Neuronal-Specific Inhibition of Endoplasmic Reticulum Mg2+/Ca2+ ATPase Ca2+ Uptake in a Mixed Primary Hippocampal Culture Model of Status Epilepticus.

Loss of intracellular calcium homeostasis is an established mechanism associated with neuronal dysfunction and status epilepticus. Sequestration of free cytosolic calcium into endoplasmic reticulum by Mg2+/Ca2+ adenosinetriphosphatase (ATPase) is critical for maintenance of intracellular calcium homeostasis. Exposing hippocampal cultures to low-magnesium media is a well-accepted in vitro model of status epilepticus. Using this model, it was shown that endoplasmic reticulum Ca2+ uptake was significantly inhibited in homogenates from cultures demonstrating electrophysiological seizure phenotypes. Calcium uptake was mainly neuronal. However, glial Ca2+ uptake was also significantly inhibited. Viability of neurons exposed to low magnesium was similar to neurons exposed to control solutions. Finally, it was demonstrated that Ca2+ uptake inhibition and intracellular free Ca2+ levels increased in parallel with increasing incubation in low magnesium. The results suggest that inhibition of Mg2+/Ca2+ ATPase-mediated endoplasmic reticulum Ca2+ sequestration contributes to loss of intracellular Ca2+ homeostasis associated with status epilepticus. This study describes for the first time inhibition of endoplasmic reticulum Mg2+/Ca2+ ATPase in a mixed primary hippocampal model of status epilepticus. In combination with animal models of status epilepticus, the cell culture model provides a powerful tool to further elucidate mechanisms that result in inhibition of Mg2+/Ca2+ ATPase and downstream consequences of decreased enzyme activity.

A cellular mechanism for dendritic spine loss in the pilocarpine model of status epilepticus.

PURPOSE: Previous studies have documented a synaptic translocation of calcineurin (CaN) and increased CaN activity following status epilepticus (SE); however, the cellular effect of these changes in CaN in the pathology of SE remains to be elucidated. This study examined a CaN-dependent modification of the dendritic cytoskeleton. CaN has been shown to induce dephosphorylation of cofilin, an actin depolymerization factor. The ensuing actin depolymerization can lead to a number of physiological changes that are of interest in SE. METHODS: SE was induced by pilocarpine injection, and seizure activity was monitored by video-EEG. Subcellular fractions were isolated by differential centrifugation. CaN activity was assayed using a paranitrophenol phosphate (pNPP) assay protocol. Cofilin phosphorylation was assessed using phosphocofilin-specific antibodies. Cofilin-actin binding was determined by coimmunoprecipitation, and actin polymerization was measured using a triton-solubilization protocol. Spines were visualized using a single-section rapid Golgi impregnation procedure. RESULTS: The immunoreactivity of phosphocofilin decreased significantly in hippocampal and cortical synaptosomal samples after SE. SE-induced cofilin dephosphorylation could be partially blocked by the preinjection of CaN inhibitors. Cofilin activation could be further demonstrated by increased actin-cofilin binding and a significant depolymerization of neuronal actin, both of which were also blocked by CaN inhibitors. Finally, we demonstrated a CaN-dependent loss of dendritic spines histologically. DISCUSSION: The data demonstrate a CaN-dependent, cellular mechanism through which prolonged seizure activity results in loss of dendritic spines via cofilin activation. Further research into this area may provide useful insights into the pathology of SE and epileptogenic mechanisms.

Epileptogenesis causes an N-methyl-d-aspartate receptor/Ca2+-dependent decrease in Ca2+/calmodulin-dependent protein kinase II activity in a hippocampal neuronal culture model of spontaneous recurrent epileptiform discharges.

Alterations in the function of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) have been observed in both in vivo and in vitro models of epileptogenesis; however the molecular mechanism mediating the effects of epileptogenesis on CaM kinase II has not been elucidated. This study was initiated to evaluate the molecular pathways involved in causing the long-lasting decrease in CaM kinase II activity in the hippocampal neuronal culture model of low Mg2+-induced spontaneous recurrent epileptiform discharges (SREDs). We show here that the decrease in CaM kinase II activity associated with SREDs in hippocampal cultures involves a Ca2+/N-methyl-d-aspartate (NMDA) receptor-dependent mechanism. Low Mg2+-induced SREDs result in a significant decrease in Ca2+/calmodulin-dependent substrate phosphorylation of the synthetic peptide autocamtide-2. Reduction of extracellular Ca2+ levels (0.2 mM in treatment solution) or the addition of dl-2-amino-5-phosphonovaleric acid (APV) 25 microM blocked the low Mg2+-induced decrease in CaM kinase II-dependent substrate phosphorylation. Antagonists of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainic acid receptor or L-type voltage sensitive Ca2+ channel had no effect on the low Mg2+-induced decrease in CaM kinase II-dependent substrate phosphorylation. The results of this study demonstrate that the decrease in CaM kinase II activity associated with this model of epileptogenesis involves a selective Ca2+/NMDA receptor-dependent mechanism and may contribute to the production and maintenance of SREDs in this model.

A significant increase in both basal and maximal calcineurin activity following fluid percussion injury in the rat.

Calcineurin, a neuronally enriched, calcium-stimulated phosphatase, is an important modulator of many neuronal processes, including several that are physiologically related to the pathology of traumatic brain injury. This study examined the effects of moderate, central fluid percussion injury on the activity of this important neuronal enzyme. Animals were sacrificed at several time-points postinjury and cortical, hippocampal, and cerebellar homogenates were assayed for calcineurin activity by dephosphorylation of p-nitrophenol phosphate. A significant brain injury-dependent increase was observed in both hippocampal and cortical homogenates under both basal and maximally-stimulated reaction conditions. This increase persisted 2-3 weeks post-injury. Brain injury did not alter substrate affinity, but did induce a significant increase in the apparent maximal dephosphorylation rate. Unlike the other brain regions, no change in calcineurin activity was observed in the cerebellum following brain injury. No brain region tested displayed a significant change in calcineurin enzyme levels as determined by Western blot, demonstrating that increased enzyme synthesis was not responsible for the observed increase in activity. The data support the conclusion that fluid percussion injury results in increased calcineurin activity in the rat forebrain. This increased activity has broad physiological implications, possibly resulting in altered cellular excitability or a greater likelihood of neuronal cell death.

A persistent change in subcellular distribution of calcineurin following fluid percussion injury in the rat.

Calcineurin, a neuronally enriched, calcium-stimulated phosphatase, is an important modulator of many neuronal processes, including several that are physiologically related to the pathology of traumatic brain injury. The effect of moderate, central fluid percussion injury on the subcellular distribution of this important neuronal enzyme was examined. Animals were sacrificed at several time points post-injury and calcineurin distribution in subcellular fractions was assayed by Western blot analysis and immunohistochemistry. A persistent increase in calcineurin concentration was observed in crude synaptoplasmic membrane-containing fractions. In cortical fractions, calcineurin immunoreactivity remained persistently increased for 2 weeks post-injury. In hippocampal homogenates, calcineurin immunoreactivity remained increased for up to 4 weeks. Finally, immunohistochemical analysis of hippocampal slices revealed increased staining in the apical dendrites of CA1 neurons. The increased staining was greatest in magnitude 24 h post-injury; however, staining was still more intense than control 4 weeks post-injury. The data support the conclusion that fluid percussion injury results in redistribution of the enzyme in the rat forebrain. These changes have broad physiological implications, possibly resulting in altered cellular excitability or a greater likelihood of neuronal cell death.

Age dependence of pilocarpine-induced status epilepticus and inhibition of CaM kinase II activity in the rat.

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.

Traumatic Brain Injury Causes a Tacrolimus-Sensitive Increase in Non-Convulsive Seizures in a Rat Model of Post-Traumatic Epilepsy.

Epilepsy is a significant but potentially preventable complication of traumatic brain injury (TBI). Previous research in animal models of acquired epilepsy has implicated the calcium-sensitive phosphatase, calcineurin. In addition, our lab recently found that calcineurin activity in the rat hippocampus increases acutely after lateral TBI. Here we use a calcineurin inhibitor test whether an acute increase in calcineurin activity is necessary for the development of late post-traumatic seizures. Adult rats were administered the calcineurin inhibitor Tacrolimus (5mg/kg; i.p.) 1 hour after lateral fluid percussion TBI and then monitored by video-electrocorticography (video-ECoG) for spontaneous seizure activity 5 weeks or 33 weeks later. At 5 weeks post-TBI, we observed epileptiform activity on the video-ECoG of brain injured rats but no seizures. By 33 weeks post-TBI though, nearly all injured rats exhibited spontaneous seizures, including convulsive seizures which were infrequent but lasted minutes (18% of injured rats), and non-convulsive seizures which were frequent but lasted tens of seconds (94% of injured rats). We also identified non-convulsive seizures in a smaller subset of control and sham TBI rats (56%), reminiscent of idiopathic seizures described in other rats strains. Non-convulsive seizures in the brain injured rats, however, were four-times more frequent and two-times longer lasting than in their uninjured littermates. Interestingly, rats administered Tacrolimus acutely after TBI showed significantly fewer non-convulsive seizures than untreated rats, but a similar degree of cortical atrophy. The data thus indicate that administration of Tacrolimus acutely after TBI suppressed non-convulsive seizures months later.

Traumatic brain injury causes an FK506-sensitive loss and an overgrowth of dendritic spines in rat forebrain.

Traumatic brain injury (TBI) causes both an acute loss of tissue and a progressive injury through reactive processes such as excitotoxicity and inflammation. These processes may worsen neural dysfunction by altering neuronal circuitry beyond the focally-damaged tissue. One means of circuit alteration may involve dendritic spines, micron-sized protuberances of dendritic membrane that support most of the excitatory synapses in the brain. This study used a modified Golgi-Cox technique to track changes in spine density on the proximal dendrites of principal cells in rat forebrain regions. Spine density was assessed at 1 h, 24 h, and 1 week after a lateral fluid percussion TBI of moderate severity. At 1 h after TBI, no changes in spine density were observed in any of the brain regions examined. By 24 h after TBI, however, spine density had decreased in ipsilateral neocortex in layer II and III and dorsal dentate gyrus (dDG). This apparent loss of spines was prevented by a single, post-injury administration of the calcineurin inhibitor FK506. These results, together with those of a companion study, indicate an FK506-sensitive mechanism of dendritic spine loss in the TBI model. Furthermore, by 1 week after TBI, spine density had increased substantially above control levels, bilaterally in CA1 and CA3 and ipsilaterally in dDG. The apparent overgrowth of spines in CA1 is of particular interest, as it may explain previous reports of abnormal and potentially epileptogenic activity in this brain region.

Mechanisms of dendritic spine remodeling in a rat model of traumatic brain injury.

Traumatic brain injury (TBI), a leading cause of death and disability in the United States, causes potentially preventable damage in part through the dysregulation of neural calcium levels. Calcium dysregulation could affect the activity of the calcium-sensitive phosphatase calcineurin (CaN), with serious implications for neural function. The present study used both an in vitro enzymatic assay and Western blot analyses to characterize the effects of lateral fluid percussion injury on CaN activity and CaN-dependent signaling in the rat forebrain. TBI resulted in an acute alteration of CaN phosphatase activity and long-lasting alterations of its downstream effector, cofilin, an actin-depolymerizing protein. These changes occurred bilaterally in the neocortex and hippocampus, appeared to persist for hours after injury, and coincided with synapse degeneration, as suggested by a loss of the excitatory post-synaptic protein PSD-95. Interestingly, the effect of TBI on cofilin in some brain regions was blocked by a single bolus of the CaN inhibitor FK506, given 1 h post-TBI. Overall, these findings suggest a loss of synapse stability in both hemispheres of the laterally-injured brain, and offer evidence for region-specific, CaN-dependent mechanisms.

Strain-related differences after experimental traumatic brain injury in rats.

The present study directly compares the effects of experimental brain injury in two commonly used rat strains: Fisher 344 and Sprague-Dawley. We previously found that Fisher rats have a higher mortality rate and more frequent seizure attacks at the same injury level than Sprague-Dawley rats. Although strain differences in rats are commonly accepted as contributing to variability among studies, there is a paucity of literature addressing strain influence in experimental neurotrauma. Therefore this study compares outcome measures in two rat strains following lateral fluid percussion injury. Fisher 344 and Sprague-Dawley rats were monitored for changes in physiological measurements, intracranial pressure, and electroencephalographic activity. We further analyzed neuronal degeneration and cell death in the injured brain using Fluoro-Jade-B (FJB) histochemistry and caspase-3 immunostaining. Behavioral studies using the beam walk and Morris water maze were conducted to characterize strain differences in both motor and cognitive functional recovery following injury. We found that Fisher rats had significantly higher intracranial pressure, prolonged seizure activity, increased FJB-positive staining in the injured cortex and thalamus, and increased caspase-3 expression than Sprague-Dawley rats. On average, Fisher rats displayed a greater amount of total recording time in seizure activity and had longer ictal durations. The Fisher rats also had increased motor deficits, correlating with the above results. In spite of these results, Fisher rats performed better on cognitive tests following injury. The results demonstrate that different rat strains respond to injury differently, and thus in preclinical neurotrauma studies strain influence is an important consideration when evaluating outcomes.

Prolonged seizure activity leads to increased Protein Kinase A activation in the rat pilocarpine model of status epilepticus.

Status epilepticus is a life-threatening form of seizure activity that represents a major medical emergency associated with significant morbidity and mortality. Protein Kinase A is an important regulator of synaptic strength that may play an important role in the development of status epilepticus-induced neuronal pathology. This study demonstrated an increase in PKA activity against exogenous and endogenous substrates during later stages of SE. As SE progressed, a significant increase in PKA-mediated phosphorylation of an exogenous peptide substrate was demonstrated in cortical structures. The increased activity was not due to altered expression of either regulatory or catalytic subunits of the enzyme. Through the use of phospho-specific antibodies, this study also investigated the effects of SE on the phosphorylation of the GluR1 subunit of the AMPA subtype of glutamate receptor. After the onset of continuous seizure activity, an increase in phosphorylation of the PKA site on the GluR1 subunit of the AMPA receptor was observed. These data suggest a potential mechanism by which SE may increase neuronal excitability in the cortex, potentially leading to maintenance of seizure activity or long-term neuronal pathology.

Age-dependent mortality in the pilocarpine model of status epilepticus.

Status epilepticus (SE) is an acute neurological emergency associated with significant morbidity and mortality. Age has been shown to be a critical factor in determining outcome after SE. Understanding the causes of this increased mortality with aging by developing an animal model to study this condition would play a major role in studying mechanisms to limit the mortality due to SE. Here we employed pilocarpine to induce SE in rats aged between 5 and 28 weeks. Similar to clinical studies in man, we observed that age was a significant predictor of mortality following SE. While no deaths were observed in 5-week-old animals, mortality due to SE increased progressively with age and reached 90% in 28-week-old animals. There was no correlation between the age of animals and severity of SE. With increasing age mortality occurred earlier after the onset of SE. These results indicate that pilocarpine-induced SE in the rat provides a useful model to study age-dependent SE-induced mortality and indicates the importance of using animal models to elucidate the mechanisms contributing to SE-induced mortality and the development of novel therapeutic interventions to prevent SE-induced death.

Modulation of CaM kinase II activity is coincident with induction of status epilepticus in the rat pilocarpine model.

PURPOSE: This study was conducted to characterize the early cellular changes in CaM kinase II activity that occur during the induction of status epilepticus (SE). METHODS: The pilocarpine model of SE was characterized both behaviorally and electrographically. At specific time points after the first discrete seizure, 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. RESULTS: After the development of SE, the data show an immediate inhibition of both cortical and hippocampal CaM kinase II activity in homogenate, but a delayed inhibition in synaptic kinase activity. The maintenance of synaptic kinase activity was due to a translocation of CaM kinase II protein to the synapse. However, despite the translocation of functional kinase, CaM kinase II activity was not maintained, membrane potential was not restored, and the newly translocated CaM kinase II did not terminate the SE event. Unlike the homogenate samples, in the crude synaptoplasmic membrane (SPM) subcellular fractions, a positive correlation is found between the duration of SE and the inhibition of CaM kinase II activity in both the cortex and hippocampus. CONCLUSIONS: The data support the hypothesis that alterations of CaM kinase II activity are involved in the early events of SE pathology.

Neuronal-specific endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration in mixed primary hippocampal culture homogenates.

Endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration is crucial for maintenance of neuronal Ca(2+) homeostasis. The use of cell culture in conjunction with modern Ca(2+) imaging techniques has been invaluable in elucidating these mechanisms. While imaging protocols evaluate endoplasmic reticulum Ca(2+) loads, measurement of Mg(2+)/Ca(2+) ATPase activity is indirect, comparing cytosolic Ca(2+) levels in the presence or absence of the Mg(2+)/Ca(2+) ATPase inhibitor thapsigargin. Direct measurement of Mg(2+)/Ca(2+) ATPase by isolation of microsomes is impossible due to the minuscule amounts of protein yielded from cultures used for imaging. In the current study, endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration was measured in mixed homogenates of neurons and glia from primary hippocampal cultures. It was demonstrated that Ca(2+) uptake was mediated by the endoplasmic reticulum Mg(2+)/Ca(2+) ATPase due to its dependence on ATP and Mg(2+), enhancement by oxalate, and inhibition by thapsigargin. It was also shown that neuronal Ca(2+) uptake, mediated by the type 2 sarco(endo)plasmic reticulum Ca(2+) ATPase isoform, could be distinguished from glial Ca(2+) uptake in homogenates composed of neurons and glia. Finally, it was revealed that Ca(2+) uptake was sensitive to incubation on ice, extremely labile in the absence of protease inhibitors, and significantly more stable under storage conditions at -80 degrees C.

Status epilepticus-induced changes in the subcellular distribution and activity of calcineurin in rat forebrain.

This study was performed to determine the effect of prolonged status epilepticus on the activity and subcellular location of a neuronally enriched, calcium-regulated enzyme, calcineurin. Brain fractions isolated from control animals and rats subjected to pilocarpine-induced status epilepticus were subjected to differential centrifugation. Specific subcellular fractions were tested for both calcineurin activity and enzyme content. Significant, status epilepticus-induced increases in calcineurin activity were found in homogenates, nuclear fractions, and crude synaptic membrane-enriched fractions isolated from both cortex and hippocampus. Additionally, significant increases in enzyme levels were observed in crude synaptic fractions as measured by Western analysis. Immunohistochemical studies revealed a status epilepticus-induced increase in calcineurin immunoreactivity in dendritic structures of pyramidal neurons of the hippocampus. The data demonstrate a status epilepticus-induced increase in calcineurin activity and concentration in the postsynaptic region of forebrain pyramidal neurons.

Calcium/calmodulin-dependent kinase II phosphorylation of the GABAA receptor alpha1 subunit modulates benzodiazepine binding.

gamma-Aminobutyric acid (GABA) is the primary neurotransmitter that is responsible for the fast inhibitory synaptic transmission in the central nervous system. A major post-translational mechanism that can rapidly regulate GABAAR function is receptor phosphorylation. This study was designed to test the effect of endogenous calcium and calmodulin-dependent kinase II (CaM kinase II) activation on both allosteric modulator binding and GABAA receptor subunit phosphorylation. Endogenous CaM kinase II activity was stimulated, and GABAA receptors were subsequently analyzed for bothallosteric modulator binding properties and immunoprecipitated and analyzed for subunit phosphorylation levels. A significant increase in allosteric-modulator binding of the GABAAR was observed under conditions maximal for CaM kinase II activation. In addition, CaM kinase II activation resulted in a direct increase in phosphorylation of the GABAA receptor alpha1 subunit. The data suggest that the CaM kinase II-dependent phosphorylation of the GABAA receptor alpha1 subunit modulated allosteric modulator binding to the GABAA receptor.