Chapter 7 - Antiepileptic drug treatment strategies in neonatal epilepsy

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Abstract

The highest risk of seizures across the lifespan is in the neonatal period. The enhanced excitability of the immature brain compared to the mature brain is related to the sequential development and expression of essential neurotransmitter signaling pathways. During the neonatal period there is an overabundance of excitatory receptors, and γ-amino-butyric acid (GABA) is potentially depolarizing, as opposed to hyperpolarizing in the older brain. While this enhanced excitability is required for regulation of activity-dependent synapse formation and refining of synaptic connections that are necessary for normal brain development, enhanced excitability predisposes the immature brain to seizures. In addition to being common, neonatal seizures are very difficult to treat; antiepileptic drugs used in older children and adults are less efficacious, and possibly detrimental to brain development. In an effort to target the unique features of neurotransmission in the neonate, bumetanide, an NKCC1 inhibitor which reduces intraneuronal Cl and induces a significant shift of EGABA toward more hyperpolarized values in vitro, has been used to treat neonatal seizures. As the understanding of the pathophysiology of genetic forms of neonatal epilepsy has evolved there have been a few successful attempts to pharmacologically target the mutated protein. This approach, while promising, is challenging due to the findings that the genetic syndromes presenting in infancy demonstrate genetic heterogeneity in regard to both the mutated gene and its function.

Introduction

Rapid changes in brain development occur during the neonatal period; dendritic and axonal outgrowth, formation of new synapses, and progressive maturation of electrical and synaptic activity set up mature functional brain networks. The neonatal period is also a period when genetically programmed activity patterns are affected by early environmental influences that result in the development of cortical maps.

The neonatal period is also the time with the greatest risk for seizures. There is considerable evidence that the neonatal brain is far more susceptible to seizures than during any other period of life. In addition to the high rate of seizures in newborns (Hauser, 1995, Lanska and Lanska, 1996, Lanska et al., 1995, Saliba et al., 1999, Uria-Avellanal et al., 2013), the propensity for seizures in the immature brain has been demonstrated in a number of experimental models, including kainic acid (Khalilov et al., 2003, Tremblay et al., 1984), electrical stimulation (Moshe, 1981), hypoxia (Jensen et al., 1991), penicillin (Swann and Brady, 1984), picrotoxin (Gomez-Di Cesare et al., 1997), GABA(B) receptor antagonists (McLean et al., 1996), and increased extracellular K+ (Dzhala and Staley, 2003b, Khazipov et al., 2004).

Experimental studies in animals suggest that many factors are developmentally regulated during the neonatal period to increase the excitability of neurons and neural networks (see Fig. 1). The enhanced excitability is a double-edged sword; on one hand, it is essential for regulation of activity-dependent synapse formation and refining of synaptic connections that are necessary for proper cognition, while on the other hand it predisposes the immature brain to aberrant excitation leading to a seizure. During the neonatal period, configuration of ion channels and neurotransmitter receptors is maximized for spontaneous activity and synchronization across multiple neuronal networks. There is an increase in density of Na+ currents, increase in Ca2 +-activated K+ channels, and the appearance of hyperpolarization-activated, cyclic nucleotide-gated channels (Picken Bahrey and Moody, 2003, Stoenica et al., 2013, Surges et al., 2006). Immature neurons show an elevated resting membrane potential and a reduction in input resistance and membrane time constant (Zhang, 2004), resulting in action potentials that are increased in amplitude and shorter duration than in older cells, allowing neurons to fire repetitive action potentials spontaneously (McCormick and Prince, 1987, Picken Bahrey and Moody, 2003). In addition, in the mammalian brain, coupling of neurons by gap junctions (electrical synapses) transiently increases during early postnatal development resulting in increased synchronization between neurons (Barnett et al., 2014, Belousov and Fontes, 2013, Belousov and Fontes, 2014).

The enhanced excitability of the immature brain compared to the mature brain is related to the sequential development and expression of essential neurotransmitter signaling pathways. In the adult brain, glutamate is the primary excitatory neurotransmitter and γ-amino-butyric acid (GABA), the principal inhibitory transmitter. The GABAA receptor is a ligand-gated anion channel that provides fast inhibition by allowing Cl efflux leading to hyperpolarization. GABAB receptors are metabotropic transmembrane receptors for GABA that are linked via G-proteins to K+ and Ca2 + channels and result in slow inhibition. Excitatory synaptic transmission is mediated by glutamate that is released from the pyramidal neurons and depolarizes and excites the target neurons via ionotropic receptors (N-methyl-d-aspartate [NMDA], α-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid [AMPA], and kainite receptors). The metabotropic glutamate receptors are members of the G-protein receptor-coupled superfamily. Because of their slow kinetics, voltage dependence, and high permeability to Ca2 + ions, NMDA receptors play a dominant role in activity-dependent synaptic plasticity and are critical for development of the brain and the processes underlying learning and neuroplasticity.

During the first few weeks of life there is enhanced glutamatergic excitation due to an overabundance of NMDA receptors (Haberny et al., 2002, Insel et al., 1990, McDonald and Johnston, 1990, McDonald et al., 1990). There are also developmental changes in the subunit composition and neurophysiology of these excitatory receptors. NMDA receptors are heteromeric with an obligate NR1 subunit. In the immature brain the predominant NR2 subunit is the NR2B subunit (Chang et al., 2009). The NMDA receptor has characteristics of both a ligand-mediated and voltage-gated channel. The ion Mg2 + lies in the pore of the channel, preventing permeability of Na+ and Ca2 + ions. When Mg2 + is released from the pore by membrane depolarization, the flow of Na+ and Ca2 + ions can occur. Compared to the NR2A subunit that is highly expressed in mature neurons, NR2B units have a reduced Mg2 + sensitivity, resulting in increased excitability (Hollmann and Heinemann, 1994). Other developmentally regulated subunits (NR2C, NR2D, and NR3A) also are increased in the first two postnatal weeks (Monyer et al., 1994).

The AMPA receptor is responsible for fast excitatory neurotransmission. AMPA receptors are heteromeric and are made up of four subunits, including combinations of the GluR1, GluR2, GluR3, or GlurR4 subunits (Hollmann and Heinemann, 1994). The AMPA receptor responds rapidly to glutamate with opening of the channel to allow Na+ to enter the cell and depolarize the membrane. In the immature rodent and human brain, AMPA receptor expression is initially low and silent synapses, or synapses with only NMDA receptors, predominate. Over the course of development, there is an increase in the AMPA to NMDA receptor-mediated current ratio as AMPA receptors are expressed more highly (for review, see Hall and Ghosh, 2008). AMPA receptors that are present in immature neurons lack the GluR2 subunit, conferring Ca2 + permeability to immature receptors (Hollmann et al., 1991, Kumar et al., 2002, Sanchez et al., 2001). The enhanced Ca2 + permeability of the AMPA receptor results in greater excitability and increases the likelihood of seizures in the immature brain (McDonald et al., 1992, Rakhade et al., 2008).

The development of GABAergic and glutamatergic synapses follows distinct timelines. During fetal development, GABAergic synapses drive the development of glutamatergic synapses (Khazipov et al., 2001). During the early postnatal period, at a time when the immature brain is highly susceptible to seizures (Jensen and Baram, 2000, Khazipov et al., 2004), GABA, which in the adult brain is the primary inhibitory neurotransmitter, exerts paradoxical depolarizing action (Dzhala and Staley, 2003b, Khazipov et al., 2004) via GABAA receptors. This has also been seen in neurons from adults with epilepsy (Huberfeld et al., 2007), raising the question of whether or not depolarizing actions of GABA may be fundamental in the development of early-life epilepsies.

The depolarizing action of GABA in immature neurons is due to elevated intracellular Cl compared to adult Cl concentrations. This is set in immature neurons primarily by the ratio of expression of two Cl cotransporters, with relatively high levels of expression of Na+–K+–Cl cotransporter (NKCC1) and low expression of the K+ cotransporter (KCC2; see Fig. 2). NKCC1 acts as a Cl loader, whereas KCC2 is a Cl extruder. As a result, EGABA is set to a value that is more positive than resting membrane potential and, upon GABA binding to GABAA receptors, Cl ions flow out from the cell. While the reversal potential for Cl is depolarizing, it is also barely above action potential threshold (Tyzio et al., 2008). However, depolarization evoked by GABAA currents in immature neurons is sufficient to activate voltage-gated Na+ or Ca2 + channels or NMDA receptors that can further depolarize the neuron, leading to an action potential (Tyzio et al., 2007). It is worth noting that not all immature cells are depolarized by GABAA and some mature cells, such as dentate gyrus granule cells, retain depolarizing GABA actions, albeit these are usually HCO3 dependent and subthreshold (Kaila et al., 1993, Misgeld et al., 1986). In addition, trauma or an insult, such as hypoxia, has been shown to further downregulate KCC2 as part of a generally adaptive response that reduces energetic costs needed to operate this cotransporter (Jin et al., 2005, Nabekura et al., 2002), thereby potentially adding a “second hit” to an already hyperexcitable system.

Depolarizing GABA in immature neurons may be excitatory in nature, yet the transmission of excitation is relatively low fidelity compared to glutamatergic transmission due to the large difference in driving forces between glutamate and GABA (Valeeva et al., 2010). This excitatory action of GABA is critical for the development and maturation of neural networks; however, there also is considerable evidence that this enhanced excitability via depolarizing GABA may contribute directly to the increased seizure susceptibility of the immature brain (Holmes et al., 2002). For example, there is a strong temporal correlation between the period of seizure susceptibility and the switch between excitation and inhibition in animal models. In addition, epileptiform activity and even frank seizures can be generated by GABAA agonism (Dzhala and Staley, 2003a, Khazipov et al., 2001). Finally, as discussed later and shown in Fig. 3, artificially shifting intracellular Cl concentration to more mature concentrations through blockade of the immature NKCC1 exchanger has been shown to alleviate seizures in animal models as well.

In addition to the immature state of GABAA receptor signaling, postsynaptic GABAB-mediated inhibition, while present very early on in embryonic development, increases until the middle of the second postnatal week (Fukuda et al., 1993, Gaiarsa et al., 1995, Kirmse and Kirischuk, 2006, Luhmann and Prince, 1991). Taken together, the immature and often excitatory state of the GABAergic signaling is a major contributor to hyperexcitability seen during this time period in development.

In summary, the immature brain's high susceptibility for seizures can be explained by the morphological and physiological events occurring during early life that are necessary for maturation and formation of adult neural networks, but also increase excitability and can therefore result in seizures. The intrinsic properties of immature neurons, overabundance of synaptic connections, and expression of Ca2 +-sensitive AMPA and NMDA receptors combined with the lack of developed inhibitory networks lead to a situation where the immature brain is at high risk for seizures. Fig. 1 illustrates the complement of voltage-gated ion channels, ligand-gated reports, and cotransporters in developing neurons.

Section snippets

Treatment Approaches

Neonatal seizures are an important sign of a potentially severe brain disorder and require immediate investigation in regard to etiology since the cause of the seizures is the most important determinant of outcome. Most neonatal seizures are reactive; ie, they are a response to an acute brain disorder and are self-limited; however, there is evidence that seizures can add to the neurological injury and they should be treated promptly. There is increasing evidence that neonatal seizures,

Acknowledgments

Supported by grants from the NIH (NINDS): NS0415951 and NS056170.

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