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. 2017 Jun 13;9:184. doi: 10.3389/fnagi.2017.00184

Figure 4.

Figure 4

The astrocytic transforming growth factor beta-1 (TGF-β1) mediates the synaptogenic action of astrocytes treated with hesperidin. Twelve days in vitro (12 DIV) cortical neurons were maintained for 3 h in the presence of Dulbecco’s minimum essential medium (DMEM)/F12 (Control; A,A′), astrocyte conditioned medium (ACM-Control; B,B′) or astrocyte conditioned medium from astrocytes treated with hesperidin (ACM-Hesperidin; D,D′), or simultaneously with a neutralizing antibody against TGF-β1 (ACM-Control + αTGF-β1; C,C′, and ACM-Hesperidin + αTGF-β1; E,E′)synapse formation was evaluated by immunocytochemistry for the synaptic markers, synaptophysin and PSD-95. ACM-Control increased the number of synapses by two times relative to control and ACM-Hesperidin enhanced synapse formation by two times in relation to ACM-Control; whereas depletion of TGF-β1 partially blocked this effect. Scale bars 20 μm (E) and 10 μm (E′). *p < 0.050 and ***p < 0.001; comparisons among multiple groups were analyzed using a one-way analysis of variance (ANOVA) followed by Newman-Keuls post hoc tests. n = 3–6 independent astrocyte cultures.