Alterations in AMPA receptor subunit expression in cortical inhibitory interneurons in the epileptic stargazer mutant mouse

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ALTERATIONS IN AMPA RECEPTOR SUBUNIT EXPRESSION IN CORTICAL INHIBITORY INTERNEURONS IN THE EPILEPTIC STARGAZER MUTANT MOUSE

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NADIA KAFUI ADOTEVI AND BEULAH LEITCH *

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Department of Anatomy, Brain Health Research Centre, Otago School of Medical Sciences, University of Otago, PO Box 913, Dunedin, New Zealand

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Key words: absence epilepsy, somatosensory cortex, AMPA receptor, stargazer, parvalbumin, feed-forward inhibition. 10

Abstract—Absence seizures arise from disturbances within the corticothalamocortical network, however the precise cellular and molecular mechanisms underlying seizure generation arising from different genetic backgrounds are not fully understood. While recent experimental evidence suggests that changes in inhibitory microcircuits in the cortex may contribute to generation of the hallmark spike-wave discharges, it is still unclear if altered cortical inhibition is a result of interneuron dysfunction due to compromised glutamatergic excitation and/or changes in cortical interneuron number. The stargazer mouse model of absence epilepsy presents with a genetic deficit in stargazin, which is predominantly expressed in cortical parvalbumin-positive (PV+) interneurons, and involved in the trafficking of glutamatergic AMPA receptors. Hence, in this study we examine changes in (1) the subunit-specific expression of AMPA receptors which could potentially result in a loss of excitation onto cortical PV+ interneurons, and (2) PV+ neuron density that could additionally impair cortical inhibition. Using Western blot analysis we found subunit-specific alterations in AMPA receptor expression in the stargazer somatosensory cortex. Further analysis using confocal fluorescence microscopy revealed that although there are no changes in cortical PV+ interneuron number, there is a predominant loss of GluA1 and 4 containing AMPA receptors in PV+ neurons in stargazers compared to non-epileptic controls. Taken together, these data suggest that the loss of AMPA receptors in PV+ neurons could impair their feedforward inhibitory output, ultimately altering cortical network oscillations, and contribute to seizure generation in stargazers. As such the feed-forward inhibitory interneurons could be potential targets for future therapeutic intervention for some absence epilepsy patients. Ó 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

*Corresponding author. Fax: +64 3 479 7254. E-mail address: [email protected] (B. Leitch). Abbreviations: AEDs, anti-epileptic drugs; AMPA, a-amino-3-hydroxy5-methyl-4-isoxazolepropionic acid; CT, corticothalamic; CTC, corticothalamocortical; E, epileptic; EEG, electroencephalogram; GABA, c-aminobutyric acid; NE, non-epileptic; NMDA, N-methyl-daspartate; PBS, phosphate buffered saline; PV+, parvalbumin-positive; RTN, reticular thalamic nucleus; SWD, spike-wave discharge; TARP, transmembrane AMPA receptor regulatory protein; TC, thalamocortical; VGlut2, vesicular glutamate transporter 2; VP, ventral posterior thalamus. http://dx.doi.org/10.1016/j.neuroscience.2016.09.052 0306-4522/Ó 2016 IBRO. Published by Elsevier Ltd. All rights reserved. 1

INTRODUCTION

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Absence epilepsy is a generalized non-convulsive form of epilepsy characterized by a sudden brief loss of consciousness and concomitant spike wave discharges (SWDs) measuring 2.5–4 Hz on an electroencephalogram (EEG) (Crunelli and Leresche, 2002). Absence seizures, which account for approximately 10% of pediatric epilepsies, can occur hundreds of times a day (Berg et al., 2010) and are associated with cognitive deficits, learning difficulties and behavioral disorders in some affected children (Pavone et al., 2001; Caplan et al., 2008; Killory et al., 2011; Tosun et al., 2011). In addition to the adverse effects on a child’s learning and social adjustment, there is an increased risk of physical injury if they occur during activities such as swimming or riding a bicycle on the road (Wirrel et al., 1996). Although absence seizures are known to arise from disturbances within the corticothalamocortical (CTC) circuitry (McCormick and Contreras, 2001), the precise underlying mechanisms are not fully understood and appear to be multifactorial as evidenced by the variability in patients’ responses to anti-epileptic drugs (AEDs). Currently available AEDs fail to control seizures or induce intolerable side effects in approximately a third of patients, and even exacerbate seizures in some cases (Regesta and Tanganelli, 1999; Glauser et al., 2010, 2013). This variability in patient response to drug treatment suggests different cellular and molecular mechanisms within specific microcircuits are potentially capable of switching the normal oscillatory firing pattern within the CTC network into pathological SWDs. Hence, there is a critical need to understand the various mechanisms that underlie generation of absence seizures, in order to identify novel therapeutic targets for treatment of absence seizures, which arise from different genetic backgrounds. Recent studies, in some rodent models of absence epilepsy, have implicated region and synapse specific changes in a-amino-3-hydroxy-5-methyl-4-isoxazolepro pionic acid (AMPA) receptor expression (Menuz and Nicoll, 2008; Kennard et al., 2011; Paz et al., 2011; Barad et al., 2012; Maheshwari et al., 2013) in seizure generation. AMPA receptors (AMPARs), formed from tetrameric combinations of GluA1–4 subunits, mediate most of the fast excitatory glutamatergic synaptic

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transmission in the brain (Opazo and Choquet, 2011). They are trafficked to synapses and modulated by a family of transmembrane AMPA receptor regulatory proteins (TARPs), including the prototypical TARPc2 stargazin (Chen et al., 2000; Tomita et al., 2005; Nicoll et al., 2006). Their presence at excitatory inputs to fast spiking inhibitory interneurons within the CTC network provides feed-forward inhibition that prevents runaway excitation within the network. GluA4 containing AMPARs are particularly abundant at synapses on parvalbumin containing (PV+) feed-forward inhibitory neurons within the CTC network; namely the inhibitory interneurons in the reticular thalamic nucleus (RTN) and somatosensory cortex (Kondo et al., 1997; Mineff and Weinberg, 2000). Recent studies have indicated that a selective loss of GluA4-AMPARs in the RTN and thus excitatory input to these PV+ inhibitory neurons (Menuz and Nicoll, 2008; Paz et al., 2011), contributes to the generation and maintenance of pathological oscillations in the thalamus. For example, the stargazer mouse, an established model of absence epilepsy in which a spontaneous recessive mutation on chromosome 15 causes a stargazin protein deficit (Noebels et al., 1990), shows a significant loss of AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSC) in RTN neurons (Menuz and Nicoll, 2008). This aberration results from a selective reduction in GluA4-AMPAR expression at corticothalamic (CT) synapses in inhibitory RTN neurons, but not at excitatory synapses on relay neurons in the ventral posterior (VP) thalamic region (Barad et al., 2012). Similarly, the Gria4-knockout mouse, which lacks GluA4 subunit expression and also presents with absence epilepsy, has been shown to have a selective impairment in CT-RTN synaptic transmission, but not in CT-TC or TC-RTN synaptic function. This ultimately leads to the increased CT excitation of the thalamic relay neurons via CT-TC-RTN-TC pathway. As a consequence, there is a selective loss of feed-forward inhibition but not of feedback inhibition in the thalamus, creating an enhanced oscillatory network that promotes the generation of SWDs (Paz et al., 2011). Collectively, the data from these mouse models of absence epilepsy implicate reduction in feed-forward inhibition but not feedback inhibition in the generation of seizure activity in the thalamus. While most studies to date, have concentrated on the changes in inhibitory microcircuits within the thalamic component of the CTC network, emerging evidence indicates that SWDs are initiated in the somatosensory cortex (Meeren et al., 2002; Polack et al., 2007). A recent study proposed that seizure exacerbation by some N-methyl-D-aspartate (NMDA) receptor (NMDAR) antagonists in stargazers is due to the reduced excitation of cortical PV+ inhibitory neurons (Maheshwari et al., 2013). This is because in the absence of AMPARmediated excitation, NMDARs, which are unaffected by the stargazin mutation, are thought to drive the excitation of inhibitory neurons (Lacey et al., 2012); as such blocking their activity could cause a further reduction in inhibition and thus enhance seizure activity. Nevertheless, the precise mechanism underlying seizure initiation in the cortex

is still unclear and could either be a result of either cortical disinhibition (Maheshwari and Noebels, 2014), enhanced tonic inhibition (Cope et al., 2009) or even enhanced excitation (Kennard et al., 2011). Furthermore, since loss of inhibitory neurons have also been implicated in several neurological diseases including epilepsy (Kann, 2015), a reduction in numbers of cortical PV+ inhibitory interneurons, in addition to any loss of excitatory input to these neurons, could contribute to a net loss of feed-forward inhibition within the cortex and runaway excitation. Currently, it is unknown whether changes in AMPAR expression in the cortex of the stargazer model are specifically linked to changes in GluA4-AMPARs in inhibitory PV+ interneurons to which thalamocortical (TC) neurons project and hence is a subunit-specific effect, or whether other subclasses of AMPAR are involved, as seen in other models of absence epilepsy (Hanada, 2014). Moreover, it is unknown whether altered cortical inhibition is also associated with changes in inhibitory interneuron number or primarily a result of their dysfunction. Hence, the aim of the current study was first, to investigate changes in the subunit-specific expression of AMPARs in stargazer cortex that could potentially result in a loss of excitation onto cortical PV+ interneurons, and second, to identify any changes in PV+ neuron density that could additionally impair cortical inhibition. To this end, we quantified the AMPAR subunit levels in whole-tissue lysates of the somatosensory cortex with Western blotting and found subunit-specific losses in the global expression of GluA1–4 in the stargazer somatosensory cortex. In addition we examined the expression levels of all AMPA receptor subunits throughout the stargazer cortical layers 1–6 with confocal fluorescence microscopy and demonstrated that although the density of cortical PV+ interneurons is unchanged between non-epileptic (NE) and stargazer mice, there is both a reduction in the somatic expression and dendritic trafficking of AMPA receptors in stargazer cortical PV+ neurons, which could impact on their feed-forward inhibitory function to induce hypersynchronous activity in the stargazer cortex.

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EXPERIMENTAL PROCEDURES

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Animals

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Breeding stock of stargazer mice obtained from the Jackson Laboratory (Bar Harbor, USA) were mated to produce epileptic stargazer (stg/stg) and NE wildtype (+/+) and heterozygote (+/stg) control littermates. Mice were maintained on a 12-h light/dark cycle with access to food and water ad libitum. 9–12 week-old adult male mice were used, with genotypes confirmed by PCR of tail DNA based on recommendations of Jackson Laboratory. All experimental procedures were performed in line with approved protocols by the University of Otago Animal Welfare Office and Ethics Committee.

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Antibodies

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The antibodies used in this study are listed in Table 1. Preadsorption control tests to confirm primary antibody

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specificity were carried out using control peptides specific to each antibody, while omission control tests were performed to test the specificity of the secondary antibodies. In the western blot analysis of tissue lysates, b-actin was used as a loading control. The antibody dilutions used were optimized in initial experiments in reference to the manufacturer’s recommendations.

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Western blotting

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Brains were extracted from adult mice following cervical dislocation, snap frozen on dry ice and stored at 80 °C until use. 300-lm thick coronal sections were cut in a freezing cryostat (Leica CM1950, Wetzlar, Germany) at a constant chamber temperature of 10 °C, with sections thaw-mounted onto glass microscope slides. Micropunches taken from the somatosensory cortex; identified on corresponding sections in the mouse brain atlas (Franklin and Paxinos, 2008), with a 1.0-mm biopsy punch (Integra Miltex, 33-31AA) were collected into lysis buffer supplemented with phenylmethyl sulfonyl fluoride (PMSF) and protease inhibitor (Sigma Aldrich P8340). Samples were homogenized through sonication, heating and centrifugation sequentially, and the supernatant transferred into new microtubes and stored at 80 °C. The protein concentration of each sample was determined using detergent-compatible protein assay (BioRad, 500-0116). Protein extracts (10–20 lg) were separated on 8.5% SDS–PAGE gels and transferred onto nitrocellulose membranes. Membranes were blocked with Odyssey blocking buffer for 45 min at room temperature, followed by overnight incubation at 4 °C with primary antibodies against GluA1–4 subunits and b-actin. Membranes were probed with secondary antibodies for 1 h at room temperature, and subsequently imaged with the Odyssey Infrared Imaging System and Odyssey v3.1 program (LI-COR Biosciences). Each protein band was analyzed and normalized against its b-actin control band. The mean of NE

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controls was calculated on individual blots, and the normalized intensities of each NE and epileptic sample expressed as a ratio relative to that mean. Data are presented as mean ± standard error of the mean (SEM). Statistical differences were assessed by unpaired Mann–Whitney U test in GraphPad Prism 6.0 (GraphPad Software, USA) with statistical significance set at p < 0.05.

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Immunofluorescence confocal microscopy

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Mice were anesthetized with pentobarbital (60 mg/kg) injected intraperitoneally, followed by transcardial perfusion with 5% heparin in phosphate buffered saline (PBS) and 4% paraformaldehyde (PFA) in 0.1 M Sorensen’s phosphate buffer (PB). Brains were extracted and post-fixed overnight in 4% PFA at 4 °C, then cryoprotected in increasing concentrations of sucrose in PBS (10%, 20% and 30%). 30-lm coronal sections were cut in a cryostat (Leica CM1950, Wetzlar, Germany), and collected into PBS. To avoid nonspecific antibody binding, sections were kept in blocking buffer (4% NGS, 0.1% BSA, 0.3% Triton X-100 in PBS) for 2 h at room temperature, before incubation in primary antibodies for 48 h at 4 °C. Sections were then labeled with secondary antibodies, mounted on polysine-coated glass slides and cover-slipped with 1,4diazabicyclo[2.2.2]octane (DABCO)-glycerol. Preadsorption controls with 5 excess antigen to antibody concentrations, and the omission of primary antibody control tests were carried out to test for the specificity of the primary and secondary antibodies respectively.

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Image acquisition and analysis

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Images were acquired using a Zeiss LSM710 confocal microscope with channel configuration; GluA1–4 (green channel, 488 nm laser excitation); PV (red channel, 543 nm laser excitation) and vesicular glutamate

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Table 1. Antibodies used in experiments

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Target Protein

Host

Source/catalog No.

Application

Dilution

Primary antibodies GluA1

Rabbit

Millipore/AB1504

GluA2 GluA2/3 GluA3 GluA4

Mouse Rabbit Rabbit Rabbit

Millipore/MAB397 Millipore/AB1506 Abcam/AB40845 Millipore/AB1508

b-actin Parvalbumin VGlut2

Mouse Mouse Guinea pig

Abcam/AB8226 Swant/235 Synaptic Sys/135 404

WB IF WB IF WB WB IF WB IF IF

1:1000 1:200 1:1000 1:200 1:1000 1:2000 1:200 1:1000 1:2000 1:500

Secondary antibodies Rabbit IgG, IRDye 680 Mouse IgG, IRDye 800CW Rabbit IgG, Alexa Fluor 488 Mouse IgG, Alexa Fluor 568 Guinea pig IgG, Alexa Fluor 633

Goat Goat Goat Goat Goat

Li-Cor/926-32210 Li-Cor/926-32221 Life Tech/A-11008 Life Tech/A-11031 Life Tech/A-21105

WB WB IF IF IF

1:10,000 1:10,000 1:1000 1:1000 1:1000

WB = Western blot, IF = immunofluorescence.

Please cite this article in press as: Adotevi NK, Leitch B. Alterations in AMPA receptor subunit expression in cortical inhibitory interneurons in the epileptic stargazer mutant mouse. Neuroscience (2016), http://dx.doi.org/10.1016/j.neuroscience.2016.09.052

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transporter 2 (Vglut2) (blue channel, 633 nm laser excitation); and channel cross-talk excluded by using band-filtering and sequential scanning (Belie¨n and Wouterlood, 2012). Fluorescence signals were maximized by optimizing the averaging, digital gain and offset. Images were analyzed, blind to the investigator, with the ImageJ software (NIH, USA) using the same settings for both control and stargazer mice. To ensure an accurate measure of the signal of interest, background fluorescence subtraction was applied; using a tissue section labeled with specific secondary antibody only (omission control) acquired with the same acquisition parameters. Thresholds were manually defined for each image channel separately, and tested with the computed channel intensities using the coloc2 plugin. All further relative quantitative analysis were performed with measurements set to ‘‘limit to threshold”. For each channel, the integrated pixel intensities of individual immunolabeled cells were compared against the mean intensity of all immunolabeled cells in profile, as a relative assessment of receptor expression. Co-labeled neurons were identified by creating a merged image of the appropriate channels using the ‘‘Channels Tool”. To determine the relative expression of AMPAR subunits within the soma of co-labeled PV+ cells, the pixel densities within the area of colocalization of each GluA subunit with PV to that of total PV expression only per co-labeled neuron was measured; with the calculated ratio (area fraction) of the average pixel densities of all analyzed co-labeled neurons presented graphically as the mean percentage area of expression of each subunit. An evaluation of the subcellular localization of AMPAR subunits’ in the dendrites of PV+ neurons was performed by quantifying the number of puncta per 10 lm dendrite length (Tao et al., 2013). Immunopositive punctae were identified at pixel intensities above the intensity threshold per each analyzed neuron dendrite; with puncta number and size plotted as a measure of the relative dendritic GluA expression. To examine possible defects in trafficking of AMPARs to synapses in stargazers, we calculated (within the same section) the ratio of GluA expression in the dendrite versus the soma of the same neuron by measuring the pixel density of GluA staining within a 10  2 lm2 rectangular region of interest (ROI) in the proximal area of a primary dendrite relative to that within a 5 lm circular ROI in the somatic region at the base of the dendrite (Maheshwari et al., 2013). In this study, statistical comparisons of AMPAR subunits’ expression were then made between epileptic stargazers (E) and NE control mice (n = 6; 3NE, 3E), with statistical differences tested using unpaired Mann–Whitney U test in GraphPad Prism 6.0. Statistical significance was set at p < 0.05.

RESULTS GluA1–4 is reduced in whole-tissue lysates of the stargazer somatosensory cortex The expression of AMPAR GluA1–4 subunits in wholetissue lysates from the somatosensory cortex of epileptic stargazers and NE control mice was analyzed using Western blotting to identify any changes in

specific subunits. All four subunits were selected for analysis as previous studies show that all AMPAR subunits are expressed in the cerebral cortex (Sato et al., 1993; Conti et al., 1994). Immunopositive bands corresponding to each GluA subunit and b-actin were detected at their expected molecular weights (100 kDa and 42 kDa respectively; Fig. 1A, B, E, F), relative to the reference protein ladder. Quantitative analysis of GluA1–4 band intensities normalized against corresponding b-actin loading controls showed that GluA2, 3 and 4 subunits were significantly reduced in the somatosensory cortex of epileptic stargazer mice compared to NE control littermates (Fig. 1C, D, G, H). Epileptic stargazer mice showed a 30% decrease in GluA2 subunit expression (0.70 ± 0.06, n = 18, p < 0.01, Fig. 1D), a 31% reduction in GluA3 subunit expression (0.69 ± 0.03, n = 18, p < 0.01, Fig. 1G), and a 40% decrease in GluA4 subunit expression (0.60 ± 0.04, n = 18, p < 0.01, Fig. 1H). Although GluA1 expression was reduced in stargazers, the difference was not statistically significant (0.91 ± 0.07, n = 19, p > 0.05, Fig. 1C). No significant differences in subunit expression were detected between wild-type and heterozygous NE controls (+/+ and +/ stg) in agreement with all previous published literature, which have consistently reported no differences between +/+ and +/stg mice (Qiao et al., 1998; Hashimoto et al., 1999; Richardson and Leitch, 2005); and thus routinely combine +/+ and +/stg tissue for control experiments (Chen et al., 2000; Khan et al., 2004; Payne et al., 2006, 2007). These data show that in stargazer somatosensory cortex there is a global loss of AMPA receptor expression. As the Western blot analyses included tissue punched from the full depth of somatosensory cortex (layers 1–6), we next wanted to determine if changes in GluA subunit expression were layer-specific.

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Cellular localization of GluA1–4 in the somatosensory cortex

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To examine the subunit-specific expression of AMPARs at cellular level, the distribution of GluA1–4 subunit expression in neurons within the different cortical layers 1–6 was analyzed using confocal immunofluorescence microscopy. Consistent with previous studies, doublelabeling of tissue sections with the VGlut2, which is predominantly a presynaptic marker of excitatory synapses (Fremeau et al., 2004; Benarroch, 2010), aided in distinguishing the cortical layers due to its intense labeling of TC terminals projecting to cortical layer 4. GluA1–4 immunolabeled neurons were visible throughout layers 2– 6 of the somatosensory cortex, with virtually no labeling in cortical layer 1 in both NE and epileptic stargazer mice (Fig. 2A-C). While GluA1 and 4 intensely labeled the soma of immunopositive neurons (Fig. 2A, C), GluA2/3 labeling was mostly diffuse and punctate around the periphery of the soma (Fig. 2B). GluA1-3 immunopositive neurons were uniformly distributed throughout layers 2–6, whereas GluA4 expressing neurons were more abundant in lower cortical layers 4–6 with a more intense and diffuse labeling in cortical layer 4. This intense labeling of cortical layer 4 could be due to the presence of mainly GluA4-containing AMPARs in the dendrites of inhibitory

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Fig. 1. Western blot analysis of GluA1–4 expression in the somatosensory cortex of epileptic (E) and non-epileptic (NE) mice. (A, B, E, F) Representative immunoblots showing expression of AMPA receptor subunits (A) GluA1, (B) GluA2, (E) GluA3 and (F) GluA4, with b-actin as a loading control in the somatosensory cortex of non-epileptic and epileptic mice. Bands were detected at their expected molecular weights of 106 kDa for GluA1, 100 kDa for GluA2 and 4, 95 kDa for GluA3, and 42 kDa for b-actin. (C, D, G ,H) Relative expression of GluA1–4 in the somatosensory cortex presented as bar graphs. Statistically significant differences in AMPA receptor subunit expression were observed in (D) GluA2 in non-epileptic (1.00 ± 0.04, n = 9) and epileptic (0.70 ± 0.06, n = 9) mice ***p < 0.001; (G) GluA3 in non-epileptic (1.00 ± 0.03 n = 9) and epileptic (0.69 ± 0.03, n = 9) mice ***p < 0.0001; (H) GluA4 in non-epileptic (1.00 ± 0.04, n = 9) and epileptic (0.60 ± 0.04, n = 9) mice **** p < 0.0001. There was however no significant difference in expression of (C) GluA1 in non-epileptic (1.00 ± 0.03, n = 9) and epileptic (0.91 ± 0.07, n = 10) mice p = 0.07. Data presented as mean ± SEM. 366 367 368 369 370 371 372 373 374 375 376

interneurons within this layer and the preferential trafficking of GluA4-AMPARs into TC synapses in layer 4, to maintain synaptic integration (Zhu, 2009). The overall pattern of labeling of GluA1–4 across cortical layers 1–6 in control mice in this study is consistent with previous observations in rat brain (Sato et al., 1993). The expression of GluA1–4 immunolabeling in the epileptic stargazer cortex was similar to the pattern of expression in NE controls with no marked differences across cortical layers. Preadsorption of GluA1–4 antibodies with their respective antigens as well as the omission of primary antibodies

eliminated virtually all immunolabeling in control tissue sections (Fig. 2D), indicating the specificity of the primary and secondary antibodies used in this study.

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Semi-quantitative analysis of GluA subunit expression within PV+ neurons in the somatosensory cortex

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As no marked differences were detected in the distribution of each GluA subunit in each of the cortical layers in stargazers compared to NE controls, we next analyzed

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Fig. 2. Expression of GluA1–4 in the somatosensory cortex. Confocal images showing (A) GluA1, (B) GluA2/3 and (C) GluA4 immunoreactivity in cortical layers 1–6 in both control (NE) and stargazer (E) mice. Images for GluA1–4 taken at 10x magnification (scale = 200 lm), with inserts (red bordered) for GluA2/3 taken at 40x magnification (scale = 20 lm). (D) Omission and preadsorption tests show elimination of immunolabeling in control tissue sections. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: Adotevi NK, Leitch B. Alterations in AMPA receptor subunit expression in cortical inhibitory interneurons in the epileptic stargazer mutant mouse. Neuroscience (2016), http://dx.doi.org/10.1016/j.neuroscience.2016.09.052

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expression of GluA subunits within specific cell types in the cortex. While some previous studies have suggested that stargazin is exclusively expressed in inhibitory PV+ neurons (Maheshwari et al., 2013), other studies have stated that stargazin immunoreactivity is also expressed in other cortical inhibitory neurons albeit at lower levels (Tao et al., 2013). Previously, we have shown a specific loss of AMPAR subunits (particularly GluA4-AMPARS) at CT-RTN synapses on inhibitory interneurons (PV+) in the thalamic RTN region of stargazers (Barad et al., 2012). Since the inhibitory function of PV+ interneurons is primarily mediated by AMPARs, loss of AMPARs and thus fast excitatory input to these PV+ inhibitory neurons (Menuz and Nicoll, 2008; Paz et al., 2011) potentially leads to loss of feed-forward inhibition within the CTC network and thus contributes to the generation of SWDs. Hence, next we analyzed the expression of GluA1–4 in cortical neurons co-labeled with an antibody specific for PV to detect any changes in AMPAR expression in PV+ interneurons throughout cortical layers 1–6. GluA1 & 4 expression in PV+ cells was intense and mostly somato-dendritic (see filled arrows; Fig. 3A, B), whereas GluA2/3 in PV+ neurons was much less abundant and mostly punctate around the periphery of the soma of co-localized neurons (see filled arrows; Fig. 3C). This staining pattern is consistent with other studies showing that a greater proportion of PV+ interneurons exhibit strong GluA1 and 4, but not GluA2/3 immunopositive signal (Catania et al., 1998). The proportion of PV+ soma co-expressing each of the GluA subunits was determined by counting co-labeled cells within regions of 5.02  104 square micrometers, across all layers of the somatosensory cortex in both NE controls and stargazers. We found that the majority of PV+ neurons were immunopositive for GluA1 and 4 subunits. Over 91% (117 of 129) of PV+ soma analyzed were immunopositive for GluA4 and 86% (101 of 117) were immunopositive for GluA1 in control mice, while 84% (121 of 144) and 82% (107 of 129) of PV+ soma expressed GluA4 or GluA1 respectively in stargazer mice. Conversely only 40% (53 of 130) of PV+ neurons analyzed displayed some punctate staining for GluA2/3 in control mice, while 36% (44 of 121) of stargazer PV+ cells also expressed some GluA2/3 (Fig. 4A). Our data indicate that there are no differences between NE and epileptic mice in the relative proportion of PV+ neurons expressing each GluA subunit type, and that the majority of PV+ neurons in both stargazers and NE controls express AMPARs containing the subunits GluA1 & 4. GluA1 & 4 subunits were also expressed in neurons that were not immunopositive for parvalbumin (see empty arrows; Fig. 3A–C indicating PV negative (PV ) soma). However, the majority of GluA1-immunolabeled neurons were also PV+ in NE controls (65%, 72 of 110) and stargazers (61%, 68 of 111) (Fig. 4B). Even more GluA4-positive soma were immunopositive for PV. In NE controls 84% (85 of 101) of GluA4+ neurons were also PV+ and in stargazer mice 79% (91 of 115) were PV+ (Fig. 4B). Further analyses of GluA1 and 4 coexpression with PV within each of cortical layer, showed

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no statistically significant differences in percentage of PV+ soma expressing GluA1 & 4 layer-specific differences in the cortical distribution between stargazers and controls (Fig. 4C, D). Collectively, these results indicate that the stargazin deficit has no effect on the relative proportion of PV+ inhibitory interneurons expressing either GluA1 or 4 subunits in stargazers compared to NE controls. Density of PV+ interneurons is unaltered in the stargazer cortex +

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Since loss of PV interneurons and the absence of PV in PV+/ and PV / mice have also been associated with increased susceptibility to epileptic seizures including absence seizures (Schwaller et al., 2004; van de Bovenkamp-Janssen et al., 2004; Gant et al., 2009), we also compared the relative density of PV+ neurons in each layer of the somatosensory cortex in NE and stargazer mice. PV+ neurons were distributed across cortical layers 2–6, with the highest density of PV+ neurons present in layer 4 and 5 but no-to-minimal expression in layer 1 in both mice groups (n = 6, Fig. 5A–C). To investigate if the reduced inhibitory output of the cortical PV+ interneurons (Maheshwari et al., 2013) could be due to a loss in number of PV+ neurons, an analysis of the expression of PV+ immunolabeled neurons in each layer of the somatosensory cortex was conducted for both mice groups. Sampling multiple regions of interest (ROI) of 2  10 1 square millimeters through all cortical layers showed a similar density of PV+ neurons in both control and stargazer mice, with the highest proportion of PV+ interneurons in cortical layer 5 (30% in both epileptic and control mice; Fig. 5C, D), 25% in layer 4, and 20% in both layers 2 and 6. These results indicate that although stargazin is predominantly expressed in PV+ neurons (Maheshwari et al., 2013; Tao et al., 2013), the stargazin deficit has no developmental effect on the relative density of PV+ neurons in each layer of the stargazer somatosensory cortex compared to controls. This result thus precludes the possibility that absence seizures in the stargazer are partly a consequence of fewer cortical PV+ interneurons in any cortical layer.

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GluA1 & 4 expression is altered in stargazer cortical PV+ neurons

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As stargazin influences the abundance of GluA4AMPARs and their synaptic expression in inhibitory PV+ neurons specifically in the RTN region of the thalamus (Barad et al., 2012), next we examined the relative expression of GluA1 and 4 subunits, (which are the predominant AMPAR subunits in cortical PV+ neurons) within the soma of these inhibitory interneurons in the somatosensory cortex. To quantify the expression of GluA1 and 4 within the soma of PV+ cells, an analysis of the ratio of the number of pixels within the area of colocalization of either GluA1 or 4 with PV to that of total PV expression, presented as the mean percentage area of expression of each subunit, was conducted (Fig. 6A– D). In stargazers, statistically significant decreases in the average expression of GluA1 (55.3 ± 2.68,

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Fig. 3. Expression of GluA1–4 in PV+ interneurons in the somatosensory cortex. (A–C) Co-localization of GluA1–4 (green channel) with cortical PV+ (red channel) interneurons shown here in cortical layer 4/5; (A) GluA1 and PV, (B) GluA4 and PV, (C) GluA2/3 and PV. Co-localized neurons are identified by yellow staining (see filled arrows) while in merged images. Open arrows indicate GluA1–4 positive neurons which are immunonegative for PV. Images taken at 40 magnification (scale = 50 lm). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Please cite this article in press as: Adotevi NK, Leitch B. Alterations in AMPA receptor subunit expression in cortical inhibitory interneurons in the epileptic stargazer mutant mouse. Neuroscience (2016), http://dx.doi.org/10.1016/j.neuroscience.2016.09.052

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Fig. 4. Representative bar graphs showing (A) percentage of PV+ neurons co-localized with GluA1–4 in the somatosensory cortex (GluA1: NE 86.84 ± 2.28 E 83.53 ± 2.384; GluA2/3: NE 40.76 ± 5.25 E 36.36 ± 6.10; GluA4: NE 90.99 ± 3.56, 86.30 ± 3.578). (B) percentage of GluA1 & 4 localized with PV in the somatosensory cortex (GluA1: NE 65.36 ± 2.20 E 64.04 ± 7.119; GluA4: NE 86.37 ± 9.05 E 78.93 ± 3.71 (C) Percentage of PV+ expressing GluA1 across cortical layers 2–5 (L2/3, NE 81.78 ± 0.88 E 80.23 ± 4.55; L4, NE 81.95 ± 1.90 E 79.85 ± 2.80; L5, NE 82.14 ± 1.48 E 80.14 ± 2.50; L6, NE 80.55 ± 1.73 E 79.59 ± 0.59). (D) Percentage of PV+ expressing GluA4 across cortical layers 2–5 (L2/3, NE 84.49 ± 1.75 E 83.0 ± 2.65; L4, NE 85.79 ± 2.36 E 82.13 ± 1.08; L5, NE 85.77 ± 1.27 E 82.61 ± 1.054; L6, NE 86.0 ± 2.36 E 84.17 ± 2.20). Data presented as mean ± SEM.

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p < 0.05, n = 102, Fig. 6B) and GluA4 (44.1 ± 2.72, p < 0.05, n = 117, Fig. 6D) were observed in the PV+ soma in stargazers compared to NE control mice. GluA2/3 expression was however not quantified due to the minimal and punctate labeling in co-labeled PV+ neurons. Reduced dendritic expression of GluA1 & 4containing AMPARs in stargazer PV+ neurons As the main role of stargazin is in trafficking AMPAR tetramers from ER to cell membrane and into the synapse (Tomita et al., 2005), we concluded our investigation into the relative levels of AMPARs in PV+ neurons in the cortex by examining whether the stargazin deficit caused reduced dendrite:soma expression and thus impaired trafficking of GluA1 and 4 containing AMPARs to synapses. We first analyzed the subcellular localization of GluA1 and 4 in the dendrites of PV+ neurons by quantifying the number of GluA1 & 4 puncta per 10 lm dendrite (according to the method of Tao et al., 2013) of co-labeled

neurons. Results from this analysis showed that although there was no statistically significant difference in GluA1 & 4 puncta sizes in both mice groups (p > 0.05, n = 3 pairs, Fig. 7D), there was a decrease in the number of puncta for both GluA1 (36% reduction, p < 0.005, n = 15, Fig. 7C) and GluA4 (30% reduction, p < 0.005, n = 12, Fig. 7C) subunits in the dendrites of stargazer PV+ neurons compared to NE controls. Secondly, to assess if trafficking of AMPARs to synapses was compromised in stargazers, we compared the ratio of GluA expression in the dendrite versus the soma of the same neuron (according to the method Maheshwari et al., 2013) by measuring the pixel density of GluA staining within a 10  2 lm2 rectangular ROI in the proximal area of a primary dendrite relative to that within a 5 lm circular ROI in the somatic region at the base of the dendrite. In co-labeled GluA4+/PV+ interneurons which met the inclusion criteria, there was a significant decrease in the dendrite:soma ratio of GluA4 staining (39%, p < 0.001, n = n = 12, Fig. 7B, E) in stargazers compared to NE control mice. However, as dendritic GluA1 staining in

Please cite this article in press as: Adotevi NK, Leitch B. Alterations in AMPA receptor subunit expression in cortical inhibitory interneurons in the epileptic stargazer mutant mouse. Neuroscience (2016), http://dx.doi.org/10.1016/j.neuroscience.2016.09.052

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Fig. 5. PV expression in the mouse somatosensory cortex. (A) Confocal images showing PV+ interneurons in cortical layers 1–6 in control and stargazer mice. Images taken at 10x magnification, scale = 200 lm. (B) Analysis of the density of PV+ interneurons showing no difference in expression across cortical layers 1–6 in between stargazer and non-epileptic mice. (C) Percentage distribution of PV+ cells through layer 1–6 showing similar pattern of expression in both mice groups.

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PV+ neurons in stargazers was almost below minimal detection levels (Fig. 7A), it was difficult to compare pixel densities in dendrites to soma. As no GluA2 & 3+/PV+ neurons met the inclusion criteria, the expression of these subunits was not analyzed in this study. Collectively, the above results indicate that there is a loss of expression of major subunits (GluA1, 4) comprising AMPARs in PV+ inhibitory interneurons, and the reduction in dendritic expression of GluA4-AMPARs relative to soma levels is indicative of reduced trafficking to synapses in cortical PV+ interneurons.

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DISCUSSION

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The current study identifies cell specific alterations in AMPAR subunit expression in the stargazer cortex. We have clearly shown that while the density of cortical inhibitory PV+ neurons is unchanged in epileptic stargazer mice compared to NE littermates, there is a loss of somatic expression and dendritic trafficking of AMPARs containing GluA1 & 4 subunits in stargazer PV+ neurons. These data complement previous work from our group demonstrating a loss of predominantly GluA4 containing AMPARs specifically in inhibitory RTN neurons (Barad et al., 2012) but not VP relay excitatory neurons, accounting for the reduction in AMPAR currents at CT-RTN synapses and a potential loss of feed-forward inhibition in the stargazer thalamus (Menuz and Nicoll,

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2008). As such, our current finding of reduced expression of the major subunits comprising AMPARs in cortical PV+ neurons could indicate a specific loss of AMPARs at TC inputs to PV+ basket cells and therefore reduced feedforward inhibition to pyramidal neurons.

Functional implications of loss of excitation to PV+ interneurons in the cortex +

Loss of feed-forward inhibition by PV neurons within CTC microcircuits has been implicated in several models of absence epilepsy arising from different genetic backgrounds (Sasaki et al., 2006; Paz et al., 2011; Maheshwari et al., 2013). The sequential coordinated firing of reciprocally connected excitatory and inhibitory neurons in the cortical circuitry is essential to achieve normal oscillations within the CTC network. Fast spiking inhibitory PV+ neurons (e.g. basket cells), which are potently excited by AMPA receptors, synapse onto the soma and proximal dendrites of excitatory pyramidal neurons (Armstrong and Soltesz, 2012). They provide feed-forward inhibition to suppress the output of the pyramidal neurons and prevent their overexcitation in order to avoid pathological network oscillations (Farrant and Nusser, 2005). However, feed-forward inhibition is fragile as the function of these PV+ neurons are vulnerable to several factors including reduced intrinsic or synaptic excitation (Paz and Huguenard, 2015). Therefore, PV+

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Fig. 6. Representative confocal images and bar graphs showing quantification of mean % area of fluorescence in co-expressed GluA/PV neurons. (A, B) GluA1 in control (NE 63.50 ± 2.45, n = 107) and stargazers (E 55.36 ± 2.68, n = 102), *p = 0.027 and (C,D) GluA4 in control (NE 52.25 ± 2.59, n = 116) and stargazers (E 44.14 ± 2.72, n = 117), *p = 0.031. 597 598 599 600

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neuron dysfunction resulting from the loss of AMPARmediated excitation, could be a potential ‘‘choke point” as proposed by Paz and Huguenard (2015), and thus represent a target for highly specific anti-epileptic therapies. Density of cortical PV+ neurons in stargazers compared to NE mice However, an alternative mechanism for disruption of inhibitory balance within the network is loss of PV+ interneurons. While one study reported a loss of inhibitory neurons in rat models of absence epilepsy that could impair cortical feed-forward inhibition (van de Bovenkamp-Janssen et al., 2004), our results indicate that there is no difference in the density of somatosensory cortical PV+ neurons in stargazers compared to NE mice.

This is consistent with other studies on epileptic models, which found no loss of neurons in the somatosensory cortex of WAG/Rij rats (Sitnikova et al., 2011) and neocortex of the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model (Sabers et al., 1996). As such, our results indicate that in the stargazer cortical disinhibition may primarily be due to impaired PV+ inhibitory (presumably basket) cell function as a consequence of loss of their specific AMPARs rather than reduction in the total number of PV+ neurons. Loss of AMPARs containing GluA1 & 4 in PV+ interneurons An examination of the global expression of each AMPAR subunit expression in whole-tissue lysates revealed that

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Fig. 7. Impaired trafficking of GluA1 & 4-AMPARs in PV+ neurons. (A) There is minimal dendritic labeling of GluA1 (yellow staining) in co-localized cortical GluA1+/PV+ neurons in stargazers compared to non-epileptic mice. (B) GluA4 trafficking is impaired in cortical PV+ neurons in the stargazer somatosensory cortex. A stronger GluA4 labeling is visible in the dendrites of co-labeled GluA4+/PV+ neurons (red-bordered inserts) of control non-epileptic mice compared to stargazer mice (white arrows). (C) Quantitative analysis of GluA puncta number in co-labeled interneurons show reduced GluA1 (NE 4.83 ± 0.44, n = 12; E 3.07 ± 0.34, n = 15; **p < 0.01) and GluA4 (NE 7.92 ± 0.54, n = 12; E 3.07 ± 0.36, n = 15; * p < 0.01) dendritic labeling in stargazers compared to NE mice. (D) Quantitative analysis of GluA puncta size showed no statistically significant difference in the sizes of both GluA1 (NE 0.69 ± 0.11; E 0.57 ± 0.10; p > 0.05) and GluA4 (NE 0.65 ± 0.23; E 0.57 ± 0.18; p > 0.05) punctae in both mice groups. (E) Quantification of the mean dendrite:soma ratio of GluA4 immunolabeling shows a significant decrease in stargazer PV+ neurons (NE 0.6377, n = 10 and E 0.3908, n = 12; ****p < 0.001).

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the GluA4 showed the largest significant reduction (40%) in the stargazer somatosensory cortex compared to NE control littermates. Consistent with the overall global loss of GluA4 detected by Western blot analyses, we also found reductions in the expression of GluA4-AMPARs in both the soma (16%) and dendrites (30–39%) of PV+ neurons in stargazers compared to NE controls. In contrast to GluA4, surprisingly the global expression of GluA1 was not significantly reduced (9%) in the stargazer cortex. However, we did find a significant decrease in its cellular expression in the soma (13%) and dendrites (36%) of PV+ neurons. If stargazin is exclusive to PV+ interneurons in the CTC network, as has been suggested (Maheshwari et al., 2013), then the stargazin deficit would account for the specific loss of GluA1 and GluA4 expression in stargazer cortical PV+ neurons (somatic and dendritic) and in mistrafficking of AMPARs to synapses (ratio of dendritic to somatic labeling). While we do not know the exact reason why changes in GluA1 were not detectable at the wholetissue level by Western blot, it could be that changes specific to PV+ neurons are masked in whole analyses by unchanged levels of GluA1 in other neuron types (e.g. pyramidal cells and non PV+ neurons). Interestingly, more than a third of GluA1-AMPARs were not co-localized with PV, in contrast to the high expression of GluA4 in these neurons compared to other cortical neurons. As such, the analyses of whole cortex tissue lysates

with Western blotting might not be sensitive enough to detect the proportion of GluA1 changes only occurring in PV+ neurons against the background of unchanged GluA1 expressed in other cortical neurons. Since most GluA4+ neurons in the cortex co-express GluA1 (Kondo et al., 1997), the similar reduction in the expression of both GluA1 (13%; and 36% respectively) and GluA4 (16%; and >30% respectively) in the soma and dendrites of PV+ neurons shown in this study may indicate the predominant loss of GluA1/4 containing AMPA receptors at synapses of PV+ interneurons. Previous studies also show that PV+ neurons predominantly express AMPARs comprising GluA1 and 4 subunits (Geiger et al., 1995; Kwok et al., 1997), consistent with our results that show a majority of PV+ neurons (>80%) express GluA1 and 4 AMPARs. GluA1/4 containing AMPARs primarily mediate fast and robust excitatory neurotransmission, and their expression has been shown to be crucial to provision of temporal and precise control of network synchrony by PV+ interneurons (Bartos et al., 2007) as the complete elimination of either GluA1 or 4 in PV+ neurons, compromises the gamma frequency oscillations (Fuchs et al., 2007) necessary to maintain the excitationinhibition balance within the circuitry. Hence, the loss of somatic and dendritic expression of these AMPAR subunits in the stargazer cortex could potentially impair the AMPAR-mediated excitation of a majority of PV+ neurons, which in turn compromises their inhibitory output.

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Changes in GluA2 & 3 expression in the cortex

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Western blot analyses of whole somatosensory cortex levels of GluA2 & 3 subunits revealed a 30% reduction in GluA2 and a 31% reduction in GluA3. While our confocal microscopy results also indicate that a proportion of PV+ neurons (36–40%) are immunopositive for GluA2/3 in the somatosensory cortex, we were unable to confirm if the subunit losses were specific to PV+ neurons due to the pattern of GluA2/3 immunolabeling and related difficulty in analyzing their expression at cellular level. Nonetheless, previous work from our group has also shown a loss of GluA2/3 in the stargazer RTN inhibitory neurons, but not excitatory VP relay cells (Barad et al., 2012), suggesting that the loss of cortical GluA2/3 seen in this study may be associated with inhibitory interneurons rather than excitatory pyramidal cells. As such, the stargazin deficit would lead to reduced expression and trafficking of all subunits comprising AMPARs in PV+ inhibitory interneurons. The expression of GluA2 containing AMPARs has been shown to be crucial for mediating the fast AMPAR synaptic currents necessary for the activation of interneurons (Stincic and Frerking, 2015) and loss of GluA2-AMPARs has been implicated in the lowering of the threshold for seizures (Friedman and Koudinov, 1999). Hence, a loss of GluA2-AMPARs may induce changes in AMPAR-mediated synaptic transmission onto PV+ neurons that contribute to the generation of SWDs. The precise role of GluA3 in seizure activity however remains largely controversial; while in vitro electrophysiology studies show normal cortical synaptic transmission and no SWDs in GluA3-knockout mice (Meng et al., 2003; Beyer et al., 2008), in vivo EEG recordings reveal enhanced susceptibility to seizures. However, it should be noted that this susceptibility was seen in only a third of sampled animals (Steenland et al., 2008), as such it is likely that the loss of GluA3 must be in concert with the loss of other AMPAR subunits to mediate seizure activity.

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Can the loss of AMPARs sufficiently impair cortical inhibition in stargazers? Our results indicate a potential impairment of cortical feed-forward inhibition in stargazer mice due to the reduction of AMPARs in cortical inhibitory PV+ neurons. However, if stargazin expression is exclusive to cortical inhibitory neurons (Maheshwari et al., 2013; Tao et al., 2013), the stargazin deficit should not affect the expression of these AMPAR subunits in excitatory pyramidal cells, which presumably express other TARPs, and thus excitation of these pyramidal cells remains intact. Hence, as feed-forward inhibition is limited in its ability to control recurrent excitation, the net result is a hyperexcitable cortex susceptible to SWDs. However, GABAergic inhibition in stargazers appears to be influenced by other factors, including the expression of excitatory NMDARs. For example, some researchers have proposed that in the absence of AMPAR-mediated excitation, there is a compensatory enhancement of

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NMDAR activation of inhibitory neurons, resulting in a corresponding increase in thalamic inhibition (Lacey et al., 2012). However, recent studies show that while both AMPARs and NMDARs mediate excitation at glutamatergic synapses in the cortex, the NMDAR component of the excitatory current onto PV+ neurons is quite small, with these neurons primarily activated by AMPAR-mediated excitation (reviewed in GonzalezBurgos et al., 2015). This is consistent with reports that in neocortical inhibitory neurons the recruitment of fast inhibition can be independent of NMDAR-mediated transmission as the blockade of AMPARs completely abolished GABAA-mediated inhibition in neocortical inhibitory neurons (Ling and Benardo, 1995). Hence, it is likely that in stargazer cortical inhibitory PV+ neurons, the compensatory NMDAR-mediated excitation may not be sufficient to overcome the cortical disinhibition resulting from the loss of AMPARs. In agreement with this observation is that NMDAR antagonists exacerbate seizures in the stargazer cortex due to a ‘‘further dose-dependent reduction in inhibition” (Maheshwari et al., 2013), and thus establishes a crucial role for the loss of AMPAR-mediated excitation onto PV+ interneurons within the cortical microcircuit in seizure activity in epileptic stargazers.

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In summary, the current study has shown reduced expression and dendritic trafficking of GluA1/4 AMPA receptors in PV+ inhibitory neurons in the somatosensory cortex of epileptic stargazers. The loss of AMPA receptors in fast spiking PV+ interneurons (presumably basket cells) provides support for the involvement of cortical disinhibition in the generation of seizures in stargazers as a result of dysfunctional feedforward microcircuits. As AMPAR-mediated excitation of PV+ inhibitory neurons is reduced, the subsequently impaired feed-forward inhibition onto excitatory pyramidal neurons fails to suppress recurrent excitation within the somatosensory cortex. This molecular switch in the excitation-inhibition balance may create enhanced network oscillations that promote the generation SWDs within the cortical microcircuit. Future ultrastructural studies would help determine if the tissue and cellular changes in AMPA receptor expression are reflected at the synapses of PV+ inhibitory neurons and could therefore impact their synaptic excitation. Furthermore, it will be important to determine if these changes in AMPAR expression are exclusive to PV+ neurons, and do not occur at excitatory synapses on excitatory pyramidal neurons, and thus selectively impair feedforward inhibition in the stargazer cortex. Electrophysiological studies may also be important to confirm the functional implications of the data presented in this study with regard to the stargazer epileptic phenotype. Clearly, although a number of different molecular mechanisms may be capable of switching CTC network from normal to pathological oscillations, the loss of cortical AMPA receptors could be an important contributory factor in some human cases of absence epilepsy and therefore elucidating cell and

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synapse specific changes is crucial to the identification and development of targeted and effective therapies.

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Acknowledgments—The authors thank the staff of the Otago Centre for Confocal Microscopy (OCCM) for excellent technical support, and the University of Otago for the Doctoral Scholarship awarded to N.K.A. This work was supported by grants from the University of Otago Research Grants (UORG) and Deans Bequest Fund awarded to B.L.

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(Accepted 30 September 2016) (Available online xxxx)

Please cite this article in press as: Adotevi NK, Leitch B. Alterations in AMPA receptor subunit expression in cortical inhibitory interneurons in the epileptic stargazer mutant mouse. Neuroscience (2016), http://dx.doi.org/10.1016/j.neuroscience.2016.09.052

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