Cerebral hypoxia is a major cause of neonatal seizures, and such seizures increase the risk of later epilepsy (Aicardi & Chevrie, 1970; Volpe, 1981; Hauser et al., 1993). Experimentally, hypoxia-induced seizures in neonatal rats are associated with immediate and subacute dysregulation of a number of ion channels that may render the hippocampus hyperexcitable during this critical maturational period (Jensen et al., 1991; Sanchez et al., 2001, 2005; Zhang et al., 2006; Sanchez et al., 2007; Rakhade et al., 2008). To date, most of these channelopathies have been identified in hippocampal CA1 pyramidal neurons, as these represent the major output of the hippocampus.
The dentate gyrus is in a unique position for information transfer from the entorhinal cortex to the hippocampus (Andersen et al., 1966), and can gate seizure propagation from the entorhinal cortex to the hippocampus proper (Walther et al., 1986; Collins et al., 1988; Heinemann et al., 1990; Dreier & Heinemann, 1991). A wealth of data from experimental models has suggested that compromise of this gating role of the dentate gyrus is critical to, or at least permissive of, long-term epileptogenesis (Dudek & Sutula, 2007). Therefore, aberrant channel function that renders principal dentate gyrus neurons hyperexcitable could contribute critically to epileptogenesis, but such changes consequent to neonatal seizure-inducing hypoxia remain largely unexplored.
Potassium channels critically regulate neuronal excitability (Pongs, 1999), and their dysfunction can result in epileptiform network activity in vitro (Traub et al., 2001; Gabriel et al., 2004) and in seizures and epilepsy in vivo (Pena & Tapia, 2000; Pena et al., 2002; Misonou et al., 2004; Binder et al., 2006). In particular, the “A-current” (IA) is a rapidly inactivating current that contributes to action potential repolarization and promotes single spike firing (i.e., inhibits burst firing) in many types of neurons and cardiac muscle (Pongs, 1999; Castro et al., 2001). IA is mediated by channels that are composed of molecular subunits from the Kv1 and Kv4 potassium channel families (Birnbaum et al., 2004). In the dentate gyrus, Kv1.1, Kv1.2, and Kv1.4 α-subunits are expressed mainly in the middle third of the molecular layer (Sheng et al., 1994; Rhodes et al., 1997), and Kv4.1, Kv4.2, and Kv4.3 are expressed in the granule cell layer (Sheng et al., 1994; Serodio & Rudy, 1998). A-type potassium channel function and regulation has been reported to be altered in animal models of status epilepticus, and may critically contribute to brain hyperexcitability and epileptogenesis (Bernard et al., 2004; Ruschenschmidt et al., 2006). Whether IA channel function is altered after perinatal seizure-inducing hypoxia and could contribute to consequent epileptogenesis has not been investigated.
In the current study, we asked if IA is pathologically altered in hippocampal dentate gyrus cells by neonatal seizure-inducing hypoxia, and may serve to alter their intrinsic firing properties, potentially contributing to limbic network hyperexcitability.
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The key findings of this study were that IA was significantly decreased in the majority of DGCs recorded 1–5 days after seizure-inducing hypoxia, and that this was associated with more rapid spiking in response to depolarizing current injection from hyperpolarized membrane potentials. Therefore, dysregulation of intrinsic firing properties secondary to altered IA could contribute to hyperexcitability of DGCs subacutely after neonatal seizure-inducing hypoxia, at a time of maturation when activity-dependent anatomic and synaptic patterning is highly labile. This conceivably could have proepileptogenic consequences or promote cognitive delay, but such functional consequences remain to be addressed. Our findings only establish altered IA in DGCs as a potential contributing factor to limbic pathophysiology consequent to seizure-inducing neonatal hypoxia.
A-type K+ channels are crucial determinants of neuronal firing patterns, and could be particularly important in controlling seizures. Reducing the amplitude of A-type currents (IA) increases seizure susceptibility (Juhng et al., 1999) and lack of A-type Kv4.2 potassium channels contributes the increased excitability and decreased seizure thresholds in methylazoxymethanol acetate–exposed rats (Castro et al., 2001). Decreased IA in hippocampal CA1 pyramidal neurons after kainate-induced status epilepticus has been observed to increase distal dendritic excitability by allowing increased back-propagation of action potentials to apical dendrites (Bernard et al., 2004). Notably, after pilocarpine-induced status epilepticus, IA in DGCs did not exhibit similar changes to that observed in other hippocampal subregions, and in fact, appeared resistant to seizure-associated changes (Ruschenschmidt et al., 2006). Nonetheless, these authors reported powerful regulation of IA recovery from inactivation in DGCs by the intracellular redox milieu, which could be profoundly altered by hypoxia or prolonged seizures. In the current study, we examined the voltage-dependence but not the time course of inactivation removal (and at room temperature), and thus did not address this mode of IA regulation. However, our finding of decreased channel availability in the hypoxia-treated group suggests that oxidation-promoted speeding of recovery from IA inactivation did not occur under our recording conditions. Under physiologic conditions, it is possible that redox changes could oppose the observed downregulation of IA in DGCs, but this has yet to be explored, as well as other mechanisms of posttranslational regulation that could be triggered by hypoxia, seizures, or both, at this early maturational stage.
Sensitivity to 4-AP is characteristic of each IA subtype, but can vary depending on the molecular composition of the channels (Bekkers, 2000; Korngreen & Sakmann, 2000; Shibata et al., 2000). The potassium channel blocker, 4-AP, significantly inhibited the IA-type channel recorded in DGCs in our study, further confirming that the transient potassium current is an IA-type current. In addition, 4-AP eliminated the difference between groups in latency to spike onset upon rapid depolarizing current injection from a hyperpolarized potential, consistent with decreased IA as underlying the more rapid spiking in the hypoxia-treated group.
IA has been shown to contribute to the regulation of action potential firing rates as well as action potential duration (Kocsis et al., 1982; Waddell & Lawson, 1990; Honmou et al., 1994). We observed that the duration of the first action potential was significantly increased by 4-AP, and consistently, AP duration was also prolonged in the hypoxia group compared to controls. Nonetheless, 4-AP had comparable effects on action potential duration and RMP in both the hypoxia and control groups, suggesting that additional 4-AP sensitive currents that contribute to these remained intact after hypoxia treatment.
Voltage-dependent potassium currents play important roles in regulating membrane excitability. IA has been implicated in determining the latency to first spike and the threshold and repolarization of action potentials (Rudy, 1988; Storm, 1990; Jagger & Housley, 2002). The first-spike latency in DGCs can be shaped by the expression, kinetic properties, and relative densities of IA. The presence of IA allows moderate hyperpolarizations (−80 mV) to evoke a well-recognized increase in the delay to first spike. In the current study, the latency to the first spike was dramatically shortened in hypoxia DGCs compared to that of P10–15 control neurons. The shortened latency would certainly increase the probability of action potential generation and change the repetitive firing pattern of DGCs. These changes could have significant impact on the maturation of limbic circuits. Therefore, the alterations in the excitability and firing pattern of DGCs in the neonatal brain could profoundly influence the activity of the whole hippocampus and vulnerability to epilepsy and other limbic pathologies.