PTX3 increases excitatory glutamatergic neurotransmission by promoting AMPA receptors insertion at the synapse.Figure 2A) Representative mEPSC traces recorded from control and PTX3-treated (1µg/ml; 48hrs) neurons. B) mEPSC frequency quantitation (Hz, Ctr=0.618±0.069; PTX3=1.991±0.313; Number of neurons: Ctr=22, PTX3=16; 3 independent experiments, Mann-Whitney test. Data are presented as a distribution plus mean±SEM). C) mEPSC amplitude quantitation and cumulative probability distribution of mEPSC amplitudes (pA, Ctr=12.76±0.813; PTX3=16.07±0.709. Number of neurons: Ctr=22, PTX3=16; 3 independent experiments, Mann-Whitney test. Data are presented as a distribution plus mean±SEM. Cumulative probability distributions are analyzed by Kolmogorov-Smirnov test D) Representative images showing 14DIV neurons stained for surface AMPAR (GluA, green), the presynaptic protein Bassoon (blue) and tubulin (red) in the different tested conditions. Arrowheads point to postsynaptic GluA clusters. Inset: Example of surface synaptic AMPARs cluster (GluA&Bsn). Scale bar: 5 µm. E) Quantification of the surface synaptic AMPARs (GluA&Bsn) normalized to the total number of Bsn shows a statistically significant increase after TTX or PTX3 exposure (Ctr=1±0.051, TTX=1.512±0.080, PTX3=1.294±0.081, PTX3 heat-inactivated=0.959±0.044; number of fields examined: 27, 36, 37, 22, 19 respectively; one-way ANOVA analysis of variance, p<0.0001, followed by post hoc Tukey test for multiple comparison as indicated in figure; 3 independent experiments, data are presented as normalized mean values±SEM). F) 1hr of PTX3 administration is not sufficient to elicit an increase in synaptic GluA content (Ctr: 1±0.065, PTX3: 1.023±0.068; number of fields: Ctr=26, PTX3=26; unpaired t-test, 3 independent experiments, data are presented as normalized mean values±SEM) G) Representative mEPSC traces recorded from control, C-term PTX3 fragment or N-term PTX3 fragment-treated cultures. H) mEPSC frequency quantitation (Hz, Ctr=0.668±0.078; C-term=0.523±0.056; N-term=1.228±0.121. Number of neurons: Ctr=23, C-term=10, N-term=23; 3 independent experiments. Kruskall-Wallis test, p<0.0001 followed by Dunn"s test as indicated, data are presented as a distribution plus mean±SEM). I) mEPSC amplitude quantitation (pA, Ctr=16.81±0.78; C-term=15.79±1.37; N-term=19.78±0.87. Number of neurons: Ctr=23, C-term=10, N-term=23; 3 independent experiments. One-way ANOVA analysis of variance, p=0.013 followed by post hoc Tukey test as indicated in figure, data are presented as a distribution plus mean±SEM) and cumulative probability distribution of mEPSC amplitudes (Kolmogorov-Smirnov J) Representative images of 14DIV neurons stained for surface AMPARs (GluA, green), the presynaptic protein Bassoon (blue) and tubulin (red) in the different tested conditions. Arrowheads point to postsynaptic GluA clusters. Scale bar: 5µm. K) Quantification of the surface synaptic AMPARs normalized to the total number of Bsn shows an increase upon N-terminal peptide application, but not upon C-terminal peptide exposure. TTX is used as positive control (Ctr=1.000±0.066. TTX=1.395±0.113, C-term=0.717±0.047, N-term=1.349±0.069. Number of fields examined: 55, 31, 18, 54, respectively; one-way ANOVA analysis of variance, p<0.0001 followed by post hoc Tukey test as indicated in figure; at least 3 independent experiments, data are presented as normalized mean values±SEM).