AMPA receptor antagonist NBQX attenuates later-life epileptic seizures and autistic-like social deficits following neonatal seizures

Abstract

Purpose: To determine whether AMPA receptor (AMPAR) antagonist NBQX can prevent early mammalian target of rapamycin (mTOR) pathway activation and long-term sequelae following neonatal seizures in rats, including later-life spontaneous recurrent seizures, CA3 mossy fiber sprouting, and autistic-like social deficits.

Methods: Long-Evans rats experienced hypoxia-induced neonatal seizures (HS) at postnatal day (P)10. NBQX (20 mg/kg) was administered immediately following HS (every 12 h × 4 doses). Twelve hours post-HS, we assessed mTOR activation marker phosphorylated p70-S6 kinase (p-p70S6K) in hippocampus and cortex of vehicle (HS + V) or NBQX-treated post-HS rats (HS + N) versus littermate controls (C + V). Spontaneous seizure activity was compared between groups by epidural cortical electroencephalography (EEG) at P70-100. Aberrant mossy fiber sprouting was measured using Timm staining. Finally, we assessed behavior between P30 and P38.

Key findings: Postseizure NBQX treatment significantly attenuated seizure-induced increases in p-p70S6K in the hippocampus (p < 0.01) and cortex (p < 0.001). Although spontaneous recurrent seizures increased in adulthood in HS + V rats compared to controls (3.22 ± 1 seizures/h; p = 0.03), NBQX significantly attenuated later-life seizures (0.14 ± 0.1 seizures/h; p = 0.046). HS + N rats showed less aberrant mossy fiber sprouting (115 ± 8.0%) than vehicle-treated post-HS rats (174 ± 10%, p = 0.004), compared to controls (normalized to 100%). Finally, NBQX treatment prevented alterations in later-life social behavior; post-HS rats showed significantly decreased preference for a novel over a familiar rat (71.0 ± 12 s) compared to controls (99.0 ± 15.6 s; p < 0.01), whereas HS + N rats showed social novelty preference similar to controls (114.3 ± 14.1 s).

Significance: Brief NBQX administration during the 48 h postseizure in P10 Long-Evans rats suppresses transient mTOR pathway activation and attenuates spontaneous recurrent seizures, social preference deficits, and mossy fiber sprouting observed in vehicle-treated adult rats after early life seizures. These results suggest that acute AMPAR antagonist treatment during the latent period immediately following neonatal HS can modify seizure-induced activation of mTOR, reduce the frequency of later-life seizures, and protect against CA3 mossy fiber sprouting and autistic-like social deficits.

Keywords: AMPA receptor antagonists; Autistic-like behavior; Early life seizures; Epileptogenesis; Hypoxic/ischemic encephalopathy.

Figure 1. NBQX treatment attenuates seizure-induced increases in the mTORC1 signaling pathway

Representative western blots of 12hr post-HS neocortical and hippocampal tissue probed with phospho-p70S6K (Thr389) and p70S6K. The ratio of p-p70S6K:p70S6K in the neocortex was significantly higher in HS+V rats (123.5 ± 3.6%, n=27) than in either C+V (100.0 ± 3.2%, n=26, p=0.0003) or HS+N rats (93.5 ± 5.4%, n=26, p<0.0001). The p-p70S6K:p70S6K ratio was also significantly increased in the hippocampus of HS+V rats (122.8 ± 5.9%, n=20) relative to that in C+V (100.0 ± 6.2%, n=20, p=0.042) and HS+N rats (96.3 ± 8.1%, n=22, p=0.003). *: p<0.05, **: p<0.01, ***: p<0.001. Error bars indicate S.E.M.

Figure 2. Increased frequency of seizure events in post-HS rats is attenuated by treatment with NBQX

(A) Representative spontaneous electrographic seizure recorded from an intracranial cortical electrode in an adult rat post neonatal HS. The cortical EEG recordings show baseline activity prior to seizure onset of seizures (arrow). Insets show the EEG trace with an expanded time scale demonstrating seizure initiation with spikes, progression to the tonic phase with high frequency, large amplitude spikes, and the termination phase of the seizure with smaller amplitude spikes and postictal slowing. Representative cortical EEG recordings from C+V (B) and HS+N (C) rats show predominantly unaltered baseline activity. Scale bars=1s, 250 μV. (D) The frequency of seizures observed per hour of video-EEG recording analyzed significantly increased in HS+V rats (3.23 ± 0.99 seizures/hr, median = 0.20 (IQR of 0.0 to 1.66) seizures/hr, n=11), compared with C+V rats (0.36 ± 0.18 seizures/hr, median = 0.0 (IQR of 0.0 to 0.11) seizures/hr, n=10, p=0.004). Rats that were treated with NBQX following HS were found to have a significantly fewer frequent seizures (0.14 ± 0.06 seizures/hr, median = 0.0 (IQR of 0.0 to 0.08) seizures/hr, n=12, p=0.0004) than HS+V rats. *: p<0.05, **: p<0.01, ***: p<0.001. Error bars indicate S.E.M.

Figure 3. NBQX blocks increased mossy fiber sprouting in hippocampal CA3 hippocampus following neonatal seizures

(A) Photomicrographs of Timm staining in hippocampi of adult, post-HS+V rats show a marked increase in aberrant mossy fiber sprouting into the stratum pyramidale and stratum oriens layers of CA3 as compared to C+V rats. NBQX treatment immediately following seizure induction in HS+N rats decreased Timm staining. Insets show higher magnification of Timm granule distribution in stratum pyramidale and stratum oriens. (scale bar = 250 μm). (B) Average Timm scores, calculated using a semi-quantitative scoring scale, from HS+V rats were significantly higher (2.683±0.15, n=6) than from either C+V (1.635±0.14, n=6, p<0.0001) or HS+N rats (2.14±0.15, n=7, p=0.036). (D) Mean density measurements of Timm staining measured in the stratum pyramidale-oriens confirmed increased aberrant mossy fiber sprouting in HS+V rats (174±11%, n=6) over C+V levels (100±8.7%, n=6, p<0.0001) and the attenuation of this increase in Timm granules following treatment with NBQX in HS+N rats (115±8%, n=7, p=0.0005). *: p<0.05, **: p<0.01, ***: p<0.001. Error bars indicate S.E.M.

Figure 4. NBQX rescues behavioral post-HS social deficits

(A) All rats showed a preference for a novel rat over a novel object in a social preference test. B) When a novel rat replace the novel object, HS+V rats failed to shift their attention from the familiar to the novel rat, showing a deficit in preference for social novelty by spending the same amount of time with the novel rat (71.0±12 sec) as the familiar rat (65.2±14.5 sec; p>0.05), as compared to C+V rats that spent more time with the novel (99.0±15.6 sec) vs. familiar rat (42.0±7.6 sec; p<0.01). NBQX treatment rescued this deficit, as post-HS NBQX-treated rats spent more time with the novel (114.3±14.1 sec) vs. familiar rat (45.6±7.6 sec; p<0.01). (C) Rat performance in the open field assay was comparable between groups, measured by time in center and (D) distance traveled, although NBQX treatment in post-HS rats caused a slight, but not significant, increase in overall distance traveled. (E) Each group demonstrated normal olfaction, including social olfaction (cages 1 and 2), with normal dishabituation to each odor (trials 1, 2, and 3 for each). C=control, H=post-HS, V=vehicle, N=NBQX. *: p < 0.05, **: p < 0.01, ***: p < 0.001. Error bars indicate S.E.M.