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Phosphorylation of PSD-95 at serine 73 in dCA1 is required for extinction of contextual fear [1]

['Magdalena Ziółkowska', 'Laboratory Of Molecular Basis Of Behavior', 'The Nencki Institute Of Experimental Biology Of Polish Academy Of Sciences', 'Warsaw', 'Malgorzata Borczyk', 'Department Molecular Neuropharmacology', 'Maj Institute Of Pharmacology Of Polish Academy Of Sciences', 'Krakow', 'Anna Cały', 'Kamil F. Tomaszewski']

Date: 2023-05

The updating of contextual memories is essential for survival in a changing environment. Accumulating data indicate that the dorsal CA1 area (dCA1) contributes to this process. However, the cellular and molecular mechanisms of contextual fear memory updating remain poorly understood. Postsynaptic density protein 95 (PSD-95) regulates the structure and function of glutamatergic synapses. Here, using dCA1-targeted genetic manipulations in vivo, combined with ex vivo 3D electron microscopy and electrophysiology, we identify a novel, synaptic mechanism that is induced during attenuation of contextual fear memories and involves phosphorylation of PSD-95 at Serine 73 in dCA1. Our data provide the proof that PSD-95–dependent synaptic plasticity in dCA1 is required for updating of contextual fear memory.

Funding: This work was supported by a National Science Centre (Poland) (Grant SONATA BIS No. 2015/19/B/NZ4/02996 and grant MAESTRO No. 2020/38/A/NZ4/00483 to KR; Grant PRELUDIUM No. 2016/21/N/NZ4/03304 to MZ; Grant PRELUDIUM No. 2015/19/N/NZ4/03611 to KŁ; Grant PRELUDIUM No. 2019/35/N/NZ4/01910 to KFT; Grant SONATA BIS No. 2017/26/E/NZ4/00637 to JW; Grant SONATA BIS No. 2019/34/E/NZ4/00387 to TW). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2023 Ziółkowska et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The present study tests the role of PSD-95(S73) phosphorylation in the dorsal hippocampus in fear memory extinction by integrated analyses of PSD-95 protein expression and phosphorylation, dCA1-targeted expression of phosphorylation-deficient PSD-95 protein (with S73 mutated to alanine, S73A), as well as examination of dendritic spines morphology with nanoscale resolution enabled by electron microscopy. We show that phosphorylation of PSD-95(S73) is necessary for contextual fear extinction-induced PSD-95 protein regulation and remodelling of glutamatergic synapses. Moreover, it is not necessary for fear memory formation but required for fear extinction even after extensive fear extinction training. Overall, our data show for the first time that the dCA1 PSD-95(S73) phosphorylation is required for extinction of the contextual fear memory.

Postsynaptic density protein 95 (PSD-95) is the major scaffolding protein at glutamatergic synapses [ 21 ]. It directly interacts with NMDARs and with AMPARs through an auxiliary protein, stargazin [ 22 , 23 ]. Interaction of PSD-95 with stargazin regulates the synaptic content of AMPARs [ 23 – 25 ]. Accordingly, PSD-95 affects stability and maturation as well as functional and structural plasticity of glutamatergic synapses [ 26 – 35 ]. Synaptic localisation of PSD-95 is controlled by a range of posttranslational modifications with opposing effects on its synaptic retention as well as synaptic function and plasticity [ 36 ]. Here, in order to test the role of dCA1 excitatory synapses in extinction of fear memories, we focused on phosphorylation of PSD-95 at Serine 73 (S73). PSD-95(S73) is phosphorylated by the calcium and calmodulin-dependent kinase II (CaMKII) [ 32 , 37 ]. Expression of phosphorylation-deficient PSD-95, with S73 mutated to Alanine [PSD-95(S73A)], blocks the reduction in the NMDAR/PSD-95 interaction during chemical LTP in a manner that is dependent on CaMKII and calpain [ 38 ]. Hence, phosphorylation of PSD-95(S73) enables PSD-95 dissociation from the complex with GluN2B, and its trafficking to regulate synaptic growth after stimulation of NMDA receptors, and is necessary for PSD-95 protein down-regulation during NMDAR-dependent long-term depression of synaptic transmission (LTD) [ 32 , 39 ]. Importantly, both authophosphorylation-deficient αCaMKII mutant mice (αCaMKII-T286A) [ 40 ] and the loss-of-function PSD-95 mutants lacking the guanylate kinase domain of PSD-95 [ 26 ] show impaired extinction of contextual fear [ 9 , 41 ], suggesting that αCaMKII and PSD-95 interact to regulate contextual fear extinction.

The ability to form, store, and update memories is essential for animal survival. In mammals, the formation, recall, and updating of memories involve the hippocampus [ 1 – 3 ]. In particular, formation of memories strengthens the Schaffer collateral-to-dorsal CA1 area (dCA1) synapses through N-methyl-D-aspartate receptor (NMDAR)-dependent forms of synaptic plasticity [ 4 – 6 ] linked with growth and addition of new dendritic spines (harbouring glutamatergic synapses) [ 7 – 10 ]. Although some studies also found long-term depression of synaptic transmission during hippocampal-dependent tasks [ 11 , 12 ]. Similarly, updating and extinction of memories induces functional, structural, and molecular alterations of dCA1 synapses [ 13 – 15 ]. Accordingly, NMDAR-dependent plasticity of dCA1 synapses is commonly believed to be a primary cellular learning mechanism. Surprisingly, the role of dCA1 synaptic plasticity in memory formation has been recently questioned. Local genetic manipulations that impair synaptic function and plasticity specifically in dCA1 affect spatial choice and incorporation of salience information into cognitive representations, rather than formation of cognitive maps and memory engrams [ 16 – 20 ]. On the other hand, the role of dCA1 synaptic plasticity in the updating and extinction of existing hippocampus-dependent memories has not been tested yet. Understanding the molecular and cellular mechanisms that underlie fear extinction memory is crucial to develop new therapeutic approaches to alleviate persistent and unmalleable fear memories.

Results

Contextual fear extinction induces phosphorylation of PSD-95(S73) in dCA1 Phosphorylation of PSD-95(S73) has been associated with regulation of PSD-95 levels during LTP and LTD [37,39]. To test whether contextual fear extinction induces phosphorylation of PSD-95(S73) in dCA1, we generated an antibody directed against this phosphorylation site (LERGNSGLGFS sequence) (Fig 3A) [37]. Mice underwent CFC and were killed 24 hours later (5US), or after 15 or 30 minutes of the contextual fear extinction session (Ext15’ or Ext30’) (Fig 3B). The levels of PSD-95, phosphorylated PSD-95(S73) [phospho-PSD-95(S73)] and their colocalization were tested on the brain sections (Fig 3C). Total PSD-95, phospho-PSD-95(S73), and their colocalization levels were higher in the Ext15’ group, but not Ext30’ group, as compared to the 5US animals (Fig 3D–3F). Thus, our data indicate that the alteration of PSD-95 protein levels during contextual fear extinction was accompanied by transiently increased phosphorylation of PSD-95(S73). The important limitation of this experiment is the fact that, using phospho-S73 antibody, we cannot exclude that other MAGUKs are detected (due to the similar LERGNSGLGFS sequence). However, the role of phospho-PSD-95(S73) in contextual fear extinction is supported by the fact that there is increased colocalization of PSD-95 and phospho-PSD-95(S73) during extinction. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Contextual fear extinction induces transient phosphorylation of PSD-95(S73) in dCA1. (A) Western blot stained with phospho-PSD-95(S73)-specific antibody detects in the hippocampus homogenates proteins with approximately 95 kDA molecular weight. M, molecular weight marker; N, naive mouse. (B) Experimental timeline and freezing levels during training. Mice underwent CFC and were killed 24 hours later (5US, n = 6) or after 15 or 30 minutes of a fear extinction session (Ext15’, n = 7; Ext30’, n = 7). (C) Representative confocal scans of the brain slices (stOri) immunostained with antibodies specific for PSD-95, phosphorylated PSD-95(S73), and their colocalization. (D-F) Quantification of the PSD-95 (two-way ANOVA, effect of training: F(2, 17) = 2.69, P = 0.097; effect of stratum: F(1,96, 33,3) = 3.83, P = 0.033), phospho-PSD-95(S73) (two-way ANOVA, effect of training: F(2, 17) = 2.20, P = 0.141; effect of stratum: F(1,24, 21,0) = 24.9, P < 0.001) and their colocalization levels (two-way ANOVA, effect of training: F(2, 17) = 4.08, P = 0.036; effect of stratum: F(2, 34) = 0.169, P = 0.845). Each dot represents one mouse. Means ± SEM are shown. The data underlying this figure and raw image for A are available from OSF (https://osf.io/cgfa9/).CFC, contextual fear conditioning; dCA1, dorsal CA1; PSD-95, postsynaptic density protein 95; S73, Serine 73; stOri, stratum oriens. https://doi.org/10.1371/journal.pbio.3002106.g003

PSD-95(S73) phosphorylation regulates PSD-95 protein levels during contextual fear extinction To test whether phosphorylation of PSD-95(S73) regulates PSD-95 protein levels in dCA1 during fear extinction, we used dCA1-targeted expression of phosphorylation-deficient PSD-95(S73A). We designed and produced adeno-associated viral vectors (AAV1/2) encoding wild-type (WT) PSD-95 protein under Camk2a promoter fused with mCherry (AAV1/2:CaMKII_PSD-95(WT):mCherry) (WT) or PSD-95(S73A) fused with mCherry (AAV1/2:CaMKII_PSD-95(S73A):mCherry) (S73A) [39] (S2 Fig). Mice underwent CFC (Fig 4A). The animals in all experimental groups showed increased freezing levels at the end of the training. Half of the mice were killed 24 hours after CFC (5US). The remaining half were killed after the 30-minute contextual fear extinction session (Ext). All animals showed high freezing levels at the beginning of the session, which decreased during the session. No effect of the virus on animal behaviour was found (Fig 4A). PPT PowerPoint slide

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TIFF original image Download: Fig 4. PSD-95(S73) is phosphorylated during fear extinction and this process is required for regulation of PSD-95 protein levels. (A) Experimental timeline and freezing during training. C57BL/6J male mice were stereotactically injected in the dCA1 with AAV1/2 encoding PSD-95(WT) (WT, n = 12) or PSD-95(S73A) (S73A, n = 12). Twenty-one days later, they underwent CFC (two-way repeated-measures ANOVA, effect of training: F(1, 30) = 269.4, P < 0.001, effect of virus: F(2, 30) = 2.815, P = 0.076) and were killed 1 day after training (5US) or they were reexposed to the training context without footshock and killed (Ext) (two-way repeated-measures ANOVA, effect of training: F(1, 15) = 65.68, P < 0.001; effect of virus: F(2, 15) = 0.993, P = 0.393). (B) Microphotography of a brain with dCA1 PSD-95(WT):mCherry expression with illustration of the brain processing scheme. (C) Summary of data showing the viruses penetrance in dCA1 (sections used for confocal and SBEM analysis) (mice: 5US/Ext, WT = 6/5; S73A = 6/6). (D) Correlative confocal-electron microscopy analysis showing that exogenous PSD-95(WT) colocalizes with PSDs. Single confocal scan of an exogenous PSD-95(WT) in dCA1, SBEM scan of the same area, superposition of confocal (orange) and SBEM images based on measured distances between large synapses (1 and 2), and thresholded synaptic PSD-95(WT) signal. Measurements: (confocal image) 1: 3.12 μm, 2: 4.97 μm; (SBEM image) 1: 2.98 μm, 2: 4.97 μm. (E, F) Analysis of total PSD-95 expression after fear extinction training. (E) Representative confocal scans of the PSD-95 immunostaining and (F) summary of data showing total PSD-95 levels (tree-way ANOVA with LSD post hoc tests for planned comparisons, effect of training × virus interaction, F(1, 19) = 4.603, P = 0.0451). Means ± SEM are shown. The data underlying this figure are available from OSF (https://osf.io/cgfa9/). CFC, contextual fear conditioning; dCA1, dorsal CA1; PSD-95, postsynaptic density protein 95; S73, Serine 73; SBEM, serial block-face scanning electron microscopy; WT, wild-type. https://doi.org/10.1371/journal.pbio.3002106.g004 For each animal, half of the brain was chosen at random for confocal analysis of the total PSD-95 protein levels, and the other half was processed for SBEM (Fig 4B). The AAVs penetrance did not differ between the experimental groups (5US versus Ext) and reached over 80% of the cells in the analysed sections of dCA1 (Fig 4C). We observed a significant increase in total PSD-95 protein levels in WT and S73A mice killed before the fear extinction session as compared to the Control group killed at the same time point (S2A–S2C Fig). Correlative light and electron microscopy confirmed that the exogenous PSD-95 colocalised with PSDs and weak signal was present in dendrites (Fig 3D). Furthermore, overexpression of PSD-95 protein (WT and S73A) resulted in decreased dendritic spines density and increased surface area of PSDs, compared to the Control group. However, total PSD surface area per tissue brick was not changed (S2D–S2G Fig). As in Thy1-GFP mice, the total PSD-95 protein levels were not changed after fear extinction in the WT group, as compared to the WT mice killed before the fear extinction session (Fig 4E and 4F). However, PSD-95 levels were up-regulated in all strata after the extinction session in the S73A mice, as compared to the WT Ext animals and the S73A 5US group (Fig 4F). Hence, exogenous PSD-95(S73A) protein impaired regulation of PSD-95 levels in dCA1 during contextual fear extinction, indicating that phosphorylation of PSD-95(S73) controls PSD-95 levels during this process.

Phosphorylation of PSD-95(S73) regulates stOri synapses during fear extinction To test whether phosphorylation of PSD-95(S73) regulates structural plasticity of excitatory synapses during contextual fear extinction, we used SBEM. We reconstructed dendritic spines and PSDs in the stOri. In total, we reconstructed 159 spines from the brains of the WT mice killed 24 hours after CFC (5US, n = 3), and 178 spines from the mice killed after fear extinction (Ext) (n = 3). For mice expressing S73A, 183 spines were reconstructed in the 5US group (n = 3) and 160 in the Ext (n = 3). Figs 4C and 5A show reconstructions of dendritic spines from representative SBEM brick scans for each experimental group. PPT PowerPoint slide

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TIFF original image Download: Fig 5. Phosphorylation of PSD-95(S73) regulates excitatory synapses during fear extinction. Male mice were stereotactically injected in the dCA1 with AAV1/2 encoding PSD-95(WT) (WT, n = 12) or PSD-95(S73A) (S73A, n = 12). Twenty-one days later, they underwent CFC and were killed 1 day after training (5US) or they were reexposed to the training context for fear extinction (Ext). (A) Exemplary reconstructions of dendritic spines and their PSDs from SBEM scans in stOri tissue bricks (3 × 3 × 3 μm). The grey background rectangles are x = 3 × y = 3 μm. (B) Summary of data showing mean density of dendritic spines (two-way ANOVA with LSD post hoc tests for planned comparisons, effect of training × genotype interaction: F(1, 18) = 9.42; P = 0.007). Each dot represents one tissue brick. (C) Exemplary reconstructions of dendritic spines and PSDs (red). PSD and dendritic spine volumes are indicated for each dendritic spine. (D-F) Summary of data showing: (D) median dendritic spine volume (Mann–Whitney test, WT: U = 9,766, P < 0.001; S73A: U = 13,217, P = 0.141), distributions of dendritic spine volumes (numbers of the analysed dendritic spines/mice are indicated) (Kolmogorov–Smirnov test, WT: D = 0.239, P < 0.001; S73A: D = 0.109, P = 0.265) and summary dendritic spine volume per tissue brick (two-way ANOVA with LSD post hoc tests for planned comparisons; effect of training, F(1, 8) = 14.6, P = 0.005; effect of genotype, F(1, 8) = 1.41, P = 0.269); (E) median PSD surface area (Mann–Whitney test, WT: U = 9,948, P < 0.001; S73A: U = 46,678, P = 0.024), distributions of PSD surface areas (numbers of the analysed dendritic spines/mice are indicated) (Kolmogorov–Smirnov test, WT: D = 0.157, P < 0.001; S73A: D = 0.128, P = 0.010), and summary PSD surface area per tissue brick (two-way ANOVA with LSD post hoc tests for planned comparisons; effect of training, F(1, 8) = 5.71, P = 0.044; effect of genotype, F(1, 8) = 1.31, P = 0.285); (F) median PSD volume (Mann–Whitney test, WT: U = 9,462, P < 0.001; S73A: U = 13,621, P = 0.431), distributions of PSD volumes (numbers of the analysed dendritic spines/mice are indicated) (Kolmogorov–Smirnov test, WT: D = 0.278, P < 0.001; S73A: D = 0.145, P = 0.054), and summary PSD volume per tissue brick (two-way ANOVA with LSD post hoc tests for planned comparisons; effect of training, F(1, 8) = 9.56, P = 0.015; effect of genotype, F(1, 8) = 2.35, P = 0.164). The data underlying this figure are available from OSF (https://osf.io/cgfa9/). CFC, contextual fear conditioning; dCA1, dorsal CA1; PSD, postsynaptic density; PSD-95, postsynaptic density protein 95; S73, Serine 73; SBEM, serial block-face scanning electron microscopy; stOri, stratum oriens; WT, wild-type. https://doi.org/10.1371/journal.pbio.3002106.g005 Dendritic spine density was lower in the WT Ext group, as compared to the WT 5US mice (Fig 5B). Furthermore, the median and summary (per volume of tissue) dendritic spine volume, PSD surface area, and PSD volume were higher after the extinction training in the WT group, as compared to the WT 5US mice. These changes were also indicated as shifts in the frequency distributions towards bigger values of all analysed metrics (Fig 5D-5F). Overall, the pattern of synaptic changes observed in the WT mice after contextual fear extinction resembled the changes found in C57BL/6J animals (Fig 2). In addition, field excitatory postsynaptic potentials (fEPSPs) were measured in stOri of the acute hippocampal slices of the WT Ext and 5US mice when Shaffer collaterals were stimulated by monotonically increasing stimuli. The input–output curves showed significant increase in the amplitude of fEPSP in the WT mice killed after fear extinction as compared to the WT 5US group (S3C Fig). As no changes in fibre volley were observed, our data indicate that contextual fear extinction resulted in global increase in synaptic strength in stOri of WT mice. On the other hand, S73A mutation impaired fear extinction–induced down-regulation of dendritic spine density (Fig 5B). We also found no significant changes of median dendritic spine volumes and PSD volumes (Fig 5D and 5F), and only a minor increase in the median PSD surface area in the S73A Ext group as compared to the S73A 5US animals (Fig 5E). These impairments were confirmed by the analyses of the distributions of metrics values (Fig 5D–5F). We also found that mutation prevented an increase of summary PSD volume and surface area, while the increase of summary dendritic spine volume after fear extinction was preserved (Fig 5D–5F, right panels). In addition, we observed no difference in fEPSP and fibre volley between the S73A mice killed before versus after fear extinction session (S3D Fig). Altogether, our data indicate that PSD-95(S73) phosphorylation regulates density, size, and strength of the excitatory synapses in stOri during contextual fear extinction.

PSD-95(S73) phosphorylation in dCA1 is required for extinction of contextual fear To test whether phosphorylation of PSD-95(S73) is necessary for consolidation of fear extinction memory, we used dCA1-targeted expression of S73A, WT, or control AAV1/2 encoding mCherry under Camk2a promoter (Control). Two cohorts of mice with dCA1-targeted expression of the Control virus, WT, or S73A underwent CFC and fear extinction training. The first cohort underwent a short extinction training with one 30-minute extinction session (Ext) and 5-minute test of fear extinction memory (Test) (Fig 6A), while the second underwent an extensive fear extinction training with three 30-minute contextual fear extinction sessions on the days 2, 3, 4 (Ext1 to 3), followed by spontaneous fear recovery/remote fear memory test on day 18, and further 3 extinction sessions on the days 18 to 20 (Ext4 to 6). Next, fear generalisation was tested in a context B (CtxB, day 22) (Fig 6D). The posttraining analysis showed that the viruses were expressed in dCA1. The control virus was expressed in 85% of the dCA1 cells, WT in 88%, and S73A in 87% (Fig 6I and 6J). PPT PowerPoint slide

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TIFF original image Download: Fig 6. Phosphorylation of PSD-95(S73) in dCA1 is required for contextual fear extinction. (A) Experimental timeline of the short fear extinction training. C57BL/6J male mice were stereotactically injected in the dCA1 with AAV1/2 encoding mCherry (Control, n = 17), PSD-95(WT) (WT, n = 17) or PSD-95(S73A) (S73A, n = 15). Twenty-one days after surgery mice underwent CFC. One day after CFC, they were reexposed to the training context in the absence of foot shock (Extinction). Consolidation of fear extinction memory was tested 1 day later in the same context (Test). (B, C) Summary of data showing percentage of freezing during (B) CFC, (C) extinction and test of the mice with dCA1-targeted expression of Control, WT, or S73A (two-way repeated-measures ANOVA with Šídák’s multiple comparisons test, effect of time: F(1, 46) = 26.13, P < 0.001, genotype: F(2, 46) = 0.540, P = 0.586; time x genotype: F(2, 46) = 1.25, P = 0.296). (D) Experimental timeline of the extensive fear extinction training. Mice with dCA1-targeted expression of Control (n = 10), WT (n = 10), or S73A (n = 9) underwent CFC, followed by six 30-minute fear extinction sessions (Ext1–6) and one exposure to novel context without footshock (CtxB). (E-H) Summary of data showing freezing levels (E) during CFC, (F) after extensive fear extinction training (two-way repeated-measures ANOVA with Dunnett’s multiple comparisons test, effect of time: F(3.681, 95.70) = 13.01, P < 0.001; genotype: F(2, 26) = 1.23, P = 0.306; time x genotype: F(10, 130) = 1.49, P = 0.147), (G) the difference in freezing between Ext1 and Ext6 (one-way ANOVA with Tukey’s multiple comparisons test, F(2, 24.94) = 4.98, P = 0.016), and (H) during the test in the context B (Brown–Forsythe ANOVA test, F(2, 17.56) = 0.902, P = 0.428). (I) The extent of viral infection. (J) Single confocal scans of the stratum pyramidale of dCA1 of the mice expressing Control, WT, and S73A and penetrance of the viruses. Means ± SEM are shown. The data underlying this figure are available from OSF (https://osf.io/cgfa9/). CFC, contextual fear conditioning; dCA1, dorsal CA1; PSD-95, postsynaptic density protein 95; S73, Serine 73; WT, wild-type. https://doi.org/10.1371/journal.pbio.3002106.g006 The analysis of the short extinction training (data pooled from 2 cohorts) showed that in all experimental groups freezing levels were low at the beginning of the training and increased after 5US delivery (Fig 6B). Furthermore, mice in all groups showed high freezing levels at the beginning of the Ext indicating similar levels of contextual fear memory acquisition. However, freezing measured during the Test was significantly decreased, as compared to the beginning of Ext, only in the Control and WT groups, not in the S73A animals (Fig 6C). The analysis of freezing levels during the extensive fear extinction training showed high levels of freezing at the end of training and beginning of Ext1 for all experimental groups (Fig 6E and 6F). In the Control and WT groups, the freezing levels decreased over consecutive extinction sessions (Ext2 to 6) and were significantly lower as compared to Ext1, indicating formation of long-term fear extinction memory. We also found no spontaneous fear recovery after 14-day delay (Ext4 versus Ext3; Control, P = 0.806; WT, P = 0.248). In the S73A group, the extensive contextual fear extinction protocol did not reduce freezing levels measured at the beginning of Ext6 sessions, as compared to Ext1, indicating no fear extinction (Fig 6F). Accordingly, we found significantly larger reduction of freezing after fear extinction training (ΔExt6-Ext1) in the controls and WT animals, as compared to the S73A group (Fig 6G). The freezing reaction was specific for the training context, as it was very low and similar for all experimental groups in the context B (Fig 6H). Thus, our data indicate that expression of the S73A in dCA1 does not affect fear memory formation, recall, or generalisation but prevents contextual fear extinction even after extensive fear extinction training.

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