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Neonatal seizures can lead to later life epilepsy and neurobehavioral deficits, and there are no treatments to prevent these sequelae. We previously showed that hypoxia-induced seizures in a neonatal rat model induce rapid phosphorylation of S831 and S845 sites of the AMPA receptor GluR1 subunit and later neuronal hyperexcitability and epilepsy, suggesting that seizure-induced post-translational modifications may represent a novel therapeutic target. To unambiguously assess the contribution of these sites, we examined seizure susceptibility in wild type mice versus transgenic knock-in mice with deficits in GluR1 S831 and S845 phosphorylation (GluR1 double phosphomutant (GluR1DPM) mice). Phosphorylation of the GluR1 S831 and S845 sites was significantly increased in the hippocampus and cortex following a single episode of pentyleneterazol (PTZ) induced seizures in postnatal day 9 (P9) wild type mouse pups, and that transgenic knock-in mice have a higher threshold and longer latencies to seizures. Like the rat, hypoxic seizures in P9 C57BL/6N wild type mice resulted in transient increases in GluR1 S831 and GluR1 S845 phosphorylation in cortex, and were associated with enhanced seizure susceptibility to later-life kainic acid induced seizures. In contrast, later-life seizure susceptibility following hypoxia-induced seizures was attenuated in GluR1 DPM mice, supporting a role for post-translational modifications in seizure-induced network excitability. Finally, human hippocampal samples from neonatal seizure autopsy cases also showed an increase in GluR1 S831 and S845, supporting the validation of this potential therapeutic target in human tissue.
Epilepsy affects approximately 65 million people worldwide and seizure susceptibility is high in the neonatal period (Hauser et al., 1993), with an estimated incidence of 2–5 per thousand live births (Ronen et al., 2007). Early life seizures can lead to development of epilepsy and other neurological deficits in adult life (Ben Ari and Holmes, 2006; Ronen et al., 2007). To date, there are only seizure-suppressing drugs but no cure to modify epileptogenesis or the associated psychiatric or cognitive comorbidities that develop in later life (Jensen, 2011). Understanding the molecular mechanisms involved in the effect of early life seizures on synaptic function, including epileptogenesis, will be critical in developing appropriate therapies targeted at preventing these long-term sequelae.
We have recently demonstrated an early and reversible enhancement of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptor (AMPAR) expression and function in hippocampal and cortical neurons following seizures in young rats (Rakhade et al., 2008; Zhou et al., 2011). Similar to the clinical disease, experimental early life seizures in rodents result in long-term epilepsy and cognitive sequelae (Chen et al., 1999; Sogawa et al., 2001; Jensen, 2011; Zhou et al., 2011), and even a single neonatal seizure may permanently alter glutamatergic synapses (Cornejo et al., 2007; Zhou et al., 2011). Furthermore, in this early neonatal period of development, AMPARs are largely Ca2+-permeable due to subunit composition (Sanchez et al., 2001; Kumar et al., 2002). Importantly, seizures in the immature rat lead to transient increases in phosphorylation at the AMPAR GluR1 subunit serine-831 (S831) and serine-845 (S845) sites and this is associated with increases in AMPAR-mediated synaptic currents (Rakhade et al., 2008).
Dynamic activity-dependent alterations and trafficking of AMPARs to and from the synaptic surface are thought to underlie changes in synaptic strength (Shepherd and Huganir, 2007; Heine et al., 2008). The strength of synaptic transmission in intact neuronal networks can be regulated by AMPAR function mediated by phosphorylation of GluR1 S831 and S845 subunit sites, as is observed in long-term potentiation (LTP) (Barria et al., 1997; Lee et al., 2000; Lee et al., 2003). Indeed, in a rat model of early life seizures, alterations in GluR phosphorylation are associated with impaired LTP, partly due to a reduction in available NMDA-only “silent synapses” due to insertion of GluR1 subunit at the synapse (Zhou et al., 2011), and autism-like behavioral abnormalities (Talos et al., 2012b). Furthermore, systemic administration of an AMPAR antagonist within the first 48 hours post seizure suppressed these early changes as well as prevented later life impairments in LTP and increased seizure susceptibility (Rakhade et al., 2008; Zhou et al., 2011).
To unambiguously identify a role for AMPAR phosphorylation in promoting long-term neurological deficits following early life seizures, we studied the effects of neonatal seizures in GluR1 double phosphomutant transgenic knock-in mice with mutations introduced at GluR1 S831 and S845 (hereafter referred to as GluR1 double phosphomutant or GluR1 DPM mice) (Lee et al., 2003). Previous reports have demonstrated that the GluR1 DPM mice have impaired spatial memory, deficits in reinforcement of repetitive learning, emotion-enhanced learning and reinforcement of addiction to cocaine and morphine (Hu et al., 2007; Billa et al., 2009).
In this study, we compared the GluR1 DPM and wild type mice to determine if the lack of ability to phosphorylate these sites subacutely altered seizure susceptibility to pentylenetatrazol (PTZ) and hypoxia. We assessed seizure-induced increases in hippocampal neuronal excitability and AMPAR mediated excitatory postsynaptic currents (EPSCs), in addition to later life seizure susceptibility. Finally, we examined phosphorylation of S831 and S845 in postmortem human brain tissue from patients with neonatal seizures compared to controls. These studies were performed to provide evidence for seizure-induced phosphorylation of GluR1 as a potential target for antiepileptogenic therapy.
Mice with serine to alanine mutations of GluR1 S831 and S845 phosphorylation sites (referred to hereafter as GluR1 DPM mice) were generated as described previously (Lee et al., 2003). Mutation sites were verified using phosphorylation-selective antibodies against GluR1. WT and GluR1 DPM (homozygous) mice with C57BL/6N hybrid genetic background were used for all experiments. All experiments were performed on mice aged postnatal day 5–40 (P5–40), which had been weaned at P21 and maintained on a 12:12 hour light/dark schedule. In experiments in which no mutant mice were used for comparing ontogenic expression of neurotransmitter receptors, wild type C57 BL6/N mice subjects were obtained from commercial vendors (Charles River Laboratories, Burlington MA). All of the electrophysiological experiments comparing WT with GluR1 DPM mice were performed blinded to the genotype of the mice being recorded. All procedures related to animal care and treatments conformed to the guidelines and policies; and were approved by the Animal Care and Use Committee of Children’s Hospital Boston.
The critical period of developmental plasticity with an imbalance between cortical excitation and inhibition has been previously described as a factor in determining the onset of neonatal seizures (Silverstein and Jensen, 2007; Rakhade and Jensen, 2009). This critical period is defined by the expression of neurotransmitter receptors and ion transporters in the neocortex. The transition between the expression pattern of these receptors and transporters from an immature to a more mature pattern has been observed to be ontogenitically conserved across rodent species. As much of prior work on hypoxic seizures (HS) has been conducted in a rat model, a goal of this study was to develop mouse models in an analogous age window. To establish the critical period of this transition and the time window for the initiation of early life seizures in the C57BL/6 mouse, at postnatal days (P)5–10, we studied the expression pattern of the ionotropic glutamate receptors for both the AMPA and NMDA-subtype including GluR1, GluR2, NR1, NR2A and NR2B. We also studied the expression of the GABA subtype receptors GABA Aα1 and GABA Aα4 as well as the chloride ion transporters NKCC1 and KCC2 (Figure 1). GluR1 expression within this time window was observed to be highest at P9 (154 ± 24%, n=8, p<0.05) with a decrease in expression with increasing age. GluR2 receptor expression continued to increase with age from P5 to P10. GluR2 receptor expression levels at P5–7 were significantly lower (48 ± 21%, n=5, p<0.05) compared to the levels at P10. Similarly, NR2A receptor expression levels at P5 were significantly lower (49 ± 6%, n=5, p<0.05) compared to the levels at P10, and increased gradually in the intervening time window. The expression levels of NR2B receptor subunit at P5 (105 ± 12%, n=5) were not significantly different from the expression levels at P10. The expression levels of KCC2 at P5 were significantly lower (21 ± 8%, n=5, p<0.05) as compared to the levels at P10. Similarly, the expression levels of GABA Aα1 at P5 were significantly lower (10 ± 5%, n= 5, p<0.05) as compared to the levels of P10. The expression levels of GABA Aα4 subunit were peaking at P9 (136 ± 9%, n=5, p<0.05) as compared to the levels of P10, and decreased significantly later in life. The neonatal age window between P7 and P9 displayed the maximal transition in these receptors and transporters. Based on the neurotransmitter and ion transporter expression patterns, we chose the time window between P7–P9 for seizure induction, given the similarities to the P10 Long Evans rat developmental expression.
For chemoconvulsant seizures PTZ (50 mg/kg i.p.) was administered to P7 mice. P7 was chosen since data showed that administration of PTZ leads to induction of spike and wave epileptic activity in the mice pups (Velisek et al., 1992). The severity of convulsive responses was videotaped and then classified by a blinded investigator according to the Racine scale: 0, no response; 1, facial jerks, pawing; 2, nodding, wet-dog shakes, myoclonic jerks; 3, forelimb clonus; 4, loss of posture, hind limb tonic-clonic movements; 5 status epilepticus and death.
For seizure induction by hypoxia, pilot data showed that optimal seizures in C57BL/6 were obtained at P9. Hypoxic seizures were induced by graded global hypoxia administered for 40 min. Briefly oxygen concentration was maintained alternately at 9% for 5 minutes, and a reduced concentration (6%, 5.5%, 5% and 5% sequentially) for 5 minute periods for a total duration of 40 min before termination of hypoxia. Seizures were recorded with video-monitoring equipment and severity and latency were measured. Littermate controls were kept at room air. For all groups, normothermic body temperature was maintained at 32–34° C on a circulating water heating pad. For both sets of seizure induction methods, the entire mouse litter was returned to their dams within one hour of initiating the experiment. Difference in seizure induction in the different groups studied was assessed using Chi-Square test.
Mouse pups exposed to early life seizures were allowed to survive into adulthood and latency to chemoconvulsant induced seizures (kainic acid (KA) 35 mg/kg i.p.) was measured at P40. Mouse pups were divided into four groups; wild type naïve mice without seizures, wild type mice that had experienced hypoxic seizures at P9, GluR1 DPM naïve mice with no seizures and GluR1 DPM mice that had experienced hypoxic seizures at P9. Kainic acid (KA) seizures have been extensively documented and reported previously, the severity of the KA seizures was classified according to Racine scale described above. Video recordings of the seizures were performed to measure the latency to the first seizure in the appropriate severity scale. Time to the first behavior was calculated to determine the latency to seizure onset, data was normalized within each litter, with the time to latency to first behavioral seizures in the wild type controls without seizures at P9 as a normalizing control.
Hippocampal slices were prepared and whole cell recordings were obtained from acute hippocampal slices prepared from mouse pups as described in detail previously (Sanchez et al., 2005a; Zhou et al., 2011). Hippocampal slices from GluR1 DPM and wild type mice were used for electrophysiological recordings at 24 hours following the PTZ-induced seizures. Mouse pups were decapitated at 24 hours after PTZ-treatment induced neonatal seizures with procedures in accordance with guidelines set by the institutional animal care and use committee, age-matched mouse pups were used as controls for both GluR1 wild type and GluR1 DPM mice. Wild type littermate mice that had not been exposed to PTZ were used as baseline controls. We focused on the sub-acute time period following induction of PTZ seizures for determining changes in hippocampal hyperexcitability, allowing for the washout of residual PTZ from the brain prior to ex-vivo measurements of hippocampal excitability (Ramzan and Levy, 1985). Mouse brains were rapidly dissected from the skull and placed for sectioning in ice-cooled cutting solution bubbled with 95% O2/5% CO2 at 4°C. Coronal hippocampal slices (300 µm thickness) were sectioned from the middle third of hippocampus with a vibratome (WPI, Sarasota, FL) in cutting solution containing 210 milli Molar (mM) sucrose, 2.5 mM KCl, 1.02 mM NaH2PO4, 0.5 mM CaCl2, 10 mM MgSO4, 26.19 mM NaHCO3, and 10 mM D-glucose, pH 7.4. Slices were incubated in oxygenated artificial CSF (ACSF; composition as described previously) (Rakhade et al., 2008; Zhou et al., 2011) and remained at 32°C for 30 min. Slices were maintained at room temperature for at least 1 hour before electrophysiological recordings were performed at 32°C.
Whole-cell patch-clamp recordings were made from CA1 pyramidal neurons in hippocampal brain slices using infrared/differential interference contrast microscopy as described previously (Zhou et al., 2011). All recordings were performed after a 1 hour incubation period, allowing for washout of any systemically administered drugs (Kapus et al., 2000). The patch-pipette internal solution contained (in mM) 110 Cs-methanesulfonate, 10 tetraethylammonium-Cl, 4 NaCl, 2 MgCl2, 10 EGTA, 10 HEPES, 4 ATP-Mg, and 0.3 GTP, pH 7.25, with QX-314 (N-2,6 dimethyl phenylcarbamoylmethyl) triethylammonium chloride) and creatine phosphokinase (17 unit/ml). Filled electrodes had resistances of 2–5 MΩ. AMPAR-mediated EPSCs were pharmacologically isolated by blocking GABA and NMDA receptors with picrotoxin (60 µM) and DL-AP-5 (100 µM), respectively. TTX (1 µM) was added to the ACSF to record miniature EPSCs (mEPSCs). All recordings were performed at 32°C. Briefly, mEPSCs were detected automatically using Clampfit 9.2 (Molecular Devices, Sunnyvale, CA), and frequency and amplitude histograms were constructed using this program as described previously (Wyllie and Nicoll, 1994).The threshold for detection of mEPSC events was set at 5 pA. This threshold remained constant throughout the analysis of whole experiments for all recordings. All detected mEPSCs were visually checked for a monotonic rising phase, and an approximately exponential decay time course.
For comparing the ontogenic expression pattern of AMPARs, GluR1 DPM mice and wild type littermates were sacrificed at postnatal day (P)5, P6, P7, P8, P9 and P10. GluR1 DPM mice and their wild type littermate controls were killed at 1, 3, 6, 12, 24 and 48 hours after PTZ-induced seizures were induced at P7. Brain tissue was dissected out immediately; and cortical and hippocampal regions were separated under a dissecting microscope. Tissue was then rapidly frozen in ethanol and stored at −80°C until used for protein extraction. Similarly procedure was followed for collection of brain tissue at 1, 12, 24 and 48 hours following hypoxia-induced seizures at P9. Membrane protein samples from the anterior 2/3rd of cortex and the entire hippocampal tissue were prepared as described previously (Wenthold et al., 1992; Talos et al., 2006). Complete Mini Protease Inhibitor Cocktail Tablet (Roche, Pleasonton CA) HALT phosphatase inhibitor tablet (Sigma-Aldrich, St. Louis MO) and phosphatase inhibitors PMSF (10 mM) were added to inhibit proteases and phosphatases. Total protein concentrations were measured using Bradford protein assay (Bio-Rad, Herculus, CA), and samples were diluted for equal amounts of protein in each sample. Samples were electrophoretically separated on 7.5% Tris-HCl gels and transferred to polyvinylidene difluoride membranes. Blots were blocked and incubated with primary and secondary antibodies. Phosphospecific antibodies raised against GluR1 S831 (1:1000 dilution), GluR1 S845 (1:1000 dilution) and GluR2 S880 (1:1000 dilution) (Millipore, Billerica, MA) were used in immunoblotting studies. The membranes were stripped using Restore Stripping buffer (Thermo Scientific, Rockford, IL) as per the manufacturers’ protocols and reprobed with antibodies raised against GluR1 subunits (Millipore Corporation, Billerica, MA, 1:1000 dilution); GluR2 (Millipore Corporation, Billerica, MA, 1:1000 dilution), NR1 and NR2 (Millipore Corporation, Billerica, MA 1:1000 dilution), Billerica, MA), NKCC1 (Millipore Corporation, Billerica, MA, 1:500 dilution) and KCC2 (Abcam Corp., Cambridge, MA, 1:500 dilution); GABA Aα1 and GABA Aα4 (Millipore Corporation, Billerica, MA, 1:1000 dilution), PSD-95 (Cell Signaling Inc., Danvers MA, 1:1000 dilution) as described previously (Talos et al., 2006; Rakhade et al., 2008). Appropriate anti-mouse or anti-rabbit IgG antibodies (Pierce; 1:5000 dilution) were used, and immunodetection was effected using Super-West Femto Maximum Sensitivity Substrate reagent (Thermo Scientific, Rockford, IL). Digital images were recorded using the Fuji Image LAS 4000 (Fujifilm, Valhalla, NY) chemiluminescence detection system. Densitometric analysis of the digital images was performed using Fuji Film MultiGauge image-analysis software to measure the optical signal density from each sample. The amount of phosphorylation observed was standardized to the amount of receptor subunit present in each sample.
Human parietal–occipital lobe specimens were collected from neonatal and pediatric autopsy populations. Cases ranged from 2 days after birth (neonatal period) to 6 months of age (n=6; 4 males and 2 females). Brain tissue was obtained from cases from the University of Maryland Brain and Tissue Bank for Developmental Disorders (Table 1). The samples were obtained from standard diagnostic postmortem examinations, and all procedures and experiments were conducted under guidelines approved by the Clinical Research Committee at all institutions. The causes of death are listed in Table 1. When possible, the postmortem interval was limited to ≤24 h, the post-mortem interval durations are provided in Table 1.
Group data were expressed as mean ± SE of mean, and n is the number of mice for a given data point. Statistical significance was defined as p<0.05.
Protein bands were visualized with enhanced chemiluminescence (Pierce) using the Image Reader LAS-3000 system and densitometric analysis was performed using Image Gauge v3.0 software (Fujifilm) as described above. Normalized values for expression of phospho-protein / total protein (for wild-type and GluR1 DPM mouse brain tissue run on the same blot with multiple time points) were expressed as a percent of the mean. Expression of neurotransmitter receptors was similarly calculated as percent of the mean compared to expression of the receptor observed at P10. Expression of β-actin was used for normalization for equal protein loading between samples. Data across multiple time points after induction of seizures were compared with matched seizure-naïve litter mate control animals. One-way ANOVA followed by post hoc Tukey’s test were used for multiple comparisons across time points. For the immunoblots comparing receptor phosphorylation in brain tissue from human subjects experiments two tailed t-tests were used for assessing statistical significance.
Latency to behavioral seizures at Stage 1– 4 were measured in minutes for individuals within each litter. The latency to seizure induction was normalized within each litter for mice subjected chemoconvulsant induced seizures. Survival curves were plotted using GraphPad Prism (GraphPad Software, La Jolla, CA). Statistical significance was assessed using Maltel-Cox log rank test comparing the survival curves.
Statistical significance for differences in the distribution of the mEPSCs for the GluR1 DPM and wild type mice was assessed using one-way ANOVA test, t test and Kolmogorov-Smirnov test (K-S test) as specified in the results.
We hypothesized that the increase in phosphorylation of GluR1 receptor subunit may be a pathological response shared in multiple models of early life seizures and may play a critical role in epileptogenesis promoting increased hyperexcitability and synaptic potentiation. Prior studies implicating a role for AMPARs in mediating early life seizures employed a model using P10 Long Evans rat (Silverstein and Jensen, 2007; Rakhade et al., 2008; Rakhade and Jensen, 2009; Zhou et al., 2011). As the GluR DPM mouse is developed on a background strain of C57BL/6N, we determined that the analogous age window in the C57BL/6N mouse was P7–P9, based on the developmental expression pattern of neurotransmitter receptors and ion transporters (Figure 1).
In P7–P9 wild type mice, we next examined the effect of seizures induced by the chemoconvulsant PTZ, on GluR S831 and Glur1 S845 phosphorylation state in the cortex and hippocampus. Systemic injections of PTZ (50 mg/kg i.p.) caused spike and wave discharges (Velisek et al., 1992) and these behavioral seizures were scored using the Racine seizure severity scale. In cortex, GluR1 S831 phosphorylation increased as early as 1 hour compared with naïve littermate controls, and this increase was maximal at 3 hours after Racine stage IV PTZ-induced seizures (152 ± 19%; n=7; p<0.01) before returning to baseline at 24 hours (Figure 2A). Similarly, GluR1 S845 phosphorylation in cortex increased by 1 hour after PTZ-induced seizures and peaked 3 hours after seizures (147 ± 16%; n=7; p<0.01), before returning to baseline at 24 hours (Figure 2B). Similar to neocortex, hippocampal tissue showed maximal increase in phosphorylation at 1 hour following PTZ seizures for both GluR1 S831 (159 ± 23%, n=6, p<0.05) (Figure 2C) and GluR1 S845 (302 ± 83%, n=6, p<0.05) (Figure 2D). Taken together, these data suggest that seizure-induced increases in phosphorylated GluR1 S831 and S845 in wild type mice were consistent with increased phosphorylation observed in Long Evans rats post-seizure (Rakhade et al., 2008).
We next evaluated the effects of seizures in the P7 GluR1 DPM transgenic mouse model. PTZ-induced seizures in the GluR1 DPM mice reached the same final level of severity (4.05 ± 0.12) compared to wild type mice (3.96 ± 0.17) (Figure 3A). However, the latency to first behavioral seizure (Stage 1) following administration of PTZ (50 mg/kg i.p.), was increased in the GluR1 DPM mice (median 188 ± 18%, n=23, p<0.001) compared to littermate wild type controls (median 100 ± 12%, n=22 (Figure 3B). Similarly, latency to onset of hind limb clonus (Stage 4 seizures) was also significantly increased (median 204, n=22, p<0.001) compared to littermate wild type controls (100 ± 17%, n=23) (Figure 3C). These data suggest that the lack of phosphorylation at the GluR1 S831 and S845 sites decreases seizure susceptibility but does not render these mice incapable of sustaining a PTZ-induced seizure.
To determine whether the alterations observed in seizure latency reflected alterations in baseline expression of AMPARs in the GluR1 DPM mice, we studied the expression of membrane GluR1 and GluR2 in the cortex of GluR1 DPM and age-matched wild type littermates. At P7, the expression of the GluR1 was not different in the GluR1 DPM mice (74.6 ± 8% normalized to expression at P10, n=6) compared to wild type mice (81 ± 6% normalized to expression at P10, n=6). Similarly, the expression of the GluR2 receptors at P7 in the GluR1 DPM mice (59 ± 6% normalized to expression at P10, n=6) was unchanged compared to wild type controls (63± 7% normalized to expression at P10, n=6). Overall, comparison of the expression of GluR1 and GluR2 subtype of receptors from P5 to P10 using western blots did not show a significant difference in their expression between the wild type and GluR1 DPM mice (Fig 3D and 3E). These data are consistent with prior observations in these transgenic mice, where there were no changes expression of the AMPA subtype of glutamate receptors in the adult mice (Lee et al., 2003), or in the visual cortex in young adult transgenic mice with GluR1S831A and GluR1 S845A mutations (Goel et al., 2011). While we did not see any changes in overall AMPAR expression, alterations in the expression of other neurotransmitter receptors and signaling proteins involved in maintaining the excitation-inhibition balance following seizures may need to be evaluated in future studies.
In the immature rat, seizure-induced phosphorylation of GluR1 S831 and S845 is associated with an increase in AMPAR-mediated EPSCs following seizures (Rakhade et al., 2008). We thus performed whole-cell patch clamp recordings in CA1 neurons in ex vivo hippocampal slices removed from mice at baseline and after seizures in vivo. Similar to the lack of changes in baseline subunit expression observed above, we found no significant change in the baseline rise time for mEPSCs in wild-type mice (2.33 ± 0.21 ms, n=10) compared to recordings from GluR1 DPM mice (2.12 ± 0.14 ms, n=9, p=0.407). In addition, the baseline decay time for mEPSCs observed in slices from wild type mice (8.56 ± 0.88 ms, n=10) was not significantly different from GluR1 DPM mice (7.01 ± 0.98 ms, n=10, p=0.256). There was no significant difference in baseline mEPSC frequency between wild-type neurons (amplitude: −14.79 ± 1.45 pA, n=12; frequency: 0.175 ± 0.04 Hz, n=12) and GluR1 DPM neurons (amplitude: −18.54 ± 1.18 pA, n=14cells, p = 0.058; frequency: 0.16 ± 0.04, n=14, p= 0.75) (also see Figure 4 D1), although this does represent a trend observed towards increased amplitude of AMPAR-mediated mEPSCs in the immature (P8) GluR1 DPM mice. Consistent with the lack of change in GluR2 expression by immunoblot, there was no significant change in the inward rectification ratios (evoked EPSC amplitude ratio at −60 to 40 mV) between the wild type and GluR1 DPM mice (WT 1.96 ± 0.38, n=7 vs. DPM 2.17 ± 0.20, n=6, t-test p = 0.651). Collectively, these data do not reveal statistically significant alterations in baseline rise time, decay time; amplitude and frequency of AMPAR mediated synaptic currents in the GluR1 DPM mice at this age. Prior results have similarly shown a lack of change in basal synaptic transmission in the adult GluR1 DPM mice (Lee et al., 2003). However, similar to our data showing a trend to increased mEPSC amplitude in the DPM mice, recent studies in layer 2/3 visual cortex at P21–23 of GluR1 S831A and GluR1 S845A mutants show an increase in the basal mEPSC amplitude in AMPAR mediated currents (Goel et al., 2011).
We next studied the ex vivo slices from mice after induction of PTZ seizures for changes in AMPAR-mediated currents in hippocampal CA1 cells to identify alterations in the excitability of the slices obtained from mice experiencing neonatal seizures (Fig 4 A,B). In wild type mice, recordings in CA1 neurons from slices removed 24 hours post seizures from wild type mice showed significantly larger amplitude mEPSCs (amplitude: −20.29 ± 1.39 pA; n=7, p=0.014; frequency: 0.31 ± 0.10 Hz, n=7, p=0.35) compared with those from slices from naive control pups (amplitude:−14.79 ± 1.45 pA, n=12 cells; frequency: 0.175 ± 0.04 Hz, n=12) (Fig 4 C1 and C3). The increased amplitude of mEPSCs at 24 hours following neonatal seizures in the wild type mice suggests an increase in the hippocampal hyperexcitability in this subacute time point, consistent with our prior results in the rat (Rakhade et al., 2008; Zhou et al., 2011). In contrast to recordings from wild type mice, recordings from slices from DPM mice removed at 24 hours post seizures showed a decrease in mEPSCs amplitude (−12.93 ± 1.07 pA, n=8, p=0.002) and frequency (0.17 ± 0.05 Hz, n=8) compared to GluR1 DPM naïve controls (amplitude −18.54±1.18 pA, n=14 cells, p = 0.005; frequency 0.16 ± 0.041, n=14, p= 0.34) (Figure 4 C2 and C4). The data suggest that while HS induces an enhancement of AMPAR function in the wild type mice, similar to the rat (Rakhade et al., 2008), these seizures result in a decrease in AMPAR function in GluR DPM mice. One possibility that we investigated was whether other GluR subunits were differentially modified, most notably the GluR2 subunit as it mediates Ca2+-permeability. Like the rat model, we observed enhanced phosphorylation of GluR2 S880 in the WT mice (132 ± 14% at 1 hour, n=7, p <0.05) following PTZ seizures when compared to naïve WT mice. Similarly, GluR1 DPM mice experiencing neonatal seizures also showed and enhancement (187 ± 19%, n=6, at 1 hour and 167 ± 9%, n=6, at 3 hours following PTZ seizures, p <0.05) compared to naïve GluR1DPM mice (100 ± 14%, n=6). In addition, the increase in GluR2 S880 phosphorylation was greater than that observed in GluR1 DPM mice (187 ± 19%, n=6, p<0.05) than in WT (132 ± 14%, n=7). Given that phosphorylation of this site results in removal of GluR2-subunit containing receptors and increased Ca2+-permeability, other signaling pathways and/or homeostatic mechanisms may be accessed to a greater degree and may underlying the paradoxical decrease in mEPSC amplitude observed in recordings in slices from GluR1 DPM mice, and merit future studies. Finally, no significant differences were observed in the paired-pulse facilitation in slices from wild type mice following PTZ seizures as compared to littermate controls, suggesting that the increase in excitability was most likely mediated by alterations in the post-synaptic component of potentiation (Figure 4E1, E2). Similarly, no significant differences were observed in the inward rectification ratios in slices from GluR1 DPM mice experiencing PTZ seizures (1.75 ± 0.12, n=7) compared to GluR1 DPM mice not experiencing seizures (2.17 ± 0.2, n=6).
In summary, while wild type mice show mEPSC potentiation similar to wild type rat (Rakhade et al., 2008), this enhancement is not observed in the GluR1 DPM mice, which actually exhibit a decrease in mEPSC amplitude following seizures. These data suggest an important role for seizure-mediated S831 and S845 phosphorylation in the acute response to seizures.
In addition to enhanced mEPSCs, another consequence of phosphorylation of GluR1 is its trafficking into the synaptic membrane (Song and Huganir, 2002; Rakhade et al., 2008; Zhou et al., 2011). Synaptic potentiation has been associated with an increase the expression of the scaffolding protein post-synaptic density protein 95 (PSD-95), and with enhanced AMPAR-mediated current amplitudes (Li et al., 1999; Stein et al., 2003; Ehrlich and Malinow, 2004). Turnover of PSD-95 protein that has been described previously in experience-dependent plasticity (El-Husseini Ael et al., 2002). There were no differences in the expression of PSD-95 in P7 GluR1DPM mice (83 ± 7%, n= 5, p = 0.46) at baseline compared to age-matched wild type controls (100 ± 17%, n=5) (Figure 5A). In P7 wild type mice, hippocampal PSD-95 expression was significantly increased as early as 1 hour following PTZ-induced neonatal seizures and was maximal 48 hours post-PTZ seizures (320%, n=6, p<0.05) compared to naïve litter mate controls (Figure 5 A–B). In contrast, this increase in PSD-95 expression was not observed in the litter mate GluR1 DPM mice at 1 hour or 48 hours following seizures (140 ± 27%, n=7, p>0.05) (Figure 5 B–C). In summary, PSD-95 is significantly increased transiently following early life seizures in wild type but not GluR1 DPM mice, supporting a role for GluR1 S831 and S845 phosphorylation in post-seizure modifications at excitatory synapses.
A more subtle but clinically relevant seizure model is that of hypoxia-induced seizures, which has been well established in immature rats (Sanchez et al., 2005b; Silverstein and Jensen, 2007; Rakhade and Jensen, 2009). Exposure to graded global hypoxia for 40 minutes in P7–P9 mice lead to development of behavioral seizures characterized by myoclonic jerks, head shaking, pawing, and eventual progression to loss of posture. The highest incidence of seizures was seen at P9, with seizures in 85% of mice exposed to hypoxia (56/66 animals monitored following graded hypoxia showed the presence of behavioral seizures). Thus, we chose P9 for hypoxic seizure induction in this study. Using this model in wild type mice, hypoxic seizures caused a phosphorylation of GluR1 S831 in cortex that was maximal 24 hours for GluR1 S831 (182 ± 27%; n=6; p<0.05) compared with normoxic controls (100 ± 17%; n=6) (Figure 6 A). Similarly, an increase in GluR1 S845 phosphorylation (158 ± 21%; n=9; p<0.05) was observed 12 hours after the hypoxic seizures in cortex (Figure 6 B). These data demonstrate that similar to the rat, hypoxic seizures during early development can result in increased phosphorylation of GluR1 subunit sites.
We next assessed whether there were differences in acute susceptibility to hypoxic seizures in GluR1 DPM and wild type mice. Compared to wild type mice, seizure incidence was lower in the GluR1 DPM mice (18/32 mice or 56% exhibited behavioral seizures, compared to 85% of wild type mice, Chi-Square test, p<0.05). As early life hypoxia-induced seizures increase later life seizure susceptibility and spontaneous seizures in the rat model (Koh and Jensen, 2001; Rakhade et al., 2011), we similarly compared later seizure susceptibility threshold to KA-induced seizures (35 mg/kg i.p.) in wild type versus GluR1 DPM mice exposed to hypoxia at P9 versus naïve litter mates. Wild type mice with prior hypoxia-induced seizures had a significantly decreased latency to developing KA-induced forelimb clonus (Racine Stage 3 seizures) (68 ± 12% of control, n=14, p<0.05) as compared to normoxic litter mate controls (100 ± 11%, n=16) (Figure 7). In contrast, in GluR1 DPM mice, early life hypoxic seizures did not result in any difference in later life seizure susceptibility as measured by latency to first forelimb clonus (154 ± 21%, n=12) compared to normoxic GluR1 DPM litter mate mice (152 ± 19%, n=12, p=0.4) (Figure 7). These data support the hypothesis that seizure-induced phosphorylation of S831 and S845 critically contributes to later life network hyperexcitability and seizure susceptibility, suggesting a role in epileptogenesis.
As we observed increased phosphorylation of the GluR1 receptor in multiple models of neonatal seizures, we examined if there was evidence for phosphorylation at the GluR S831 or S845 site in human postmortem tissue from cases of neonatal seizures. Western blot analysis of GluR1 receptor subunit expression and its phosphorylation was performed using post mortem hippocampal tissue obtained from infants that had been diagnosed with neonatal seizures secondary to hypoxic encephalopathy (n=3) (see Table 1) and compared to age-matched controls (control tissue obtained from infants < 6 months age at death). Hippocampal samples from neonates with confirmed seizures, showed increased phosphorylation of the GluR1 S831 (296 ± 83%, n=3, p<0.05) compared to brain tissue from autopsy controls (100 ± 19%, p<0.05) and S845 receptor subunit (232 ± 72%, n=3, p<0.05) compared to brain tissue from autopsy controls (100 ± 16%, p<0.05) (Figure 8). These results show phosphorylation of GluR1 S831 and S845 can be measured in human postmortem brain tissue, and supports the experimental animal data that this post-translational modification may be a therapeutic target in human neonatal seizures.
The present study is the first direct evidence to suggest a critical role for post-translational modification of GluR1 S831 and S845 in the genesis of seizure-induced network excitability, and that this may represent a potential therapeutic target in human brain tissue. Early life seizures in wild type C57BL/6N mice pups lead to increased phosphorylation of the GluR1 subunit at S831 and S845 and commensurate increases in AMPAR-mediated mEPSCs in the hippocampal CA1 neurons. Furthermore, seizure-induced phosphorylation of GluR1 S831 and S845 was associated with increases in the synaptic scaffolding protein PSD-95. GluR1 DPM mice lacking the ability to phosphorylate S831 and S845 are less susceptible to PTZ and hypoxia-induced seizures, and lacked the seizure-induced AMPAR potentiation, PSD-95 overexpression, as well as an attenuated long term hyperexcitability that was observed in wild type mice. These data implicate GluR1 phosphorylation as an important step upstream of mechanisms involved in initiation and maintenance of seizure-induced network hyperexcitability. Importantly, there was also evidence of increased phosphorylation of these subunits in human postmortem brain tissue from cases of neonatal seizures compared to control cases.
Infants experiencing neonatal seizures have a significantly higher incidence of development of epilepsy, autism and other cognitive and neurobehavioral disabilities. The pathological processes underlying these changes are likely to be multifactorial, but the imbalance between excitation and inhibition in the neuronal circuits plays a critical role in epileptogenesis (McNamara et al., 2006; Rakhade and Jensen, 2009). Given the relative preponderance of GluR2-lacking, Ca2+-permeable AMPARs during this window, the effects of any change in synaptic plasticity signaling cascades are likely to be accentuated. Here we report a new model and methods for inducing hypoxia-induced seizures in C57BL/6 mice, as previous studies were performed in rats which demonstrated functional enhancement of AMPARs associated with increased phosphorylation of GluR1 S831 and S845 within 24 hours following hypoxic seizures (Rakhade et al., 2008; Zhou et al., 2011). Unambiguous proof of a role for these novel post-translational modifications in epilepsy required re-establishing this model in a transgenic mouse lacking the ability to phosphorylate these sites (Lee et al., 2003). In P7–9 wild type mice, seizures induced by either PTZ or global hypoxia, increased phosphorylation of GluR1 S831 and S845. The increase in GluR1 phosphorylation was observed as early as 1 hour following the PTZ seizures, providing a mechanism for early alterations in the transition of the normal hippocampal circuits into hyperexcitable circuits.
Enhanced excitability and synaptic potentiation mediated by increased synaptic AMPARs and occluded LTP have been previously observed in the hippocampal CA1 neurons immediately following early life seizures in the rat model of hypoxia-induced seizures (Jensen et al., 1998; Rakhade et al., 2008; Zhou et al., 2011). These alterations in synaptic potentiation and hippocampal excitability are not observed following induction of hypoxia alone; seizures are required for initiating these changes (Zhou et al., 2011). In rats, neonatal seizures can lead to later life alterations in plasticity at glutamatergic synapses in the hippocampus (Cornejo et al., 2007), accompanied by alterations in synaptic neurotransmitters, silent synapses and deficits in spatial memory, impaired LTP and learning (Mikati et al., 2005; Zhou et al., 2011). In addition, this study establishes a model of hypoxia-induced neonatal seizures in wild type mice that exhibit similar consequences in post-translational GluR1 modifications and seizure susceptibility as the rat model (Rakhade et al., 2008). Chemically induced seizures in wild type mice also lead to these alterations in hippocampal synaptic excitability and susceptibility to later life seizures.
GluR1 phosphorylation has been shown to result in changes in AMPAR kinetics and amplitude, synaptic trafficking and insertion of AMPARs in the synaptic membrane (Shepherd and Huganir, 2007), and plays a critical role in mediating LTP and long term depression (LTD) following appropriate stimuli (Mammen et al., 1997; Derkach et al., 1999; Derkach et al., 2007). GluR1 phosphorylation at S845 leads to an increase in the reinsertion of GluR1 subunits at the post-synaptic density, and phosphorylation at S831 leads to an increase in the conductance of AMPARs during the induction of LTP. The first two postnatal weeks in rodents is a critical period in development, with multiple changes that affect the balance of excitation and inhibition in the brain (Rakhade and Jensen, 2009). Here, we have observed that acute seizures increase AMPAR-mediated mEPSC amplitude in wild type mice, similar to the results previously described in the rat model of neonatal seizures (Rakhade et al., 2008). However, neonatal seizures in the GluR1 DPM mice result in a decrease in AMPAR-mediated mEPSC amplitude, this paradoxical effect suggesting the phosphorylation of GluR1 receptors plays an important role for mediating the excitability observed 24 hours following the initial seizures. The decrease in mEPSC amplitude observed following PTZ-induced seizures in the GluR1DPM mice, may involve interactions between phosphorylation events that promote homeostatic events such as trafficking and stabilization of the AMPARs via internalization and lysosomal degradation of these receptors (Shepherd and Huganir, 2007; Heine et al., 2008; He et al., 2009). We hypothesize that the synaptic changes observed following neonatal seizures may be due to complex interactions between the AMPAR phosphorylation and trafficking, in combination with homeostatic mechanisms involved in maintaining the excitation-inhibition imbalance. Indeed, previous studies have suggested that GluR1 S845 phosphorylation plays an important role in stabilizing Ca2+-permeable AMPARs and preventing their lysosomal degradation (Ehlers, 2000; Goel and Lee, 2007; Man et al., 2007; He et al., 2009; Goel et al., 2011). The lack of GluR1 S845 phosphorylation in the GluR1DPM mice may significantly enhance the lysosomal degradation of the internalized GluR1 containing receptors, and may affect the perisynaptic AMPARs that are available for ‘ready insertion’ in response to neuronal activity (He et al., 2009). Furthermore, enhanced GluR2 phosphorylation at S880 observed following neonatal seizure may contribute to receptor internalization and expression of Ca2+-permeable AMPARs at the synaptic surface (Rakhade et al., 2008).
Recent studies utilizing single molecule tracking to detect the movement of AMPARs have shown that GluR1-containing AMPARs freely diffuse in and out of the synapse within the post-synaptic density (PSD) (Heine et al., 2008; Petrini et al., 2009) and anchoring them at the synapse may require PDZ domain interactions. Future studies may reveal the mechanisms involved in mediating this effect on AMPAR-mediated mEPSCs following PTZ-induced seizures, compensatory changes in the excitation-inhibition imbalance may provide information regarding the specific mechanism involved in the paradoxical change observed. Animal models of seizures induced by use of chemoconvulsants in early life have shown multifactorial changes including alterations in the GABARs and AMPARs (Zhang et al., 2004; Silva et al., 2005; Friedman et al., 2007).
GluR1 DPM mice retain the ability to exhibit seizures in the presence of PTZ but display an increased latency to the onset of these behavioral seizures. There does not appear to be significant changes in baseline expression of AMPAR GluR1 or GluR2 subunits or in baseline mEPSCs in hippocampal neurons studied in the GluR1DPM and wild type mice. Furthermore, we did not observe a difference in the rise time, decay time, and frequency of AMPAR mediated mEPSCs between the naïve wild type and GluR1DPM mice at P8. Consistently, previous reports comparing GluR1 DPM mice to wild type did not show any significant abnormalities in anatomical structure, receptor subunit distribution, baseline synaptic transmission and transport of receptors to the synaptic surface (Lee et al., 2003).
Phosphorylation of the GluR1 subunit has been reported to be critical for multiple synaptic potentiation events including trafficking of GluR1 containing receptors, stabilization at the synaptic surface, maintenance of LTP, as well as lysosomal internalization and degradation of Ca2+-permeable AMPARs (Shepherd and Huganir, 2007; Heine et al., 2008; Talos et al., 2012a). Previous reports have suggested that increased GluR2 S880 phosphorylation may result in the enhanced persistence of Ca2+-permeable AMPARs following neonatal seizures (Rakhade et al., 2008) and status epilepticus (Rajasekaran et al., 2012). The increased phosphorylation of GluR2 S880 following chemoconvulsant induced seizures can further influence the AMPAR-mediated synaptic currents and synaptic excitability following neonatal seizures.
Here, we propose that the differences observed in synaptic potentiation in GluR1 DPM mice following the initial neonatal seizures are important in initiating, and also perhaps the maintenance, of the susceptibility to seizures observed in later life. Following induction of seizures at P7–9, seizure susceptibility to KA-provoked seizures in adulthood was increased in wild type mice. In contrast, later seizure susceptibility was unchanged in naïve GluR1 DPM mice compared to those exposed to early life seizures. Susceptibility to KA-induced seizures in later life represents a surrogate for long-term susceptibility to developing epileptic seizures and has been previously used in rodent models (Huang et al., 1999; Koh and Jensen, 2001; Koh et al., 2004). The lack of change in seizure susceptibility in GluR1 DPM mice suggests that these mice may be less prone to development of later life epilepsy. Future studies evaluating the development of spontaneous recurrent seizures following induction of neonatal seizures in the GluR1 DPM and wild type mice may provide significant evidence regarding the role of these post-translational modifications in epileptogenesis.
Stabilization of GluR1 receptors during the maintenance of LTP is thought to be dependent on its attachment to the post-synaptic density proteins (Lisman and Raghavachari, 2006). In models of synaptic potentiation, Ca2+ influx following enhanced activity leads to activation of kinases like CaMKII and PKA, which in turn bind to the intracellular tails of the receptor at the PSD (Yoshimura et al., 2000; Lisman et al., 2002). CaMKII and other associated proteins have been proposed to act as slot proteins for AMPAR insertion (Lisman and Zhabotinsky, 2001), and previous reports have shown enhanced activity of CaMKII in the post-synaptic membrane following neonatal seizures in animal models (Cornejo et al., 2007; Rakhade et al., 2008). Recent data has led to the recognition of distinct process involved in exocytosis of the intracellular AMPARs to extra/perisynaptic sites, lateral diffusion to synaptic sites and retention at synapses via scaffolding proteins (Petrini et al., 2009; Opazo and Choquet, 2011). Increased phosphorylation of the GluR1 receptor following neonatal seizures may affect some of these processes and alter the balance between excitation and inhibition, promoting development of seizures. PSD-95 over-expression has been found to drive the recruitment of AMPARs to synaptic sites (Stein et al., 2003; Ehrlich and Malinow, 2004). The enhanced phosphorylation of GluR1 receptors and the increased expression of PSD-95 following early life PTZ-induced seizures suggest that mechanisms similar to those involved in synaptic potentiation may be involved in maintaining hyperexcitability and seizure susceptibility. Constant interplay between these synaptic mechanisms leading to potentiation of neuronal networks, and homeostatic mechanisms involved in maintaining the excitation-inhibition balance may be involved in either promoting epileptogenesis or preventing the susceptibility to later life seizures respectively.
A novel finding in this study was that the phosphorylation of GluR1 S831 and S845 was increased in the postmortem samples of hippocampi of 3 patients who had experienced neonatal seizures and had succumbed to related complications during infancy compared to 3 age-matched autopsy control cases. The increase in GluR1 phosphorylation in the human subjects who had experienced neonatal seizures suggests that this phenomenon observed in the animal models recapitulates the alterations that are relevant to the human disease condition. Children suffering from neonatal seizures have a significantly increased risk for developing epilepsy and cognitive disabilities in later life; in fact the lifetime incidence of these associated problems reflects a far greater disease burden than the neonatal seizures themselves (Mizrahi and Kellaway, 1998; Mizrahi, 1999; Ronen et al., 2007). The molecular mechanisms involved in the development of these long-term sequelae are incompletely understood despite intense investigation. The current study highlights the potential role of post-translational modification of a critical neurotransmitter receptor in epileptogenesis it may represent a potential target in at least a subset of the population experiencing neonatal seizures. This study provides a proof of concept for the therapeutic implications of these findings in the human disease process of epileptogenesis in the immature brain.
This work was supported by National Institutes of Health Grants NS 031718 (F.E.J.) and DP1 OD003347 (F.E.J.) (from the Office of the Director), Intellectual Developmental Disabilities Research Center Grant P30 HD18655 (NICHD). Human tissue was obtained from the NICHD Brain and Tissue Bank for Developmental Disorders at the University of Maryland, Ref No. NO1-HD-09-0011. We would like to thank Michelle Johnson for assistance with animal handling and immunoblot experiments. We thank members of the Jensen lab for valuable discussion. CZ and HS contributed to the electrophysiology experiments.
The authors declare that they have no competing financial interests