Decreased A-currents in hippocampal dentate granule cells after seizure-inducing hypoxia in the immature rat

Abstract

Purpose: Cerebral hypoxia is a major cause of neonatal seizures, and can lead to epilepsy. Pathologic anatomic and physiologic changes in the dentate gyrus have been associated with epileptogenesis in many experimental models, as this region is widely believed to gate the propagation of limbic seizures. However, the consequences of hypoxia-induced seizures for the immature dentate gyrus have not been extensively examined.

Methods: Seizures were induced by global hypoxia (5-7% O2 for 15 min) in rat pups on postnatal day 10. Whole-cell voltage-clamp recordings were used to examine A-type potassium currents (IA ) in dentate granule cells in hippocampal slices obtained 1-17 days after hypoxia treatment.

Key findings: Seizure-inducing hypoxia resulted in decreased maximum IA amplitude in dentate granule cells recorded within the first week but not at later times after hypoxia treatment. The decreased IA amplitude was not associated with changes in the voltage-dependence of activation or inactivation removal, or in sensitivity to inhibition by 4-aminopyridine (4-AP). However, consistent with the role of IA in shaping firing patterns, we observed in the hypoxia group a significantly decreased latency to first spike with depolarizing current injection from hyperpolarized potentials. These differences were not associated with changes in resting membrane potential or input resistance, and were eliminated by application of 10 m 4-AP.

Significance: Given the role of IA to slow action potential firing, decreased IA could contribute to long-term hippocampal pathology after neonatal seizure-inducing hypoxia by increasing dentate granule cell excitability during a critical window of activity-dependent hippocampal maturation.

Keywords: A-current; Epilepsy; Hippocampal slice; Patch clamp.

Figure 1. Decreased I_A in DGCs after seizure-inducing hypoxia

Figure 1 shows I_A recorded in representative DGCs in slices from a control and hypoxia-treated rat. (A) Raw currents are shown in the left and middle panels, and subtracted currents are shown in the right panels (see Methods). For the cells shown, the peak subtracted amplitude activated at 30 mV was 963 pA for control compared to 608 pA for hypoxia. (B): Summary current-voltage relationships for I_A in DGCs from the control and hypoxia-treated groups showed significantly decreased I_A amplitudes in the hypoxia-treated group compared to controls (control, n=26; hypoxia, n=38, P<0.0001, ANOVA).

Figure 2. Voltage-dependence of I_A was unchanged in DGCs after seizure-inducing hypoxia

(A, B) Traces show the voltage-protocols and example subtracted currents used to measure the voltage-dependence of I_A activation (A) and removal of inactivation (B). (C, D) Summary data and Boltzmann fits (see Methods) showed no differences between control and hypoxia-treated groups in the voltage-dependence of activation (C) or removal of steady-state inactivation (D).

Figure 3. 4-AP inhibition of I_A in DGCs is slowed but intact after seizure-inducing hypoxia

(A) Representative raw traces using the I_A activation protocol with test step to +30 mV are shown before and after 5-minute application of 10 mM 4-AP. (B, C) Summary data illustrate greater apparent inhibition of I_A by 4-AP after 5-minute application in the control group compared to the hypoxia-treated group. However, prolonged 4-AP application (>15 minutes) resulted in complete inhibition of I_A in both groups (see Results).

Figure 4. Decreased spike latency in DGCs upon rapid depolarization

(A) Raw voltage traces are shown to compare spikes evoked by stepwise current injection from rest (solid lines) and from −80 mV (dotted lines). In the control neuron (left), current injection from −80 mV resulted in increased latency to first spike compared to rest consistent with the removal of I_A inactivation by membrane hyperpolarization and subsequent I_A activation. In the neuron from the hypoxia-treated group (right), there was no significant increase in latency to first spike upon current injection from −80 mV compared to rest consistent with decreased I_A activation. (B) Decreased I_A was associated with increased action potential duration in DGCs after hypoxia. The bar graphs show summary action potential half-widths (left) and amplitudes (right) in each group with and without 4-AP. Action potential half-widths were significantly increased in the hypoxia-treated group compared to controls consistent with slowed repolarization consequent to decreased I_A. However, 4-AP comparably increased action potential half-widths in both groups, suggesting that other 4-AP-sensitive currents that underlie action potential repolarization were unaffected by hypoxia treatment. Action potential amplitudes were not significantly different between groups. (C) Summary bar graphs show no differences in resting membrane potential between control and hypoxia-treated groups. 4-AP comparably depolarized RMP in both groups (P<0.001, paired t-test), and this effect also was not different between the control and hypoxia-treated groups. (D) Current-clamp recordings before and after 4-AP showed that the 4-AP-induced depolarization of the resting potential observed in both groups was sufficient to elicit spontaneous action potential firing in DGC neurons.