Allopregnanolone increases mature excitatory synapses along dendrites via protein kinase A signaling

Allopregnanolone (APα; 5α-pregnan-3α-ol-20-one) is synthesized in both the periphery and central nervous system and is known to be a potent positive allosteric modulator of the GABAA receptor. Because APα was suggested to improve the symptoms of depression and Alzheimer's disease (AD), which involve synaptic dysfunction and loss, we examined whether APα affects excitatory synapses. Drebrin, which is an actin-binding protein, forms a unique stable actin structure in dendritic spines, and drebrin levels correlate positively with cognitive levels in AD and mild cognitive impairment. We investigated whether APα increases excitatory synapse density along dendrites of mature hippocampal neurons using drebrin-imaging-based evaluation of mature synapses. We prepared primary cultures of hippocampal neurons and either transfected them with GFP or immunostained them against drebrin. Morphological analysis of GFP-transfected neurons revealed that a 24-h exposure to 0.3 or 1 μM APα significantly increased dendritic spine density without any morphological changes to spines. Drebrin cluster density was also increased by 0.3 and 1 μM APα. The protein kinase A (PKA) inhibitor H-89 inhibited the APα-induced increase in drebrin cluster density. These data demonstrate that APα increases mature excitatory synapses via activation of PKA. Therefore, the PKA-cAMP response element-binding protein (CREB) signaling pathway is likely to be involved in the APα-induced increase of mature excitatory synapses. Another possibility is that the PKA-dependent increase in AMPA receptors at dendritic spines mediates the APα function. In conclusion, our study indicates that APα may improve neuropsychiatric disorder outcomes via increasing the numbers of mature excitatory synapses.