File:The Abundance of Excitatory Synaptic Sites in Mature Brain Tissue Is Promoted by Treatment with Myo-Inositol.jpg

English: "Myo-inositol is synaptogenic in mature hippocampal organotypic slice cultures. (A) Organotypic slice cultures were prepared from the hippocampus of mouse pups and cultured for 90 d. A subset of cultured slices was treated from 60 to 90 div with myo-inositol at 1 mM. (B) Representative confocal images of CA3 stratum lucidum of organotypic hippocampal slice cultures for 90 div under control conditions (B, Left) or after treatment with myo-inositol at 1 mM from 60 to 90 div (B, Right). Slice cultures were stained for presynaptic Bassoon (red) or the excitatory postsynaptic marker PSD-95 (green). (C–E) Quantification of immunostainings from the hippocampal CA3 area as in B showed that myo-inositol increases the density of presynaptic Bassoon (C) and postsynaptic PSD-95-positive specializations (D) and of sites where these pre- and post-synaptic markers colocalize (E). (F and G) Quantification of images as in B determined that myo-inositol does not change the size of Bassoon-positive sites (F) but enlarges PSD-95-positive specializations (G) in the hippocampal CA3 area (C–G, one-way ANOVA with Tukey’s multiple comparison test; N = 25 control and N = 10 myo-inositol slices from two independent experiments)." "Finally, we asked whether myo-inositol is not only bioactive during development but also in mature brain tissue. We tested this in interface organotypic slice cultures prepared from the hippocampus of neonate mice, a preparation in which the structural and synaptic organization of the original tissue is preserved (23). Organotypic slices can be cultured for prolonged periods of time ex vivo, and we selected them as a system because they allow precise control over treatment conditions to obtain results complementary to our in vitro studies. They reach maximal functional connectivity by 3 wk in culture, similar to the time course in vivo, and acquire by the third wk in culture the complex morphological appearance of dendritic spines found in the mature brain (24, 25). We therefore considered that these slice cultures reached maturity by 60 d in culture. At this time point, we started treating them for 30 d with myo-inositol at 1 mM, or vehicle control (Fig. 4A). At the end of the treatment period, slice cultures were subjected to immunohistochemical staining to measure the abundance and density of presynaptic Bassoon and the postsynaptic excitatory marker PSD-95 (Fig. 4B). We analyzed the CA3 area as it is among the brain regions that show the most pronounced differential gene expression during aging, including genes involved in synaptic signaling and cell communication (26). This area may hence be particularly responsive to synaptogenic interventions. Quantification of synaptic immunostainings in the CA3 stratum lucidum showed that myo-inositol addition to mature organotypic hippocampal slices increased presynaptic Bassoon puncta density by 46 ± 22 % (Student’s t test; P = 0.048) and promoted the density of postsynaptic PSD-95 puncta by 61 ± 19 % (Student’s t test; P = 0.004) (Fig. 4 C and D). This corresponded with a striking 3.1-fold increase in synaptic sites where both markers colocalized (Student’s t test; P < 0.0001) (Fig. 4E). Moreover, myo-inositol addition to the cultured slices enlarged PSD-95-positive specializations, while the size of presynaptic Bassoon puncta remained unchanged (Fig. 4 F and G). The use of organotypic slice cultures provided the opportunity of additionally studying possible acute effects on microglia, the brain-resident immune cells which play roles in synapse formation and turnover during development (27). Immunostaining with the microglial marker Iba1 found that its staining intensity decreased in mature organotypic slice cultures after they were treated with myo-inositol (SI Appendix, Fig. S11). This is indicative of lower microglia activity which may reduce synapse elimination and complement synaptogenic effects of myo-inositol on neurons. Together, these results showed that myo-inositol treatment elevated synapse number when added to mature brain tissue."