Transmembrane α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor regulatory protein expression during the development of absence seizures in genetic absence epilepsy rats from Strasbourg

The transmembrane α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor (AMPAR) regulatory proteins (TARPs), γ2 (stargazin), γ3, γ4, γ5, γ7, and γ8, are a family of proteins that regulate AMPAR trafficking, expression, and biophysical properties that could have a role in the development of absence seizures. Here, we evaluated the expression of TARPs and AMPARs across the development of epilepsy in the genetic absence epilepsy rats from Strasbourg (GAERS) model of idiopathic generalized epilepsy (IGE) with absence seizures. Pre‐epileptic (7‐day‐old), early epileptic (6‐week‐old), and chronically epileptic (16‐week‐old) GAERS, and age‐matched male nonepileptic control rats (NEC) were used. Electroencephalographic (EEG) recordings were acquired from the 6‐ and 16‐week‐old animals to quantify seizure expression. Somatosensory cortex (SCx) and whole thalamus were collected from all the animals to evaluate TARP and AMPAR mRNA expression. Analysis of the EEG demonstrated a gradual increase in the number and duration of seizures across GAERS development. mRNA expression of the TARPs γ2, γ3, γ4, γ5, and γ8 in the SCx, and γ4 and γ5 in the thalamus, increased as the seizures started and progressed in the GAERS compared to NEC. There was a temporal association between increased TARP expression and seizures in GAERS, highlighting TARPs as potential targets for developing novel treatments for IGE with absence seizures.


| INTRODUCTION
Genetic absence epilepsy rats from Strasbourg (GAERS) are one of the most studied models of idiopathic generalized epilepsy (IGE).Absence seizures, a pathognomonic seizure type of IGE, appear in humans during early childhood and are characterized by spike-wave discharges (SWDs) on the accompanying electroencephalogram (EEG).GAERS gradually develop seizures as they mature, starting to displaying SWDs in epidural recordings at approximately 4-6 weeks of age; the seizures continue to increase in frequency and duration until adulthood (4-6 months of age). 1,2In contrast, nonepileptic control rats (NEC) were derived from the same original Wistar strains as GAERS but selectively inbred not to express seizures at any age. 1 The αamino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) mediate the majority of glutamatergic fast excitatory synaptic neurotransmission in the brain, and evidence in animal models links the dysfunction or pharmacological manipulation of AMPARs with absence seizures. 1,3The AMPARs are heterotetramers with different combinations of the GluA1-GluA4 subunits. 4hanges in the functional properties and synaptic expression of AMPARs play a critical role in synaptic plasticity. 5he transmembrane AMPAR regulatory proteins (TARPs), γ2 (stargazin), γ3, γ4, γ5, γ7, and γ8, are a family of proteins that regulate AMPAR trafficking, expression, and biophysical properties that are found in the brain. 6,7Previous studies have shown that γ2 mRNA expression is increased in the somatosensory cortex (SCx) and thalamus of epileptic GAERS compared to controls. 8Furthermore, γ2 mRNA and protein expression are increased in the SCx and thalamus as seizures begin to develop in GAERS. 8,9In addition, membrane protein expression of the AMPAR subunits GluA1 and GluA2 are also increased in epileptic GAERS in the SCx. 9 Therefore, we hypothesized that TARP and AMPAR expression will be altered in GAERS and will show a temporal evolution that correlates with the time course of seizure expression.To address this question, we investigated AMPAR and TARP expression in GAERS, compared with age-and sex-matched NEC, before the appearance of seizures (preepileptic), at the age of appearance of the first spontaneous seizures (early epileptic), and once the animals reached the mature (chronically epileptic) state.

| Ethics statement
Experimental procedures were approved by the Florey Animal Ethics Committee (#1011823) and adhered to the Australian code for the care and use of animals for scientific purposes.All the experiments were conducted by researchers blinded to the experimental conditions.Animals were individually housed with alternating 12-h cycles of light and dark.Food and water were provided ad libitum for the whole duration of the study.

| Sample size analysis and experimental cohorts
Power analyses based on our previously published GAERS studies indicated that n = 6 per group was required to detect a 30% difference between the groups considering α = .05and power = 95%.Seven-day pre-epileptic (NEC, n = 9; GAERS, n = 9), 5-week early epileptic (NEC, n = 8; GAERS, n = 10), and 15-week-old epileptic (NEC, n = 8; GAERS, n = 8) male rats were used to evaluate whether changes in TARP and AMPAR expression are causative or a consequence of epilepsy.

| EEG electrode implantation surgery
Seven-day-old male GAERS and NEC pups were obtained for brain dissections, but these animals did not have EEG recordings.GAERS and NEC aged 5 weeks (early epileptic) and 15 weeks (epileptic) underwent surgery to implant EEG recording electrodes to confirm and characterize the seizure phenotype.Rats were anesthetized with isoflurane (Ceva isoflurane, Piramal Enterprises), and EEG surgery was performed under an aseptic technique as described previously. 10EEG recording electrodes (EM12/20/SPC, Plastics One) were inserted via burr holes without penetrating the dura, one on each side of the frontotemporal regions (anteroposterior [AP], 1.7 mm; mediolateral [ML], ±2.5 mm) and the parietal regions (AP, 5.6 mm; ML, ±2.5 mm). 11Ground and reference electrodes were implanted on each side of the occipital bone above the cerebellum and secured using dental cement (VX-SC1000GVD5/VX-SC1000GMLLQ, Vertex) around the electrodes and over the skull.Buprenorphine (.05 mg/kg, Indivior) was used as an analgesic at the start of the surgery, and every 12 h for 3 days after the surgical procedure.

| EEG acquisition and seizure analyses
One week after the surgery, animals underwent two sessions of 24 h of continuous video-EEG recording during 1 week while freely moving in their cages.EEG recordings were obtained using Profusion 3 software (Compumedics) unfiltered and digitized at 512 Hz.All EEG recordings were using Assyst semiautomated seizure detection software. 10An EEG seizure was defined as SWDs of more than three times baseline amplitude, 5-12-Hz frequency, and duration of >2 s. 10,12 The start and end of each seizure were determined by manually marking the beginning and end of each EEG seizure on Assyst.The total number of seizures, total seizure duration, and average seizure duration were quantified for all animals.Two blinded independent experts visually confirmed seizure events.The SWDs on the EEG were accompanied behaviorally by automatisms such as head nodding, chewing, vibrissae twitching, and blinking as previously described. 1

| Tissue collection
Brain tissue was collected from 7-day-old (no EEG recordings), 6-week-old (early epileptic), and 16-week-old (epileptic) NEC and GAERS.Animals were anesthetized using isoflurane at 5% then culled using a lethal dose of Lethabarb (150 mg/kg ip; pentobarbitone sodium; Virbac).Using the Paxinos Atlas, SCx and whole thalamus from NEC and GAERS were rapidly dissected, snap-frozen in liquid nitrogen, and stored at −80°C.

| Statistical analysis
All EEG parameters and qPCR data were normally distributed, as per the Shapiro-Wilk test.Student two-tailed t-test was used to analyze the EEG and molecular data.Pearson correlations between the mRNA expression, number of seizures, and average and total seizure duration were also performed.Statistical analysis was done using Prism 9 (GraphPad Software).Statistical significance in all cases was set at p < .05.

| Epilepsy progression in GAERS is manifested by an increased number and duration of SWDs as the GAERS age
EEG analyses revealed that the number of seizures (Figure 1A; p < .0001),average seizure duration (Figure 1B; p < .0001),and seizure ration (Figure 1C; p < .0001)are increased in chronically epileptic GAERS (16 weeks of age) compared to early epileptic GAERS (6 weeks of age).Analyses of the EEG data corroborated that none of the NEC developed SWDs.

| AMPAR and TARPs show increased expression that follows epilepsy onset in the SCx of GAERS
In the SCx, γ2 and γ8 mRNA expression is not significantly different in pre-epileptic 7-day-old GAERS compared to NEC (Figure 2A); however, at the age where epilepsy began to develop (i.e., 6 weeks), GAERS displayed increased mRNA expression of both γ2 and γ8 TARPs (Figure 2C; p < .05for both comparisons), and this increase was maintained when the animals were chronically epileptic at 16 weeks of age (Figure 2C; p < .05for γ2 and p < .001for γ8).Interestingly, γ3 and γ5 mRNA expression are decreased in 7-day-old GAERS (Figure 2A; p < .0001,p < .01,respectively); however, with epilepsy onset, the γ3 expression increased significantly (Figure 2C; p < .05),and γ5 expression gradually increased until both γ3 and γ5 expression were significantly more expressed in chronically epileptic GAERS than age-matched NEC (Figure 2E; p < .01 for both comparisons).AMPAR GluA4 subunit was only differentially expressed in 16-week-old chronically epileptic GAERS (Figure 2E; p < .01).Spearman correlation showed that γ2 expression correlates with the total seizure duration of chronically epileptic GAERS (p < .05,r = .7129).

| DISCUSSION
This study shows that the increased mRNA expression of the TARPs occurs from the age at which seizures develop in GAERS.This corroborates previous studies from our laboratory, where mRNA expression of γ2 was increased in the SCx and thalamus as seizures began to develop in GAERS. 8,9TARPs differentially regulate the properties of AMPARs.4][15] It is intriguing to see changes in the expression patterns across epilepsy development, from mRNA expression downregulation, in the case of γ3 and γ5 in pre-epileptic GAERS, to increased mRNA expression as epilepsy develops in the case of the majority of the TARPs, γ2, γ3, γ4, γ5, and γ8, suggesting a potential association with seizure development and increased TARP expression.Upregulation and increased function of AMPARs in the SCx may be particularly important for the development of seizures in GAERS, as previous studies have shown that SWDs are initiated by hyperexcitable neurons in layer V/VI of the SCx, and that pharmacological inhibition of AMPARs in the SCx prevents the development of absence seizures.Linkage and association analysis in people with IGE revealed that CACNG3, the human ortholog that codes for γ3, is a susceptibility gene for the development of absence seizures.TARP γ4 is preferentially found in the thalamus, and its expression peaks during brain development but decreases as maturation is reached. 6,14Functionally, γ4 radically slows the decay rate of AMPARs and is believed to dictate developmental receptor kinetics. 6The slower decay kinetics can prolong the postsynaptic effects of glutamate, increasing excitatory neurotransmission, which could be a factor in developing absence seizures.The type II TARP γ5 is heavily expressed in the thalamus, where it is known to augment glutamate currents, increase AMPAR conductance, regulate channel behavior, and reduce sensitivity to block by polyamines of AMPARs that contain the GluA2 subunit, which are characteristics that could promote seizures in GAERS.
The GluA4 is the main AMPAR subunit expressed in thalamus and in corticothalamic neurons, 16 and we found that the GluA4 subunit was significantly reduced in the thalamus of epileptic GAERS.Importantly, AMPARs that contain a GluA4 subunit have the fastest desensitization rate to glutamate.Abnormal hypersynchronous thalamocortical activity, which precedes the development of SWDs, has been described in knockout mice with mutations of the Gria4 gene that codes for GluA4.Moreover, GluA4 deficiency in the Gria4 mutant results in a specific and selective reduction in the strength of the connection in nucleus reticularis of the thalamus synapses. 17Based on previous evidence, the reduced expression of GluA4 in epileptic GAERS could possibly increase the duration of response to excitatory glutamate neurotransmission, which in turn might increase burst firing in reticular neurons, enhancing circuit synchrony, a characteristic that underlies SWDs in IGE.
Taken together, our results indicate that changes in TARP expression occur as seizures begin to manifest in GAERS, which suggests a potential temporal association between increased TARP expression and seizures in this rat model of IGE, highlighting TARPs as potential targets for developing novel antiseizure treatments.We acknowledge a limitation of this work is that EEG recordings could not be done in 7-day-old GAERS.Future studies should investigate whether this temporal association is causal to the development of seizures in GAERS.Recent studies have focused on developing TARP γ8 antagonists, 18 including a recent study that showed the effectiveness of LY3130481 (a novel selective antagonist of AMPARs containing γ8) in reducing GAERS absence seizures. 18However, in the relatively novel field of TARPs in IGE, there are many mechanisms and biological implications in the development of absence epilepsy that need to be elucidated.Further studies should also examine TARP protein expression within SCx layers and specific thalamic nuclei, and to determine whether inhibiting seizures changes TARP expression.E. has received research grants from Supernus Pharmaceuticals, Praxis, Eisai, and Kaoskey outside the submitted work.T.J.O.reports grants and consulting fees paid to his institution from Eisai, UCB Pharma, Biogen, ES Therapeutics, Kinoxis Therapeutics, Epidarex, and Zynerba.None of the other authors has any conflict of interest to disclose.We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

F I G U R E 1
Progression of spike-wave discharges (SWDs) across genetic absence epilepsy rats from Strasbourg (GAERS) development in young (5 weeks old) versus adult (16 weeks old) rats.Adult rats displayed increased (A) total number of seizures on 24-h electroencephalographic recording, (B) average duration of SWDs, and (C) total SWD duration per 24-h recording compared to 6-week-old (young) GAERS.Student two-tailed t-test (****p < .0001).Data are expressed as mean ± SEM.
ACKNOWLEDGMENTS P.M.C.-E. is supported by an Early Career Fellowship from the National Health and Medical Research Council (APP1087172), the US Department of Defense Epilepsy Research Program (EP200022), and a Department of Health and Aged Care (Medical Research Future Fund) Stem Cell Therapies Mission grant (MRF2015957).T.J.O. is supported by a Program Grant (APP1091593) and Investigator Grant (APP1176426) from the National Health and Medical Research Council of Australia and the Victorian Medical Research Acceleration Fund.Open access publishing facilitated by Monash University, as part of the Wiley - Monash University agreement via the Council of Australian University Librarians.CONFLICT OF INTEREST STATEMENT P.M.C.-