- Open Access
Allopregnanolone-induced rise in intracellular calcium in embryonic hippocampal neurons parallels their proliferative potential
BMC Neuroscience volume 9, Article number: S11 (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.
Animals and primary hippocampal neuron culture
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+] microfluorimetry and imaging
[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.
APα induces a rapid and transient change in [Ca2+]iin hippocampal neurons
APα induced a rapid (within seconds) and transient (over the course of minutes) increase in [Ca2+]i in E18 rat hippocampal neurons (Figure 1A) as observed by ratiometric imaging of the Ca2+-sensitive fluorescent dye Fura-2 AM (Figure 1A). [Ca2+]i was calculated using a linear regression standard curve of the 340 nm/380 nm ratios of fluorescence generated by a standard series of calcium concentrations (Figure 1B). The multiple correlation coefficient (R-square = 0.9942) indicated a linear relationship between fluorescence ratio and [Ca2+]i (Figure 1B). Based on this linear relationship, it was possible to generate an approximate [Ca2+]i value. Three distinct 'calcium responses' in neurons exposed to APα emerged from this analysis: high response, neurons exhibiting a ≥0.78 of 340 nm/380 nm ratio; low response, neurons exhibiting ≤0.73 of 340 nm/380 nm ratio; and no response (Figure 1C). In the low response population, APα (250 nM) induced a [Ca2+]i rise of 38 ± 2.6 nM, whereas in the high response population, the same concentration of APα induced a [Ca2+]i rise of 118 ± 32 nM (Figure 1D; p < 0.05 compared to control neurons).
APα-induced [Ca2+]i rise is dependent upon dose and days in vitroand 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). Analysis for dose-response and stereospecificity were conducted in neurons at 3 DIV. Application of 10 nM APα showed an insignificant increase in [Ca2+]i. A linear and significant increase was observed from 50 nM, (13 ± 7 nM [Ca2+]i, p < 0.05 versus vehicle control), and 100 nM APα (62 ± 12 [Ca2+]i, p < 0.01). Maximal response was observed when 250 nM APα was applied (135 ± 14 nM [Ca2+]i, p < 0.01), which could not be further increased using 500–1,000 nM APα (Figure 2A). The estimated EC50 value for APα was 124 ± 15 nM.
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.
Different types of [Ca2+]iresponses
To determine the developmental profile for low and high responders, we determined the change in the magnitude of APα-induced [Ca2+]i in low and high responders, and the portion of the population of responders across DIV. We first investigated whether the magnitude of the APα-induced [Ca2+]i changed with time in culture. High-response, low-response, and non-responsive neurons were consistently observed from DIV 0 to DIV 11. As summarized in Figure 3A, the magnitude of APα-induced [Ca2+]i in low responders (340/380 nm fluorescence ratio lower than 0.73 and reflect <45 nM increase in free calcium; Figure 1B) showed no significant change during the entire 11-day experimental period. In contrast, the magnitude of APα-induced [Ca2+]i in high-response neurons (340/380 nm fluorescent ratio >0.78, reflecting a >65 nM increase in free calcium, p < 0.01) increased from 65 to 125 ± 10 nM in the first 3 days, and reached a plateau for the remaining days of culture. We then determined whether the number of high-response neurons versus low-response neurons changed during time in culture and present the data as a percentage of total cells (Figure 3B; *p < 0.01 and **p < 0.001). The number of high-response neurons increased rapidly and linearly, and reached the highest percentage of 65 ± 7% on DIV 3, gradually decreasing to less than 40% on DIV 7. The number of low-response neurons also increased early in the course of culturing and reached the greatest percentage of 27 ± 5% on DIV 2, decreasing to 17 ± 3% on DIV 3 (no significant difference with DIV 0). However, on DIV 4, the percentage of low-response neurons returned to a level similar to that observed on DIV 2 and then gradually decreased. DIV 3 was the time-point at which the highest number of high-response neurons, and the lowest number of low-response neurons, were observed. These data indicate that the magnitude of calcium responses, and the numbers of APα-induced [Ca2+]i-responsive neurons, vary with the days of culture and that DIV 3 represents a pivotal time-point in the response to APα.
APα-induced [Ca2+]i rise reflects an influx of extracellular Ca2+and is regulated by an L-type calcium channel
Free [Ca2+]i increase could result from either an influx of extracellular calcium or a release of calcium from an intracellular pool stored in organelles, including the endoplasmic reticulum and mitochondria. To investigate whether the APα-induced [Ca2+]i rise required an influx of extracellular Ca2+, neurons at DIV 3–4, when 84% of neurons should be responsive to APα (65% high responders), were treated with 500 nM APα in a calcium-free medium. To sustain comparable osmolarity and ionic strength, 2 mM NaCl was substituted for 1 mM CaCl2. In the absence of extracellular calcium, APα did not induce a rise in [Ca2+]i (Figure 4A). This result suggested that the APα-induced [Ca2+]i rise could be attributed mainly, if not completely, to the influx of extracellular Ca2+. To discern which calcium channel was required for the calcium influx, neurons were exposed to the inorganic-calcium transport inhibitor La3+ (10 μM) [36, 37]. Exposure to La3+ reduced the APα-induced [Ca2+]i rise from 78 ± 5 nM to 42 ± 7 nM (Figure 4B; p < 0.05). When nifedipine, a more specific blocker to L-type calcium channels was used, the APα-induced [Ca2+]i rise decreased from 82 ± 14 nM to 20 ± 4 nM (Figure 4C; p < 0.01). These data indicate that the APα-induced [Ca2+]i rise is dependent upon extracellular Ca2+ and is regulated by the L-type calcium channel.
GABAA receptor mediates the [Ca2+]irise induced by APα
It is well known that APα acts as an allosteric modulator of the GABAA receptor/chloride channel by altering chloride flux and thereby hyperpolarizing (in mature neurons) or depolarizing (in immature neurons) the neuronal membrane [15, 16, 38–42]. To determine whether the APα-induced [Ca2+]i rise in hippocampal neurons required the GABAA receptor, two GABAA receptor blockers, picrotoxin (a non-competitive antagonist) and bicuculline (the prototypical competitive antagonist that directly competes with GABA for binding to the receptor complex), were applied at 100 μM and 30 μM, respectively. Both GABAAreceptor blockers completely abolished the APα-induced [Ca2+]i rise (Figure 5A,B). These data indicate that the APα-induced [Ca2+]i rise requires activation of GABAA receptor and the L-type calcium channel in rat hippocampal neuron cultures.
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 AM:
Fura-2 acetooxymethyl ester
γ-aminobutyric acid type A
Hank's balanced salt solution
standard error of the mean.
Baulieu EE, Robel P, Schumacher M: Neurosteroids: beginning of the story. Int Rev Neurobiol. 2001, 46: 1-32.
Compagnone NA, Mellon SH: Neurosteroids: biosynthesis and function of these novel neuromodulators. Front Neuroendocrinol. 2000, 21: 1-56.
Plassart-Schiess E, Baulieu EE: Neurosteroids: recent findings. Brain Res Brain Res Rev. 2001, 37: 133-140.
Gee KW, Bolger MB, Brinton RE, Coirini H, McEwen BS: Steroid modulation of the chloride ionophore in rat brain: structure-activity requirements, regional dependence and mechanism of action. J Pharmacol Exp Ther. 1988, 246: 803-812.
Gee KW, Chang WC, Brinton RE, McEwen BS: GABA-dependent modulation of the Cl- ionophore by steroids in rat brain. Eur J Pharmacol. 1987, 136: 419-423.
Belelli D, Herd MB, Mitchell EA, Peden DR, Vardy AW, Gentet L, Lambert JJ: Neuroactive steroids and inhibitory neurotransmission: mechanisms of action and physiological relevance. Neuroscience. 2006, 138: 821-829.
Belelli D, Lambert JJ: Neurosteroids: endogenous regulators of the GABA(A) receptor. Nat Rev Neurosci. 2005, 6: 565-575.
Belelli D, Peden DR, Rosahl TW, Wafford KA, Lambert JJ: Extrasynaptic GABAA receptors of thalamocortical neurons: a molecular target for hypnotics. J Neurosci. 2005, 25: 11513-11520.
Herd MB, Belelli D, Lambert JJ: Neurosteroid modulation of synaptic and extrasynaptic GABA(A) receptors. Pharmacol Ther. 2007, 116: 20-34.
Hosie AM, Wilkins ME, da Silva HM, Smart TG: Endogenous neurosteroids regulate GABAA receptors through two discrete transmembrane sites. Nature. 2006, 444: 486-489.
Lambert JJ, Belelli D, Peden DR, Vardy AW, Peters JA: Neurosteroid modulation of GABAA receptors. Prog Neurobiol. 2003, 71: 67-80.
Cherubini E, Rovira C, Gaiarsa JL, Corradetti R, Ben Ari Y: GABA mediated excitation in immature rat CA3 hippocampal neurons. Int J Dev Neurosci. 1990, 8: 481-490.
Wang YF, Gao XB, Pol van den AN: Membrane properties underlying patterns of GABA-dependent action potentials in developing mouse hypothalamic neurons. J Neurophysiol. 2001, 86: 1252-1265.
Perrot-Sinal TS, Auger AP, McCarthy MM: Excitatory actions of GABA in developing brain are mediated by l-type Ca2+ channels and dependent on age, sex, and brain region. Neuroscience. 2003, 116: 995-1003.
Ben-Ari Y, Gaiarsa JL, Tyzio R, Khazipov R: GABA: a pioneer transmitter that excites immature neurons and generates primitive oscillations. Physiol Rev. 2007, 87: 1215-1284.
Pol van den AN: Developing neurons make the switch. Nat Neurosci. 2004, 7: 7-8.
Brinton RD: Cellular and molecular mechanisms of estrogen regulation of memory function and neuroprotection against Alzheimer's disease: recent insights and remaining challenges. Learn Mem. 2001, 8: 121-133.
Son MC, Brinton RD: Regulation and mechanism of L-type calcium channel activation via V1a vasopressin receptor activation in cultured cortical neurons. Neurobiol Learn Mem. 2001, 76: 388-402.
Zhao L, Brinton RD: Vasopressin-induced cytoplasmic and nuclear calcium signaling in embryonic cortical astrocytes: dynamics of calcium and calcium-dependent kinase translocation. J Neurosci. 2003, 23: 4228-4239.
Berninger B, Marty S, Zafra F, da Penha Berzaghi M, Thoenen H, Lindholm D: GABAergic stimulation switches from enhancing to repressing BDNF expression in rat hippocampal neurons during maturation in vitro. Development. 1995, 121: 2327-2335.
Gniadecki R, Gajkowska B: Intracellular calcium pool emptying induces DNA synthesis in HaCaT keratinocytes. Exp Dermatol. 2003, 12: 453-459.
Means AR: Calcium, calmodulin and cell cycle regulation. FEBS Lett. 1994, 347: 1-4.
Lu KP, Means AR: Regulation of the cell cycle by calcium and calmodulin. Endocr Rev. 1993, 14: 40-58.
Campagne van Lookeren M, Gill R: Cell cycle-related gene expression in the adult rat brain: selective induction of cyclin G1 and p21WAF1/CIP1 in neurons following focal cerebral ischemia. Neuroscience. 1998, 84: 1097-1112.
Yoshikawa K: Cell cycle regulators in neural stem cells and postmitotic neurons. Neurosci Res. 2000, 37: 1-14.
Whitaker M, Patel R: Calcium and cell cycle control. Development. 1990, 108: 525-542.
Ashworth R, Bolsover SR: Spontaneous activity-independent intracellular calcium signals in the developing spinal cord of the zebrafish embryo. Brain Res Dev Brain Res. 2002, 139: 131-137.
Owens DF, Flint AC, Dammerman RS, Kriegstein AR: Calcium dynamics of neocortical ventricular zone cells. Dev Neurosci. 2000, 22: 25-33.
Wang JM, Johnston PB, Ball BG, Brinton RD: The neurosteroid allopregnanolone promotes proliferation of rodent and human neural progenitor cells and regulates cell-cycle gene and protein expression. J Neurosci. 2005, 25: 4706-4718.
Brinton RD: The neurosteroid 3 alpha-hydroxy-5 alpha-pregnan-20-one induces cytoarchitectural regression in cultured fetal hippocampal neurons. J Neurosci. 1994, 14: 2763-2774.
Wang JM, Irwin WR, Liu L, Chen S, Brinton RD: Regeneration in a degenerating brain: potential of allopregnanolone as a neuroregenerative agent. Curr Alzheimer Res. 2007, 4: 510-517.
Wang JM, Liu L, Irwin WR, Chen S, Brinton RD: Regenerative potential of allopregnanolone. Brain Res Rev. 2008, 57: 398-409.
Wang JM, Singh C, Irwin WR, Liu L, Chen S, Thompson RF, Brinton RD: Allopregnanolone reverses the neurogenic and cognitive deficits of triple transgenic Alzheimer's mice. Alzheimer's Dementia. 2007, 3: S194-
Nilsen J, Chen S, Brinton RD: Dual action of estrogen on glutamate-induced calcium signaling: mechanisms requiring interaction between estrogen receptors and src/mitogen activated protein kinase pathway. Brain Res. 2002, 930: 216-234.
Wu TW, Wang JM, Chen S, Brinton RD: 17Beta-estradiol induced Ca2+ influx via L-type calcium channels activates the Src/ERK/cyclic-AMP response element binding protein signal pathway and BCL-2 expression in rat hippocampal neurons: a potential initiation mechanism for estrogen-induced neuroprotection. Neuroscience. 2005, 135: 59-72.
Henry PD: Atherogenesis, calcium and calcium antagonists. Am J Cardiol. 1990, 66: 3I-6I.
Raeburn D: Calcium entry blocking drugs: their classification and sites of action in smooth muscle cells. Med Biol. 1987, 65: 175-180.
Son M, Dietrich A, Brinton RD: Allopregnanolone induces a rapid transient rise in intracellular calcium in embryonic hippocampal neurons. Program No. 272.8. 2002 Abstract Viewer/Itinerary Planner. 2002, Washington, DC: Society for Neuroscience
Gee KW, Lan NC, Bolger MB, Wieland S, Belelli D, Chen JS: Pharmacology of a GABAA receptor coupled steroid recognition site. Adv Biochem Psychopharmacol. 1992, 47: 111-117.
Gee KW, McCauley LD, Lan NC: A putative receptor for neurosteroids on the GABAA receptor complex: the pharmacological properties and therapeutic potential of epalons. Crit Rev Neurobiol. 1995, 9: 207-227.
Grobin AC, Matthews DB, Montoya D, Wilson WA, Morrow AL, Swartzwelder HS: Age-related differences in neurosteroid potentiation of muscimol-stimulated 36Cl(-) flux following chronic ethanol treatment. Neuroscience. 2001, 105: 547-552.
Liu QY, Chang YH, Schaffner AE, Smith SV, Barker JL: Allopregnanolone activates GABA(A) receptor/Cl(-) channels in a multiphasic manner in embryonic rat hippocampal neurons. J Neurophysiol. 2002, 88: 1147-1158.
Dayanithi G, Tapia-Arancibia L: Rise in intracellular calcium via a nongenomic effect of allopregnanolone in fetal rat hypothalamic neurons. J Neurosci. 1996, 16: 130-136.
Lambert JJ, Belelli D, Hill-Venning C, Peters JA: Neurosteroids and GABAA receptor function. Trends Pharmacol Sci. 1995, 16: 295-303.
Rupprecht R, di Michele F, Hermann B, Strohle A, Lancel M, Romeo E, Holsboer F: Neuroactive steroids: molecular mechanisms of action and implications for neuropsychopharmacology. Brain Res Brain Res Rev. 2001, 37: 59-67.
Sanford LD, Parris B, Tang X: GABAergic regulation of the central nucleus of the amygdala: implications for sleep control. Brain Res. 2002, 956: 276-284.
Pakarinen ED, Moerschbaecher JM: Effects of competitive and noncompetitive GABA(A) antagonists on the acquisition of a discrimination in squirrel monkeys. Behav Pharmacol. 1995, 6: 156-166.
Impagnatiello F, Pesold C, Longone P, Caruncho H, Fritschy JM, Costa E, Guidotti A: Modifications of gamma-aminobutyric acidA receptor subunit expression in rat neocortex during tolerance to diazepam. Mol Pharmacol. 1996, 49: 822-831.
Chavez ME, Salado-Castillo R, Sanchez-Alavez M, Quirarte GL, Prado-Alcala RA: Post-training injection of GABAergic antagonists into the striatum produces retrograde amnesia. Neurobiol Learn Mem. 1995, 63: 296-300.
Teshima K, Kim SH, Allen CN: Characterization of an apamin-sensitive potassium current in suprachiasmatic nucleus neurons. Neuroscience. 2003, 120: 65-73.
Chapouthier G, Venault P: GABA-A receptor complex and memory processes. Curr Top Med Chem. 2002, 2: 841-851.
Blair LA, Levitan ES, Marshall J, Dionne VE, Barnard EA: Single subunits of the GABAA receptor form ion channels with properties of the native receptor. Science. 1988, 242: 577-579.
Owens DF, Boyce LH, Davis MB, Kriegstein AR: Excitatory GABA responses in embryonic and neonatal cortical slices demonstrated by gramicidin perforated-patch recordings and calcium imaging. J Neurosci. 1996, 16: 6414-6423.
Obrietan K, Gao XB, Pol Van Den AN: Excitatory actions of GABA increase BDNF expression via a MAPK-CREB-dependent mechanism – a positive feedback circuit in developing neurons. J Neurophysiol. 2002, 88: 1005-1015.
Obrietan K, Pol van den AN: GABA activity mediating cytosolic Ca2+ rises in developing neurons is modulated by cAMP-dependent signal transduction. J Neurosci. 1997, 17: 4785-4799.
Obrietan K, Pol van den AN: GABA neurotransmission in the hypothalamus: developmental reversal from Ca2+ elevating to depressing. J Neurosci. 1995, 15: 5065-5077.
Ben-Ari Y, Khalilov I, Represa A, Gozlan H: Interneurons set the tune of developing networks. Trends Neurosci. 2004, 27: 422-427.
Tyzio R, Holmes GL, Ben-Ari Y, Khazipov R: Timing of the developmental switch in GABA(A) mediated signaling from excitation to inhibition in CA3 rat hippocampus using gramicidin perforated patch and extracellular recordings. Epilepsia. 2007, 48 (Suppl 5): 96-105.
Ikeda Y, Nishiyama N, Saito H, Katsuki H: Furosemide-sensitive calcium rise induced by GABAA-receptor stimulation in cultures of embryonic rat striatal neurons. Jpn J Pharmacol. 1997, 74: 165-169.
Ikeda Y, Nishiyama N, Saito H, Katsuki H: GABAA receptor stimulation promotes survival of embryonic rat striatal neurons in culture. Brain Res Dev Brain Res. 1997, 98: 253-258.
Obata K: Excitatory and trophic action of GABA and related substances in newborn mice and organotypic cerebellar culture. Dev Neurosci. 1997, 19: 117-119.
Delpire E: Cation-chloride cotransporters in neuronal communication. News Physiol Sci. 2000, 15: 309-312.
Sung KW, Kirby M, McDonald MP, Lovinger DM, Delpire E: Abnormal GABAA receptor-mediated currents in dorsal root ganglion neurons isolated from Na-K-2Cl cotransporter null mice. J Neurosci. 2000, 20: 7531-7538.
Brooks-Kayal AR, Shumate MD, Jin H, Rikhter TY, Kelly ME, Coulter DA: gamma-Aminobutyric acid(A) receptor subunit expression predicts functional changes in hippocampal dentate granule cells during postnatal development. J Neurochem. 2001, 77: 1266-1278.
Behringer KA, Gault LM, Siegel RE: Differential regulation of GABA A receptor subunit mRNAs in rat cerebellar granule neurons: importance of environmental cues. J Neurochem. 1996, 66: 1347-1353.
Montpied P, Yan GM, Paul SM, Morrow AL: Transient increase in cerebellar transcriptional activity precedes the expression of GABA(A) receptor alpha6 subunit mRNA during postnatal maturation. Dev Neurosci. 1998, 20: 74-82.
Poulter MO, Brown LA: Transient expression of GABAA receptor subunit mRNAs in the cellular processes of cultured cortical neurons and glia. Brain Res Mol Brain Res. 1999, 69: 44-52.
Holopainen IE, Lauren HB: Neuronal activity regulates GABAA receptor subunit expression in organotypic hippocampal slice cultures. Neuroscience. 2003, 118: 967-974.
Didelon F, Mladinic M, Cherubini E, Bradbury A: Early expression of GABA(A) receptor delta subunit in the neonatal rat hippocampus. J Neurosci Res. 2000, 62: 638-643.
Lambert JJ, Harney SC, Belelli D, Peters JA: Neurosteroid modulation of recombinant and synaptic GABAA receptors. Int Rev Neurobiol. 2001, 46: 177-205.
Rahman M, Lindblad C, Johansson IM, Backstrom T, Wang MD: Neurosteroid modulation of recombinant rat alpha5beta2gamma2L and alpha1beta2gamma2L GABA(A) receptors in Xenopus oocyte. Eur J Pharmacol. 2006, 547: 37-44.
Maitra R, Reynolds JN: Subunit dependent modulation of GABAA receptor function by neuroactive steroids. Brain Res. 1999, 819: 75-82.
Maric D, Liu QY, Maric I, Chaudry S, Chang YH, Smith SV, Sieghart W, Fritschy JM, Barker JL: GABA expression dominates neuronal lineage progression in the embryonic rat neocortex and facilitates neurite outgrowth via GABA(A) autoreceptor/Cl- channels. J Neurosci. 2001, 21: 2343-2360.
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://0-www.biomedcentral.com.brum.beds.ac.uk/1471-2202/9?issue=S2.
The authors declare that they have no competing interests.
JMW analyzed the data and drafted the manuscript. RDB designed the experiments, coordinated the study and edited the manuscript.
About this article
Cite this article
Wang, J.M., Brinton, R.D. Allopregnanolone-induced rise in intracellular calcium in embryonic hippocampal neurons parallels their proliferative potential. BMC Neurosci 9, S11 (2008) doi:10.1186/1471-2202-9-S2-S11
- Hippocampal Neuron
- GABAA Receptor
- Neural Progenitor
- Intracellular Calcium Concentration