Robert E. Blair

Activation of the Cannabinoid Type-1 Receptor Mediates the Anticonvulsant Properties of Cannabinoids in the Hippocampal Neuronal Culture Models of Acquired Epilepsy and Status Epilepticus

Cannabinoids have been shown to have anticonvulsant properties, but no studies have evaluated the effects of cannabinoids in the hippocampal neuronal culture models of acquired epilepsy (AE) and status epilepticus (SE). This study investigated the anticonvulsant properties of the cannabinoid receptor agonist R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolol[1,2,3 de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone (WIN 55,212-2) in primary hippocampal neuronal culture models of both AE and SE. WIN 55,212-2 produced dose-dependent anticonvulsant effects against both spontaneous recurrent epileptiform discharges (SRED) (EC50 = 0.85 μM) and SE (EC50 = 1.51 μM), with total suppression of seizure activity at 3 μM and of SE activity at 5 μM. The anticonvulsant properties of WIN 55,212-2 in these preparations were both stereospecific and blocked by the cannabinoid type-1 (CB1) receptor antagonist N-(piperidin-1-yl-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamidehydrochloride (SR141716A; 1 μM), showing a CB1 receptor-dependent pathway. The inhibitory effect of WIN 55,212-2 against low Mg2+-induced SE is the first observation in this model of total suppression of SE by a selective pharmacological agent. The clinically used anticonvulsants phenytoin and phenobarbital were not able to abolish low Mg2+-induced SE at concentrations up to 150 μM. The results from this study show CB1 receptor-mediated anticonvulsant effects of the cannabimimetic WIN 55,212-2 against both SRED and low Mg2+-induced SE in primary hippocampal neuronal cultures and show that these in vitro models of AE and SE may represent powerful tools to investigate the molecular mechanisms mediating the effects of cannabinoids on neuronal excitability.

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.

Temporal characterization of changes in hippocampal cannabinoid CB1 receptor expression following pilocarpine-induced status epilepticus

Several reports have focused on the involvement of the endocannabinoid system in hyperexcitability, particularly in seizure and epilepsy models. Our laboratory recently characterized a novel plasticity change of the cannabinoid type 1 (CB(1)) receptor in hippocampi of epileptic rats following pilocarpine-induced status epilepticus (SE). This long-term redistribution included selective layer-specific changes in CB(1) receptor expression within distinct hippocampal subregions. However, the temporal characteristics of this redistribution during the development of epilepsy had not been examined. Therefore, this study was initiated to evaluate the time course by which pilocarpine-induced SE produced changes in CB(1) receptor expression. Immunohistochemical analysis demonstrated that within 1 week following SE, there was a pronounced loss in CB(1) receptor expression throughout the hippocampus, while staining in many interneurons was preserved. By 1 month post-SE, pilocarpine-treated animals began to display epileptic seizures, and CB(1) receptor expression was characteristic of the redistribution observed in long-term epileptic rats, with decreases in CB(1) receptor immunoreactivity in the stratum pyramidale neuropil and dentate gyrus inner molecular layer, and increases in the strata oriens and radiatum of CA1-3. Observed changes in CB(1) receptor expression were confirmed at multiple time points by western blot analysis. The data indicate that overall decreases in expression following SE preempt a long-lasting CB(1) receptor redistribution, and that differential responses occur within the hippocampus to initial CB(1) receptor losses. This suggests a role for dysregulation of the endocannabinoid system during epileptogenesis and indicates that the CB(1) receptor redistribution temporally correlates with the emergence of epileptic seizures.

Development of a prolonged calcium plateau in hippocampal neurons in rats surviving status epilepticus induced by the organophosphate diisopropylfluorophosphate

Organophosphate (OP) compounds are among the most lethal chemical weapons ever developed and are irreversible inhibitors of acetylcholinesterase. Exposure to majority of OP produces status epilepticus (SE) and severe cholinergic symptoms that if left untreated are fatal. Survivors of OP intoxication often suffer from irreversible brain damage and chronic neurological disorders. Although pilocarpine has been used to model SE following OP exposure, there is a need to establish a SE model that uses an OP compound in order to realistically mimic both acute and long-term effects of nerve agent intoxication. Here we describe the development of a rat model of OP-induced SE using diisopropylfluorophosphate (DFP). The mortality, behavioral manifestations, and electroencephalogram (EEG) profile for DFP-induced SE (4 mg/kg, sc) were identical to those reported for nerve agents. However, significantly higher survival rates were achieved with an improved dose regimen of DFP and treatment with pralidoxime chloride (25 mg/kg, im), atropine (2 mg/kg, ip), and diazepam (5 mg/kg, ip) making this model ideal to study chronic effects of OP exposure. Further, DFP treatment produced N-methyl-D-aspartate (NMDA) receptor-mediated significant elevation in hippocampal neuronal [Ca(2+)](i) that lasted for weeks after the initial SE. These results provided direct evidence that DFP-induced SE altered Ca(2+) dynamics that could underlie some of the long-term plasticity changes associated with OP toxicity. This model is ideally suited to test effective countermeasures for OP exposure and study molecular mechanisms underlying neurological disorders following OP intoxication.

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.

Calcium-dependent epileptogenesis in an in vitro model of stroke-induced "epilepsy".

Summary:  Purpose: Stroke is the most common cause of acquired epilepsy. The purpose of this investigation was to characterize the role of calcium in the in vitro, glutamate injury–induced epileptogenesis model of stoke‐induced epilepsy.

Long-term decrease in calbindin-D28K expression in the hippocampus of epileptic rats following pilocarpine-induced status epilepticus

Acquired epilepsy (AE) is characterized by spontaneous recurrent seizures and long-term changes that occur in surviving neurons following an injury such as status epilepticus (SE). Long-lasting alterations in hippocampal Ca(2+) homeostasis have been observed in both in vivo and in vitro models of AE. One major regulator of Ca(2+) homeostasis is the neuronal calcium binding protein, calbindin-D28k that serves to buffer and transport Ca(2+) ions. This study evaluated the expression of hippocampal calbindin levels in the rat pilocarpine model of AE. Calbindin protein expression was reduced over 50% in the hippocampus in epileptic animals. This decrease was observed in the pyramidal layer of CA1, stratum lucidum of CA3, hilus, and stratum granulosum and stratum moleculare of the dentate gyrus when corrected for cell loss. Furthermore, calbindin levels in individual neurons were also significantly reduced. In addition, the expression of calbindin mRNA was decreased in epileptic animals. Time course studies demonstrated that decreased calbindin expression was initially present 1 month following pilocarpine-induced SE and lasted for up to 2 years after the initial episode of SE. The results indicate that calbindin is essentially permanently decreased in the hippocampus in AE. This decrease in hippocampal calbindin may be a major contributing factor underlying some of the plasticity changes that occur in epileptogenesis and contribute to the alterations in Ca(2+) homeostasis associated with AE.

Development of status epilepticus, sustained calcium elevations and neuronal injury in a rat survival model of lethal paraoxon intoxication

Paraoxon (POX) is an active metabolite of organophosphate (OP) pesticide parathion that has been weaponized and used against civilian populations. Exposure to POX produces high mortality. OP poisoning is often associated with chronic neurological disorders. In this study, we optimize a rat survival model of lethal POX exposures in order to mimic both acute and long-term effects of POX intoxication. Male Sprague-Dawley rats injected with POX (4mg/kg, ice-cold PBS, s.c.) produced a rapid cholinergic crisis that evolved into status epilepticus (SE) and death within 6-8min. The EEG profile for POX induced SE was characterized and showed clinical and electrographic seizures with 7-10Hz spike activity. Treatment of 100% lethal POX intoxication with an optimized three drug regimen (atropine, 2mg/kg, i.p., 2-PAM, 25mg/kg, i.m. and diazepam, 5mg/kg, i.p.) promptly stopped SE and reduced acute mortality to 12% and chronic mortality to 18%. This model is ideally suited to test effective countermeasures against lethal POX exposure. Animals that survived the POX SE manifested prolonged elevations in hippocampal [Ca(2+)]i (Ca(2+) plateau) and significant multifocal neuronal injury. POX SE induced Ca(2+) plateau had its origin in Ca(2+) release from intracellular Ca(2+) stores since inhibition of ryanodine/IP3 receptor lowered elevated Ca(2+) levels post SE. POX SE induced neuronal injury and alterations in Ca(2+) dynamics may underlie some of the long term morbidity associated with OP toxicity.

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.