Allopregnanolone-induced rise in intracellular calcium in embryonic hippocampal neurons parallels their proliferative potential
© Wang and Brinton. 2008
Published: 3 December 2008
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© Wang and Brinton. 2008
Published: 3 December 2008
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 (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.
In 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.
Results 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.
Collectively, 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.
Allopregnanolone (APα; 3α-hydroxy-5α-hydroxy-pregnan-20-one; also known as tetrahydroprogesterone) is a derivative of progesterone that is produced in both the periphery and the central nervous system via enzymatic conversions of progesterone [1–3]. In mature neurons, APα is known to act as an allosteric modulator of the γ-aminobutyric acid type A (GABAA) receptor, binding to a specific site within the GABAA receptor at physiological concentrations (6–35 nM) [4, 5] to increase chloride influx, thereby hyperpolarizing the neuronal membrane potential, and decreasing neuron excitability [6–11]. In marked contrast, the flux of chloride in developing neurons is opposite to that of mature neurons. 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 [12–16].
Calcium signalling plays a key role in neural function and neural development [17–20]. Increases in intracellular calcium concentration ([Ca2+]i) also control cell cycle protein expression and promote cell proliferation [21–26]. Therefore, 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 consequently may influence early developmental events, including neurogenesis and synaptogenesis [16, 27–29].
Previously we found that APα rapidly induced neurite regression in cultured hippocampal neurons , which we later identified as a prelude to entry into the cell cycle and mitosis . Recently, we demonstrated that APα regulates the expression of genes encoding cell cycle-related molecules and enhances human cortical neural progenitor, and rat hippocampal neuronal progenitor cell proliferation in vitro , and in vivo in triple transgenic Alzheimer's disease mice dentate gyrus [31–33]. Moreover, the L-type calcium blocker nifedipine abolished the APα-induced cell proliferation. We therefore hypothesized that the APα-induced neural progenitor cell proliferation is mediated by calcium influx via GABAA receptor-activated L-type calcium channels. To test this hypothesis, we investigated the impact of APα on calcium dynamics using Fura2 fluorescent ratio calcium imaging in rat E18 hippocampal neurons.
Timed-pregnant Sprague-Dawley rats were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN, USA). Rats were housed under controlled conditions of temperature (22°C), humidity (30–50%), and light (14 hour light:10 hour dark); water and food were available ad libitum. All experiments conformed to the Animal Welfare Act, Guide to Use and Care of Laboratory Animals, and the US Government Principles of the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training guidelines on the ethical use of animals. In addition, the minimal number of required animals was used for these experiments and suffering was minimized. Primary cultures of dissociated hippocampal neurons were performed as previously described [29, 30]. Briefly, hippocampi were dissected from the brains of E18 rat fetuses, treated with 0.02% trypsin in Hank's balanced salt solution (HBSS, Invitrogen, Grand Island, NY, USA) for 5 minutes at 37°C and dissociated by repeated passage through a series of constricted, fire-polished Pasteur pipettes. Cells were plated onto poly-D-lysine-coated 22 mm diameter cover slips at a density of 2–4 × 104 cells per cm2, and grown in Neurobasal medium without phenol red (NBM; Gibco/Life Technologies, St. Petersburg, FL, USA) supplemented with 2% B27 (Gibco/Life Technologies), 10 U/ml penicillin, 10 μg/ml streptomycin, 0.5 mM glutamine and 25 μM glutamate. Cultures were maintained at 37°C in a humidified 5% CO2 atmosphere until the day of imaging.
All steroids used in this study were purchased from Steraloids.Inc (Newport, Rhode Island, USA). They were: allopregnanolone (APα; 5α-pregnan-3α-ol-20-one); pregnanolone (3α5βAP; 5β-pregnan-3α-ol-20-one); epipregnanolone (3β5βAP; 5β-pregnan-3β-ol-20-one); epiallopregnanolone (3β5αAP; 5α-pregnan-3β-ol-20-one); and progesterone (P4; 4-pregnane-3,20-dione).
[Ca2+] in hippocampal neurons was determined by ratiometric imaging of the Ca2+-sensitive fluorescent dye, Fura-2 acetooxymethyl ester (Fura-2 AM, Molecular Probes, Eugene, OR, USA), as previously described [34, 35]. Briefly, cells were loaded with 2 μM Fura-2 AM in HEPES-buffered solution (HBS; 100 mM NaCl, 2.0 mM KCl, 1.0 mM CaCl2, 1.0 mM MgCl2, 1.0 mM NaH2PO4, 4.2 mM NaHCO3, 12.5 mM HEPES and 10.0 mM glucose, pH 7.4) for 45 minutes at 37°C, 5% CO2. Cells were then washed in HBS to remove excess Fura-2 AM and incubated in HBS for another 30 minutes to equilibrate. The cover slip with cells loaded with Fura-2 AM was then mounted in a perfusion chamber and placed on an inverted microscope (Olympus IMT-2). Baseline [Ca2+] was obtained over 1 minute prior to the initiation of stimulus, which was maintained for the duration of the imaging. Stimulation was initiated by perfusion (2 ml/minute), or bolus addition (200 μl in total of 1,000 μl), to a static bath of the indicated compounds. Fura-2 was successively excited by a xenon light source at 340 nm and 380 nm by means of two narrow beam band-pass filters selected by a computer-controlled filter wheel. The emitted fluorescence was filtered through a 520 nm filter, captured with an intensified CCD camera (COHU, San Diego, CA, USA), and analyzed with InCyt Im2 software (Intracellular Imaging, Inc., Cincinnati, OH, USA). The [Ca2+] was calculated by comparing the ratio of fluorescence at 340 nm and 380 nm against a standard curve. The curve was made from five different [Ca2+] standards available from Molecular Probes. Data from regions of interest were displayed in real-time and logged to hard disk. Data are presented as representative traces averaged from at least 10 cells per experiment. Responses to treatments were quantified by determining the difference between the average [Ca2+]i for the period of time of maximal response during the drug exposure and the average [Ca2+]i for 30 seconds prior to exposure. Changes in [Ca2+]i are presented as mean ± standard error of the mean (SEM) from three or more independent experiments with ≥10 cells per experiment. Statistical comparisons utilized one-way ANOVA followed by Newman-Keul's post hoc analysis.
To determine the specificity of APα on the increase of [Ca2+]i, the effects of several APα isomers, and its parent molecule, progesterone, were compared at a concentration of 250 nM. APα induced an [Ca2+]i increase with an average of 133 ± 11 nM. No significant [Ca2+]i response was observed for 3β5αAP, 3β5βAP, or progesterone at the same concentration. Only 3α5βAP induced a significant, but lower, [Ca2+]i rise (73 ± 8 nM, p < 0.01; Figure 2B,C). These data indicate that the APα-induced [Ca2+]i increase is sterospecific for APα, and its 5β-, but not 3β-isomers.
To determine the proportion of hippocampal neurons responsive to APα, and the duration of responsiveness, the number of APα-induced [Ca2+]i-responsive neurons were assessed each day for 11 days (216 neurons/day). The percentage of APα-induced [Ca2+]i rise in neurons increased linearly from day 0 to day 4 (DIV), followed by a stepwise decrease in the number of neurons that, by day 11, had returned to day 0 levels (Figure 2D). At DIV 0, 25 ± 5% of the neurons exhibited a APα-induced [Ca2+]i increase; at DIV 1, 61 ± 7%; at DIV 2, 84 ± 4%. By DIV 3, the number of APα-responding neurons reached an asymptote at 90 ± 6% (Figure 2D; *p < 0.01 and **p < 0.001) and remained at that level until DIV 4. From DIV 7–10, the number of responding neurons declined and remained stable at approximately 63%. By DIV 11, the number of neurons responding to APα (30 ± 4%) declined to the level observed at DIV 0.
Analyses using intracellular ratiometric Fura2 calcium imaging demonstrated that APα specifically induced a rapid, transient, and dose-dependent [Ca2+]i rise in E18 rat hippocampal neurons in primary culture. The APα-induced [Ca2+]i rise was not observed in calcium-free medium and was blocked by the L-type calcium channel blockers La3+ and nifedipine. In addition, the GABAA receptor inhibitors picrotoxin and bicuculline completely abolished the APα-induced [Ca2+]i rise. These findings in hippocampal neurons parallel those described by Dayanithi and Tapia-Arancibia  in fetal rat hypothalamic neurons, suggesting that the APα-induced rise in [Ca2+]i is a generalized effect in developing neurons.
The APα-induced rapid response in [Ca2+]i rise was specific to APα as the β-isomers of APα, namely 3β5βAP and 3β5αAP, and its parent molecule, progesterone, were without effect, whereas 3α5βAP induced a [Ca2+]i rise that was 50% of the effect of APα. These data suggest the structure with the most important impact on stereospecificity may reside at the 3 position of the neurosteroid. Indeed, both an extracellular application, and 3α-substitutions of the A ring of the steroid nucleus, have been reported to be prerequisites for many of the interactions of the neurosteroid with GABAA receptor/Cl- channels, because neither intracellular applications, nor 3β-substituted steroids applied extracellularly, show agonistic effects of GABA [4, 44, 45]. Recent analyses by Hosie et al.  indicate that APα can bind to two sites on the GABAA receptor, one that potentiates, and one that directly activates the GABAA receptor. The potentiating binding site of APα resides in a cavity formed by the α-subunit transmembrane domains. The direct activating binding site of APα located among interfacial residues between the α and β subunits and is enhanced by steroid binding to the potentiation site . Data presented here demonstrate that the APα-induced [Ca2+]i rise can be abolished by two GABAA receptor blockers, namely bicuculline [46–49] and picrotoxin [50–52], strongly supporting the notion that the APα-induced [Ca2+]i rise is a GABAA receptor-mediated process and most likely through the direct activating binding site.
GABAA receptor is an ion channel that allows either influx or efflux of chloride ions (Cl-), depending upon the prevailing transmembrane [Cl-] gradient. Because immature neurons have a higher intracellular [Cl-], activation of the GABAA receptor by GABA, or other agonists, for example, muscimol, causes efflux of Cl- and thus membrane depolarization [15, 53]. This depolarization is sufficient to open L-type voltage-gated calcium channels, leading to calcium influx [54–56]. GABA and GABA-induced calcium influx have been linked to trophic actions important for developmental processes, including the expression of brain-derived neurotrophic factor . Thus, GABAA receptor-mediated depolarization may be the trigger for a spontaneous, activity-independent [Ca2+]i rise in early precursor cells, or subventricular zone radial precursor cells, thereby influencing early developmental events, including neurogenesis and synaptogenesis [16, 28, 29]. In the present study, we have demonstrated that the APα-induced [Ca2+]i rise can be blocked by a voltage-gated calcium channel blocker, La3+, as well as a more specific L-type calcium channel blocker, nifedipine. Further, we recently demonstrated that the L-type calcium channel blocker nifedipine specifically abolished the APα-induced proliferation of rat hippocampal neuronal progenitor cells . Taken together, these data suggest that the APα-induced [Ca2+]i rise, regulated by the L-type calcium channel and evoked by GABAA receptor, may be the signalling initiation mechanism for APα-induced neuroprogenitor cell proliferation and cell cycle gene expression.
As development progresses, the effect of APα associated with GABAA receptor binding gradually switches from excitatory to inhibitory [15, 16, 57, 58]. The timing of the shift from depolarizing to hyperpolarizing via GABAA receptor varies across brain regions, but is generally complete by the second week of life in the rat and mouse [12, 59–61]. Remarkably, APα induction of [Ca2+]i rise in cultured hippocampal neurons closely parallels the developmental time course of Na+-K+-2Cl- co-transporter expression [62, 63]. In primary cultures of the E18 rat hippocampal neurons, a mixture of E16–E18 neurons with differing phenotypes will exist. Therefore, it is not surprising that high and low APα-induced calcium responses were observed in this study. The high and low calcium responses may be indicative of the developmental stage of the neuron in culture.
The GABAA receptor is composed of a pentamer of structurally homologous subunits that may be drawn from the α1–6, β1–3, γ1–3, δ, ε, θ, ρ1–3, and π subunit families. The precise subunit composition of different GABAA receptor isoforms is an important determinant of their pharmacological and biophysical properties [4, 64–69], but the exact combination heterogeneity exist. For example, a study indicated that the subunit combinations comprising α1β1γ2 and α3β1γ2 required a three- to seven-fold lower concentration for APα to enhance GABA-evoked current to the same degree as other combinations , while another study demonstrated that the potencies of APα to enhance GABA response were significantly higher in the α5β2γ2 receptor versus α1β2γ2 . Another study suggested that the efficacy of APα to enhance GABA response depended on the γ subunit subtype: α1β1γ3 > α1β1γ2 = α1β1γ1 . Interestingly, during migration from the subventricular zone to the cortical plate, neurons became predominantly GABAergic, and their dominant GABAA receptor subunit expression pattern changed from α4β1γ1 to α3β3γ2 or α3β3γ3, coincident with an increasing potency of GABA on GABAA receptor-mediated depolarization . Although heterogeneity of the subunit combination exists, all these studies suggest that the effects of APα depend on subunit combinations of the GABAA receptor and the combination is changing during development. Therefore, the different GABAA receptor subunit combinations might be the underlying mechanism for the variety of APα-induced [Ca2+]i responses in cultured hippocampal neurons. Along with DIV, the combination of the GABAA receptor subunits is changing and a possible time-point at which one GABAA receptor subunit combination switches to another is 3–4 DIV. However, exact matching of neuronal high and low calcium responses to the expression of specific GABAA receptor subunit combinations, and investigation of the role of each combination, needs to be further addressed.
These data demonstrate that APα induced a dose and developmentally regulated rise in [Ca2+]i that was stereospecific, rapid, and transient in E18 rat hippocampal neurons in primary culture. Moreover, our data indicated that the APα-induced rise in [Ca2+]irequires both GABAA receptor and L-type calcium channel. Together with earlier findings, we propose that the APα-induced rise in [Ca2+]i provides a mechanism whereby APα can promote proliferation of rodent, and also possible of human, neural progenitor cells.
intracellular calcium concentration
days in vitro
Fura-2 acetooxymethyl ester
γ-aminobutyric acid type A
Hank's balanced salt solution
standard error of the mean
This research was supported by grants from the Institute for Study of Aging/Alzheimer's Drug Discovery Foundation, the Kenneth T and Eileen L Norris Foundation and the LK Whittier Foundation to RDB. The excellent technical contributions of Angela Dietrich are acknowledged.
This article has been published as part of BMC Neuroscience Volume 9 Supplement 2: 2008 Proceedings of the 8th International Conference on Alzheimer's Disease Drug Discovery The full contents of the supplement are available online at http://www.biomedcentral.com/1471-2202/9?issue=S2.
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.