Figure 2.

Leptin signaling activates Fyn, which is required for synaptogenesis. (A) Representative Western blot of immunoprecipitated Fyn from hippocampal cultures treated with leptin (50 nM). (B) Quantification of pTyr⁴¹⁶ intensity normalized to total immunoprecipitated Fyn (n = 3). (C) Representative Western blot of HEK293T cells transfected with V5-Fyn ± shFyn. (D-G) DIV6 hippocampal neurons were transfected with a fluorescent Clover-βactin and the indicated constructs. Neurons were stimulated with leptin (50 nM) on DIV8 and fixed on DIV11 to 12 for spine density or pre/post synaptic juxtaposition experiments. (D) Representative fluorescent images of dendrite segments. Dendritic spine density was measured by hand using ImageJ with the NeuronJ plugin. (E) Quantification of dendritic spine density from a minimum of 2 to 3 dendritic segments from 15 neurons. (F) Representative fluorescent images of hippocampal cultures immunostained for the presynaptic marker vGlut1. (G) Quantification of juxtaposition of dendritic spines and vGlut1 puncta averaged from 30 to 80 spines from 10 neurons. All experiments were repeated in 3 independent culture preparations and expressed as the mean ± SEM, *P < 0.05, **P < 0.01, compared to control or wild-type. White bar = 5µm.

Abbreviations: DN, dominant negative; IP, immunoprecipitate; min, minutes; pTyr416, phosphotyrosine 416.