In order to assess any differences in synaptic sensitivity to ethanol application, it is important to first understand whether the development of synapses (synaptic number) is comparable between N40 and D40 iNs. Therefore, we immunolabeled N40 and D40 AD-iNs at DIV40 to measure the number of synapses in each neuron by counting Synapsin 1+ puncta in close proximity to MAP2+ mature neuronal cell bodies and dendrites (Fig. 2A and B). We observed no significant (p = 0.9) difference in the number of MAP2-correlated Synapsin-positive puncta, and no differences in the mean Synapsin puncta intensity, mean Synapsin puncta area, or MAP2 fluorescence intensity (Fig. 2C). However, the average MAP2+ cellular area of D40 AD-iNs was significantly larger when compared to N40 (Fig. 2C, ***p ≤ 0.001). Ultimately, our analysis failed to uncover any evidence of significant differences in synaptic numbers between N40 and D40 iNs. Thus, even though MOR D40 influences average MAP2 area, it does not impact the development of synapses in human AD-iNs.
