Allopregnanolone-induced rise in intracellular calcium in embryonic hippocampal neurons parallels their proliferative potential

Jun Ming Wang,Roberta Diaz Brinton

doi:10.1186/1471-2202-9-s2-s11

Abstract

BackgroundFactors 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 (APα; 5α-pregnan-3α-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 APα-induced proliferation of neural progenitors is abolished by a calcium channel blocker, nifedipine, indicating a calcium dependent mechanism for the proliferation.MethodsIn the present study, we investigated the effect of APα on the regulation of intracellular calcium concentration in E18 rat hippocampal neurons using ratiometric Fura2-AM imaging.ResultsResults indicate that APα 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 3β-hydroxy-5α-hydroxy-pregnan-20-one, and 3β-hydroxy-5β-hydroxy-pregnan-20-one, as well as progesterone, were without significant effect. APα-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 APα-induced intracellular calcium concentration rise.ConclusionCollectively, these data indicate that APα 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 APα-induced intracellular calcium concentration increase serves as the initiation mechanism whereby APα promotes neurogenesis.

Highlights

Factors that regulate intracellular calcium concentration are known to play a critical role in brain function and neural development, including neural plasticity and neurogenesis
GABAA receptor-mediated depolarization may be the trigger that leads to activity-independent [Ca2+]i rise in early precursor cells, or neural progenitors and stem cells, and may influence early developmental events, including neurogenesis and synaptogenesis [16,27,28,29]
APα-induced [Ca2+]i rise is dependent upon dose and days in vitro and is stereoisomer specific To characterize APα-induced [Ca2+]i signalling in embryonic hippocampal neurons, three issues were addressed: dose-response; stereospecificity; and developmental profile of response during days in vitro (DIV)

Summary

Introduction

Factors that regulate intracellular calcium concentration are known to play a critical role in brain function and neural development, including neural plasticity and neurogenesis. Because of the high intracellular chloride content in immature neurons, APα provokes an efflux of chloride through the GABAA receptor, depolarization of the membrane, opening voltage dependent L-type calcium channels, leading to an influx of calcium from the extracellular medium [12,13,14,15,16]. GABAA receptor-mediated depolarization may be the trigger that leads to activity-independent [Ca2+]i rise in early precursor cells, or neural progenitors and stem cells, and may influence early developmental events, including neurogenesis and synaptogenesis [16,27,28,29]

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Conclusion