Jerome Niquet

Rapid surface accumulation of NMDA receptors increases glutamatergic excitation during status epilepticus.

After 1h of lithium-pilocarpine status epilepticus (SE), immunocytochemical labeling of NMDA receptor NR1 subunits reveals relocation of subunits from the interior to the cell surface of dentate gyrus granule cells and CA3 pyramidal cells. Simultaneously, an increase in NMDA-miniature excitatory postsynaptic currents (mEPSC) as well as an increase in NMDA receptor-mediated tonic currents is observed in hippocampal slices after SE. Mean-variance analysis of NMDA-mEPSCs estimates that the number of functional postsynaptic NMDA receptors per synapse increases 38% during SE, and antagonism by ifenprodil suggests that an increase in the surface representation of NR2B-containing NMDA receptors is responsible for the augmentation of both the phasic and tonic excitatory currents with SE. These results provide a potential mechanism for an enhancement of glutamatergic excitation that maintains SE and may contribute to excitotoxic injury during SE. Therapies that directly antagonize NMDA receptors may be a useful therapeutic strategy during refractory SE.

Hypoxic neuronal necrosis: Protein synthesis-independent activation of a cell death program

Hypoxic necrosis of dentate gyrus neurons in primary culture required the activation of an orderly cell death program independent of protein synthesis. Early mitochondrial swelling and loss of the mitochondrial membrane potential were accompanied by release of cytochrome c and followed by caspase-9-dependent activation of caspase-3. Caspase-3 and -9 inhibitors reduced neuronal necrosis. Calcium directly induced cytochrome c release from isolated mitochondria. Hypoxic neuronal necrosis may be an active process in which the direct effect of hypoxia on mitochondria may lead to the final common pathway of caspase-3-mediated neuronal death.

Seizure-induced Neuronal Death in the Immature Brain

The response of the developing brain to epileptic seizures and to status epilepticus is highly age-specific. Neonates with their low cerebral metabolic rate and fragmentary neuronal networks can tolerate relatively prolonged seizures without suffering massive cell death, but severe seizures in experimental animals inhibit brain growth, modify neuronal circuits, and can lead to behavioral deficits and to increases in neuronal excitability. Past infancy, the developing brain is characterized by high metabolic rate, exuberant neuronal and synaptic networks and overexpression of receptors and enzymes involved in excitotxic mechanisms. The outcome of seizures is highly model-dependent. Status epilepticus may produce massive neuronal death, behavioral deficits, synaptic reorganization and chronic epilepsy in some models, little damage in others. Long-term consequences are also highly age- and model-dependent. However, we now have some models which reliably lead to spontaneous seizures and chronic epilepsy in the vast majority of animals, demonstrating that seizure-induced epileptogenesis can occur in the developing brain. The mode cell death from status epilepticus is largely (but not exclusively) necrotic in adults, while the incidence of apoptosis increases at younger ages. Seizure-induced necrosis has many of the biochemical features of apoptosis, with early cytochrome release from mitochondria and capase activation. We speculate that this form of necrosis is associated with seizure-induced energy failure.

Short-Term Plasticity of Hippocampal Neuropeptides and Neuronal Circuitry in Experimental Status Epilepticus

Summary:  Purpose: We used a model of self‐staining status epilepticus (SSSE), induced by brief intermittent stimulation of the perforant path in unanesthetized rats, to study the mechanism of initiation and of maintenance of SSSE and the role of neuropeptides in those processes.

Vulnerability of postnatal hippocampal neurons to seizures varies regionally with their maturational stage.

The mechanism of status epilepticus-induced neuronal death in the immature brain is not fully understood. In the present study, we examined the contribution of caspases in our lithium-pilocarpine model of status epilepticus in 14 days old rat pups. In CA1, upregulation of caspase-8, but not caspase-9, preceded caspase-3 activation in morphologically necrotic cells. Pretreatment with a pan-caspase inhibitor provided neuroprotection, showing that caspase activation was not an epiphenomenon but contributed to neuronal necrosis. By contrast, upregulation of active caspase-9 and caspase-3, but not caspase-8, was detected in apoptotic dentate gyrus neurons, which were immunoreactive for doublecortin and calbindin-negative, two features of immature neurons. These results suggest that, in cells which are aligned in series as parts of the same excitatory hippocampal circuit, the same seizures induce neuronal death through different mechanisms. The regional level of neuronal maturity may be a determining factor in the execution of a specific death program.

Rational polytherapy in the treatment of acute seizures and status epilepticus.

We used a model of severe cholinergic status epilepticus (SE) to study polytherapy aimed at reversing the effects of seizure‐induced loss of synaptic GABAA receptors and seizure‐induced gain of synaptic NMDA receptors. Combinations of a benzodiazepine with ketamine and valproate, or with ketamine and brivaracetam, were more effective and less toxic than benzodiazepine monotherapy in this model of SE.

Status Epilepticus Triggers Caspase-3 Activation and Necrosis in the Immature Rat Brain

Summary:  The mode and mechanism of neuronal death induced by status epilepticus (SE) in the immature brain have not been fully characterized. In this study, we analyzed the contribution of neuronal necrosis and caspase‐3 activation to CA1 damage following lithium‐pilocarpine SE in P14 rat pups. By electron microscopy, many CA1 neurons displayed evidence of early necrosis 6 hours following SE, and the full ultrastructural features of necrosis at 24–72 hours. Caspase‐3 was activated in injured (acidophilic) neurons 24 hours following SE, raising the possibility that they died by caspase‐dependent “programmed” necrosis.

Trafficking of NMDA receptors during status epilepticus: Therapeutic implications

We used two models of status epilepticus (SE) to study trafficking of N‐methyl‐d‐aspartate (NMDA) receptors. SE is associated with increased surface expression of NR1 subunits of NMDA receptors, and with an increase of NMDA synaptic and extrasynaptic currents suggesting an increase in number of functional NMDA receptors on dentate granule cells. The therapeutic implications of these results are discussed.

Distinct caspase pathways mediate necrosis and apoptosis in subpopulations of hippocampal neurons after status epilepticus

Status epilepticus in the immature brain induces neuronal injury in the hippocampal formation, but the mode and mechanism of death are poorly understood. Our laboratory has recently investigated the role of caspase‐3, ‐8, and ‐9 in neuronal injury, using a lithium–pilocarpine model of status epilepticus in 2‐week‐old rat pups. Our results showed that dying neurons in the dentate gyrus and CA1‐subiculum area do not share the same mechanism of death. In CA1‐subiculum, caspase‐8 upregulation preceded caspase‐3 activation in morphologically necrotic neurons. The pan‐caspase inhibitor Q‐VD‐OPH reduced CA1 damage, showing that caspases contribute to status epilepticus–induced necrosis. In the dentate gyrus, dying neurons were caspase‐9 and ‐3 immunoreactive and morphologically apoptotic. It is not clear why the same seizures cause different types of cell death in neurons that are connected in series along the same hippocampal circuit, but the apoptotic dentate neurons express doublecortin, and do not express calbindin‐D28k, suggesting that their immaturity may be a factor in producing an apoptotic mode of death.

Molecular Basis of Self-Sustaining Seizures and Pharmacoresistance During Status Epilepticus: The Receptor Trafficking Hypothesis Revisited

During status epilepticus (SE), seizures become selfsustaining (Wasterlain, 1974), and pharmacoresistance to benzodiazepines develops progressively, and can result in a 20-fold reduction of response to diazepam after 30 min of experimental seizures (Kapur & Macdonald, 1997; Mazarati et al., 1998a,b). This time-dependent pharmacoresistance is not mediated by classical transporter mechanisms (Lçscher, 2007), but may reflect seizureinduced internalization of synaptic c-aminobutyric acid (GABA)A receptors (GABAAR) containing the b2–3 and/ or c2 subunits (Goodkin et al., 2005, 2008; Naylor et al., 2005).