EMBO/soda-nel huggingface dataset:
Wbp2 is required for normal glutamatergic synapses in the cochlea and is crucial for hearing (2016)
Figure 1

Figure 1. Wbp2 mutation and Wbp2 expression in the cochlea

A, Diagram showing the design of the mutated Wbp2 allele. A promoterless cassette including LacZ and neo genes was inserted in the second intron of the Wbp2 gene flanked by FRT sites (green triangles). LoxP sites (red triangles) flank the critical exon (exon2) of the Wbp2 gene (exons in yellow).

B, Western blot showing no detectable Wbp2 protein in 4 week old mutant brain compared to wt littermate controls. 5μg of the protein lysate were subjected to 10% SDS-PAGE. β-tubulin was used as loading control. Wbp2 l and Wbp2 s refer to the long and short isoforms respectively.

C, Quantitative real-time PCR showing severe knockdown of Wbp2 transcription in 4 week old mutants (n=3) inner ears and eyes, compared to wt littermate controls (n=3). Heterozygotes show intermediate levels. Hprt was used as a control and levels are normalised to wt levels.

D, F, X-gal staining of Wbp2 hets at P14 showing Wbp2 expression (blue) in all the main cochlear structures: the stria vascularis (black arrowhead in D), spiral prominence (empty arrowhead in D), Reissner’s membrane (arrow in D), strong expression in the spiral ganglion cells (red arrowheads in E), IHCs and OHCs in the organ of Corti (arrowheads in F), pillar cells and spiral limbus (empty arrowhead in F). Scale bars: D: 50μm, E,F: 20μm. ihc: inner hair cells; ohc: outer hair cells. No X-gal staining is observed in wt controls (not shown). The X-gal reaction is always cytoplasmic.

Data information: All data plotted as mean ±SD. Two-tailed t test: Wbp2 ear: het p=0.03, hom p=0.000000033; Wbp2 eye: het p=0.01, hom p=5.35537E-13.
Wbp2 is required for normal glutamatergic synapses in the cochlea and is crucial for hearing (2016)
Figure 4

Figure 4. Analysis of the Wbp2 mouse isoforms in the brain and in the cochlea

A. Splice forms of WBP2, numbered according to the Ensembl numbering scheme. The GRAM domain is marked in blue, the WW domains in green, and the locations of the variants in black (the p.Ala160Thr and p.Met163Leu variants are too close to show separately in this view).

B. Agarose gel trace of cDNA obtained from mouse wt P28 inner ear (IE), P28 brain, adult and P4 organ of Corti (OC). Results show expression of two Wbp2 isoforms in the brain at P28 and very faint band for the short isoform together with a strong band for the long isoform in the inner ear at P28. If we look at just the organ of Corti (adult and P4), we observe a strong band for the long isoform in the P4 OC and an almost undetectable band for the short isoform, which was not even picked up by sequencing (see C). Wbp2 l: long isoform (550 bp); Wbp2 s: short isoform (480 bp).

C. Cartoon illustrating the results from the sequencing of mouse wt cDNA performed at P4 and P28. While in the brain we detect both Wbp2 isoforms and in the organ of Corti only the long isoform, in the whole inner ear sample we detect the presence of the long isoform with a small band for the short one.

D. Western blot showing the predominant presence of the Wbp2 long isoform in the cochlea at P28, with a short trace of the short isoform showing up only when a higher concentration of protein lysate (20μg) is loaded. Both isoform are absent in the Wbp2-deficient mouse. Gapdh was used as a loading control. Wbp2 l: long isoform; Wbp2 s: short isoform.
Wbp2 is required for normal glutamatergic synapses in the cochlea and is crucial for hearing (2016)
Figure 5

Figure 5. Afferent innervation in Wbp2-deficient mice

A. At P14, afferent terminals below IHCs are slightly swollen in the mutants (neurofilament labelling in green, arrowheads to compare; CtBP2 labels ribbons and IHC nuclei in red). Scale bars: 10 μm. At P28,Neurofilament/CtBP2 labelling in the organ of Corti of 4 week old Wbp2-deficient mice and littermate controls shows severe swelling of IHC afferent terminals in the mutants, especially in the 24kHz region (yellow arrowheads). The pre-synaptic ribbons do not look as well aligned to the terminals in the mutants (white arrows). At this stage, we also observe swelling of OHC afferent terminals in the 24kHz region (empty arrowheads). Scale bars: 5 μm. ihc: IHC nucleus; p: pillar side; m: modiolar side.

B. Counts of pre-synaptic ribbons per IHC in the 8, 18 and 24kHz regions, showing no difference between mutants and controls at P28.

C. TEM of the organ of Corti performed at P28 showing swollen afferent terminals below inner and outer hair cells (arrowheads for comparisons between mutants and controls). Scale bars: 5 μm.

D. Neurofilament/CtBP2 labelling shows swollen and retracting terminals (white arrows), especially in the 24kHz regions in the mutants at 8 weeks (arrows). Scale bars: 10μm.

E. Counts of pre-synaptic ribbons in the 8, 18 and 24kHz regions, showing no difference in their number per IHC in the mutants compared to littermate controls at 8 weeks.

Data information: all data are shown as mean ±SD and statistically analysed by two-tailed Student t test. Synaptic count at 4 weeks: Wildtype: 24kHz 16.9 ± 2.4, 16kHz 13.67±2, 9kHz 13.0 ±1.8; Mutants: 24kHz 18.17 ± 1.18 (p=0.27), 16kHz 15.89±1.8 (p=0.26), 9 kHz 15 ± 2 (p=0.07). Synaptic count at 8 weeks: Wildtypes: 24kHz: 15.35 ± 3.75, 18kHz 17.73 ± 1.79, 8kHz: 11.25 ± 0.92. Mutants: 24kHz: 15.78 ± 3.46 (p=0.9), 18kHz 15.78 ± 0.17 (p=0.26), 8kHz: 12.3 ± 2.26 (p=0.6). (35 hair cells per mean).
Wbp2 is required for normal glutamatergic synapses in the cochlea and is crucial for hearing (2016)
Figure 6

Figure 6. GluR2/3 expression and synaptic defects in Wbp2-deficient mice

A. We arrayed synapse images aligned by ascending size of the post-synaptic site after GluR2/3 and CtBP2 labelling. This is a composite image made of several synapses taken from a single IHC from a single wt and a single hom, representing double labelling experiments performed on 3 mutants and 3 controls. Synapses in the mutants show abnormal morphology and smaller green patches, suggesting reduced expression of the GluR2/3 AMPA receptor subunits. Scale bar at bottom right: 1μm.

B-G. TEM images of IHC ribbon synapses of wt (B, arrowheads for synaptic vesicles) and homs (C-G) at 4 weeks of age, showing a representative array of synaptic phenotypes in the 24 kHz cochlear region of mutants. While in C and D the ribbons look slightly abnormal in size with misplaced synaptic vesicles (arrowheads in C), we also observe orphan post-synaptic densities surrounded by floating synaptic vesicles (arrowheads in E), ribbons with synaptic membranes (arrow in F) that have detached from the IHC membrane (the asterisk labels the original position of the synapse before detachment) and are floating in the swollen nerve terminal (F), ribbons (arrow in G) that are detached from their densities (asterisk in G). Scale bars: B-E: 200 nm; F,G: 500 nm. nt: nerve terminal; psd: post-synaptic density; snt: swollen nerve terminal.
Wbp2 is required for normal glutamatergic synapses in the cochlea and is crucial for hearing (2016)
Figure 7

Figure 7. The Wbp2 molecular pathway

A, Diagram showing the Wbp2 molecular pathway, including its downstream targets and their functional relationship. The blue arrows, light blue lines and green lines link data from the literature (in vivo and in vitro); the orange squares and red arrows indicating up- or downregulation show our experimental observations, as reported in this study.

B, Quantitative real-time PCR showing reduced mRNA levels for Esr1, Esr2 and Pgr and upregulation of Shank3 and Psd-95 in cochleae of 4 week old Wbp2-deficient mice compared to littermate controls (n=3 for each genotype). Hprt is used as relative control. Error bars: ±SD. Two-tailed student t test: p=0.03 for Psd-95; p=0.007 for Shank3; p=0.03 for Esr2; p=0.0016 for Esr1; p=0.037 for Pgr.

C, Synapses from one mutant and one control IHC at 4 weeks of age after Psd-95 and CtBP2 labelling, showing stronger Psd-95 expression in the mutants compared to controls, representing double labelling experiments performed on 3 mutants and 3 controls. P indicates direction of pillar cells. Scale bar: 10μm.

D, Quantification of Psd95 fluorescence in IHC synapses, representing expression in the apical (9kHz best frequency region of the cochlea) and basal (24kHz best frequency region of the cochlea) regions at 4 weeks old . Data from 2 wt and 2 homs were analysed (16 synapses per cochlear region per mouse). AU, Arbitrary Units.

Wt: 24kHz 22.41±10.70, 9kHz 11.045±2.128; Mutants: 24kHz 66.73±13.70, p=0.069; 9kHz: 26.02±5.79 p=0.075.

Data information: data are shown as mean ±SD and statistically analysed by two-tailed Student t test.
miR-124-dependent tagging of synapses by synaptopodin enables input-specific homeostatic plasticity (2022)
Figure 1

Figure 1. Cultured hippocampal neurons exhibit non-uniform homeostatic synaptic plasticity upon TTX treatment.

(A) Homer1c-GFP signal (magenta) and immunostaining for surface AMPARs (cyan) and SP (green) in neurons treated for 48h with TTX, or untreated (UT). Scale bars: 10 µm (low magnification), 5 µm (insets). Dotted lines indicate the outline of dendrites. Arrowheads indicate SP+ synapses.

(B) Percentage of SP+ synapses for untreated (UT) and TTX-treated neurons (dot plots represent different cells; UT: n = 26; TTX: n = 33, n indicates the number of cells, from 2 cultures). % SP+ spines: **P = 0.019 (Mann Whitney test).

(C) AMPAR synaptic fluorescence intensity for SP+ versus SP- synapses in UT and TTX-treated neurons. AMPAR synaptic fluorescence intensity was normalized to SP- or small synapses, respectively, from UT condition. (UT: SP-, n = 1455, SP +, n = 516; TTX: SP-, n = 1529, SP+, n = 724, n indicates the number of synapses, from 2 cultures). **P < 0.01, ****P < 0.0001, ns, not significant, P > 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test).

(D) Average AMPAR synaptic fluorescence intensity in UT or TTX-treated neurons, regardless of the expression of SP (all synapses) (UT: n = 26; TTX: n = 33, n indicates the number of cells, from 3 cultures). AMPAR intensities were normalized to UT condition. **P = 0.0014 (Mann Whitney test).

(E) Cumulative probability distribution of AMPAR synaptic fluorescence intensity for untreated neurons (UT, light blue), 48h TTX-treated neurons (TTX, dark blue) and scaled UT neurons (dotted black line, scaled factor = 1.26). The scale factor was obtained from by computing the ratio between TTX and UT average values. UT vs TTX, ****P < 0.0001; UT-scaled vs TTX, ***P = 0.0005 (Kolmogorov-Smirnov test).

(F) Representative traces of AMPAR-mediated miniature currents (mEPSCs) recorded from neurons cultured in BrainPhys supplemented with TTX for 48 h (TTX, dark gray), or untreated (UT, light gray).

(G) Mean mEPSC amplitudes for each condition (UT: n = 65; TTX: n = 68, n indicates the number of cells, from 10 cultures). ****P < 0.0001 (Mann Whitney test).

(H) Cumulative probability distributions of mEPSC amplitudes for untreated neurons (UT, light grey), TTX-treated neurons (TTX, black) and untreated scaled to TTX condition (dotted black line, scale factor = 1.27). The scale factor was obtained from the linear regression of the ranked mEPSC amplitudes for UT vs TTX condition. UT vs TTX, *P = 0.0412, UT scaled vs TTX, *P = 0.0316 (Kolmogorov-Smirnov test).

Data information: Data are represented as mean ± SEM.
miR-124-dependent tagging of synapses by synaptopodin enables input-specific homeostatic plasticity (2022)
Figure 2

Figure 2. Higher immobilization of surface diffusing AMPARs at SP+ vs SP- synapses.

(A) Example images of a FRAP experiment: SP- and SP+ spines from the same cultured hippocampal neuron transfected with SEP-GluA2 (gray and color-coded) + RFP-SP (red) 10s before, and 0, 370, 750 s after the photobleaching. Scale bars: 1 µm. Dotted lines represent the outline of dendrites. Arrowheads indicate dendritic spines which were targeted for photobleaching.

(B) SEP-GluA2 fluorescence intensity at SP- vs SP+ spines. Each pair of dot plots represents average fluorescence for SP- and SP+ spines from a same neuron (4 cultures). **P = 0.0061 (two-tailed paired Student's t test).

(C) Quantification of FRAP dynamics for SP- vs SP+ spines. Recovery curves represent SEP-GluA2 fluorescence average per cell (n = 14 cells, from 4 cultures). The two traces were fitted using double exponential components equations and the convergence of the traces to a common fit was tested using the extra sum of squares F test. The F test indicates that the traces are best fitted by two divergent models (P < 0.0001).

(D) Quantification of the recovery fraction 750 s after the photobleaching. Each pair of dot plots represents average recovery for SP- and SP+ spines from a same neuron (4 cultures). **P = 0.0067 (Wilcoxon matched-pairs signed rank test).

Data information: Data are represented as mean ± SEM.
miR-124-dependent tagging of synapses by synaptopodin enables input-specific homeostatic plasticity (2022)
Figure 3

Figure 3. The synaptic recruitment of AMPARs induced by TTX treatment is controlled by SP.

(A) Immunoblot analysis of SP in lysates from COS-7 cells which were untransfected, transfected with RFP-SP + empty vector or co-transfected with SP-shRNA-GFP + RFP-SP (knock-down) or shRNA-resistant RFP-SP (rescue). β-actin and GFP signals illustrate equal protein loading and SP-shRNA expression level, respectively.

(B) Quantification of SP expression from 6 different experiments, normalized to the β-actin signal. *P < 0.05, ns, not significant, P > 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test).

(C) Homer-DsRed (green) and immunostained endogenous SP (red) in dendrites from neurons transfected with either empty vector, scramble shRNA or SP-shRNA with GFP reporter (blue). Scale bar: 10 µm.

(D) Average SP intensity for same conditions (EV: n = 47; Scr-shRNA: n = 53; SP-shRNA: n = 62, n indicates the number of cells, from 3 cultures; normalized to empty vector condition). **P < 0.01, ****P < 0.0001 (Kruskal-Wallis test followed by Dunn's multiple comparison test).

(E) Fraction of SP+ synapses for same conditions. (EV: n = 47; Scr-shRNA: n = 53; SP-shRNA: n = 62, n indicates the number of cells from 3 cultures). *P < 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test).

(F) Homer1c-DsRed (red) and immunostained surface AMPARs (green) in dendrites from neurons transfected with either SP-shRNA-GFP or empty vector (EV) with GFP reporter (blue) and treated with TTX or left untreated (UT). Scale bar: 5 µm.

(G) AMPAR synaptic fluorescence intensity normalized to untreated empty vector (EV) condition (EV: UT, n = 42, TTX, n = 33; SP-shRNA: UT, n = 30, TTX, n = 31, n indicates the number of cells, from 3 cultures). *P < 0.05, **P < 0.01, ns, not significant, P > 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test).

Data information: Data represent mean ± SEM.
miR-124-dependent tagging of synapses by synaptopodin enables input-specific homeostatic plasticity (2022)
Figure 5

Figure 5. miR-124 overexpression inhibits TTX-induced HSP.

(A) Micrographs showing Homer1c-GFP (green) and immunostaining for surface AMPARs (blue) and endogenous SP (red) in neurons transfected with miR-124 or control miR-67 (miR-Ctrl), and treated with TTX, or left untreated (UT). Scale bar: 5 µm.

(B) Percentage of SP+ synapses for same conditions as in (A) (miR-Ctrl: UT, n = 25, TTX, n = 26; miR-124: UT, n = 30, TTX, n = 30, n indicates the number of cells, from 3 cultures). ****P < 0.0001, ns, not significant, P > 0.05 (two-way ANOVA test followed by Tukey's multiple comparison test).

(C) Homer1c-GFP intensity for same condition as in (A), normalized to untreated miR-Ctrl (miR-Ctrl: UT, n = 25, TTX, n = 26; miR-124: UT, n = 30, TTX, n = 30, n indicates the number of cells, from 3 cultures). ns, not significant, P > 0.05 (two-way ANOVA test followed by Tukey's multiple comparison test).

(D) AMPAR synaptic fluorescence intensity for same condition as in (A), normalized to untreated miR-Ctrl (miR-Ctrl: UT, n = 25, TTX, n = 26, miR-124: UT, n = 30, TTX, n = 30, n indicates the number of cells, from 3 cultures). ***P < 0.001, *P < 0.01, ns, not significant, P > 0.05 (two-way ANOVA test followed by Tukey's multiple comparison test).

(E) Representative traces of AMPAR-mediated miniature currents (mEPSCs) recorded from neurons expressing miR-124 or miR-Ctrl in TTX-treated or untreated neurons.

(F) AMPAR-mEPSC average amplitudes for same condition as in (E) (miR-Ctrl: UT, n = 16, TTX, n = 15, miR-124: UT, n = 16, TTX, n = 15, n indicates the number of cells, from 4 cultures). ****P < 0.0001, ns, not significant, P > 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test).

Data information: Data are represented as mean ± SEM.
miR-124-dependent tagging of synapses by synaptopodin enables input-specific homeostatic plasticity (2022)
Figure 6

Figure 6. SP but not GluA2 synaptic recruitment upon TTX is controlled by endogenous miR-124.

(A) Micrographs showing dendrites from neurons transfected with Homer1c-DsRed (magenta) and SEP-GluA2 constructs containing wild-type (WT) or mutated (MUT) 3'UTR (green) and treated with TTX for 48 h, or left untreated (UT). Scale bar: 10 µm.

(B) SEP-GluA2 synaptic fluorescence intensity for each condition, normalized to GluA2-3'UTR-WT untreated neurons (3'UTR-WT: UT, n = 29, TTX, n = 37; 3'UTR-MUT: UT, n = 18, TTX, n = 14; n indicates the number of cells, from 3 cultures). *P < 0.05, ns, not significant, P > 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test).

(C) Micrographs showing dendrites from neurons transfected with Homer1c-BFP (magenta), GFP-SP-shRNA (gray) and a rescue RFP-SP construct containing wild-type (WT) or mutated (MUT) 3'UTR (geen) and treated with TTX, or left untreated (UT). Scale bar: 10 µm.

(D) Percentage of SP+ synapses for each condition (3'UTR-WT: UT, n = 26, TTX, n = 28; 3'UTR-MUT: UT, n = 26, TTX, n = 24; n indicates the number of cells, from 3 cultures). **P < 0.01, ***P < 0.001, ns, not significant, P > 0.05 (two-way ANOVA test followed by Tukey's multiple comparison test).

(E) Micrographs showing Homer1c-GFP (magenta) and immunostaining for surface AMPARs (cyan) and endogenous SP (green) in neurons transfected with or without 50 nM SP TSB-LNA and treated with TTX, or left untreated (UT). Scale bar: 10 µm.

(F) Percentage of SP+ synapses for each condition (Control: UT, n = 36, TTX, n = 52; SP TSB-LNA: UT, n = 62, TTX, n = 29; n indicates the number of cells from 3 cultures). *P < 0.05, **P < 0.01, ns, not significant, P > 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test).

(G) AMPAR synaptic fluorescence intensity for each condition, normalized to control untreated neurons (Control: UT, n = 30, TTX, n = 36; SP TSB-LNA: UT, n = 37, TTX, n = 20; n indicates the number of cells from 2 cultures). *P < 0.05, ns, not significant, P > 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test).

Data information: Data are represented as mean ± SEM.
miR-124-dependent tagging of synapses by synaptopodin enables input-specific homeostatic plasticity (2022)
Figure 7

Figure 7. Local SP translation is enhanced upon TTX treatment and occurs preferentially at proximity of large synapses.

(A) Micrographs showing puro-PLA staining of newly synthesized SP (green) in neurons immunostained for MAP-2 (magenta) and DAPI (blue) and treated with TTX for 24 h or left untreated. The images on bottom panels show no staining in the absence of puromycin treatment or when omitting SP primary antibody. Scale bars: 30 µm (large view), 5 µm (insets).

(B) Puro PLA labeling strategy showing ligation and amplification steps.

(C) Density of SP puro-PLA puncta, normalized to the untreated condition (UT) (UT: n = 62; TTX: n = 57, n represents the number of cells, from 3 cultures). *P < 0.05 (Mann-Whitney test).

(D) SP puro-PLA cluster fluorescence intensity normalized to the untreated condition (UT) (UT: n = 62; TTX: n = 57, n indicates the number of cells, from 3 cultures). ****P < 0.0001 (Mann-Whitney test).

(E) Micrographs showing puro-PLA staining of newly synthesized SP (green) in neurons transfected with Homer1c-GFP (magenta) and treated with TTX for 24 h or left untreated. Scale bar: 5 µm. Arrowheads indicate puro-PLA+ spines.

(F) Percentage of SP puro-PLA+ synapses (UT, n = 26, TTX, n = 33; n indicates the number of cells, from 2 cultures). **P < 0.01, ns, not significant (Mann-Whitney test).

(G) Homer1c-GFP integrated fluorescence intensity at SP puro-PLA- vs SP puro-PLA+ synapses (n = 32 cells, from 2 cultures). ****P < 0.0001, ns, not significant (Wilcoxon matched-pairs signed rank test).

Data information: Data are represented as mean ± SEM.
miR-124-dependent tagging of synapses by synaptopodin enables input-specific homeostatic plasticity (2022)
Figure 8

Figure 8. Non-uniform regulation of spine size by SP-3'UTR miR-124 binding region.

(A) Confocal images showing dendrites from CA3 pyramidal cells transfected with RFP-SP (green) containing wild-type (WT) or mutated (MUT) 3'UTR + SP-shRNA-BFP (magenta). Arrowheads indicate SP+ spines. Scale bar: 10 µm.

(B) Graph plot showing spine head volume vs SP integrated intensity (n = 5 cells). Each plot represents a single spine for which SP integrated intensity has been normalized to average SP intensity of SP+ spines in the same neuron. Plots for SP- and SP+ spines appear in grey and magenta, respectively. The black dotted line represents linear regression (R2 = 0.25, P = 0.0004).

(C) Paired data showing spine head volume for SP- vs SP+ spines. Each pair of plot represents average values for SP- vs SP+ spines for a given neuron. *P = 0.0031 (two-tailed Student t test).

(D) Percentage of SP+ spines in CA3 pyramidal cells transfected with SP SP-shRNA-BFP and rescue RFP-SP containing wild-type (WT) vs mutated (MUT) 3'UTR (SP-3'UTR-WT: n = 5 cells, from 5 slices; SP-3'UTR-MUT: n = 14 cells, from 5 slices). **P = 0.0029 (Mann Whitney test).

(E) Cumulative probability distributions of spine head volumes for neurons expressing RFP-SP-3'UTR-WT (SP-WT, light magenta) or RFP-SP-3'UTR-MUT (SP-MUT, dark magenta). The black dotted curve represents cumulative probability distribution corresponding to RFP-SP-3'UTR-WT condition scaled to RFP-SP-3'UTR-MUT (scale factor = 1.61). ****P < 0.0001 by Kolmogorov-Smirnov test. The inset shows average spine head volume for same conditions (SP-3'UTR-WT: n = 220 from 5 cells; SP-3'UTR-MUT: n = 616, from 14 cells, n indicates the number of spines). **P < 0.01 (Mann Whitney test).

(F) Plot showing the rank-ordered spine head volumes for neurons expressing RFP-SP-3'UTR-WT vs RFP-SP-3'UTR-MUT. The rank-order plot was obtained by sorting from smallest to largest spine head volumes in SP-WT vs SP-MUT and plotting them against each other. The extra sum of squares F test indicates that the first values are better fitted with a second order polynomial quadratic curve (in magenta, SP-MUT = 0.02 - 0.42 x SP-WT + 25.32 x SP-WT2; R2 = 0.99; ****P < 0.0001) than with a linear regression (not shown, SP-MUT = - 0.05 + 2.42 x SP-WT).

Data information: Data are represented as mean ± SEM.