File:Synapse Formation Is Promoted by Myo-Inositol in a Dose-Dependent Manner.jpg

English: "Synapse size and number are increased by myo-inositol in a dose-dependent manner, and it promotes neuronal responsiveness to synaptogenic Neuroligin 1. (A) Representative confocal images of dendritic segments of control primary rat neurons at 14 div (A, Top), or of neurons treated with myo-inositol at 1 mM (A, Center) or 13 mM (A, Bottom). Immunostainings show presynaptic Bassoon (green), postsynaptic Homer (red), and dendritic MAP2 (blue). (B–F) Quantification of images as in A determined that myo-inositol increases in a dose-dependent manner the density of Bassoon (B) and Homer (C) puncta and of the synaptic sites where they colocalize (D). Myo-inositol also increased the size of Bassoon and Homer puncta (E and F), with data distribution shown in Violin plots as in Fig. 1 (one-way ANOVA with Tukey’s multiple comparison test; N = 16 to 18 dendritic segments per condition). (G) Confocal images of rat hippocampal neurons cultured in the presence of vehicle control or myo-inositol at 1 mM as indicated. Neurons were cocultured with HEK293-expressing CFP as transfection marker (cyan) that served as negative control, or with HEK293 cells coexpressing CFP and Neuroligin 1. At 14 div., immunostaining was performed for the presynaptic active zone marker Bassoon (green) and the dendritic marker MAP2 (blue). Insets show enlarged cocultured HEK293 cells. (H) Quantification of images as in G showed that contact with HEK293 cells expressing Neuroligin 1 induced neurons to assemble Bassoon-positive specializations, as expected. Myo-inositol treatment increases this synaptogenic response by a further 50 ± 13 %. Violin plots show data distribution (one-way ANOVA with Tukey’s multiple comparison test; N = 57 to 72 HEK293 cells per condition from three independent experiments)." "We next cultured neurons in regular medium or added myo-inositol starting at 4 div, when these primary neurons begin to differentiate in vitro. Synaptic analyses were performed at 14 div by immunostaining for the presynaptic active zone protein Bassoon and the excitatory postsynaptic Homer, together with staining for MAP2 to trace dendrites, followed by confocal microscopy (Fig. 2A). We validated the Homer antibody for labeling excitatory postsynaptic sites by determining that the puncta it detected colocalized to the same extent with Bassoon as the excitatory postsynaptic marker PSD-95 and that 80 ± 3 % of PSD-95 puncta were positive for Homer (SI Appendix, Fig. S4). Quantitative analysis determined that the density of Bassoon-positive presynaptic sites along dendrites was increased by 38 ± 11 % (Student’s t test; P = 0.002) upon treatment with 1 mM myo-inositol compared to vehicle-treated control neurons (Fig. 2B). The number of Homer-labeled postsynaptic sites was similarly elevated in the presence of 1 mM myo-inositol by 40 ± 19 % (Student’s t test; P = 0.039) (Fig. 2C). Synapses are defined by the apposition of pre- and post-synaptic sites, and 1 mM myo-inositol strongly promoted the number of postsynaptic Homer sites colocalized with Bassoon by 50 ± 18 % (Student’s t test; P = 0.010) (Fig. 2D). With these effects, myo-inositol shared the synaptogenic effects of DHA (SI Appendix, Fig. S5) (18, 19). Myo-inositol acted in a dose-dependent manner and had even higher effects at 13 mM (Fig. 2 B–D). Moreover, treatment of neurons with this carbocyclic sugar enlarged Bassoon- and Homer-positive specializations (Fig. 2 E and F)."