Figure 1. MC4R activation is required for the maintenance of mature spines and functional synapses. a, Expression of MC4R and its ligands in adult mouse hippocampus. mRNAs of different brain parts were prepared and subjected to real time-PCR. The data were presented as relative ratio of mRNA against that of cerebral cortex (agouti was used as a negative control). b, Top row, Staining of surface MC4R in hippocampal neurons at 22 DIV. F-actin was labeled with rhodamine-phalloidin. Scale bar, 5 μm. Bottom row, cultured hippocampal neurons (17 DIV) were transfected with SEP-MC4R and mKOrange constructs, and were examined at 21 DIV. Scale bar, 10 μm. c, d-Tyr MTII increased intracellular cAMP level. Hippocampal neurons were treated with d-Tyr MTII for 30 min (*p < 0.05 versus no treatment, one-way ANOVA with Student-Newman–Keuls test). d, Reduction of MC4R protein in HEK293T cells upon shMC4R knockdown (by ∼83.0 ± 4.2%; ***p < 0.001, n = 3 experiments, Student's t test). The cells were cotransfected with EGFP-tagged rat MC4R expression construct, together with either shMC4R (+) or pSUPER vector (−). e, Cultured hippocampal neurons were cotransfected with shMC4R or its scrambled shRNA (Scr-MC4R) with GFP plasmid. f–h, Hippocampal neurons were cotransfected with shMC4R and constructs expressing shRNA-resistant human MC4R (WT) or its point mutants (D90N or I125K). f, Representative images. g, h, Quantification of spine density (g) and the percentage of mature spines (spines with a mushroom head) (h). Data were expressed as mean ± SEM; **p < 0.01 and ***p < 0.001 versus shMC4R; #p < 0.05 and ### <0.001 versus shMC4R+WT (one-way ANOVA with Student-Newman–Keuls test). i–k, Overexpression of hMC4R-WT and its point mutants (D90N or I250K) in cultured hippocampal neurons. i, Representative images. j, k, Quantification of spine density (j) and percentage of mature spines (k). *p < 0.05 and ***p < 0.001 versus control (Con); ##p < 0.01 and ###p <0.001 versus WT (one-way ANOVA with Student-Newman–Keuls test). Scale bars, 10 μm.