Indeed, reducing SOD1 expression has been reported to slow diseas

Indeed, reducing SOD1 expression has been reported to slow disease progression of transgenic mice and rats expressing human mutant SOD1 Olaparib nmr (Ralph et al., 2005, Raoul et al., 2005 and Smith et al., 2006). A further glimmer of hope has emerged from a successful phase I safety trial using antisense oligonucleotides against SOD1 in patients carrying mutant SOD1 (Miller et al., 2013). A similar strategy targeting the potentially toxic RNA species or RAN translation of it can be envisioned for the more frequent instances of disease from hexanucleotide expansion in C9ORF72. Several lines of evidence indicate that broad defects in protein homeostasis

may contribute to ALS pathogenesis: (1) all ALS patients have one of the following protein inclusions in affected motor neurons: TDP-43, FUS/TLS, or SOD1; (2) ALS-linked mutations are identified in several selleck chemicals llc genes involved in ER stress, autophagy, and the ubiquitin-proteasome pathway; (3) ALS-linked mutations in ubiquilin-2, CHMP2B, and VCP can lead to TDP-43 aggregation; (4) dysfunctions in ERAD and autophagy are observed in mouse models expressing mutant SOD1; and (5) autophagy appears to be activated and upregulated in motor neurons of sporadic ALS patients. It is not clear how a decline in general protein

degradation machinery might cause aggregation of specific proteins in different neurodegenerative diseases. However, it is conceivable that increasing (or delaying age-dependent decline in) proteostasis could, in principle, prevent or slow down the formation of protein inclusions—or 17-DMAG (Alvespimycin) HCl at least accumulation of some or all of the toxic protein species. Initial hints that this approach could be beneficial came from report of modest delay in disease progression following treatment of a very small number of mice with arimoclomol, an inducer of heat shock proteins HSP70 and HSP90 (Kieran et al., 2004). Phase 2/3 clinical trials are currently underway for this approach. Dampening the UPR by deleting a downstream

X-box-binding protein (XBP-1) was reported to provide a modest survival benefit (∼20 days) to a small cohort (n = 7) of SOD1G86R mice, but the apparent benefit was disappointingly found only in female mice (Hetz et al., 2009). Finally, pharmacological activation of autophagy was reported in another small cohort of mice (n = 10 per drug treatment) to improve cognitive and motor phenotype in male mice overexpressing wild-type TDP-43 (Wang et al., 2012). Independent replications of the above experiments with larger cohorts that are powered to provide statistical significance—and extended to multiple ALS/FTD mouse models—are now needed to validate the therapeutic potential of these approaches.

, 2006) Without photoconversion, Dendra2 emits green fluorescenc

, 2006). Without photoconversion, Dendra2 emits green fluorescence. UV illumination

converts the pre-existing green fluorescent proteins to red so that they can be distinguished from newly synthesized proteins. We chased the degradation of SAX-3 by measuring the intensity Selleckchem DAPT change of red fluorescence at different time points postphotoconversion. At 7 hr after photoconversion, an average 35% of photoconverted SAX-3(WT)::Dendra was degraded in AVM at L1–L2 stages, and temperature rise had little effect on its degradation rate (Figures 6B and 6C). In contrast, over 50% of SAX-3(P37S)::Dendra was degraded at 20°C, and the level of degradation was further increased by temperature shift to 22.5°C (Figure 6C). In touch neurons, a fraction of SAX-3(P37S) was misfolded and either diffused or formed aggregates in the cytosol, whereas most SAX-3(WT) was in a native form and predominantly located on the cell surface (Figure 5C). Therefore, the difference of degradation rates between SAX-3(P37S) and SAX-3(WT) suggests that misfolded KU 55933 SAX-3 is overall more vulnerable to degradation than native SAX-3. Further, we found that the ebax-1(ju699) null mutation significantly reduced

the degradation of SAX-3(P37S) ( Figure 6D). Interestingly, a similar reduction was also observed in the degradation rate of SAX-3(WT) in ebax-1 mutants, suggesting that in vivo a pool of wild-type proteins,

possibly those in nonnative forms, relies on EBAX-1 for degradation. Supporting this finding, around 44% of AVM neurons in ebax-1 mutants showed aggregation of SAX-3(WT)-GFP, a 3-fold increase not over neurons in wild-type animals ( Figure S6E). Additionally, we found that after enriching misfolded proteins by proteasome inhibition in live worms, a fraction of SAX-3(WT) was detected in the EBAX-1 immunoprecipitant ( Figure S6F). In HEK293T cells expressing EBAX-1, ubiquitinated SAX-3(WT) was accumulated after proteasome activity was blocked for 4 hr ( Figure S6G). When the function of Hsp90 was further inhibited, the level of SAX-3 ubiquitination was increased ( Figure S6G), and SAX-3(WT) was recognized by EBAX-1 ( Figure S6H). Together, these data indicate that EBAX-1 can target misfolded wild-type SAX-3 as well as metastable mutant SAX-3. Consistent with the dependence of misfolded SAX-3 on EBAX-1 for degradation, we found that the AVM guidance defect in the sax-3(ky200) mutant showed strong sensitivity to the protein level of EBAX-1. Loss of endogenous EBAX-1 worsened the guidance defect caused by sax-3(ky200) at 20°C but did not further enhance the defect at 22.5°C. Overexpression of EBAX-1 significantly suppressed the guidance defect at 22.5°C ( Figure 7A).

These would not rely upon the global re-expression of intracellul

These would not rely upon the global re-expression of intracellular molecules, such as BDNF signaling or Mecp2 itself (Chang et al., C646 2006; Guy et al., 2007; Kline et al., 2010). Importantly, NR2A transcription, translation and posttranslational modifications

are regulated by multiple factors, including but not limited to Mecp2 binding (Sanz-Clemente et al., 2010). For example, novel NR2A receptor antagonists (Liu et al., 2004; de Marchena et al., 2008), as well as the casein kinase pathway (Sanz-Clemente et al., 2010), can now be assayed. Ketamine—an NMDA receptor antagonist acting preferentially on PV cells (Behrens et al., 2007)—has recently been reported to reverse functional deficits in key forebrain nodes of the default mode network in Mecp2 KO mice (Kron et al., 2012). Other factors, such as the Otx2 homeoprotein, have been found to maintain PV-cells in a mature state (Beurdeley et al., 2012). Knockdown strategies regulating Otx2 content may also be fruitful in treating the Mecp2 KO mice. Maturation of visual cortical circuits is reportedly impaired in another autism model, the Angelman syndrome mouse deficient in Ube3a (Yashiro et al., 2009), which can also be reversed

by sensory deprivation. Our results indicate that ongoing endogenous neural activity may ensure the stability of cortical circuits. At a synaptic level, spontaneous transmitter release is required to maintain Selumetinib clinical trial postsynaptic receptors (McKinney et al., 1999; Saitoe et al., 2001), while spontaneous action potentials observe spike-timing-dependent plasticity rules for synapse strengthening and maintenance of connectivity (Gilson et al.,

2009; Kolodziejski et al., 2010). DR or NR2A disruption, while degrading orientation tuning even further (Figure 6), is sufficient to rescue both spontaneous neural activity and normal Tryptophan synthase visual acuity in Mecp2 KO mice. Retinogeniculate circuits are instead unaffected by late DR, responding as if deprived in the Mecp2 KO mouse (Noutel et al., 2011). Our findings ultimately reveal that vision in Rett syndrome patients may serve as a robust biomarker of both cortical status and its response to therapy. To date, visual processing and vision, in general, have never been analyzed in a systematic manner in RTT patients. Available data in the literature are limited and mixed ( Saunders et al., 1995; von Tetzchner et al., 1996) and a few studies have suggested some abnormal visual processing in RTT patients ( Bader et al., 1989, Stauder et al., 2006; von Tetzchner et al., 1996). This is a missed opportunity, given that eye gaze is one of the relatively well-preserved functions in non-verbal RTT girls, making vision testing feasible. Preliminary data indicate a clear correlation between visual processing and the clinical stage of RTT patients (G. DeGregorio, O. Khwaja, W. Kaufmann, M.F., and C.A. Nelson, unpublished data).

These different findings could be reconciled by a model in which

These different findings could be reconciled by a model in which HVCX neurons accumulate feedback information slowly (hours to days) and where feedback-driven changes in these cells first appear as a subtle modification of synaptic input, rather than changes in

action potential output. Testing this model requires a way of longitudinally monitoring synapses on identified Lumacaftor solubility dmso neurons before and after manipulation of auditory feedback changes song output, a goal currently impractical to achieve using electrophysiological methods. In vivo, multiphoton imaging of fluorescently labeled neurons can resolve individual dendritic spines, which are postsynaptic components of excitatory synapses in the vertebrate brain (De Robertis and Bennett, 1955 and Palay, 1956), and this method has been used in a variety of longitudinal studies to measure experience-dependent changes to synapses (for reviews, see Alvarez and Sabatini, 2007, and Holtmaat and Svoboda, 2009). Recently, this method has also been used to show that auditory experience of a vocal model stabilizes

and enlarges HVC dendritic spines in juvenile songbirds over a period of days (Roberts et al., 2010), advancing it as a suitable method for detecting relatively slow feedback-related changes to synapses in the HVC of adult songbirds. Here, we used longitudinal in vivo two-photon imaging of dendritic spines in deafened adult zebra finches to test the idea that synapses on HVC PNs are ABT-199 ic50 sensitive to changes in auditory

feedback. To label and identify HVC projection neurons for in vivo imaging, a GFP-lentivirus was injected into HVC, and differently colored retrograde tracers were injected into the two downstream targets of HVC, the striatal region Area X and the song premotor nucleus RA, in young adult male zebra finches (Figure 1A and see Figure S1A available online; 80 to 150 days posthatch (dph), mean age was 97 ± 5 days, all reported errors are SEM unless otherwise noted). Birds were maintained on a reverse day-night cycle and imaging sessions were conducted during the birds’ subjective nighttimes, to minimize interference with singing behavior Phosphoprotein phosphatase (2 sessions per night separated by a 2 hr interval). Images were obtained through a cranial window and collection of imaging data was restricted to neurons with dendritic spines, because both populations of HVC PNs are spinous (Mooney, 2000). Neurons were identified as either HVCX or HVCRA cells by the presence of blue or red retrograde label or, in the absence of retrograde label, by the measurement of soma size, which differed significantly for the two PN types (Figures 1A and S1B). After collecting 1–2 nights of baseline imaging data, birds were deafened by bilateral removal of the cochleae, and data collection was continued as long as possible (13 birds were imaged for an average of 7.2 ± 4.1 nights postdeafening).

001 for both), as previously shown ( Marquardt et al , 2005) In

001 for both), as previously shown ( Marquardt et al., 2005). In medial LMC neuron growth cones labeled by e[Isl1]:GFP electroporation, however, we observed that the majority of patches contained both ephrin-A5 and EphA3 protein ( Figures 7D–7F; p = 0.124 and 0.236). These observations thus suggest a vastly different distribution EphAs and ephrin-As in medial LMC versus lateral LMC growth Apoptosis inhibitor cones. To examine the effect of ephrin expression on the distribution pattern of Ephs and ephrins, we knocked down ephrin-A5 expression in medial LMC neurons. Similar to the control

medial LMC growth cones, those treated with scrambled [eA5]siRNA via in ovo electroporation, showed obvious copatching of ephrin-A5 and EphA3 ( Figures 7G–7I; p = 0.538 and 0.169). In contrast, in medial LMC neuron growth cones electroporated with ephrin-A5 siRNA, ephrin-A5-containing patches were occasionally observed, but they no longer contained any obvious EphA3 protein ( Figures 7J–7L; p < 0.001 for

both), a configuration similar to that found in lateral LMC neurons. These findings suggest that ephrin expression levels control the subcellular distribution pattern of Ephs and ephrins, and their consequence is a shift between cis-attenuation and trans-signaling modes, PCI-32765 order increasing the precision of axon trajectory selection. Concurrent trans-reverse and trans-forward signaling versus cis-attenuation have been proposed as two divergent modes of Eph and ephrin interaction. To understand their relative contribution to axon guidance in vivo, we studied them in the context of the choice of LMC motor axon trajectory

in the limb and showed that (1) limb trajectory selection by LMC axons is specified by ephrins in LMC neurons, (2) in addition to their signaling in trans, ephrins expressed in LMC neurons contribute to guidance of LMC axons by cis-attenuation of Eph receptor signaling, and (3) the balance between cis- and trans-interaction appears to be determined by ephrin protein levels. Here, we discuss the role of the molecular symmetry of ephrin-A and ephrin-B cis-attenuation function in the fidelity of LMC axon guidance, secondly the possible mechanisms and modes of Eph cis-attenuation by ephrins, and in-cis receptor-ligand interactions as a common strategy for axon guidance signaling refinement. Based on our gain and loss of ephrin function experiments, we propose that a molecular symmetry of ephrin cis- and trans-signaling in LMC neurons controls LMC axon guidance ( Figure 7M). Our model builds on the previous in vitro observation that ephrin-A5 in LMC axons can elicit attractive EphA:ephrin-A reverse signaling ( Marquardt et al., 2005) in parallel to forward ephrin:Eph signaling ( Eberhart et al., 2002, Helmbacher et al., 2000, Kania and Jessell, 2003 and Luria et al., 2008). This model is based on our observation that increasing or decreasing ephrin levels in LMC neurons leads to, correspondingly, attenuated or augmented sensitivity to ephrins provided in trans.

To explain the robust results obtained by Bai et al (2010), it i

To explain the robust results obtained by Bai et al. (2010), it is also possible that the BAR domain may promote some see more form of clathrin-independent endocytosis, considering that rescue experiments with exogenous proteins are likely to result in at least some degree of overexpression ( Bai et al., 2010).

Although our data and previous studies emphasize the major similarity of the defects produced by the absence of either endophilin or synaptojanin 1, one notable difference was observed. In contrast to what we have found here at endophilin TKO synapses, the amplitude of mEPSCs was increased relative to control at synaptojanin 1 KO synapses. Interestingly, a similar discrepancy was observed in Drosophila, in which other properties of endophilin and synaptojanin Selleck BMS 354825 mutant synapses were similar ( Dickman et al., 2005). Determining whether

this discrepancy is due to a different impact of the lack of endophilin and of synaptojanin on postsynaptic functions is an interesting question for future investigations. Studies of endophilin’s bilayer-deforming properties had suggested that it helps bend the membrane at CCPs, perhaps starting early in the process and then shaping their neck (Farsad et al., 2001, Gallop et al., 2006 and Ringstad et al., 1999). However, imaging data have demonstrated that endophilin is recruited only shortly before fission, when most of the curvature of the bud and of its neck is already acquired (Ferguson et al., 2009 and Perera et al., 2006). Proteins suited to bind curved bilayers may function as curvature inducers or sensors depending on several Metalloexopeptidase parameters, including their concentration, bilayer chemistry, and a variety of regulatory mechanisms (Antonny, 2006). Both curvature-sensing and -generating properties of endophilin were directly demonstrated (Chang-Ileto et al., 2011, Cui et al., 2009, Farsad et al., 2001 and Madsen et al., 2010). Curvature sensing may predominate in the initial recruitment of endophilin at CCP necks, although additional polymerization may facilitate curvature stabilization and neck elongation. Our observation that the endophilin

BAR construct is targeted to the CCPs supports this possibility. Consistent with this scenario, absence of the endophilin homolog Rvs167 in yeast leads to endocytic invaginations that bounce back and forth and often do not proceed to fission, suggesting a role of Rvs167 in stabilizing a preformed invagination (Kaksonen et al., 2005). An action of endophilin before fission, even if one of its main effects becomes manifested only after fission, also agrees with the finding that a plasma-membrane-tethered endophilin-chimeric construct rescued the absence of endophilin in worms (Bai et al., 2010). The role of Hsc70 and its cochaperone auxilin in the disassembly of the clathrin lattice is well established (Massol et al., 2006, Xing et al., 2010 and Yim et al., 2010).

We found that cadherin-9-Fc but not control Fc binds the surface

We found that cadherin-9-Fc but not control Fc binds the surface of 293T cells expressing cadherin-9

in a calcium-dependent manner indicating that, like other classic cadherins, cadherin-9 undergoes homophilic binding (Figures 5B and 5C) (Shimoyama et al., 2000). One consequence of cadherin binding is recruitment and activation of β-catenin (Arikkath and Reichardt, 2008 and Stepniak et al., 2009). To determine if cadherin-9-mediated adhesion recruits β-catenin, we expressed cadherin-9 in 293T cells and found that β-catenin is recruited to the interface between cadherin-9 cells (Figure 5D). Additionally, epitope-tagged cadherin-9 colocalizes with β-catenin in neurons (Figure 5E). These results suggest that cadherin-9 is capable of intracellular signaling via catenin proteins. We next analyzed the location of cadherin-9 protein in neurons. Consistent with in situ hybridization results selleck chemicals showing that cadherin-9 is expressed selectively Apoptosis Compound Library high throughput by DG and CA3 neurons, we found that endogenous cadherin-9 protein is found in puncta along axons and dendrites in a subset of cultured hippocampal neurons (Figures 5F and 5G). Cadherin-9 puncta do not directly overlap with synaptic vesicles or the postsynaptic density but are frequently found adjacent to synaptic active zones (Figure 5H), similar to what has been observed for other

cadherins (Fannon and Colman, 1996 and Uchida et al., 1996). Antibody incompatibility prevented us from determining precisely which cell types are cadherin-9 positive in culture and from analyzing endogenous cadherin-9 protein

in vivo. However, we were able to examine the location of epitope-tagged cadherin-9 in DG neurons in the brain. DG neurons of P5 rats were infected with a lentivirus expressing cadherin-9 and GFP using a 2A peptide to drive expression of both genes from a single promoter (Figure S3A). Because the 2A peptide remains attached to cadherin-9, it was used as an epitope tag to visualize cadherin-9 with anti-2A these antibodies. We found that at P14 cadherin-9-2A localizes to dendrites and axons of DG neurons (Figures S3B–S3E). Although cadherin-9-2A was found diffusely throughout the dendrites, it localized to discrete bands at mossy fiber terminals adjacent to but not overlapping presynaptic vesicles marked by VGlut1 (Figures 5I and S3B–S3E), consistent with a role as a synaptic organizer. To examine the role of cadherin-9 in synapse formation, we generated a cadherin-9 shRNA that markedly reduces expression of a tagged cadherin-9 construct in 293T cells (Figure 6A). The construct does not significantly affect expression of several other cadherins expressed in the hippocampus, including cadherin-2 (N-cadherin), cadherin-8, and cadherin-10, based on western blot and quantitative PCR analysis (Figures 6B and 6C, and S4).

We also found no effect of learning on song coding or auditory sc

We also found no effect of learning on song coding or auditory scene processing in the higher-level AC, in contrast with previous reports that used the European Starling (e.g., Gentner and Margoliash, 2003 and Meliza and Margoliash, 2012), which may suggest differences in cortical plasticity between PI3K inhibitor species with open-ended (European Starling) and close-ended (zebra finch) learning periods.

We propose and model a cortical circuit based on feedforward inhibition that recapitulates salient aspects of the neural coding transformations observed between the primary and higher-level AC. Although the results of the simulation are in close agreement with our physiologic and pharmacologic findings, the model makes assumptions regarding the identity and connectivity of excitatory and inhibitory neurons, and the relative timing of excitatory and inhibitory inputs. The model also assumes that excitatory and inhibitory inputs to BS neurons are perfectly cotuned in frequency,

because in the model excitation is directly supplied and inhibition is indirectly supplied by the same neuron in the primary AC. Although we do not explicitly verify these assumptions, they are supported by previous studies showing that the higher-level AC receives direct Gefitinib ic50 synaptic input from the primary AC and is richly interconnected by local interneurons (Vates et al., 1996), and that neurons

in the songbird (Mooney and Prather, 2005) and mammalian (Atencio and Schreiner, 2008) cortex can be segregated based on action potential width into excitatory (broad) and inhibitory (narrow) populations. Our data show that primary AC and NS neurons in the higher-level AC have similar spike train patterns, firing rates, selectivity, and STRFs, in support of NS neurons receiving direct excitatory very input from the primary AC. Spectrally cotuned but temporally offset excitation and inhibition have been demonstrated in the mammalian auditory cortex (Wehr and Zador, 2003). Our proposed model captures our experimental findings and makes testable hypotheses about how the auditory cortex is organized to transform behaviorally relevant information. Across organisms and sensory modalities, examples of sparse coding (Crochet et al., 2011, DeWeese et al., 2003, Stopfer et al., 2003 and Weliky et al., 2003), contextual sparsification (Haider et al., 2010 and Vinje and Gallant, 2000), and feedforward inhibition (Tiesinga et al., 2008, Vogels et al., 2011 and Wehr and Zador, 2003) are common.

The converse was true of the hippocampal spectra

The converse was true of the hippocampal spectra LDK378 cost averages with the beta bandwidth exhibiting a difference favoring all successive presentations over the initial presentation (all t(25) > 3.4; p < 0.0025), and the gamma bandwidth exhibiting no differences (Figure 3B). Thus, although the polarity of the responses differed between the monkey entorhinal cortex and hippocampus, both structures signaled immediate novelty similar to the prominent signal seen in

humans. One of the most prominent task-related signals we have seen in the monkey hippocampus from single cell recording was a strong differentiation between correct and error trials (trial outcome) during the reward and ITI periods of an object-place associative HIF-1 activation learning task (Wirth et al., 2009). Similar trial outcome signals have also been reported by us in the entorhinal cortex during the location-scene association task used in the present study (E.L. Hargreaves, unpublished data). This information can be used to strengthen correct and/or rewarded associations and modify incorrect and/or unrewarded ones during learning. We first asked whether the prominent outcome signals seen at the single unit level of analysis

in monkeys were also reflected in the LFP. For all new stimuli, frequency spectra averages of the “correct” and “error” trials were analyzed during a postresponse trial epoch spanning 1,500 ms across the reward and ITI periods. Multiple regressions generated β values for power of both the gamma and beta bandwidths, which were then compared in group analyses using parametric statistics (Figures 4A and 4B). An additional exclusion criterion was applied to these analyses requiring that sessions had a minimum of seven error responses for adequate weighting of the β coefficients. For the entorhinal cortex, significant differences

between correct and error trials were seen for both the gamma (t(41) = 4.25; p < 0.0001) and beta (t(41) = 3.63; p < 0.0007) bands (Figure 4A). The direction of the Ergoloid difference for both bandwidths favored the error trials with positive β values contrasted to the correct trials negative β values. Consistent with our single unit findings in the hippocampus (Wirth et al., 2009), significant differences between correct and error trial β values were seen for the gamma band (t(24) = 3.09; p < .0036), but not the beta band. Like the entorhinal cortex, the gamma band difference in the hippocampus favored the error trials with positive β values (Figure 4B). To examine trial outcome signals in the human MTL, we analyzed the entorhinal and hippocampal ROIs using the same multiple regression to generate β values for the correct and error trial responses to new stimuli for each subject. We observed significant differences in both the entorhinal cortex (t(30) = 3.19; p < 0.0034; Figure 4C) and hippocampal (t(30) = 4.75; p < 0.0001; Figure 4D) ROIs.

We measured concentration-response curves in the A665C mutant at

We measured concentration-response curves in the A665C mutant at the beginning of the application of oxidizing conditions (peak, when the receptors were still reduced) and in steady-state oxidizing conditions, when we assumed trapping was complete. We obtained the EC50 from fits to the Hill equation: IImax=[Glu]n[Glu]n+EC50n,where n is the Hill coefficient, and [Glu] is glutamate concentration. We measured trapping after the application of CuPhen in different concentrations of glutamate, plotting

the immediate active fraction of the current against different concentrations of glutamate. This was normalized against the current following oxidizing conditions plus 10 mM glutamate. A log normal function was fitted to the data. The GluA2 receptor contains 11 cysteines, 4 of them involved in disulfide bonds (C63 with C315 and C718 with C773C). In order to obtain a construct running monomerically under denaturing conditions, CFTR activator we serially removed free cysteines, eventually constructing the 7 × Cys(−) mutant by introducing the following mutations into the GluA2 WT receptor: C89S, C190A, C436S, C425S, check details C528S, C589S, and C815S (Figure S3A). This channel remained functional and had similar properties to WT (Figure S3B). All cysteine mutants studied by western blotting were made on this background. All mutations were introduced by overlap PCR and confirmed by double-stranded

sequencing. HEK293T cells were plated in 10 cm dishes and transfected with different plasmids (5 μg) using polyethylenimine (PEI; 1 mg/μl). After Astemizole 72 hr, cells were collected in PBS, centrifuged 5 min at 1,000 × g, and pellets were resuspended in a buffer containing 300 mM NaCl, 50 mM Tris (pH 8), 1% DDM (Anatrace), and a protease inhibitor mixture (Roche). For treatments under reducing and oxidizing

conditions, dishes were rinsed with PBS followed by incubation with 100 mM DTT or 100 μM CuPhen in serum-free medium for 30 min before lysis. After sonication, the lysates were rotated (10 rpm) for 1 hr at 4°C and subsequently centrifuged at 20,000 × g to obtain cleared lysates. Protein extracts (50 μg) were then separated by 4%–12% Bis-Tris Glycine SDS/PAGE and transferred to nitrocellulose membranes. Blots were immunostained overnight at 4°C, or for 5 hr at RT, with anti-GluA2 N terminus (1:1,000; Millipore) or anti-β-actin (1:2,000; Cell Signaling) primary antibodies. Following exposure to appropriate peroxidase-conjugated secondary antibodies (Biozol), blots were visualized with chemiluminescence reagent (SuperSignal West Pico; Thermo Scientific). Densitometric analysis was performed using ImageJ ( Schneider et al., 2012). The signal from β-actin was used as a loading control, and the results were normalized as the ratio of dimer band intensity versus the total intensity of dimer and monomer bands.