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• W2149311653 abstract "During brain development, the neocortex shows periods of enhanced plasticity, which enables the acquisition of knowledge and skills that we use and build on in adult life. Key to persistent modifications of neuronal connectivity and plasticity of the neocortex are molecular changes occurring at the synapse. Here we used isobaric tag for relative and absolute quantification to measure levels of 467 synaptic proteins in a well-established model of plasticity in the mouse visual cortex and the regulation of its critical period. We found that inducing visual cortex plasticity by monocular deprivation during the critical period increased levels of kinases and proteins regulating the actin-cytoskeleton and endocytosis. Upon closure of the critical period with age, proteins associated with transmitter vesicle release and the tubulin- and septin-cytoskeletons increased, whereas actin-regulators decreased in line with augmented synapse stability and efficacy. Maintaining the visual cortex in a plastic state by dark rearing mice into adulthood only partially prevented these changes and increased levels of G-proteins and protein kinase A subunits. This suggests that in contrast to the general belief, dark rearing does not simply delay cortical development but may activate signaling pathways that specifically maintain or increase the plasticity potential of the visual cortex. Altogether, this study identified many novel candidate plasticity proteins and signaling pathways that mediate synaptic plasticity during critical developmental periods or restrict it in adulthood. During brain development, the neocortex shows periods of enhanced plasticity, which enables the acquisition of knowledge and skills that we use and build on in adult life. Key to persistent modifications of neuronal connectivity and plasticity of the neocortex are molecular changes occurring at the synapse. Here we used isobaric tag for relative and absolute quantification to measure levels of 467 synaptic proteins in a well-established model of plasticity in the mouse visual cortex and the regulation of its critical period. We found that inducing visual cortex plasticity by monocular deprivation during the critical period increased levels of kinases and proteins regulating the actin-cytoskeleton and endocytosis. Upon closure of the critical period with age, proteins associated with transmitter vesicle release and the tubulin- and septin-cytoskeletons increased, whereas actin-regulators decreased in line with augmented synapse stability and efficacy. Maintaining the visual cortex in a plastic state by dark rearing mice into adulthood only partially prevented these changes and increased levels of G-proteins and protein kinase A subunits. This suggests that in contrast to the general belief, dark rearing does not simply delay cortical development but may activate signaling pathways that specifically maintain or increase the plasticity potential of the visual cortex. Altogether, this study identified many novel candidate plasticity proteins and signaling pathways that mediate synaptic plasticity during critical developmental periods or restrict it in adulthood. Plasticity in the neocortex allows us to learn and adapt to our environment and occurs with active training and passive exposure. In particular during critical periods of development, neuronal connections of the neocortex are highly malleable. Understanding the molecular mechanisms that regulate critical period plasticity is highly relevant because dysregulation of neocortical plasticity during development underlies many disorders of the brain, ranging from a lazy eye to schizophrenia. Knowledge about the molecular events that regulate plasticity may eventually let us control neocortical plasticity during development or reactivate it in adulthood for clinical purposes. The primary visual cortex (V1) 1The abbreviations used are:V1primary visual cortexiTRAQisobaric tag for relative and absolute quantitationMDmonocular deprivationODocular dominanceFDRfalse discovery rateECMextracellular matrix. is the most frequently used brain area for studying neocortical plasticity. Plasticity of ocular dominance is an especially convenient experimental model. Prolonged occlusion of one eye (monocular deprivation, MD) during the critical period results in a physiological (1Gordon J.A. Stryker M.P. Experience-dependent plasticity of binocular responses in the primary visual cortex of the mouse.J. Neurosci. 1996; 16: 3274-3286Crossref PubMed Google Scholar) and anatomical (2Antonini A. Fagiolini M. Stryker M.P. Anatomical correlates of functional plasticity in mouse visual cortex.J. Neurosci. 1999; 19: 4388-4406Crossref PubMed Google Scholar) overrepresentation of inputs from the open eye at the cost of inputs from the deprived eye. Dark rearing, whereby animals are raised in total darkness from birth, results in a delayed critical period for plasticity of ocular dominance (OD) (3Cynader M. Prolonged sensitivity to monocular deprivation in dark-reared cats: effects of age and visual exposure.Brain Res. 1983; 284: 155-164Crossref PubMed Scopus (3) Google Scholar). Because these functional and anatomical changes are well described and can be induced with relative ease, OD plasticity in V1 is highly suitable for identifying cellular and molecular mechanisms involved in neocortical plasticity and its critical period. primary visual cortex isobaric tag for relative and absolute quantitation monocular deprivation ocular dominance false discovery rate extracellular matrix. Studies in rodents have provided increasing knowledge on the genes and proteins involved in OD plasticity, and the use of both forward- and reverse genetics (4Heimel J.A. Hermans J.M. Sommeijer J.P. Levelt C.N. Genetic control of experience-dependent plasticity in the visual cortex.Genes Brain Behav. 2008; 7: 915-923Crossref PubMed Scopus (29) Google Scholar, 5Nedivi E. Molecular analysis of developmental plasticity in neocortex.J. Neurobiol. 1999; 41: 135-147Crossref PubMed Scopus (41) Google Scholar) has been instrumental in this. Also changes in gene expression observed by microarray studies (6Majdan M. Shatz C.J. Effects of visual experience on activity-dependent gene regulation in cortex.Nat. Neurosci. 2006; 9: 650-659Crossref PubMed Scopus (138) Google Scholar, 7Tropea D. Kreiman G. Lyckman A. Mukherjee S. Yu H. Horng S. Sur M. Gene expression changes and molecular pathways mediating activity-dependent plasticity in visual cortex.Nat. Neurosci. 2006; 9: 660-668Crossref PubMed Scopus (184) Google Scholar) investigating plasticity-manipulating paradigms have generated valuable insights into the molecular processes underlying visual cortex plasticity. In order to study the molecular events intrinsic to the synapse, however, direct approaches to quantitatively address the synaptic proteome are necessary. This can be achieved by assessing fractions biochemically enriched for synaptic membranes. Such an approach has the important advantage that localized events can be revealed that are otherwise hidden in the complexity of molecular changes occurring in other subcellular compartments or in non-neuronal cell types. Here we performed proteomic analyses using an isobaric tag for relative and absolute quantitation (iTRAQ) and tandem mass spectrometry. We used this approach to identify proteins in the synaptic membrane fraction whose levels are altered by visual experience or age. This method allowed for the labeling of peptides derived from four different experimental paradigms and permitted parallel identification and comparative quantification. We analyzed the synaptic membrane proteome of the binocular area of V1 from mice: (i) during the critical period, (ii) during the critical period while OD plasticity was being induced, (iii) in young adult mice after the critical period, and (iv) in young adult mice in which the critical period was delayed with dark rearing. Direct comparison of these groups enabled us to study the effects of monocular deprivation and age on the synaptic membrane proteome fraction and analyze how dark rearing affected the age-induced changes. Throughout the study, male C57BL/6JOlaHsd mice from Harlan Netherlands were used. Mice in group 1 (“P30,” binocular visual cortex isolated at P30), group 2 (“P30-MD,” binocular visual cortex isolated at P30 after 4 days of monocular deprivation) and group 3 (“P46,” binocular visual cortex isolated at P46) were housed on a standard 12 h light-dark cycle. Right eyelids of P30-MD mice were sutured at P26 under isoflurane anesthesia as previously described (8Heimel J.A. Hartman R.J. Hermans J.M. Levelt C.N. Screening mouse vision with intrinsic signal optical imaging.Eur. J. Neurosci. 2007; 25: 795-804Crossref PubMed Scopus (57) Google Scholar). Mice in group 4 (“P46-DR,” binocular visual cortex isolated at P46 after dark rearing) were housed in the dark from before birth until decapitation. Because decapitation for this group was performed in the dark, tissue collection for P30, P30-MD, and P46 mice was done just before the end of the dark period of the light-dark cycle, to avoid fast effects of light exposure on protein expression. All experiments involving mice were approved by the institutional animal care and use committee of the Royal Netherlands Academy of Arts and Sciences. In order to prepare protein extracts enriched for synaptic membranes, binocular visual cortex was dissected, snap-frozen in liquid nitrogen, and stored at −80 °C until protein isolation. Bilateral binocular V1 was collected, except for P30-MD mice, for which only the binocular visual cortex contralateral to the deprived eye was isolated. Pools of dissected visual cortex (n = 8 hemicortices per treatment, corresponding to four mice in the groups P30, P46, and P46-DR, or to eight P30-MD mice, randomized with regard to litter composition) were homogenized in ice-cold 0.32 m sucrose buffer with 5 mm HEPES at pH 7.4 and protease inhibitor (Roche), and centrifuged at 1000 × g for 10 min at 4 °C to remove debris. Supernatant was loaded on top of a discontinuous sucrose gradient consisting of 1.2 m and 0.85 m sucrose. After ultracentrifugation at 110000 × g for 2 h at 4 °C, the fraction at the interface of 0.85 m and 1.2 m sucrose, containing the synaptosomes, was collected, resuspended, and pelleted by ultracentrifugation at 70000 × g for 30 min at 4 °C. The pellet was subsequently resuspended in a hypotonic HEPES solution and lysed. The resulting synaptic membrane fraction was recovered by ultracentrifugation using the discontinuous sucrose gradient as described earlier. The interface fraction containing the synaptic membranes was collected and pelleted by ultracentrifugation at 70000 × g for 30 min at 4 °C after which the material was redissolved in 5 mm HEPES. For iTRAQ labeling, protein concentrations were determined by means of a Bradford assay (Bio-Rad) after which for each sample, 150 μg of protein was transferred to a fresh tube and dried by SpeedVac. Synaptic membranes were dissolved in detergent (0.85% RapiGest Waters Corporation, Milford, MA), alkylated with methyl methanethiosulfonate, and digested with trypsin as described (9Van den Oever M.C. Goriounova N.A. Wan L.K. Van der Schors R.C. Binnekade R. Schoffelmeer A.N. Mansvelder H.D. Smit A.B. Spijker S. De Vries T.J. Prefrontal cortex AMPA receptor plasticity is crucial for cue-induced relapse to heroin-seeking.Nat. Neurosci. 2008; Crossref PubMed Scopus (162) Google Scholar, 10Li K.W. Miller S. Klychnikov O. Loos M. Stahl-Zeng J. Spijker S. Mayford M. Smit A.B. Quantitative proteomics and protein network analysis of hippocampal synapses of CaMKIIalpha mutant mice.J. Proteome. Res. 2007; 6: 3127-3133Crossref PubMed Scopus (46) Google Scholar). Peptides were tagged with the respective iTRAQ reagents (114 = P30; 115 = P30-MD; 116 = P46; 117 = P46-DR). To accommodate four separate pools of tissue of each of the four experimental conditions, a total of four times 4-plex iTRAQ experiments were performed. Dried iTRAQ samples were separated in the first dimension by a polysulfoethyl A strong cation exchange column (PolyLC), and the second dimension on an analytical capillary C18 column (150 mm × 100 μm i.d. column). The eluate from the C18 column was mixed with matrix (7 mg α-cyano-hydroxycinnaminic acid in 1 ml 50% acetonitril, 0.1% trifluoroacetic acid, 10 mm dicitrate ammonium), delivered at 1.5 μl/min and deposited onto an Applied Biosystems matrix-assisted laser desorption ionization plate by means of a robot (Dionex) once every 15s for a total of 384 spots. MALDI plate analysis was performed on a 4800 Proteomics Analyzer (Applied Biosystems, Forster City, CA). Peptide collision-induced dissociation was performed at 1 kV with nitrogen collision gas. Tandem MS (MS/MS) spectra were collected from 5000 laser shots. Peptides with a signal to noise ratio over 50 at the MS mode were selected for MS/MS, at a maximum of 30 MS/MS per spot. The precursor mass window was set to a relative resolution of 180. Peaklists were extracted using GPS software (AB Sciex, version 3.6). MS/MS spectra search was performed against the mouse SwissProt (release 7 February 2007; ∼15,000 sequences) and and NCBInr (release October 2007; ∼150,000 sequences) databases using Mascot (version 2.2, Matrix Science) and GPS Explorer (version 3.6, Applied Biosystems) software. Searches were performed with cysteine modification by methyl methanethiosulfonate as fixed modifications, oxidation of methionine as variable modification, a precursor mass tolerance of 150 ppm, and a fragment mass tolerance of 0.4 Da while allowing a single site of miscleavage. The false positive rates of peptide identification estimated from decoy database searches were ∼0.05 for all searches (supplemental Table S1). For subsequent analysis only those peptides were included that mapped unique to one protein. Proteins were considered for quantification if at least one unique peptide had a C.I. ≥ 95% and at least two peptides in three out of four experiments were identified. iTRAQ areas (m/z 114–117) were extracted from raw spectra and corrected for isotopic overlap using GPS explorer. As a low iTRAQ signal is less reliable for quantitation, only peptides with iTRAQ signals above 2000 were included. To compensate for potential variations in the starting amounts of the samples, individual peak areas of each iTRAQ signature peak were log transformed to yield a normal distribution, and normalized to the mean peak area for every sample. The average iTRAQ peak area of all unique peptides annotated to a certain protein was used to determine protein abundance per treatment. In order to obtain better insight in the concerted changes in protein levels and the underlying biological events, we also inspected proteins that were identified with less stringent criteria (C.I. >85%, 1 or more peptide in each set for quantification). We clearly indicated such proteins in figures and tables. We derived no conclusions about individual proteins identified with these less stringent criteria unless we confirmed these findings by Western blot analysis. To compare the abundance of proteins across four parallel iTRAQ-based experiments (sets A–D), within each experiment peptide quantity values were standardized to scores around zero by subtracting the mean peak of all four samples. Data from all experimental sets were then combined, and analyzed by Student's t test (independent samples, two-tailed) for each of the four biologically relevant comparisons. As the t test does not take into account the effect of multiple testing, we used the Statistical Analysis of Microarrays package (11Tusher V.G. Tibshirani R. Chu G. Significance analysis of microarrays applied to the ionizing radiation response.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 5116-5121Crossref PubMed Scopus (9802) Google Scholar), a resampling-based method, to estimate the false-positive rate. By creating randomized data distributions SAM estimates the rate of false positive discoveries. The q-value calculated by SAM for each protein reflects the number of empirically determined false-positives at the significance level of the respective protein. Therefore the false discovery rate (FDR) levels in our results hold information about a single protein and should not be interpreted as a global FDR level. Changes in expression levels were considered significant when the p value was below 0.05 and the respective FDR below 15%. Protein-level quantification data are listed in supplemental Table S4. Peptide-level identification and iTRAQ quantification data of the four iTRAQ experiments in this study are listed in supplemental Tables S7A–D. To establish whether functional categories of proteins were over- or underrepresented among the proteins with increased or decreased levels under different experimental conditions, we categorized all 467 proteins by function (mitochondrial, or regulating the actin cytoskeleton or neurofilament, tubulin, or septin cytoskeletons, synaptic efficacy or signal transduction, supplemental Table S5). Next we performed Chi-square tests followed by Benjamini-Hochberg correction for multiple testing to investigate whether any of the functional categories were over- or -underrepresented under a specific experimental condition. All mass spectra used in this study are publicly available at the PRIDE PRoteomics IDEntifications database under Accession numbers: 16649–16656 (http://www.ebi.ac.uk/pride/q.do?accession=1664916656) (12Vizcaíno J.A. Côté R. Reisinger F. Barsnes H. Foster J.M. Rameseder J. Hermjakob H. Martens L. The Proteomics Identifications database: 2010 update.Nucleic Acids Res. 2010; 38: D736-D742Crossref PubMed Scopus (211) Google Scholar). Western blots were performed on the four synaptic membrane protein extracts that were also used for the iTRAQ experiments. In addition we analyzed by immunoblotting two to four independent samples from pools of four animals each, kept under the same experimental conditions as the animals for iTRAQ experiments. The required amount of protein to be applied onto the gel was determined individually for each antibody in a set of test runs. Depending on the antibody, between 1 and 5 μg was used. Samples for Western blotting were prepared according to the manufacturer's protocol (NuPage®, Invitrogen) and loaded onto a NuPAGE 4–12% continuous Bis-Tris gel (Invitrogen). Before transfer to PVDF-paper, the gel was soaked in transfer buffer containing 20% MetOH and 0.1% NuPAGE antioxidant for 15 min. PVDF-paper was incubated in 100% methanol for 5 min, in MQ water and subsequently in transfer buffer. Subsequently, proteins were transferred to the PDV-paper overnight at 4 °C. After transfer, the PVDF-paper was rinsed with water, air dried, and kept at 4 °C overnight. It was then reactivated with 100% methanol, washed with MQ water and subsequently with Tris-buffered saline (TBS). After blocking with 1% casein solution in TBS for 1 h, paper was incubated with either of the following primary antibodies in 0.3% casein solution in TBS with 0.1% Tween (TBST) for 2 h at room temperature: mouse-α-Sema4D (BD Transduction Labs, 610670/553005, 1:500), rb-α-SOS-1 (Santa Cruz, Santa Cruz, CA; 1:1000), m-α-Clathrin light chain (SySy, Goettingen, Germany; 113011, 1:250), rb-α-NCAM (Millipore, AB5032, 1:1000), rb-α-Synapsin (Millipore, Billerica, MA; 1:4000), rb-α-Septin-8 (gift of B. Zieger, 1:1000), rb-α-GAT-1 (Millipore, AB1570, 1:1000), rb-α-Ube4b (gift from M. Coleman, Cambridge UK, 1:50), m-α-14–3-3 beta (Santa Cruz, sc-59417, 1:1000), m-α-14–3-3 eta (Millipore, AB9736, 1:2000), rb-α-GABA(A)-R alpha1 (Millipore, AB5609, 1:1000). Paper was then washed with TBST and incubated for 1 h at room temperature with an infrared IRDye®800CW-labeled secondary antibody (goat-α-mouse-IR (926–32210) or goat-α-rabbit-IR (926–32211), LI-COR Biosciences, Lincoln, NE; 1:5000 in TBST with 0.01% SDS to reduce background). From secondary antibody incubation onwards, papers were protected from light. Papers were washed with TBST and then with TBS, after which they were scanned for secondary antibody fluorescence using the Odyssey® Infrared Imager (LI-COR Biosciences). Relative amounts of fluorescence were quantified using the Odyssey 2.1 software package (LI-COR Biosciences). To test whether the Western blot confirmed the results obtained with iTRAQ, we determined the significance of the Western blotting data by Student's t test (one-tailed, independent samples). In order to identify synaptic proteins involved in mediating OD plasticity in the visual cortex or regulating its critical period, we performed quantitative proteomics using iTRAQ on fractions containing synaptic membranes derived from the binocular visual cortex from groups of mice kept under four different experimental conditions (Fig. 1A). The first group (P30) contained mice during the peak of the critical period, at P30. The second group (P30-MD) contained mice from the same age that were monocularly deprived from P26 for a period of 4 days. In this group we only used the binocular cortex contralateral to the deprived eye. The third group (P46) contained adolescent mice, in which the peak of the critical period had passed, at P46. The fourth group (P46-DR) contained mice of the same age that were dark reared, and in which the critical period should thus have been delayed. We used four independent sets of mice per experimental condition in order to adequately replicate our findings (sets A–D). We identified a total of 467 proteins with a confidence of more than 95% that were detected under all four conditions, in all four sets and quantified with two or more peptides in at least three sets. We compared the synaptic membrane proteome of P30 with that of P30-MD, P46, and P46-DR, and P46 with P46-DR. Overall, we found that between the different experimental conditions synaptic protein levels differed only to a moderate degree (Fig. 1B). The total numbers of proteins that had significantly (p < 0.05, t test, and false discovery rate (FDR <15%)) different levels between conditions ranged from 35 to 84 (out of the 467 proteins that were considered). Their average changes ranged between 1.16- and 1.28-fold, depending on the experimental condition. Only a small number of proteins had changed levels of more than 1.25-fold (Fig. 1B). To validate the results, we performed Western blots for 14 proteins and conditions under which we detected significant changes using iTRAQ (supplemental Fig. S1). Of these 14, we confirmed 11, showing expression level changes concordant with the iTRAQ experiment. In all these 11 cases the level changes as observed by Western blot of the samples that were also used for iTRAQ analyses occurred in the same direction as the samples prepared independently (not shown). In most cases, changes in the levels as assessed by Western blot were larger than observed by iTRAQ, which was also described previously (9Van den Oever M.C. Goriounova N.A. Wan L.K. Van der Schors R.C. Binnekade R. Schoffelmeer A.N. Mansvelder H.D. Smit A.B. Spijker S. De Vries T.J. Prefrontal cortex AMPA receptor plasticity is crucial for cue-induced relapse to heroin-seeking.Nat. Neurosci. 2008; Crossref PubMed Scopus (162) Google Scholar) This is partially caused by the fact that iTRAQ suffers to some extent from the compression of the quantitation ratios to a ratio of 1 when used with complex samples, such as our synaptic membrane preparation (13Ow S.Y. Noirel J. Cardona T. Taton A. Lindblad P. Stensjö K. Wright P.C. Quantitative overview of N2 fixation in Nostoc punctiforme ATCC 29133 through cellular enrichments and iTRAQ shotgun proteomics.J. Proteome. Res. 2009; 8: 187-198Crossref PubMed Scopus (63) Google Scholar). To analyze the effects of MD on synaptic proteins, we compared their relative expression levels in the binocular cortex of P30 and P30-MD mice. During MD, synapses may become stabilized or instead, replaced by new synapses. To obtain insight into which changes in protein levels relate to which of these events, we made two comparisons. First, we compared the significant (p < 0.05 t test, FDR <15%) changes in protein levels induced by MD (Figs. 2A–2C, indicated in gray) with those that occurred with age (P46/P30 ratio, indicated by black bars) and are thus expected to correlate with synapse maturation, which is usually associated with an increase in synapse size, efficacy, and stability. Second, we compared the changes induced by MD (P30 MD/P30 ratio) with those induced by DR (P46-DR/P46 ratio, indicated by green bars), which are expected to correlate with reduced synapse maturity. We found that among the MD regulated proteins, there was an anticorrelation between the changes in levels of proteins caused by dark rearing and by age (corr = −0.50, p < 0.005) (Fig. 2D) indicating that the changes in levels of these proteins indeed represent partially opposing biological events. Interestingly, significant changes in protein levels induced by MD correlated strongly with changes occurring with age (corr = 0.72, p < 0.000001), whereas they showed no significant correlation with changes induced by DR (corr = −0.12, p = 0.473). This suggests that V1 of mice monocularly deprived for 4 days showed a relative increase of mature synapses compared with that of visually undeprived mice. We categorized the regulated proteins in groups based on their cellular function, allowing us to obtain a better understanding of the functional implications of the observed changes. Shown in Figs. 2A–2C are changes in levels of proteins in those three categories in which more than five proteins were found to be affected by MD: (a) proteins associated with the cytoskeleton, (b) proteins involved in signal transduction, and (c) proteins known to regulate synaptic efficacy. The latter include neurotransmitter receptors, proteins regulating their trafficking, and proteins involved in vesicle release and recycling. Together, these groups of proteins represent approximately two thirds of all proteins regulated by MD. All proteins regulated by MD, including those that did not fit into one of these three categories are in supplemental Table S2. The latter mostly represent proteins with unknown functions in the central nervous system. For completeness, the relative levels, standard deviations, p values, FDRs and numbers of peptides used for quantitation of all proteins identified with a confidence higher than 85% in all four sets and under all four experimental conditions are included in supplemental Table S4. A relatively large number of proteins (p < 0.05 Chi-square test with Benjamini-Hochberg correction compared with all identified proteins, supplemental Table S5) of which synaptic expression was increased after MD are involved in regulating the actin cytoskeleton (Fig. 2A). Several of these also showed higher levels in DR (P46-DR compared with P46), suggesting that these proteins are associated with more immature synapses. These included, among others, the developmentally regulated brain protein Drebrin (14Imamura K. Shirao T. Mori K. Obata K. Changes of drebrin expression in the visual cortex of the cat during development.Neurosci. Res. 1992; 13: 33-41Crossref PubMed Scopus (23) Google Scholar) and Basp1, which has similar and partially overlapping functions in neurite outgrowth (15Frey D. Laux T. Xu L. Schneider C. Caroni P. Shared and unique roles of CAP23 and GAP43 in actin regulation, neurite outgrowth, and anatomical plasticity.J. Cell Biol. 2000; 149: 1443-1454Crossref PubMed Scopus (246) Google Scholar) as the growth associated protein GAP-43. Other proteins, including Profilin-2, Septin-3, α-Adducin and AIP1, also showed higher levels with age (P30 to P46) suggesting that they are associated with more mature synapses. MD resulted in increased levels of various kinases at synaptic membranes (Fig. 2B), including several that have been previously implicated in plasticity in V1, such as PKC-α and -γ (16Schrader L.A. Perrett S.P. Ye L. Friedlander M.J. Substrates for coincidence detection and calcium signaling for induction of synaptic potentiation in the neonatal visual cortex.J. Neurophysiol. 2004; 91: 2747-2764Crossref PubMed Scopus (4) Google Scholar) and the regulatory subunit RII-β of PKA (17Fischer Q.S. Beaver C.J. Yang Y. Rao Y. Jakobsdottir K.B. Storm D.R. McKnight G.S. Daw N.W. Requirement for the RIIbeta isoform of PKA, but not calcium-stimulated adenylyl cyclase, in visual cortical plasticity.J. Neurosci. 2004; 24: 9049-9058Crossref PubMed Scopus (53) Google Scholar). We also found an increase of Rasal1, an inhibitor of Ras-signaling. In contrast, we observed a strongly (1.59×) decreased level of StARD13, an inhibitor of Rho-signaling. Only a small set of proteins involved in regulating synaptic strength (Fig. 2C) was altered with MD. Among these were Clathrin light chains A and B, which were both up-regulated after MD. We confirmed this by Western blot analysis (supplemental Fig. S1). This suggests that endocytosis may be activated by MD, which is further supported by our observation that the endocytosis associated proteins Amphiphysin, AP-2 α-1 and AP-2 μ-1 were also significantly (t test, p < 0.05) increased after MD, albeit with an FDR of over 15% (supplemental Table S2). The other proteins in this group showing altered levels after MD did not consistently point toward well-defined biological events. To gain better insight into the broader biological context of the observed changes we also investigated the proteins that were quantified with less stringent criteria (C.I. >85% with 1 or more peptides in all four sets). These proteins are marked in Fig. 2 by a light gray bar (if quantified by fewer peptides) and/or an asterisk (if the C.I. was between 85 and 95%). Overall, these added proteins fit well in the overall molecular portrait. Among the cyto" @default.
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• W2149311653 date "2011-05-01" @default.
• W2149311653 modified "2023-10-10" @default.
• W2149311653 title "The Synaptic Proteome during Development and Plasticity of the Mouse Visual Cortex" @default.
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