Status epilepticus (SE) is a major neurological disorder associated with significant morbidity and mortality. SE causes significant damage to limbic system and patients surviving SE have a very high probability for developing acquired epilepsy and cognitive deficits [1
]. It is therefore important to stop SE and limit brain damage as early as possible. Unfortunately, SE has been found to be refractory to many treatments and there is rapid development of tolerance to standard anticonvulsant drugs as seizure duration lengthens [2
]. Thus, there is a need to develop newer drugs and treatment strategies for prompt treatment of SE.
Exposure of cultured hippocampal neurons to a low Mg2+
medium elicits high frequency epileptiform discharges in neurons that exceed 3 Hz in spike frequency. This model has been widely used to study SE mechanisms in an in vitro setting [3
]. It is considered as a model of refractory SE, since standard anticonvulsant drugs such as phenytoin and phenobarbital do not stop the high frequency epileptiform discharges [7
]. In addition, similar to humans [8
], benzodiazepines such as lorazepam stop SE-like activity only initially and then display pharmacoresistance resulting in its loss of efficacy as SE continues [7
]. Molecular changes such as loss of GABAA
receptors following SE in animals are also observed in this preparation [4
]. This in vitro model allows for precise control of extracellular milieu while mimicking electrographic properties, neuronal population synchronicity, and anticonvulsant sensitivity identical to SE observed in vivo and is therefore ideally suited to evaluate the effects of investigational compounds on SE-like activity.
The endocannabinoid system has come to forefront as novel target for treatment of seizures. This system comprises of at least two cannabinoid receptors (CB1 and CB2), the endogenous ligands (anandamide and 2-arachidonyl glycerol) and the protein machinery for the synthesis, degradation and reuptake of endocannabinoids. CB1 receptors are abundant in hippocampus and are located on both excitatory and inhibitory nerve terminals. Activation of inhibitory G-protein coupled presynaptic CB1 receptors results in decreased neurotransmitter release (Reviewed in: [9
]). This action is thought to underlie the potent anticonvulsant effects of CB1 agonists in models of acquired epilepsy [10
]. Plasticity changes in the endocannabinoid system have also been found following febrile seizure [13
]. Cannabinoids have also been found to stop SE-like activity in hippocampal neuronal cultures [7
]. It has also been reported that a CB1 receptor-dependent endocannabinoid tone plays an important role in modulating seizure frequency and duration and preventing the development of SE-like activity in epileptic neurons [14
]. Thus, the endocannabinoid system plays a critical role in modulating neuronal excitability.
The development of medicinal cannabinoids has been greatly tempered by psychoactivity and abuse potential associated with CB1 agonists. Altering endocannabinoid levels by targeting enzymes that synthesize and degrade the endogenous ligands is thought to be an attractive strategy for therapeutic modulation of endocannabinoid system [9
]. In this regard, the endocannabinoid reuptake inhibitors and fatty acid amide hydrolase (FAAH) inhibitors have been found to exert effects similar to CB1 receptor agonists but are not associated with abuse liability [16
]. Acetaminophen or paracetamol has recently been suggested as a prodrug for activating the endocannabinoid system [17
]. This commonly used analgesic/ antipyretic drug was reported to undergo deacetylation and then conjugation with arachidonic acid to yield N-arachidonoyl-phenolamine or AM 404- a potent endocannabinoid reuptake inhibitor [18
]. AM 404 has been reported to increase levels on anandamide [19
]. This conversion of acetaminophen to AM 404 is thought to underlie its pain alleviating effect since CB1 receptor antagonist blocked its analgesic effect in various pain models [20
]. In addition, acetaminophen has also been reported to exhibit anxiolytic properties that are thought to be mediated by endocannabinoids and CB1 receptors [23
]. We have previously demonstrated that endocannabinoids block SE-like activity in vitro [3
]. Further, AM 404 has been shown to prevent naloxone induced seizures [24
]. In light of these studies, we investigated if acetaminophen via its conversion to AM 404 and subsequent increases in endocannabinoid levels would block low Mg2+
induced SE-like activity in cultured hippocampal neurons.