Regulation of Ca(2+) concentrations is essential to maintain the structure and function of the axon initial segment (AIS). The so-called cisternal organelle of the AIS is a structure involved in this regulation, although little is known as to how this organelle matures and is stabilized. Here we describe how the cisternal organelle develops in cultured hippocampal neurons and the interactions that facilitate its stabilization in the AIS. We also characterize the developmental expression of molecules involved in Ca(2+) regulation in the AIS. Our results indicate that synaptopodin (synpo) positive elements considered to be associated to the cisternal organelle are present in the AIS after six days in vitro. There are largely overlapping microdomains containing the inositol 1,4,5-triphosphate receptor 1 (IP(3)R1) and the Ca(2+) binding protein annexin 6, suggesting that the regulation of Ca(2+) concentrations in the AIS is sensitive to IP(3) and subject to regulation by annexin 6. The expression of synpo, IP(3)R1 and annexin 6 in the AIS is independent of the neuron activity, as it was unaffected by tetrodotoxin blockage of action potentials and it was resistant to detergent extraction, indicating that these proteins interact with scaffolding and/or cytoskeleton proteins. The presence of ankyrin G seems to be required for the acquisition and maintenance of the cisternal organelle, while the integrity of the actin cytoskeleton must be maintained for the expression IP(3)R1 and annexin 6 to persist in the AIS.
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