Sompong Sombati

Cannabinoid CB1 receptor antagonists cause status epilepticus- like activity in the hippocampal neuronal culture model of acquired epilepsy

Status epilepticus (SE) is a major medical emergency associated with a significant morbidity and mortality. Little is known about the mechanisms that terminate seizure activity and prevent the development of status epilepticus. Cannabinoids possess anticonvulsant properties and the endocannabinoid system has been implicated in regulating seizure duration and frequency. Endocannabinoids regulate synaptic transmission and dampen seizure activity via activation of the presynaptic cannabinoid receptor 1 (CB1). This study was initiated to evaluate the role of CB1 receptor-dependent endocannabinoid synaptic transmission towards preventing the development of status epilepticus-like activity in the well-characterized hippocampal neuronal culture model of acquired epilepsy using patch clamp electrophysiology. Application of the CB1 receptor antagonists SR141716A (1 microM) or AM251 (1 microM) to epileptic neurons caused the development of continuous epileptiform activity, resembling electrographic status epilepticus. The induction of status epilepticus-like activity by CB1 receptor antagonists was reversible and could be overcome by maximal concentrations of CB1 agonists. Similar treatment of control neurons with CB1 receptor antagonists did not produce status epilepticus or hyperexcitability. These findings suggest that CB1 receptor-dependent endocannabinoid endogenous tone plays an important role in modulating seizure frequency and duration and preventing the development of status epilepticus-like activity in populations of epileptic neurons. The regulation of seizure activity and prevention of status epilepticus by the endocannabinoid system offers an important insight into understanding the basic mechanisms that control the development of continuous epileptiform discharges.

Aging is associated with elevated intracellular calcium levels and altered calcium homeostatic mechanisms in hippocampal neurons

Aging is associated with increased vulnerability to neurodegenerative conditions such as Parkinsons and Alzheimers disease and greater neuronal deficits after stroke and epilepsy. Emerging studies have implicated increased levels of intracellular calcium ([Ca(2+)](i)) for the neuronal loss associated with aging related disorders. Recent evidence demonstrates increased expression of voltage gated Ca(2+) channel proteins and associated Ca(2+) currents with aging. However, a direct comparison of [Ca(2+)](i) levels and Ca(2+) homeostatic mechanisms in hippocampal neurons acutely isolated from young and mid-age adult animals has not been performed. In this study, Fura-2 was used to determine [Ca(2+)](i) levels in CA1 hippocampal neurons acutely isolated from young (4-5 months) and mid-age (12-16 months) Sprague-Dawley rats. Our data provide the first direct demonstration that mid-age neurons in comparison to young neurons manifest significant elevations in basal [Ca(2+)](i) levels. Upon glutamate stimulation and a subsequent [Ca(2+)](i) load, mid-age neurons took longer to remove the excess [Ca(2+)](i) in comparison to young neurons, providing direct evidence that altered Ca(2+) homeostasis may be present in animals at significantly younger ages than those that are commonly considered aged (> or =24 months). These alterations in Ca(2+) dynamics may render aging neurons more vulnerable to neuronal death following stroke, seizures or head trauma. Elucidating the functionality of Ca(2+) homeostatic mechanisms may offer an understanding of the increased neuronal loss that occurs with aging, and allow for the development of novel therapeutic agents targeted towards decreasing [Ca(2+)](i) levels thereby restoring the systems that maintain normal Ca(2+) homeostasis in aged neurons.

Time course and mechanism of hippocampal neuronal death in an in vitro model of status epilepticus: role of NMDA receptor activation and NMDA dependent calcium entry.

The hippocampus is especially vulnerable to seizure-induced damage and excitotoxic neuronal injury. This study examined the time course of neuronal death in relationship to seizure duration and the pharmacological mechanisms underlying seizure-induced cell death using low magnesium (Mg2+) induced continuous high frequency epileptiform discharges (in vitro status epilepticus) in hippocampal neuronal cultures. Neuronal death was assessed using cell morphology and fluorescein diacetate-propidium iodide staining. Effects of low Mg2+ and various receptor antagonists on spike frequency were assessed using patch clamp electrophysiology. We observed a linear and time-dependent increase in neuronal death with increasing durations of status epilepticus. This cell death was dependent upon extracellular calcium (Ca2+) that entered primarily through the N-methyl-d-aspartate (NMDA) glutamate receptor channel subtype. Neuronal death was significantly decreased by co-incubation with the NMDA receptor antagonists and was also inhibited by reduction of extracellular (Ca2+) during status epilepticus. In contrast, neuronal death from in vitro status epilepticus was not significantly prevented by inhibition of other glutamate receptor subtypes or voltage-gated Ca2+ channels. Interestingly this NMDA-Ca2+ dependent neuronal death was much more gradual in onset compared to cell death from excitotoxic glutamate exposure. The results provide evidence that in vitro status epilepticus results in increased activation of the NMDA-Ca2+ transduction pathway leading to neuronal death in a time-dependent fashion. The results also indicate that there is a significant window of opportunity during the initial time of continuous seizure activity to be able to intervene, protect neurons and decrease the high morbidity and mortality associated with status epilepticus.

Prolonged exposure to WIN55,212-2 causes downregulation of the CB1 receptor and the development of tolerance to its anticonvulsant effects in the hippocampal neuronal culture model of acquired epilepsy

Cannabinoids have been shown to cause CB1-receptor-dependent anticonvulsant activity in both in vivo and in vitro models of status epilepticus (SE) and acquired epilepsy (AE). It has been further demonstrated in these models that the endocannabinoid system functions in a tonic manner to suppress seizure discharges through a CB1-receptor-dependent pathway. Although acute cannabinoid treatment has anticonvulsant activity, little is known concerning the effects of prolonged exposure to CB1 agonists and development of tolerance on the epileptic phenotype. This study was carried out to evaluate the effects of prolonged exposure to the CB1 agonist WIN55,212-2 on seizure activity in a hippocampal neuronal culture model of low-Mg(2+) induced spontaneous recurrent epileptiform discharges (SREDs). Following low-Mg(2+) induced SREDs, cultures were returned to maintenance media containing 10, 100 or 1000 nM WIN55,212-2 from 4 to 24 h. Whole-cell current-clamp analysis of WIN55,212-2 treated cultures revealed a concentration-dependent increase in SRED frequency. Immunocytochemical staining revealed that WIN55,212-2 treatment induced a concentration-dependent downregulation of the CB1 receptor in neuronal processes and at both glutamatergic and GABAergic presynaptic terminals. Prolonged exposure to the inactive enantiomer WIN55,212-3 in low-Mg(2+) treated cultures had no effect on the frequency of SREDs or CB1 receptor staining. The results from this study further substantiate a role for a tonic CB1-receptor-dependent endocannabinoid regulation of seizure discharge and suggest that prolonged exposure to cannabinoids results in the development of tolerance to the anticonvulsant effects of cannabinoids and an exacerbation of seizure activity in the epileptic phenotype.

Alterations in neuronal calcium levels are associated with cognitive deficits after traumatic brain injury

Traumatic brain injury (TBI) survivors often suffer from a post-traumatic syndrome with deficits in learning and memory. Calcium (Ca(2+)) has been implicated in the pathophysiology of TBI-induced neuronal death. However, the role of long-term changes in neuronal Ca(2+) function in surviving neurons and the potential impact on TBI-induced cognitive impairments are less understood. Here we evaluated neuronal death and basal free intracellular Ca(2+) ([Ca(2+)](i)) in acutely isolated rat CA3 hippocampal neurons using the Ca(2+) indicator, Fura-2, at seven and thirty days after moderate central fluid percussion injury. In moderate TBI, cognitive deficits as evaluated by the Morris Water Maze (MWM), occur after injury but resolve after several weeks. Using MWM paradigm we compared alterations in [Ca(2+)](i) and cognitive deficits. Moderate TBI did not cause significant hippocampal neuronal death. However, basal [Ca(2+)](i) was significantly elevated when measured seven days post-TBI. At the same time, these animals exhibited significant cognitive impairment (F(2,25)=3.43, p<0.05). When measured 30 days post-TBI, both basal [Ca(2+)](i) and cognitive functions had returned to normal. Pretreatment with MK-801 blocked this elevation in [Ca(2+)](i) and also prevented MWM deficits. These studies provide evidence for a link between elevated [Ca(2+)](i) and altered cognition. Since no significant neuronal death was observed, the alterations in Ca(2+) homeostasis in the traumatized, but surviving neurons may play a role in the pathophysiology of cognitive deficits that manifest in the acute setting after TBI and represent a novel target for therapeutic intervention following TBI.

An in vitro model of stroke-induced epilepsy: elucidation of the roles of glutamate and calcium in the induction and maintenance of stroke-induced epileptogenesis.

Stroke is a major risk factor for developing acquired epilepsy (AE). Although the underlying mechanisms of ischemia-induced epileptogenesis are not well understood, glutamate has been found to be associated with both epileptogenesis and ischemia-induced injury in several research models. This chapter discusses the development of an in vitro model of epileptogenesis induced by glutamate injury in hippocampal neurons, as found in a clinical stroke, and the implementation of this model of stroke-induced AE to evaluate calciums role in the induction and maintenance of epileptogenesis. To monitor the acute effects of glutamate on neurons and chronic alterations in neuronal excitability up to 8 days after glutamate exposure, whole-cell current-clamp electrophysiology was employed. Various durations and concentrations of glutamate were applied to primary hippocampal cultures. A single 30-min, 5-microM glutamate exposure produced a subset of neurons that died or had a stroke-like injury, and a larger population of injured neurons that survived. Neurons that survived the injury manifested spontaneous, recurrent, epileptiform discharges (SREDs) in neural networks characterized by paroxysmal depolarizing shifts (PDSs) and high-frequency spike firing that persisted for the life of the culture. The neuronal injury produced in this model was evaluated by determining the magnitude of the prolonged, reversible membrane depolarization, loss of synaptic activity, and neuronal swelling. The permanent epileptiform phenotype expressed as SREDs that resulted from glutamate injury was found to be dependent on the presence of extracellular calcium. The epileptic neurons manifested elevated intracellular calcium levels when compared to control neurons, independent of neuronal activity and seizure discharge, demonstrating that alterations in calcium homeostatic mechanisms occur in association with stroke-induced epilepsy. Findings from this investigation present the first in vitro model of glutamate injury-induced epileptogenesis that may help elucidate some of the mechanisms that underlie stroke-induced epilepsy.

The novel antiepileptic drug carisbamate (RWJ 333369) is effective in inhibiting spontaneous recurrent seizure discharges and blocking sustained repetitive firing in cultured hippocampal neurons

This study was initiated to investigate effects of the novel neuromodulator carisbamate (RWJ 333369) in the hippocampal neuronal culture model of status epilepticus and spontaneous epileptiform discharges. Whole-cell current clamp techniques were used to determine the effects of carisbamate on spontaneous recurrent epileptiform discharges (SREDs, in vitro epilepsy), depolarization-induced sustained repetitive firing (SRF) and low Mg(2+)-induced continuous high frequency spiking (in vitro status epilepticus). This in vitro model is an important tool to study the effects of anticonvulsant drugs (AEDs) on SREDs that occur for the life of the neurons in culture. Carisbamate dose dependently blocked the expression and reoccurrence of SREDs. The ED(50) value for its antiepileptic effect was 58.75+/-2.43 microM. Inhibition of SRF is considered a common attribute of many AEDs. Carisbamate (100 microM) significantly decreased SRF in hippocampal neurons. All these effects of carisbamate were reversed during a 5 to 30 min drug washout period. When exposed to low Mg(2+) medium cultured hippocampal neurons exhibit high frequency spiking. This form of in vitro status epilepticus is not effectively blocked by conventional AEDs that are known to be effective in treating status epilepticus in humans. Carisbamate, like phenytoin and phenobarbital, had little or no effect on low Mg(2+)-induced continuous high frequency spiking. These results characterize the effects of carisbamate in the hippocampal neuronal culture model of epileptiform discharges and suggest that the ability of carisbamate to inhibit depolarization-induced SRF may account in part for some of its anticonvulsant effect.

In vitro status epilepticus but not spontaneous recurrent seizures cause cell death in cultured hippocampal neurons

It is established that the majority but not all of the seizure-induced cell death is associated with status epilepticus while spontaneous recurrent seizures associated with epilepsy do not cause neuronal death. Extracellular effects and compensatory changes in brain physiology complicate assessment of neuronal death in vivo as the result of seizures. In this study we utilized a well-characterized in vitro hippocampal neuronal culture model of both continuous high-frequency epileptiform discharges (status epilepticus) and spontaneous recurrent epileptiform discharges (acquired epilepsy) to investigate the direct effects of continuous and episodic electrographic epileptiform discharges on cell death in a carefully controlled extracellular environment. The results from this study indicate that continuous high-frequency epileptiform discharges can cause neuronal death in a time-dependent manner. Episodic epileptiform seizure activity occurring for the life of the neurons in culture was not associated with increased neuronal cell death. Our data confirm observations from clinical and some animal studies that spontaneous recurrent seizures do not initiate cell death. The hippocampal neuronal culture model provides a powerful in vitro tool for carefully evaluating the effects of seizure activity alone on neuronal viability in the absence of various confounding factors and may provide new insights into the development of novel therapeutic agents to prevent neuronal injury during status epilepticus.

Development of pharmacoresistance to benzodiazepines but not cannabinoids in the hippocampal neuronal culture model of status epilepticus.

Status epilepticus (SE) is a life-threatening neurological disorder associated with a significant morbidity and mortality. Benzodiazepines are the initial drugs of choice for the treatment of SE. Despite aggressive treatment, over 40% of SE cases are refractory to the initial treatment with two or more medications. It would be a major advance in the clinical management of SE to identify novel anticonvulsant agents that do not lose their ability to treat SE with increasing seizure duration. Cannabinoids have recently been demonstrated to regulate seizure activity in brain. However, it remains to be seen whether they develop pharmacoresistance upon prolonged SE. In this study, we used low Mg(2+) to induce SE in hippocampal neuronal cultures and in agreement with animal models and human SE confirm the development of resistance to benzodiazepine with increasing durations of SE. Thus, lorazepam (1 microM) was effective in blocking low Mg(2+) induced high-frequency spiking for up to 30 min into SE. However, by 1 h and 2 h of SE onset it was only 10-15% effective in suppressing SE. In contrast, the cannabinoid type-1 (CB1) receptor agonist, WIN 55,212-2 (1 microM) in a CB1 receptor-dependent manner completely abolished SE at all the time points tested even out to 2 h after SE onset, a condition where resistance developed to lorazepam. Thus, the use of cannabinoids in the treatment of SE may offer a unique approach to controlling SE without the development of pharmacoresistance observed with conventional treatments.

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

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.