Ca2+ currents are thought to enhance glutamate excitotoxicity. To investigate whether reduced expression of the Ca2+ limiting GluR2(B) subunit enhances seizure-induced vulnerability to either CA1 or CA3 neurons, we delivered GluR2(B) oligodeoxynucleotides (AS-ODNs) to the dorsal hippocampus of adult rats before inducing kainate (KA) seizures. After knockdown, no changes in behavior, electrographic activity, or histology were observed. In contrast, GluR2(B) knockdown and KA-induced status epilepticus produced accelerated histological injury to the ipsilateral CA3a-b and hilar subregions. At 8 to 12 h, the CA3a was preferentially labeled by both silver and TUNEL methods. TUNEL staining revealed 2 types of nuclei. They were round with uniform label, features of necrosis, or had DNA clumping or speckled chromatin deposits within surrounding cytosol, features of apoptosis. At 16 to 24 h, many CA3a-c neurons were shrunken, eosinophilic, argyrophilic, or completely absent. Immunohistochemistry revealed marked decreases in GluR2(B) subunits throughout the hippocampus, NR1 immunoreactivity was also reduced but to a lesser extent. In contrast, GluR1 and NR2A/B immunohistochemistry was relatively uniform except in regions of cell loss or within close proximity to the CA1 infusion site. At 144 h, the CA3 was still preferentially injured although bilateral CA1 injury was also observed in some AS-ODN-, S-ODN-, and KA-only-treated animals. Glutamate receptor antibodies revealed generalized decreases in the CA3 with all probes tested at this delayed time. In contrast, GluR2(B) expression was increased within CA1 irregularly shaped, injured neurons. Therefore, hippocampal deprivation of GluR2(B) subunits is insufficient to induce cell death in mature animals but may accelerate the already known CA3/hilar lesion, possibly by triggering apoptosis within CA3 neurons. CA1 and DG survive the first week despite their loss of GluR2(B) subunits, suggesting that other intrinsic properties such as increased Na+ conductance and reduced ability of the GluR2(B) subunit to interact with certain cytoplasmic proteins may be responsible for the augmented cell death rather than changes in AMPA receptor-mediated Ca2+ permeability. Alternatively, changes in allosteric interactions that affect other receptor classes of high density at the mossy fiber synapse (e.g. KA receptors) may augment KA neurotoxicity. Latent GluR2(B) increases in CA1 injured neurons support a role for AMPA receptor subunit alterations in seizure-induced tolerance.

The effects of single versus multiple episodes of status epilepticus on the expression of AMPA receptors during a critical growth spurt are unknown. To determine whether the pattern of hippocampal AMPA receptor subunit expression depends upon the age of the animal, timing and number of perinatal seizures, we characterized maturational changes in AMPA receptor protein levels of the hippocampus with immunohistochemistry and Western blotting in rats of juvenile ages with and without a history of neonatal seizures. Kainic acid (KA) was used to induce a single episode of status epilepticus (1 × KA) in rats on P20 or P30. Animals with a history of multiple seizures (3 × KA) were given KA on P6, P9, and then on P20 or P30. After 1 × KA, in P20 and P30 rats that are preferentially sensitive to CA1 damage, GluR1 immunoreactivity was depleted remarkably in CA1 stratum pyramidale and stratum lucidum and only morphologically healthy cells were faintly labeled. At P30, GluR2 subunit expression was nearly absent in the healthy cells and increased within the injured CA1 neuronal population. Western blot analysis confirmed that the GluR1/GluR2 ratio was decreased at P20 and further decreased at P30. A history of perinatal seizures (3 × KA) prevented the age-dependent alterations in the CA1. Except for areas of cell loss, NR1 and NR2A/B antibody labeling was relatively stable throughout the hippocampus at both ages and conditions examined. Data suggest that (i) Ca²⁺ permeable AMPA receptors may not be responsible for neuronal injury or irreversible cell loss and that (ii) the expression of AMPA receptors after status epilepticus depends upon the age of the animal, the timing of the first insult and subsequent formation of AMPA receptor subunit compositions within specific populations of hippocampal neurons.

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NMDA receptors (NMDARs) are important for the propagation of seizures. To understand the role of NR1 subunits in the propagation of seizures we knocked down the NR1 subunit by intracranial injection of antisense deoxyoligonucleotides (NR1-AS-ODNs) into the right hippocampus during a window of maximal seizure susceptibility in development. Control missense and sense ODNs followed by focal injection of NMDA (2.5-25 nmoles) into the hippocampal CA1 and sensorimotor cortex of P15 rat pups resulted in behavioral and electrographic (EEG) seizures. After NR1 knockdown, low- and high-doses produced little or no spike activity in the hippocampus and overlying sensorimotor cortex as predicted. Despite reduced activity in the hippocampal and cortical EEG, intracranial NMDA or peripheral kainate (KA)-induced seizures led to paradoxical cell death of CA1 neurons, which is not typically observed in this age group. Histological changes were modest or absent in the cortex away from the infusion site. Signal specificity of the targeted CA1 or cortex was observed in autoradiograms, immunohistochemistry and Western blots. After knockdown, Ca2+ influx was suppressed as both NMDA and muscimol-stimulated Ca2+ permeability of the immature CA1 was blocked in ex-vivo slices measured with FURA-2AM optical dye imaging. Data suggest that certain constituent levels of NMDA receptors distributed on excitatory and/or inhibitory interneurons may be developmentally required for survival of CA1 pyramidal neurons during a critical period when ictal activity is present. Moreover, selective NR1 subunit downregulation simultaneously reduces NMDA and GABA A receptor Ca2+ ion permeability properties that may contribute to a premature cell death mechanism.

Accumulating lines of evidence suggest that glycogen synthase kinase-3β (GSK-3β) is involved in aging. However, the effects of GSK-3β on cardiac aging and the underlying mechanisms remain to be elucidated. Autophagy, a protective mechanism in aging, decreases with age. We hypothesized that GSK-3β attenuates cardiac aging via Ulk1, a regulator of autophagy, and studied constitutively active GSK-3βS9A knock-in mice (βKI), GSK-3βS9A/Ulk1+/- bigenic mice (Bigenic), and GSK-3β+/- mice (βKO) up to 24 months (M) of age. Left ventricular (LV) weight/body weight (LVW/BW, mg/g) was not significantly different among wild-type mice (WT), βKI and βKO at 6M. It was lower in βKI (2.4±0.1, p<0.005) and higher in βKO (4.8±0.8, p<0.05) than in WT (3.8±0.2) at 24M. Cardiomyocyte cross-sectional area (CSA, μm2) was smaller in βKI (360±9, p<0.001) but bigger in βKO (540±11, p<0.01) than in WT (502±5) at 24M. The LVW/BW was greater (3.5±0.2, p<0.001) and the CSA was bigger (527±4, p<0.001) in Bigenic than in βKI at 24M. These data demonstrate that GSK-3β inhibits age-dependent cardiac hypertrophy via Ulk1. Cardiac fibrosis (%) was more in βKO (5.4±0.1, p<0.001) and less in βKI (2.4±0.1, p<0.001) than in WT (4.0±0.3) at 24M. There was much more fibrosis in Bigenic (5.5±0.6, p<0.001) than in βKI at 24M. These data show that GSK-3β reduces age-related cardiac fibrosis via Ulk1. LV end-systolic elastance (Ees, mmHg/μl) and chamber stiffness constant (CSC, μl-1) were not significantly different among WT, βKI, and βKO at 6M. At 24M, the Ees was lower in βKO (4±1, p<0.05) and higher in βKI (12±3, p<0.05) than in WT (7±0), and the CSC was higher in βKO (0.19±0.01, p<0.001) and lower in βKI (0.06±0.01, p<0.001) than in WT (0.14±0.01). The beneficial effects of GSK-3β on cardiac function were abolished in the Bigenic, indicating that GSK-3β prevents age-specific cardiac dysfunction via Ulk1. The level of p62, a protein degraded by autophagy, was lower in βKI and higher in βKO than in WT. The numbers of autophagosomes and autolysosomes were significantly greater in βKI/tfLC3 (tandem fluorescent mRFP-GFP-LC3) mice than in tfLC3 or βKI/tfLC3/Ulk1+/- mice. These data suggest that GSK-3β activates autophagy via Ulk1. In conclusion, GSK-3β attenuates cardiac aging by activating Ulk1-dependent autophagy.