Allopregnanolone-induced rise in intracellular calcium in embryonic hippocampal neurons parallels their proliferative potential.
BACKGROUND
Factors that regulate intracellular calcium concentration are known to play a critical role in brain function and neural development, including neural plasticity and neurogenesis. We previously demonstrated that the neurosteroid allopregnanolone (APalpha; 5alpha-pregnan-3alpha-ol-20-one) promotes neural progenitor proliferation in vitro in cultures of rodent hippocampal and human cortical neural progenitors, and in vivo in triple transgenic Alzheimer's disease mice dentate gyrus. We also found that APalpha-induced proliferation of neural progenitors is abolished by a calcium channel blocker, nifedipine, indicating a calcium dependent mechanism for the proliferation.


METHODS
In the present study, we investigated the effect of APalpha on the regulation of intracellular calcium concentration in E18 rat hippocampal neurons using ratiometric Fura2-AM imaging.


RESULTS
Results indicate that APalpha rapidly increased intracellular calcium concentration in a dose-dependent and developmentally regulated manner, with an EC50 of 110 +/- 15 nM and a maximal response occurring at three days in vitro. The stereoisomers 3beta-hydroxy-5alpha-hydroxy-pregnan-20-one, and 3beta-hydroxy-5beta-hydroxy-pregnan-20-one, as well as progesterone, were without significant effect. APalpha-induced intracellular calcium concentration increase was not observed in calcium depleted medium and was blocked in the presence of the broad spectrum calcium channel blocker La3+, or the L-type calcium channel blocker nifedipine. Furthermore, the GABAA receptor blockers bicuculline and picrotoxin abolished APalpha-induced intracellular calcium concentration rise.


CONCLUSIONS
Collectively, these data indicate that APalpha promotes a rapid, dose-dependent, stereo-specific, and developmentally regulated increase of intracellular calcium concentration in rat embryonic hippocampal neurons via a mechanism that requires both the GABAA receptor and L-type calcium channel. These data suggest that APalpha-induced intracellular calcium concentration increase serves as the initiation mechanism whereby APalpha promotes neurogenesis.
Wang
Jun Ming
JM
Department of Pharmacology and Pharmaceutical Sciences and Program in Neuroscience, University of Southern California, Los Angeles, CA 90089, USA. jwang@pathology.umsmed.edu
Brinton
Roberta Diaz
RD
D000777:Anesthetics / Q000494:pharmacology; D000818:Animals; D001640:Bicuculline / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D005260:Female; D018756:GABA Antagonists / Q000494:pharmacology; D006624:Hippocampus / Q000166:cytology / Q000196:embryology; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D009543:Nifedipine / Q000494:pharmacology; D010852:Picrotoxin / Q000494:pharmacology; D011247:Pregnancy; D011280:Pregnanolone / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012196:Review Literature as Topic
D000777:Anesthetics; D001640:Bicuculline; D002118:Calcium; D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D018756:GABA Antagonists; D009543:Nifedipine; D010852:Picrotoxin; D011280:Pregnanolone