Decreased glutamate receptor 2 expression and enhanced epileptogenesis in immature rat hippocampus after perinatal hypoxia-induced seizures

Abstract

Hypoxic encephalopathy is the most common cause of neonatal seizures and can lead to chronic epilepsy. In rats at postnatal days 10-12 (P10-12), global hypoxia induces spontaneous seizures and chronically decreases seizure threshold, thus mimicking clinical aspects of neonatal hypoxia. We have shown previously that the acute and chronic epileptogenic effects of hypoxia are age-dependent and require AMPA receptor activation. In this study, we aimed to determine whether hypoxia-induced seizures and epileptogenesis are associated with maturational and seizure-induced changes in AMPA receptor composition and function. Northern and Western blots indicated that glutamate receptor 2 (GluR2) mRNA and protein expression were significantly lower in neocortex and hippocampus at P10-12 compared with adult. After hypoxia-induced seizures at P10, GluR2 mRNA was significantly decreased within 48 hr, and GluR2 protein was significantly decreased within 96 hr. AMPA-induced Co(2+) uptake by neurons in hippocampal slices indicated higher expression of Ca(2+)-permeable AMPA receptors in immature pyramidal neurons compared with adult. In slices obtained 96 hr after hypoxia-induced seizures, AMPA-induced Co(2+) uptake was significantly increased compared with age-matched controls, and field recordings revealed increased tetanus-induced afterdischarges that could be kindled in the absence of NMDA receptor activation. In situ end labeling showed no acute or delayed cell death after hypoxia-induced seizures. Our results indicate that susceptibility to hypoxia-induced seizures occurs during a developmental stage in which the expression of Ca(2+)-permeable AMPA receptors is relatively high. Furthermore, perinatal hypoxia-induced seizures induce increased expression of Ca(2+)-permeable AMPA receptors and an increased capacity for AMPA receptor-mediated epileptogenesis without inducing cell death.

Fig. 1.

GluR2 mRNA expression was found to be relatively low during the age window of susceptibility to hypoxia-induced seizures. Shown is a representative Northern blot comparing GluR2 mRNA levels in P10–12 rat brain with those of the adult. Note the lower expression of GluR2 mRNA in both the hippocampus and neocortex of rat at ages P10 and P12 compared with adult (P60). GAPDH was used as a control and was not significantly different across lanes.

Fig. 2.

GluR2 mRNA was significantly decreased within 48 hr after hypoxia-induced seizures at P10. Shown is a representative Northern blot comparing GluR2 mRNA levels in P14 rat brain from control animals and animals that experience hypoxia-induced seizures at P10. In hippocampus, GluR2 mRNA showed a progressive, significant decline with time, to a maximum decrease to 50% of control at 48 hr after hypoxia (p < 0.05, ANOVA).

Fig. 3.

GluR2 mRNA was significantly decreased in the hippocampal pyramidal cell layers after perinatal hypoxia-induced seizures. In situ hybridization showed that GluR2 mRNA was significantly decreased in the pyramidal cell layers within 48 hr after hypoxia-induced seizures at P10. The most significant decrease was observed in CA1/CA2 pyramidal cells (arrows).

Fig. 4.

GluR2 protein expression was found to be relatively low at an age when susceptibility to hypoxia-induced seizures is highest. Shown is a representative Western blot comparing hippocampal GluR2 protein expression in P10 rat with that in the adult (P60). GluR2 expression was clearly less in immature rat hippocampus compared with adult.

Fig. 5.

GluR2 protein expression was significantly decreased within 96 hr after hypoxia-induced seizures. Shown are Western blots comparing GluR1 and GluR2 expression. GluR2 protein expression was significantly decreased in hippocampus within 96 hr after hypoxia treatment. In contrast, GluR1 expression did not change significantly after hypoxia-induced seizures.

Fig. 6.

Co²⁺ staining showed that the lower GluR2 expression in the immature hippocampal pyramidal cell layers was associated with increased numbers of divalent-permeable AMPA receptors. A, High-power micrograph of the CA1/CA2 pyramidal cell layer in a section of Co²⁺-stained P10 hippocampus; B, same for a P60 animal. Clearly, a large number of pyramidal neurons in the P10 hippocampus exhibited AMPA-induced Co²⁺ uptake, whereas no uptake was observed in pyramidal cells of the adult hippocampus. Scale bar, 20 μm.

Fig. 7.

Co²⁺ staining revealed increased AMPA-induced Co²⁺ uptake by pyramidal neurons in P14 hippocampal slices from animals that experienced hypoxia-induced seizures at P10 (B) compared with age-matched controls (A). The numbers of Co²⁺-positive neurons intersecting a 250 μm straight line segment drawn through the CA1 pyramidal cell layer of each section were counted and averaged (6 sections per animal). The average numbers of Co²⁺-positive neurons counted in this manner were 4.3 ± 0.8 for the control group (n = 5 animals) and 12.5 ± 2.3 for the hypoxia-treated group (n = 5; p< 0.02). Additionally, coapplication of the selective antagonist 1-napthyl-acetyl-spermine (100 μm) clearly inhibited AMPA-induced Co²⁺ uptake (C), indicating that the Co²⁺ entry was at least in part through divalent-permeable AMPA receptors. Scale bar, 20 μm.

Fig. 8.

I–V relationships for kainate-evoked currents showed increased inward rectification in CA1 pyramidal neurons from animals that experienced hypoxia-induced seizures. I–V curves were generated by subtracting the current responses to continuous voltage ramps without kainate from those with kainate in the bath. Representative subtracted currents are shown for one neuron from the control group (A) and one from the hypoxia-treated group (B). The magnitude of rectification in the I–V curves was determined by taking the ratio of the slopes fitted over the region of +20 to +40 mV to that over the region of −60 to −40 mV.C, The bar graphs indicate the mean rectification ratios determined in this manner and illustrate the increase in the magnitude of inward rectification forI–V curves obtained from the hypoxia-treated group.

Fig. 9.

Repeated tetanic stimulation in the presence of the NMDA receptor antagonist APV (100 μm) revealed that hippocampal slices from rat pups that experienced hypoxia-induced seizures 4–9 d earlier (at P10) had the capacity for purely AMPA receptor-mediated epileptogenesis, whereas control slices did not. Shown are representative field recordings from a control slice and a slice from a same-age hypoxia-treated animal. Tetanus-induced afterdischarges were always observed in the presence of APV but increased with repeated stimulation only in the majority of slices (7 of 10) from hypoxia-treated animals. This type of increase was observed in only 1 of 10 control slices.

Fig. 10.

ISEL revealed no cell injury at any point within 1 week after perinatal hypoxia-induced seizures at P10. High-power micrographs of the hippocampal CA1 region show no ISEL-positive cells at 24 hr (A), 48 hr (B), and 7 d (C) after hypoxia-induced seizures.D, Positive control hippocampal CA1 region from a rat that experienced kainate-induced status epilepticus at P45, 3 d earlier.