Transfection of p65/RelA promoted Hes1 expression and counteracted the effects of amyloid beta (Aβ) on the morphology and GABAergic connectivity of cultured hippocampal neurons. (A) 7 days in vitro (DIV) neurons (300,000 cells) were transfected with a plasmid encoding myc-tagged p65/RelA. After 16 h, the cells were lysed and processed for real time-PCR, showing that Hes1 expression levels increased by 35%. (B-D) Cultured hippocampal neurons (40,000 cells/cm2, 7 DIV) were co-transfected with enhanced green fluorescent protein (EGFP) and myc-p65/RelA plasmids, treated with Aβ (5 μM) and incubated for a further 16 h, before analyzing dendritic patterning (B, C) and GABAergic connectivity (B, D). (B) Representative micrographs of 7 DIV hippocampal neurons treated with Aβ and/or transfected with p65/RelA. EGFP immunostaining (green) and the transfected p65/RelA mostly located to the nucleus (purple). Vesicular inhibitory amino acid transporter (VIAAT) was evident as punctuated red dots. Lower panels show the boxed regions at higher magnification. (C) Morphometric analysis of treated neurons. p65/RelA overexpression increased the length (left panel) but decreased the number (right panel) of primary dendrites, thereby counteracting the effects of Aβ. (D) p65/RelA overexpression increased the number of GABAergic terminals in cultured neurons and overrode the decrease in GABAergic terminals produced by Aβ. *P < 0.05, **P < 0.01, and ***P < 0.001.