Hypothalamic neurospheres differentiate to mature neurons expressing appetite-related neuropeptides.

Images of hypothalamic neurons cultures obtained from plated neurospheres after 18 days in differentiation conditions. NPY vesicular immunoreactivity (arrow-heads) (A) and NPY-positive neurons (arrows) (A-merge); AGRP vesicular immunoreactivity (arrow-heads) (C) and AGRP-positive neurons (arrows) (C-merge); POMC vesicular immunoreactivity (arrow-heads) (E) and POMC-positive neurons (arrows) (E-merge); CART vesicular immunoreactivity (arrow-heads) (G) and CART-positive neurons (arrows) (G-merge); Orexin-A immunoreactivity (I) and Orexin-A positive neurons (arrows) (I-merge). (B, D, F, H, J) Relative mRNA expression of feeding-related neuropeptides in hypothalamic neuronal cultures. The expression of NPY, CART and Orexin-A increases in differentiated hypothalamic cultures while the expression of AGRP and POMC is unchanged. Mean ± SEM. ns p>0.05, * p<0.05, ** p<0.01 and *** p<0.001, compared to non-differentiated hypothalamic neurospheres. n = 4/5; t-test. DAPI, nuclear staining; non-dif, non-differentiated hypothalamic neurospheres; 18d-dif, hypothalamic neurons culture after 18 days of differentiation.