Dmytro Isaev

Role of extracellular sialic acid in regulation of neuronal and network excitability in the rat hippocampus.

The extracellular membrane surface contains a substantial amount of negatively charged sialic acid residues. Some of the sialic acids are located close to the pore of voltage-gated channel, substantially influencing their gating properties. However, the role of sialylation of the extracellular membrane in modulation of neuronal and network activity remains primarily unknown. The level of sialylation is controlled by neuraminidase (NEU), the key enzyme that cleaves sialic acids. Here we show that NEU treatment causes a large depolarizing shift of voltage-gated sodium channel activation/inactivation and action potential (AP) threshold without any change in the resting membrane potential of hippocampal CA3 pyramidal neurons. Cleavage of sialic acids by NEU also reduced sensitivity of sodium channel gating and AP threshold to extracellular calcium. At the network level, exogenous NEU exerted powerful anticonvulsive action both in vitro and in acute and chronic in vivo models of epilepsy. In contrast, a NEU blocker (N-acetyl-2,3-dehydro-2-deoxyneuraminic acid) dramatically reduced seizure threshold and aggravated hippocampal seizures. Thus, sialylation appears to be a powerful mechanism to control neuronal and network excitability. We propose that decreasing the amount of extracellular sialic acid residues can be a useful approach to reduce neuronal excitability and serve as a novel therapeutic approach in the treatment of seizures.

Selective impairment of GABAergic synaptic transmission in the flurothyl model of neonatal seizures.

Neonatal seizures can result in long‐term adverse consequences including alteration of seizure susceptibility and impairment in spatial memory. However, little is known about the effects of neonatal seizures on developmental changes occurring in synaptic transmission during the first postnatal weeks. The purpose of the present study was to examine the effect of neonatal seizures on several aspects of γ‐aminobutyric acid (GABA)ergic and glutamatergic synaptic transmission in the developing rat hippocampus. Flurothyl was used to induce multiple recurrent seizures in rat pups during the first postnatal days. Whole‐cell patch‐clamp recordings from the hippocampal CA3 pyramidal cell and extracellular recordings from the CA3 pyramidal cell layer were made in slice preparations. In rats that experienced neonatal seizures the amplitude of spontaneous inhibitory postsynaptic currents at P15–17 was decreased by 27% compared with controls, whereas neither frequency nor the kinetic properties were altered. Neonatal seizures did not affect the timing of the developmental switch in the GABAA signaling from excitatory to inhibitory. None of the studied parameters of glutamatergic postsynaptic currents was different between the flurothyl and control groups, including the amplitude and frequency of the spontaneous excitatory postsynaptic currents, the ratio of the amplitudes and frequencies of the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) and N‐methyl‐d‐aspartate (NMDA)‐mediated spontaneous postsynaptic currents, and the kinetics of AMPA and NMDA mediated postsynaptic currents in the age groups P8–10 and P15–17. We suggest that the selective depression of the amplitude of GABAergic synaptic responses may contribute to the adverse neurological and behavioral consequences that occur following neonatal seizures.

Recurrent neonatal seizures result in long-term increases in neuronal network excitability in the rat neocortex

Neonatal seizures are associated with a high likelihood of adverse neurological outcomes, including mental retardation, behavioral disorders, and epilepsy. Early seizures typically involve the neocortex, and post‐neonatal epilepsy is often of neocortical origin. However, our understanding of the consequences of neonatal seizures for neocortical function is limited. In the present study, we show that neonatal seizures induced by flurothyl result in markedly enhanced susceptibility of the neocortex to seizure‐like activity. This change occurs in young rats studied weeks after the last induced seizure and in adult rats studied months after the initial seizures. Neonatal seizures resulted in reductions in the amplitude of spontaneous inhibitory postsynaptic currents and the frequency of miniature inhibitory postsynaptic currents, and significant increases in the amplitude and frequency of spontaneous excitatory postsynaptic currents (sEPSCs) and in the frequency of miniature excitatory postsynaptic currents (mEPSCs) in pyramidal cells of layer 2/3 of the somatosensory cortex. The selective N‐methyl‐d‐aspartate (NMDA) receptor antagonist d‐2‐amino‐5‐phosphonovalerate eliminated the differences in amplitude and frequency of sEPSCs and mEPSCs in the control and flurothyl groups, suggesting that NMDA receptors contribute significantly to the enhanced excitability seen in slices from rats that experienced recurrent neonatal seizures. Taken together, our results suggest that recurrent seizures in infancy result in a persistent enhancement of neocortical excitability.

Surface charge impact in low-magnesium model of seizure in rat hippocampus

Putative mechanisms of induction and maintenance of seizure-like activity (SLA) in the low Mg(2+) model of seizures are: facilitation of NMDA receptors and decreased surface charge screening near voltage-gated channels. We have estimated the role of such screening in the early stages of SLA development at both physiological and room temperatures. External Ca(2+) and Mg(2+) promote a depolarization shift of the sodium channel voltage sensitivity; when examined in hippocampal pyramidal neurons, the effect of Ca(2+) was 1.4 times stronger than of Mg(2+). Removing Mg(2+) from the extracellular solution containing 2 mM Ca(2+) induced recurrent SLA in hippocampal CA1 pyramidal layer in 67% of slices. Reduction of [Ca(2+)](o) to 1 mM resulted in 100% appearance of recurrent SLA or continuous SLA. Both delay before seizure activity and the inter-SLA time were significantly reduced. Characteristics of seizures evoked in low Mg(2+)/1 mM Ca(2+)/3.5 K(+) were similar to those obtained in low Mg(2+)/2 Ca(2+)/5mM K(+), suggesting that reduction of [Ca(2+)](o) to 1 mM is identical to the increase in [K(+)](o) to 5 mM in terms of changes in cellular excitability and seizure threshold. An increase of [Ca(2+)](o) to 3 mM completely abolished SLA generation even in the presence of 5 mM [K(+)](o). A large variation in the ability of [Ca(2+)](o) to stop epileptic discharges in initial stage of SLA was found. Our results indicate that surface charge of the neuronal membrane plays a crucial role in the initiation of low Mg(2+)-induced seizures. Furthermore, our study suggests that Ca(2+) and Mg(2+), through screening of surface charge, have important anti-seizure and antiepileptic properties.

Altered short-term plasticity in the prefrontal cortex after early life seizures.

Seizures during development are a relatively common occurrence and are often associated with poor cognitive outcomes. Recent studies show that early life seizures alter the function of various brain structures and have long-term consequences on seizure susceptibility and behavioral regulation. While many neocortical functions could be disrupted by epileptic seizures, we have concentrated on studying the prefrontal cortex (PFC) as disturbance of PFC functions is involved in numerous co-morbid disorders associated with epilepsy. In the present work we report an alteration of short-term plasticity in the PFC in rats that have experienced early life seizures. The most robust alteration occurs in the layer II/III to layer V network of neurons. However short-term plasticity of layer V to layer V network was also affected, indicating that the PFC function is broadly influenced by early life seizures. These data strongly suggest that repetitive seizures early in development cause substantial alteration in PFC function, which may be an important component underlying cognitive deficits in individuals with a history of seizures during development.

Thrombin facilitates seizures through activation of persistent sodium current.

An epileptic seizure is frequently the presenting sign of intracerebral hemorrhage (ICH) caused by stroke, head trauma, hypertension, and a wide spectrum of disorders. However, the cellular mechanisms responsible for occurrence of seizures during ICH have not been established. During intracerebral bleeding, blood constituents enter the neuronal tissue and produce both an acute and a delayed effect on brain functioning. Among the blood components, only thrombin has been shown to evoke seizures immediately after entering brain tissue. In the present study, we tested the hypothesis that thrombin increases neuronal excitability in the immature brain through alteration of voltage‐gated sodium channels.

Long-term Suppression of GABAergic Activity by Neonatal Seizures in Rat Somatosensory Cortex

Here we studied the long-term effects of neonatal seizures on inhibitory synaptic transmission in somatosensory cortex. We found that recurrent flurothyl-induced seizures result in a marked reduction in amplitude of spontaneous inhibitory postsynaptic currents (IPSCs) and increases of miniature IPSCs interevent intervals. These results indicate that decreasing the inhibitory synaptic strength following neonatal seizures in neocortical neurons is not due to a postsynaptic mechanism.

Blockade of endogenous neuraminidase leads to an increase of neuronal excitability and activity-dependent synaptogenesis in the rat hippocampus

Polysialic acids are widely distributed in neuronal tissue. Due to their position on glycoproteins and gangliosides on the outer cell membranes and anionic nature, polysialic acids are involved in multiple cell signaling events. The level of sialylation of the cellular surface is regulated by endogenous neuraminidase (NEU), which catalyses the hydrolysis of terminal sialic acid residues. Using the specific blocker of endogenous NEU, N‐acetyl‐2,3‐dehydro‐2‐deoxyneuraminic acid (NADNA), we show that downregulation of the endogenous NEU activity causes a significant increase in the level of hippocampal tissue sialylation. Acute application of NADNA increased the firing frequency and amplitude of spontaneous synchronous oscillations, and frequency of multiple unit activity in cultured hippocampal slices. The tonic phase of seizure‐like activity in the low‐magnesium model of ictogenesis was significantly increased in slices pretreated with NADNA. These data indicate that the degree of synchronization is influenced by the amount of active NEU in cultured hippocampal slices. Pretreatment with NADNA led to an increase of the density of simple and perforated synapses in the hippocampal CA1 stratum radiatum region. Co‐incubation of slices with NADNA and high concentrations of calcium eliminated the effect of the NEU blocker on synaptic density, suggesting that synaptogenesis observed following downregulation of the endogenous NEU activity is an activity‐dependent process.

Alteration of synaptic plasticity by neonatal seizures in rat somatosensory cortex

Seizures in newborns are associated with a high risk for subsequent epilepsy and adverse neurodevelopmental consequences. Understanding the mechanisms by which neonatal seizures adversely disturb the immature brain is important in developing therapeutic strategies. Using the convulsant agent flurothyl to mimic repetitive neonatal seizures we show that early-life seizures result in long-term alteration in the maintenance phase of long-term potentiation (LTP) in layer IV to layer II/III synapses of the somatosensory cortex without alteration of basal synaptic transmission, the induction phase of LTP and short-term depression. Such alterations may have a role in functional deficits seen following neonatal seizures.

Novel Potent Orthosteric Antagonist of ASIC1a Prevents NMDAR-Dependent LTP Induction.

Acid sensing ion channels 1a (ASIC1a) are of crucial importance in numerous physiological and pathological processes in the brain. Here we demonstrate that novel 2-oxo-2H-chromene-3-carboxamidine derivative 5b, designed with molecular modeling approach, inhibits ASIC1a currents with an apparent IC50 of 27 nM when measured at pH 6.7. Acidification to 5.0 decreases the inhibition efficacy by up to 3 orders of magnitude. The 5b molecule not only shifts pH dependence of ASIC1a activation but also inhibits its maximal evoked response. These findings suggest that compound 5b binds to pH sensor of ASIC1a acting as orthosteric noncompetitive antagonist. At 100 nM, compound 5b completely inhibits induction of long-term potentiation (LTP) in CA3-CA1 but not in MF-CA3 synapses. These findings support the knockout data indicating the crucial modulatory role of ASIC1a channels in the NMDAR-dependent LTP and introduce a novel type of ASIC1a antagonists.