (TTX; Sigma) to block synaptic transmission to allow for measurement of isolated [Ca²⁺]_i dynamics in the absence of seizure and synaptic activity. Multiple neuronal culture fields were evaluated to determine basal [Ca²⁺]_i measurements, each for 60 s with image/ratio values acquired at 5-s intervals. For evaluation of the glutamate-induced Ca²⁺ loads, basal [Ca²⁺]_i levels were recorded for 3 min at 30-s intervals before glutamate treatment. The recording solution was replaced with 1 ml of pBRS containing glutamate (50 µM; Sigma), and glycine (10 µM) was added to the cells. After 2 min of exposure, the glutamate was removed, and the cells were washed twice in pBRS. The cells were monitored for up to 30-min post-glutamate treatment at 30-s intervals.

Calcium Calibration. The [Ca²⁺]_i values in the paper are presented as 340/380 ratio values for Fura-2 and Fura-FF, because absolute [Ca²⁺]_i concentrations can vary depending on the fluorescent indicator used (Hyrc et al., 1997; Pal et al., 2000; Raza et al., 2001). It has been reported that ratio values provide more objective presentation of [Ca²⁺]_i (Pal et al., 2000; Raza et al., 2001). However, we have performed Ca²⁺ calibration determinations on both the Fura-2 and Fura-FF data to provide estimates of absolute [Ca²⁺]_i concentrations from the 340/380 ratio values using established procedures (Pal et al., 2001). An in vitro calcium calibration curve was constructed for each indicator and used to convert fluorescent ratios to [Ca²⁺]_i concentrations using ionomycin (10 µM) and prepared calibration buffers (Invitrogen, Carlsbad, CA). [Ca²⁺]_i concentrations were calculated from the background corrected 340/380 ratios using the following equation (Grynkiewicz et al., 1985):

(1)

where R is the 340/380 ratio at any time; R_max is the maximum measured ratio in saturating Ca²⁺ solution (39 µM free Ca²⁺); R_min is the minimal measured ratio Ca²⁺ free solution; S_f2 is the absolute value of the corrected 380-nm signal at R_min; S_b2 is the absolute value of the corrected 380-nm signal at R_max; and the K_d values used were 224 nM and 20 µM for Fura-2 and Fura-FF, respectively (Pal et al., 2001; Sun et al., 2004).

For basal [Ca²⁺]_i experiments, Fura-2 was used as the indicator, because Fura-FF is not sensitive in the low nanomolar range (Hyrc et al., 1997). For the experiments evaluating Ca²⁺ homeostasis, both Fura-2 and Fura-FF were used because of the large change in [Ca²⁺]_i produced by the glutamate exposure. Basal [Ca²⁺]_i concentrations for control, missense-treated, antisense-treated, and antisense-recovered neurons were determined from the ratio values and were 107.1 ± 3.4, 95.1 ± 3.4, 138.4 ± 7.9, and 110.3 ± 4.6 nM, respectively. For the calcium homeostasis experiments, peak glutamateinduced [Ca²⁺]_i values in missense-treated and antisense-treated neurons using Fura-2 were calculated to be 1.99 ± 0.16 and 1.88 ± 0.36 µM, respectively. Peak glutamate-induced [Ca²⁺]_i concentrations in missense-treated and antisense-treated neurons employing Fura-FF were determined to be 29.3 ± 1.4 and 33.8 ± 2.7 µM. The values obtained in this study for peak glutamate-induced [Ca²⁺]_i values using Fura-2 and Fura-FF were essentially identical to values obtained previously in both in vitro and in vivo epileptic neurons (Pal et al., 2000; Raza et al., 2001). Although the peak [Ca²⁺]_i concentration varied between Fura-2 and Fura-FF, as previously observed (Pal et al., 2000), the ability of the indicators to reflect the neurons' Ca²⁺-buffering capability was similar. Although Fura-2 gave a lower absolute calcium concentration upon calibration, the shapes of its decay curves were almost identical to those of Fura-FF.

Data Analysis. [Ca²⁺]_i data were collected using the UltraVIEW Imaging System and statistically analyzed and plotted using SigmaPlot (version 8.0) software. Experiments were repeated at least three times from different batches of neuronal cultures. The significance of the data were tested by Student's t test, one-way ANOVA, or one-way repeated measures (RM) ANOVA where applicable The Tukey's test was used as the post hoc analysis for multiple comparisons. Statistical analysis was performed using SigmaStat 2.0 (Jandel Corp., San Rafael, CA). P < 0.05 was considered statistically significant for all data analysis. To compare the kinetics of the [Ca²⁺]_i decay curves, ratio values from individual neurons obtained after glutamate exposure were normalized to "Percent of [Ca²⁺]_i Load" using the following equation:

(2)

where Ratio_{t = x} is the 340/380 ratio at any time following the glutamate exposure; Ratio_{t = 0} is the 340/380 ratio at the peak [Ca²⁺]_i load; and Ratio_Basal is the average 340/380 ratio before glutamate exposure.

	Results

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Suppression of CaMK-II Activity and Protein Expression in Hippocampal Neurons in Culture. These studies were initiated to evaluate whether the decreased activity and expression of CaMK-II caused by antisense oligonucleotide suppression specific for the subunit of CaMK-II were reversible. Hippocampal neurons in culture were exposed to either missense or antisense oligonucleotides specific for the subunit of CaMK-II. CaMK-II-dependent substrate phosphorylation of the synthetic peptide Syntide II was carried out to evaluate the activity of CaMK-II following oligonucleotide treatment (Fig. 1A). Immunocytochemical staining was performed to determine the localization of the subunit of CaMK-II in cultures treated with missense or antisense oligonucleotides (Fig. 1B). Antisense oligonucleotide treatment significantly decreased CaMK-II activity and protein expression in comparison to control and missense-treated neurons (Fig. 1, A and B). Removal of the antisense oligonucleotide treatment followed by a 3-day recovery period resulted in a restoration of CaMK-II activity and protein expression to missense control levels (Fig. 1, A and B, bottom), demonstrating that the antisense oligonucleotide suppression of CaMK-II was reversible.

View larger version (17K): [in this window] [in a new window]   Fig. 1. Antisense oligonucleotide knockdown of CaMK-II causes decrease in both expression and activity of CaMK-II. Hippocampal neurons in culture were treated with CaMK-II missense and antisense oligonucleotides for 3 days (missense and antisense). To evaluate the recovery from the antisense oligonucleotide, oligonucleotides were removed from the culture media, and neurons were allowed to recover for 3 days (antisense recovery). A, following these treatment paradigms, cells were harvested from the culture plates, and CaMK-II-dependent phosphorylation of exogenously added Syntide II was determined (see Materials and Methods). CaMK-II activity was significantly decreased in the antisense-treated cultures but not in the missense-treated or antisense recovery conditions. The data represent the means ± S.E.M. and are expressed as percentage of control (untreated cultures) CaMK-II activity. (*, P < 0.01, Student's t test, n = 5). B, immunocytochemistry was performed with a monoclonal antibody directed against the subunit of CaMK-II. Exposure of the neurons to antisense oligonucleotides resulted in a substantial decrease in CaMK-II immunocytochemical staining. After 3 days of recovery from antisense oligonucleotide, immunocytochemical staining of CaMK-II returned to control levels.

View larger version (35K): [in this window] [in a new window]   Fig. 2. Induction of SREDs following antisense oligonucleotide knockdown of -CaMK-II. Whole-cell current-clamp recordings were obtained from neurons undergoing four different treatment regimens in culture. Representative recordings from control (A) and missense-treated (B) neurons displayed nonsynchronous background neuronal activity characterized by intermittent action potentials. C, neurons treated with antisense oligonucleotides displayed SREDs. A continuous 30-min recording from this neuron revealed the presence of characteristic SREDs. The bottom panel recording (a) corresponds to an expansion of the first SRED from C above and demonstrates the presence of individual paroxysmal depolarization shifts that comprise a high-frequency discharge activity of a single SRED. D, neurons that recovered from antisense oligonucleotide treatment demonstrated absence of SREDs and displayed a return to control conditions with nonsynchronous back-ground neuronal activity.

Suppression of CaMK-II Activity Results in Elevation of Basal [Ca²⁺]_i. Since alteration of [Ca²⁺]_i has been implicated in epileptogenesis (Delorenzo et al., 2005), we wanted to investigate whether the suppression of CaMK-II, with subsequent induction of SREDs, affected neuronal [Ca²⁺]_i. To evaluate the effects CaMK-II knockdown on [Ca²⁺]_i in neurons, [Ca²⁺]_i imaging was conducted on control, missense-treated, antisense-treated, and antisense-recovered neurons using the fluorescent Ca²⁺ indicator Fura-2. Figure 3 shows basal [Ca²⁺]_i measurements from control (n = 124), missense-treated (n = 164), and antisense-treated (n = 164) neurons. Treatment with missense oligonucleotide did not alter basal [Ca²⁺]_i, with average 340/380 ratios from control and missense-treated neurons at 0.26 and 0.25, respectively. Neurons from antisense-treated cultures had significantly higher basal [Ca²⁺]_i compared with both control and missense-treated neurons, with an average ratio value of 0.31 (p < 0.001, ANOVA with Tukey's post hoc test), demonstrating that treatment with antisense oligonucleotide with subsequent suppression of CaMK-II function caused an increase in basal neuronal [Ca²⁺]_i (Fig. 3A).

View larger version (35K): [in this window] [in a new window]   Fig. 3. Knockdown of CaMK-II results in changes of basal [Ca2+]i. Ratio values of 340/380 were obtained from control (n = 124; white bar), missense-treated (n = 164; black bar), antisense-treated (n = 164; gray bar), and antisense-recovered neurons (n = 83; checked bar). Both control and missense-treated neurons demonstrate low basal [Ca2+]i. Basal [Ca2+]i measured from antisense-treated neurons was significantly higher than basal [Ca2+]i measured from both control and missense-treated neurons. Recovery of neurons from antisense oligonucleotide treatment restored basal [Ca2+]i to control levels. Data are represented by mean ratio value ± S.E.M. *, P < 0.001, ANOVA with Tukey's post hoc test, control versus missense-treated versus antisense-treated; , P < 0.001, ANOVA with Tukey's post hoc test, antisense-recovered versus antisense-treated neurons.

View larger version (29K): [in this window] [in a new window]   Fig. 4. Histograms demonstrating percentage distribution of basal [Ca2+]i within bins (0.025 increments) of ratio values less than 0.30 in black and those greater than 0.30 in white from control (A), missense-treated (B), antisense-treated (C), and antisense-recovery neurons (D). For control, missense-treated, and antisense recovery groups, 80.8, 78.1, and 81.9% fell below the 0.30 ratio value level (black), respectively. Antisense treatment resulted in a redistribution of basal [Ca2+]i where only 51% of neurons had ratio values less than 0.30.

Alteration of Ca²⁺ Homeostasis in Neurons with Decreased Expression of CaMK-II. To evaluate the ability of the neurons to regulate [Ca²⁺]_i homeostasis, a Ca²⁺ load was induced by brief exposure to glutamate resulting in an immediate and marked increase in [Ca²⁺]_i using established techniques (Pal et al., 2001; Sun et al., 2004). Both Fura-2 and Fura-FF were employed to evaluate changes in [Ca²⁺]_i caused by glutamate exposure. [Ca²⁺]_i measurements were taken from missense-treated and antisense-treated neuronal cultures. Following brief glutamate exposure (50 µM, 2 min), the glutamate-induced Ca²⁺ load and the ability of each treatment group to regulate [Ca²⁺]_i was analyzed. Analysis of the glutamate-induced [Ca²⁺]_i load in missense-treated and antisense-treated neurons indicated that the increased [Ca²⁺]_i during the glutamate exposure was not significantly different in both Fura-2 and Fura-FF-loaded cultures. The change in the 340/380 ratios before and during glutamate in cultures loaded with Fura-2 was 1.07 ± 0.05 for missense-treated cultures and 0.93 ± 0.09 for antisense-treated cultures (Fig. 5A; P = 0.1, Student's t test). In Fura-FF-loaded cultures, the change in the 340/380 ratios before and during glutamate was 0.19 ± 0.01 for missense-treated cultures and 0.20 ± 0.02 for antisense-treated cultures (Fig. 5B; P = 0.8, Student's t test). Thus the impairment of the antisense-treated neurons to buffer [Ca²⁺]_i was not due to an increased [Ca²⁺]_i load produced by glutamate.

View larger version (31K): [in this window] [in a new window]   Fig. 5. The altered ability of CaMK-II knockdown neurons to restore basal [Ca2+]i was not due to an increased Ca2+ load. A, peak increase in Fura-2 ratio during a 2-min exposure to 50 µM glutamate in missense-treated (n = 29; white bar) and antisense-treated (n = 29; black bar) neurons. No statistically significant differences were observed between missense-treated and antisense-treated neurons. B, peak increase in Fura-FF ratio during a 2-min exposure to 50 µM glutamate in missense-treated (n = 17; white bar) and antisense-treated (n = 9; black bar) neurons. No statistically significant differences were observed between missense-treated and antisense-treated neurons. C, Ca2+ decay curves obtained with Fura-2 were normalized as percentage of Ca2+ load and demonstrate the impaired ability of antisense-treated neurons (n = 28; ) to buffer [Ca2+]i. compared with missense-treated neurons (n = 32; ). D, Ca2+ decay curves obtained with Fura-FF were normalized as percentage of Ca2+ load and demonstrate the impaired ability of antisense-treated neurons (n = 9; ) to buffer [Ca2+]i compared with missense-treated neurons (n = 17; ). For both indicators, the percentage of Ca2+ load remained significantly higher for antisense-treated neurons at all time points after glutamate was washed from the neurons. *, P < 0.001, RM ANOVA with Tukey's post hoc test. Data are represented as mean percentage Ca2+ load ± S.E.M.

To determine whether [Ca²⁺]_i in antisense-treated neurons eventually returned to basal levels after the glutamate-induced Ca²⁺ load, neurons were imaged 2 h after glutamate exposure. Missense-treated and antisense-treated neurons were exposed to glutamate (50 µM, 2 min). One hour later, neurons were loaded with Fura-2 for 30 min and then washed three times with recording solution and incubated an additional 15 min for esterase cleavage. Neurons were then placed on the microscope, and 340/380 ratio values were obtained. Two hours after glutamate exposure, ratio values from missense-treated neurons (n = 21) had dropped to 0.24, whereas ratio values from antisense-treated neurons (n = 30) had dropped to 0.31 (Fig. 6). To ensure that basal [Ca²⁺]_i was consistent with what was previously reported, basal [Ca²⁺]_i was obtained from the same batches of neurons and were not statistically different from the values obtained 2 h post-glutamate. However, analysis with the Student's t test revealed that the 2-h post-glutamate values from antisense-treated neurons were still significantly elevated over missense-treated neurons (p < 0.001).

View larger version (15K): [in this window] [in a new window]   Fig. 6. Recovery of [Ca2+]i to basal levels 2-h post glutamate exposure in both missense-treated (A) and antisense-treated neurons (B). Neurons were exposed 2 min to 50 µM glutamate and then analyzed with Fura-2 2 h after glutamate was washed from the neurons. At 2 h, ratio values from both missense-treated and antisense-treated neurons were not significantly different from basal [Ca2+]i. At the 2-h time point, ratio values obtained from antisense-treated neurons (n = 30; ) were significantly higher than those from missense-treated neurons (n = 21; ). *, P < 0.001, Student's t test. Data are represented as mean ± S.E.M.

View larger version (39K): [in this window] [in a new window]   Fig. 7. Restoration of Ca2+ homeostasis in neurons recovered from antisense oligonucleotide. Neurons recovered from CaMK-II antisense oligonucleotide were loaded with Fura-2, and 340/380 ratio values were obtained. Antisense-recovered neurons demonstrate the ability to buffer [Ca2+]i following a Ca2+ load produced by a 2-min exposure to 50 µM glutamate. Ca2+ decay curves were normalized to percentage of Ca2+ load. Antisense-treated neurons (n = 32; ) show a decreased rate of decline of [Ca2+]i compared with missense-treated (n = 28; ) and antisense-recovered neurons (n = 29; ) in culture. [Ca2+]i in antisense-treated neurons remained elevated for up to 30 min following the glutamate exposure, whereas [Ca2+]i in missense-treated and antisense-recovered neurons declined rapidly approaching basal levels. The data represent the mean percentage Ca2+ load ± S.E.M. *, P < 0.001, RM ANOVA with Tukey's post hoc test, antisense-treated versus antisense-recovered.

View larger version (16K): [in this window] [in a new window]   Fig. 8. Abolishment of SREDs did not restore [Ca2+]i homeostasis. TTX (400 nM) was added to the pBRS and glutamate solutions to inhibit synaptic activity before and during Ca2+ imaging. A, in the presence of TTX, basal [Ca2+]i in antisense-treated neurons (n = 106; black bar) remained elevated above missense-treated neurons (n = 97; white bar). *, P < 0.001, Student's t test, antisense + TTX versus missense + TTX. Data are represented as mean ratio value ± S.E.M. B, following a glutamate-induced Ca2+ load, antisense-treated neurons (n = 19; ) remained elevated above missense-treated neurons (n = 24; ) in the presence of TTX for up to 30 min after glutamate exposure. Data are represented as mean percentage Ca2+ load ± S.E.M. *, P < 0.01, RM ANOVA with Tukey's post hoc test.

To exclude the possibility that ongoing SRED activity was accounting for observed changes in [Ca²⁺]_i homeostasis, TTX was added to the cultures before [Ca²⁺]_i imaging to inhibit basal electrophysiological activity and epileptiform discharges. Addition of TTX lowered basal [Ca²⁺]_i in both missense-treated and antisense-treated cultures (0.23 and 0.26, respectively). However, with the abolishment of seizure and background activity, basal [Ca²⁺]_i levels from antisense-treated cultures still remained significantly elevated above [Ca²⁺]_i from missense-treated cultures (Fig. 8A; P < 0.001, Student's t test). Likewise, in the presence of TTX, the ability of antisense-treated neurons to recover from a glutamate-induced Ca²⁺ load is altered compared with missense-treated neurons (Fig. 8B). Antisense-treated neurons recovered to 40.1 ± 7.2% of the Ca²⁺ load, whereas missense-treated neurons recovered to 11.1 ± 4.6% of the Ca²⁺ load. Thus, alterations in the [Ca²⁺]_i homeostatic mechanisms associated with decreased CaMK-II activity and protein expression were not solely dependent on the presence of SREDs in this preparation.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Alterations in Ca²⁺ homeostasis and CaMK-II levels have been observed in various models of epilepsy (Bronstein et al., 1993; Butler et al., 1995; DeLorenzo et al., 1998, 2005; Blair et al., 1999; Churn et al., 2000a). Because epileptogenesis has been associated with increased [Ca²⁺]_i and alterations in Ca²⁺ homeostatic mechanisms (reviewed in Delorenzo et al., 2005), it was important to determine whether changes in CaMK-II activity were associated with changes in Ca²⁺ homeostasis and the development of epileptogenesis. The present study provides evidence that suppression of CaMK-II activity is associated with both alterations in Ca²⁺ homeostasis and the development of SREDs in a reversible manner. Fluorescence imaging of [Ca²⁺]_i, with both Fura-2 and Fura-FF, was used to determine [Ca²⁺]_i in neurons treated with an antisense oligonucleotide to knock down levels of CaMK-II. Addition of antisense oligonucleotide decreased CaMK-II activity and immunocytochemical expression and resulted in the development of SREDs, a characteristic of epilepsy. In the antisense-treated neurons, basal [Ca²⁺]_i was elevated over both control and missense-treated neurons. Reversal of the CaMK-II knockdown following removal of the oligonucleotide restored CaMK-II activity and basal [Ca²⁺]_i and abolished epileptiform activity (SREDs), demonstrating a cause and effect relationship between CaMK-II levels and the development of SREDs and changes in basal [Ca²⁺]_i. [Ca²⁺]_i in antisense-treated neurons were still elevated over [Ca²⁺]_i in missense-treated neurons incubated in TTX to abolish both basal synaptic and seizure activity, demonstrating that CaMK-II changes in [Ca²⁺]_i was effecting the underlying Ca²⁺ homeostatic mechanisms that regulate basal [Ca²⁺]_i independent of neuronal activity.

In addition to evaluating the effects of CaMK-II knock-down on [Ca²⁺]_i, this study evaluated the effects of altering CaMK-II activity on Ca²⁺ homeostatic mechanisms that regulate the neuron's ability to handle a Ca²⁺ load. After a glutamate-induced Ca²⁺ load, antisense-treated neurons were not able to buffer [Ca²⁺]_i back to basal levels as efficiently as control neurons, similar to findings observed in hippocampal neuronal culture models of both SE and stroke-induced AE (Pal et al., 1999; Sun et al., 2004). Abolishing both basal synaptic and seizure activity with TTX did not affect the altered Ca²⁺ homeostatic mechanisms observed with decreased CaMK-II activity. To determine the reversibility of the CaMK-II knockdown-induced changes on Ca²⁺ homeostatic mechanisms and the development of SREDs, antisense-treated neurons were allowed to recover from the antisense oligonucleotide treatment. SREDs were abolished, Ca²⁺ homeostasis was restored, and recovered neurons were as effective as controls in their ability to effectively buffer [Ca²⁺]_i back to basal levels after a glutamate-induced Ca²⁺ load. The results from this study employed both the high and lower affinity Ca²⁺ indicators Fura-2 and Fura-FF. Both missense-treated and antisense-treated neurons loaded with each indicator showed similar responses to the glutamate-induced Ca²⁺ load. The data suggest a correlation between the suppression of CaMK-II activity with alterations in Ca²⁺ homeostatic mechanisms and expression of SREDs in hippocampal neurons in culture, indicating that CaMK-II is playing a role in regulating Ca²⁺ homeostasis during epileptogenesis in this model of epileptiform activity.

A decrease in function of CaMK-II has been shown to occur in a number of models of epilepsy (Bronstein et al., 1993; Butler et al., 1995; Churn et al., 2000a,b). However, this is the first study that shows a direct relationship between a specific suppression of CaMK-II activity and a loss in the ability of neurons to maintain Ca²⁺ homeostatic mechanisms in association with the development of SREDs. CaMK-II is a ubiquitous enzyme that phosphorylates and regulates the activity of many receptors, including N-methyl-D-aspartate, GABA_A, and inositol triphosphate, all of which play a role in epilepsy (Cardy and Taylor, 1998; Huang et al., 2005). CaMK-II activation has been associated with increased excitability, including long-term potentiation of excitatory synapses (reviewed by Bronstein et al., 1993). However, decreased CaMK-II activity has been consistently found in various models of epilepsy. The present study shows that decreasing the expression of CaMK-II results in a hyperexcitable state in neurons in culture. Further studies are needed to determine the role of decreased CaMK-II activity in inducing SREDs and altering Ca²⁺ homeostasis.

It can be deduced from the results of the present study that the phosphorylation of CaMK-II substrates might be linked to the observed alterations in Ca²⁺ homeostasis. Further studies need to be conducted to isolate the specific substrate(s) involved in these processes. Interestingly, unlike the SE-induced model of AE (Sombati and Delorenzo, 1995), knocking down levels of CaMK-II does not permanently alter Ca²⁺ homeostasis or permanently induce SREDs. Our results indicate that a 3-day recovery period is sufficient to fully restore CaMK-II activity, abolish SREDs, and restore the ability of the neurons to regulate Ca²⁺ homeostasis. Thus, this model of reversible AE may provide unique opportunities to identify the important cellular mechanisms mediating the induction and maintenance of AE and indicate that the effects of CaMK-II in regulating Ca²⁺ homeostasis plays an important role in the development of AE in hippocampal neurons in culture. Although using cultured hippocampal neurons is a well established model for studying epilepsy, it is important to confirm the results of studies in culture with in vivo models. Further studies are needed to determine the in vivo effects of CaMK-II knockdown to understand the role of CaMK-II in epilepsy.

Calcium plays a pivotal role in normal neuronal function (Llinas et al., 1992; Berridge, 1998). Normal neuronal [Ca²⁺]_i is maintained around 100 nM (Mody and MacDonald, 1995). This concentration is less than one-ten thousandth of the free extracellular Ca²⁺ concentration ([Ca²⁺]_e) (Putney, 1999). In light of the important signaling function of Ca²⁺ and the excitotoxic implications of excess [Ca²⁺]_i, neurons have an intricate system to regulate [Ca²⁺]_i. Evidence indicates that Ca²⁺ homeostasis is markedly altered acutely during the injury and epileptogenic phases of AE (Raza et al., 2004) and even in the chronic spontaneous recurrent seizures (epilepsy) phases of AE (Pal et al., 2000, 2001; Raza et al., 2001, 2004). These observations provide direct evidence that the alterations of normal Ca²⁺ homeostasis plays a role in epileptogenesis and the maintenance of the epileptic phenotype (Delorenzo et al., 2005).

Complex regulatory processes are mediated by Ca²⁺ homeostatic mechanisms in neurons (Carafoli et al., 1997). Increased or prolonged entry of extracellular Ca²⁺ could contribute to the altered Ca²⁺ homeostatic mechanisms in epilepsy (Carafoli et al., 1997). [Ca²⁺]_i homeostasis is regulated by a number of cellular mechanisms that include calcium-induced calcium release from intracellular stores, sequestration into the endoplasmic reticulum by the sarco/endoplasmic reticulum calcium ATPase (SERCA) (Carafoli et al., 1997), mitochondrial uptake of Ca²⁺ (Kunz et al., 1999), and efflux via the plasma membrane Na⁺/Ca²⁺ exchanger (Ryan, 1999). In addition, a number of [Ca²⁺]_i buffering systems that include the binding proteins calbindin, calretinin, and parvalbumin (Nagerl et al., 2000) are important regulators of [Ca²⁺]_i. In the CaMK-II knockdown neurons, Ca²⁺ homeostasis was disrupted as indicated by elevated basal [Ca²⁺]_i and the impaired ability to buffer a Ca²⁺ load. These results are similar to previous observations of altered [Ca²⁺]_i in other models of epilepsy, including the low Mg²⁺-induced SE model of AE (Pal et al., 1999) and the stroke model of AE (Sun et al., 2004). Studies have implicated decreased activity of SERCA leading to increases in [Ca²⁺]_i in whole animal and in vitro models of epilepsy (Parsons et al., 2000; Pal et al., 2001). Enhanced calcium-induced calcium release via the inositol triphosphate receptor has also been shown to be a contributor to increased [Ca²⁺]_i and altered Ca²⁺ homeostatic mechanisms in epilepsy (Pal et al., 2001). Further studies are needed to evaluate what cellular pathway(s) is involved in the loss of Ca²⁺ homeostasis in the CaMK-II antisense knockdown model of AE.

CaMK-II activity plays a role in maintaining Ca²⁺ homeostasis in both skeletal and cardiac muscle, with a positive correlation between CaMK-II activity and SERCA activity. In these tissues, CaMK-II has been shown to regulate SERCA by two different mechanisms. One mechanism involves CaMK-II phosphorylation of the SERCA accessory protein, phospholamban, which enhances the uptake of Ca²⁺ by SERCA (Colyer, 1998). Although phospholamban is not present in neurons (Plessers et al., 1991), there is evidence that a protein analogous to phospholamban exists in neurons and may be required for neuronal SERCA activity (Dou and Joseph, 1996). Furthermore, SERCA activity in skeletal muscle is increased by direct phosphorylation by CaMK-II (Xu et al., 1993). This relationship has yet to be studied in neurons and could provide the link between decreased CaMK-II activity and altered Ca²⁺ homeostasis in our model of epilepsy.

A better understanding of the molecular mechanisms that underlie the relationship between altered Ca²⁺ dynamics and the induction and maintenance of AE is important for understanding the process of epileptogenesis (Delorenzo et al., 2005). Although further studies are needed, it is apparent that elucidating the molecular basis contributing to altered Ca²⁺ regulatory mechanisms in the CaMK-II knockdown model of AE may provide an insight into the long-term plasticity changes associated with epilepsy and offer specific molecular targets for preventing and possibly reversing the pathophysiological condition of AE.

	Footnotes

 
This work was supported by a National Institutes of Health, National Institute of Neurological Disorders and Stroke Grants R01-NS23350 (to R.J.D.) and P50-NS25630 (to R.J.D.) and the Milton L. Markel Alzheimer's Disease Research and the Sophie and Nathan Gumenick Neuroscience Research Funds. D.S.C. and S.N.H. are supported by the National Institutes of Health Training Grant T32-NS007288-20.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.110403.

ABBREVIATIONS: SREDs, spontaneous recurrent epileptiform discharges; AE, acquired epilepsy; SE, status epilepticus; CaMK-II, calcium/calmodulin-dependent protein kinase II; DIV, days in vitro; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid); pBRS, physiological bath recording solution; SERCA, sarco(endo)plasmic reticular calcium ATPase; TTX, tetrodotoxin; PBS, phosphate-buffered saline; RM, repeated measures; ANOVA, analysis of variance.

Address correspondence to: Dr. Robert J. DeLorenzo, Virginia Commonwealth University School of Medicine, P.O. Box 980599, Richmond, VA 23298. E-mail: rjdeloren{at}hsc.vcu.edu

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