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(The FASEB Journal. 2006;20:1377-1386.)
© 2006 FASEB

Neuroprotective effect of mifepristone involves neuron depolarization

A. M. Ghoumari*,1, C. Piochon{dagger}, C. Tomkiewicz{ddagger}, B. Eychenne1, C. Levenes{dagger}, I. Dusart{dagger}, M. Schumacher* and E. E. Baulieu*

* INSERM UMR788 and University Paris XI, Bicêtre, France;

{dagger} UMR7102 CNRS-Université Pierre et Marie Curie, Paris, France; and

{ddagger} INSERM U490, Paris, France

1 Correspondence: INSERM U488, 80 rue du Général Leclerc, Kremlin-Bicêtre 94276, France. E-mail: ghoumari{at}kb.inserm.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In several regions of the developing nervous system, neurons undergo programmed cell death. In the rat cerebellum, Purkinje cell apoptosis is exacerbated when cerebellar slices are cultured during the first postnatal week. To understand the mechanism of this developmental apoptosis, we took advantage of its inhibition by the steroid analog mifepristone. This effect did not involve the classical steroid nuclear receptors. Microarray analysis revealed that mifepristone down-regulated mRNA levels of the Na+/K+-ATPase {alpha}3 subunit more than three times. Consistent with the down-regulation of the Na+/K+-ATPase, mifepristone caused Purkinje cell membrane depolarization. Depolarizing agents like ouabain (1 µM), tetraethylammonium (2 mM), and veratridine (2 µM) protected Purkinje cells from apoptosis. These results suggest a role of excitatory inputs in Purkinje cell survival during early postnatal development. Indeed, coculturing cerebellar slices with glutamatergic inferior olivary neuron preparations allowed rescue of Purkinje cells. These findings reveal a new neuroprotective mechanism of mifepristone and support a pivotal role for excitatory inputs in the survival of Purkinje neurons. Mifepristone may be a useful lead compound in the development of novel therapeutic approaches for maintaining the resting potential of neurons at values favorable for their survival under neuropathological conditions.—Ghoumari, A. M., Piochon, C., Tomkiewicz, C., Eychenne, B., Levenes, C., Dusart, I., Schumacher, M., Baulieu, E. E. Neuroprotective effect of mifepristone involves neuron depolarization.


Key Words: Purkinje cells • Na+/K+-ATPase • rat cerebella


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PURKINJE CELLS DIE by apoptosis in organotypic culture when cerebellar slices are taken between P1 and P7 (1 , 2) . Although protein kinase C (PKC), the mitochondrial pathway of apoptosis, and microglial cells are involved in this developmental Purkinje cell death, the underlying cellular and molecular signaling mechanisms remain unknown (2 3 4) . As Purkinje cells in organotypic cultures die precisely at a time when increased neuronal apoptosis is observed in vivo and when intense dendrite remodeling and synaptogenesis take place (4 5 6) , the event is likely to reflect a physiologically relevant developmental process.

We previously demonstrated that mifepristone, well known as an antagonist of the glucocorticosteroid and progesterone receptors (7) , protects Purkinje cells from apoptotic death in organotypic slice cultures of postnatal rat and mouse cerebella by a novel mechanism that involves neither classical intracellular steroid receptors nor the antioxidant properties of the steroid analog (8) . Mifepristone had already been shown to protect hippocampal neurons from apoptosis after traumatic brain injury or during oxidative stress (9 , 10) .

The strong neuroprotective effect of mifepristone was expected to give clues to the age-dependent Purkinje cell death and to reveal important developmental features of this neuron. We show that mifepristone prevents the increase in Na+/K+-ATPase {alpha}3 subunit expression and activity that normally follows the culture process, thus maintaining the Purkinje cells in a depolarized status resulting in their survival. Indeed, various depolarizing procedures such as high external K+, blockade of K+ channels and of the Na+/K+-ATPase, and activation of Na+ channels were able to mimic the protective effects of mifepristone on Purkinje cells. The catalytic {alpha}3 subunit isoform of the Na+/K+-ATPase is expressed in the nervous system, particularly in rat Purkinje cells (11 12) , and blocking the pump leads to a rapid depolarization of Purkinje cells (13) .

Most important, the restoration of excitatory synaptic afferents from inferior olivary neurons allowed partial rescue of Purkinje cells. The present findings reveal a novel mechanism involved in the neuroprotective effects of mifepristone, and provide further support for a pivotal role of excitatory inputs, provided at least in part by climbing fiber innervation, in the survival of postnatal Purkinje neurons.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Slice cultures and cocultures
Cerebellar slices were prepared from postnatal day 3 (P3) Sprague-Dawley rats (Janvier, Le Genest St. Isle, France). For each experiment, at least 3 animals and 18 slices were used. After decapitation, brains were dissected out into cold Gey’s balanced salt solution containing 5 mg/ml glucose (Glc) (GBSS-Glu) and meninges were removed. Cerebellar parasagittal slices (350 µm thick) were cut on a Macllwain tissue chopper and transferred onto membranes of 30 mM Millipore culture inserts with 0.4 µm pore size (Millicell, Millipore, Bedford, MA, USA).

Transverse slices (350 µm thick) of the ventral medial portion of the anterior medulla containing the inferior olivary neurons (14) were obtained from the same P3 Sprague-Dawley rat and cut on a Macllwain tissue chopper. Cerebellar slices and the slices containing inferior olivary neurons were cocultured on membranes of 30 mM Millipore culture inserts. Even if the ventral medial portion of the anterior medulla contains neurons other than inferior olivary neurons, in this study we use the terminology of inferior olivo-cerbellar coculture as used in a study by Audinat et al. (14) .

Slices were maintained in culture in 6-well plates containing 1 ml of medium at 35°C in an atmosphere of humidified 5% CO2. The medium was composed of 50% basal medium with Earle’s salts (Invitrogen, Gaithersburg, MD, USA), 25% Hanks’ balanced salts solution (Life Technologies, San Diego, CA, USA), 25% horse serum (Life Technologies), L-glutamine (1 mM), and 5 mg/ml Glc.

Chemicals
The principal steroids and chemical compounds used were RU486 (mifepristone: 17ß-hydroxy-11ß-(4-methylamino-phenyl)-17{alpha})-(1-propynyl)estra-4,9-dien-3 one), ouabain, tetraethylammonium (TEA), veratridine, tetrodotoxin (TTX), nifedipine, flunarizine (Sigma, St. Louis, MO, USA), and KCl (Merck, Rahway, NJ, USA). Doses with maximal efficiency retained were 1–50 µM for all compounds except for TEA (1–5 mM) and KCl (30 mM). Cerebellar slices were treated with these compounds the day of culture and maintained for 5 days in vitro (5DIV). Medium with the respective steroids or drugs was replaced once after 2 or 3 days.

RNA isolation and cDNA probe synthesis
Cerebellar slices from rat P3 were cultured in the presence or not of 20 µM RU486. After 1 h incubation, total RNA isolation, poly(A)+ RNA enrichment, and cDNA probe synthesis were carried out for treated or untreated cells with the Atlas Pure Total RNA labeling system (Clontech, Palo Alto, CA, USA), as specified by the manufacturer. Prior to purification of poly(A)+ RNA, total RNA was treated with DNase I. Poly(A)+ RNA enrichment was done on 50 µg of total RNA.

Hybridization of labeled cDNA to Atlas plastic microarrays
Plastic microarrays (Atlas Plastic Mouse 5K microarray-S2838, Clontech, Palo Alto, CA, USA) were prehybridized for 30 min at 60°C with prewarmed hybridization solution. Equal counts of labeled probe from treated or untreated cells were added independently, after denaturation in a boiling water bath (95–100°C) for 5 min, onto two plastics microarrays. The reaction was allowed to proceed overnight at 60°C in roller bottles. The next day the plastic microarrays were washed twice with a high-salt wash solution at 58°C for 5 min. Two additional washes with a low salt solution were done at 58°C for 5 min. The plastic microarrays were then exposed to a PhosphorImaging screen for 24 h and scanned at a resolution of 50 µm on a PhosphorImager (Storm 840, Molecular Dynamics, Sunnyvale, CA, USA). Analysis of differential gene expression was performed with the Atlasimage 2.0.1 software (BD Biosciences, San Jose, CA, USA). This experiment was repeated twice and the genes regulated by RU486 in both experiments were selected.

Immunoblot assay
Cerebellar slice cultures were cultured in the absence (controls) or presence of 20 µM RU486 for 30 min, 1 h, 3 h, or 9 h. Slices were then washed with PBS and dissolved in Laemmli buffer. Identical amounts of proteins from each sample (25 µg) were separated by electrophoresis using 10% polyacrylamide gel, then transferred onto PVDF membranes by semidry transfer. After blocking with 5% dry milk, the membranes were incubated overnight at 4°C with a primary monoclonal anti-Na+/K+-ATPase alpha3 antibody (Ab) (1/1000 dilution; Ozyme, St. Quentin En Yvelines, France). After washing with Tween20-PBS buffer, membranes were incubated for 1 h with peroxidase-conjugated AffiniPure goat anti-mouse (1/20000 dilution; Jackson Immunoresesearch Laboratories, Inc., West Grove, PA, USA). The blots were developed with an enhanced chemiluminescence+plus detection kit (Amersham, Little Chalfont, UK).

Na+/K+-ATPase activity
Purified rat Na+/K+-ATPase was prepared from the organotypic slice cultures of P3 rat cerebella by an optimized method of the method of Jørgensen (15) and the one of Muszbek (16) . The slices were untreated (Ctr) or treated separately with 20 µM RU486 or 1 µM ouabain for 3 h. Enzyme preparations were stored in ice-cold 0.25M sucrose, 30 mM histidine (pH 7.2). Homogenates were centrifuged twice with renewing buffer for 45 min at 60,000 rpm. Protein concentration was determined by the Lowry method using BSA as a standard. Fifty micrograms of each sample were transferred to tubes for enzyme assays with ATP (1 mM), MgCl2 (3 mM), NaCl (130 mM), KCl (20 mM), and histidine (30 mM). After 30 min at room temperature, the reaction was stopped with 250 µl TCA; the inorganic phosphate (Pi) complex with phosphomolybdate was measured using spectrophotometer at 623 nM. Enzyme activity is expressed in µmoles Pi/min per milligram protein and mean values ± SE are calculated for groups of at least 3 animals.

Antibodies and staining procedures
Rabbit polyclonal and mouse monoclonal antibodies against calbindin D-28K (diluted 1/10,000, Swant, Bellinzona, Switzerland) were used to visualize Purkinje cells, and Ab against guinea pig VGluT2 (1/350, Chemicon, Temecula, CA, USA) was used to label climbing fibers but also mossy and parallel fibers (1 , 17 , 18) . These first antibodies were revealed, respectively, with secondary antibodies against goat anti-rabbit CY3 Ab (1/200 dilution; Jackson ImmunoResearch Laboratories, Inc.), goat antimouse Alexa Fluor488 (1/1000 dilution, Molecular Probes, Leiden, Netherlands), and donkey anti-guinea pig CY3 (1/200 dilution, ImmunoResearch Laboratories). Staining procedures were performed as described previously (1 , 2) .

Quantification of Purkinje cell survival
To determine the Purkinje cell survival in the cultures, neurons were immunostained with the anticalbindin Ab and counted under a fluorescence microscope (axiovert 135M; Zeiss, Oberkochen, Germany) as described previously (8) . Under these conditions, we counted the total number of surviving Purkinje cells per slice and calculated the means. Images of the immunostained Purkinje cells in organotypic slice cultures of rat and mice cerebella were acquired using an image analyzing system, confocal Zeiss LSM 410 (Zeiss). Images were acquired with a nonconfocal configuration (488 nM excitation).

Electrophysiology
Cerebellar slices were prepared from Sprague-Dawley rats (P3-P4). The cerebellum was rapidly removed and submerged in ice-cold bicarbonate-buffered solution (BBS) bubbled with 95% O2, 5% CO2. Sagittal slices 180 µm thick were cut with a vibroslicer (LEICA VT-1000S) and incubated at room temperature (20–22°C) for at least 1 h prior to electrophysiological recordings either in standard BBS supplemented with 0.5 {per thousand} alcohol (as the vehicle) for controls or in standard BBS + 25 µM RU486 for test experiments. Slices were then transferred in a recording chamber superfused at a rate of 1.5 ml/min with oxygenated BBS containing, in mM: NaCl, 130; KCl, 2.5; MgCl2, 1; CaCl2, 2; NaHCO3, 26; NaH2PO4, 1.3; Glc, 10; final pH 7.35 at 20°C.

Purkinje neurons were visually identified from their position, size, and shape using Nomarski differential interference optics [40x water immersion objective (Zeiss) plus a 2.25 x zoom (Nikon)] mounted on an upright Axioskop fibrous sheath microscope (Zeiss). Patch-pipettes were pulled with a 2-stage puller (Sutter Instrument, Movato, CA, USA) from borosilicate capillary glass tubing. Pipettes were fire polished to a final resistance of 3–5 M{Omega} when filled with the following internal solution (in mM): KCl, 150; HEPES, 10; EGTA, 1; MgCl2, 4.6; CaCl2, 0.1; ATP-Na, 4; GTP-Na, 0.4; pH was adjusted to 7.3 with KOH. In some experiments 150 mM KCl was replaced by 150 mM K-gluconate to prevent chloride currents. As no difference was observed between cells patched with K-gluconate compared to those patched with KCl-based internal solution, results were pooled. Purkinje cells were recorded by patch-clamp in whole-cell configuration using an AXOPATCH 200A amplifier (Axon Instruments, Union City, CA, USA). Patch and estimation of capacitance and series resistance were made in voltage-clamp mode and series resistances were partially compensated (60–70%). Recordings were then made in the current-clamp mode. Acquisition and storage were made on a PC running the ACQUIS1 software (Biological). The Mann-Whitney procedure was used for statistical comparison of means; P is given as the probability of the null hypothesis. Statistical values are given as mean ± SE.

Measurement of intracellular Ca2+ levels
Cytoplasmic free calcium levels were analyzed in different regions of the P3 slices using calcium Fluo-4 (Molecular Probes, Inc., Eugene, OR, USA). P3 cerebellar slices were made in culture in the absence (Ctr) or presence of 20 µM RU486. At the same time, 10 µM calcium Fluo-4 was added to cultures for 1 h at 37°C, then washed with serum-free Glc-supplemented Eagle basal medium medium to remove Fluo-4 in excess. The fluorescent signal (excitation at 480 nM; emission, 510 nM) was visualized using an image analyzing system, confocal Zeiss LSM 410 and measured using the NIH image software. The calcium Fluo-4 staining density was quantified on a continuous scale of 0–255 (darkest). To minimize differences among the respective measurements, we set as control an arbitrary concentration of staining 100. The Fluo-4 staining density was evaluated as a percentage of (light pixels/light+dark pixels)

Statistical analysis
Data were expressed as mean for at least 18 cerebellar slices (n=18) from three animals (n=3) and in three independent experiments ± SE. The significance of differences between means was evaluated by Newman-Keuls tests after 1-way ANOVA and by the Mann-Whitney procedure.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Mifepristone inhibits the increase in Na+/K+-ATPase mRNA, protein, and activity in cerebellar slices
To study the neuroprotective mechanisms of mifepristone, P3 rat cerebella were cut into slices and immediately cultured in the presence or absence of 20 µM of the steroid analog. After 1 h in culture, RNA extracted from control and mifepristone-treated slices was hybridized on plastic microarrays. Analysis of changes in gene expression revealed that mifepristone down- or up-regulated the expression of several genes. The most prominent change observed was the > 3-fold decrease in the expression of the gene encoding the Na+/K+-ATPase {alpha}3 subunit when compared to control (ratio of mifepristone/control=0.31).

Immunoblotting assays showed a corresponding decrease in Na+/K+-ATPase {alpha}3 subunit protein levels in the cerebellar slices after 1 h of treatment with mifepristone when compared to untreated slices (Fig. 1 A). The reduction in the Na+/K+-ATPase {alpha}3 subunit protein could still be observed after 3 h and slightly increased after 9 h of culture (Fig. 1A ). The enzymatic activity of the Na+/K+-ATPase was also decreased ~3-fold after treating the cerebellar slices for 3 h with mifepristone (control: 126±41; mifepristone: 43.5±12 nmol Pi/min/mg of protein) (Fig. 1B ). Most important, Na+/K+-ATPase activity in cerebellar slices treated with mifepristone was comparable to that observed in vivo (46.9±4.7 nmol Pi/min/mg of protein). These results suggested that mifepristone may prevent an increase in Na+/K+-ATPase expression and activity resulting from the culture process and that this effect may be related to its neuroprotective action.


Figure 1
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  		Figure 1. RU486 down-regulates the Na+/K+-ATPase alpha3 expression and activity. A) Immunoblotting assay was performed on the Na+/K+-ATPase alpha 3 isoform. Time courses of Na+/K+-ATPase alpha3 protein expression were determined in cerebellar slices treated or not by 20 µM RU486 for 1/2, 1, 3, and 9 h. B) Enzymatic activity of the pump was measured in RU486- and ouabain-treated P3 cerebellar slices and in untreated P3 cerebellar slices. This Na+/K+-ATPase alpha3 activity was determined by measuring inorganic phosphate (Pi) complexes with phosphomolybdate at 623 nM. Enzyme activity is expressed in nanomoles Pi/min per milligram protein and mean values ± SE were calculated for groups of at least 3 animals. RU486 maintains the Na+/K+-ATPase activity at the in vivo concentration. Thus, RU486 inhibits the increase rather than strongly inhibits Na+/K+-ATPase activity. **P ≤ 0.01 as indicated by Newman-Keuls tests after 1-way ANOVA.

Inhibiting the increase of Na+/K+-ATPase activity in cerebellar slices prevents Purkinje cell death
We then examined whether a direct down-regulation of the Na+/K+-ATPase activity by ouabain in cultured P3 cerebellar slices in which nearly all Purkinje cells die also allowed rescue of these neurons. We first verified that addition of a low concentration of ouabain (1 µM) to the medium at the beginning of the culture for 3 h partially reduced the activity of the pump to 34.3 ± 11 nmol Pi/min/mg of protein, comparable to the normalization observed after mifepristone treatment (Fig. 1B ). Cerebellar slices from P3 rats were then cultured for 5 days in vitro (5DIV) in the absence or presence of 20 µM mifepristone or 1 µM ouabain (Fig. 2 ). Purkinje cell survival was assessed by counting the total number of calbindin D-28K immunostained cells in the slices. Results confirmed the strong protective effect of mifepristone on Purkinje cells in P3 cerebellar slices (Fig. 2B, D ). The neuroprotective effect mifepristone could be mimicked by reducing the Na+/K+-ATPase activity with ouabain (Fig. 2C, D ). As expected, very few Purkinje cells survived in untreated slices (~10 Purkinje cells/slice, Fig. 2A ). However, the mean number of surviving Purkinje cells was 2087 ± 105 and 1300 ± 80 in slices treated respectively with mifepristone or ouabain (Fig. 2D ). Thus, inhibiting the culture-dependent increase of Na+/K+-ATPase activity by either mifepristone or a low concentration of ouabain protects Purkinje cells from the apoptotic process described in cultures of young rat cerebellar slices (2) . We also noted that adding mifepristone or ouabain to the culture medium for periods as short as 3 h was sufficient to prevent some Purkinje cells from death (data not shown).


Figure 2
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  		Figure 2. Inhibiting the increase of Na+/K+-ATPase alpha3 either by RU486 or by ouabain prevents Purkinje cell death in organotypic slice cultures of rat cerebellum. Slices of 3-day-old (P3) rats were cultured for 5 days in vitro (5DIV). Slice cultures were immunostained with anticalbindin D28-K Ab to label Purkinje cells. A) Untreated control slices (Ctr): very few Purkinje cells were present. B) Slices treated with 20 µM RU486. C) Slices treated with 1 µM ouabain, the specific Na+/K+-ATPase inhibitor. D) Quantitative analysis of Purkinje cell survival after treatment with RU486 or with ouabain. In the treated slices with either RU486 or with ouabain, high Purkinje cell survival was observed. Scale bars: 200 µm.

Mifepristone induces Purkinje cell depolarization
It has been reported that inhibition of the Na+/K+-ATPase activity in neurons leads to their depolarization (13 , 19) . We therefore compared the membrane potentials between Purkinje cells of P3 cerebellar slices cultured in the absence or presence of mifepristone. In control slices, the resting membrane potential (RP) of Purkinje cells fluctuated over time between –60/–70 mV and more depolarized values, i.e., –49.4 mV on average (see below). Such a typical "two-state" behavior (–70/–50 mV) was never observed in Purkinje cells treated with mifepristone (n=18). Therefore, to allow reliable comparison between untreated and mifepristone-treated cells, mean resting potential of control cells was calculated from the more depolarized state. Mean resting potential value of mifepristone treated cells was –30.9 ± 2.8 mV (n=18), being significantly above the mean resting potential in control cells (–49.4±2.8 mV; n=14; P<0.001, Fig. 3 C). Therefore, treatment with mifepristone caused a persistent depolarization of Purkinje cells.


Figure 3
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  		Figure 3. Depolarized resting potential and lack of firing behavior in RU486-treated cells. Purkinje cell membrane potential was recorded in current clamp mode. A) Typical recordings of a RU486-treated cell. B) Control cells. Figures illustrate the lack of repetitive firing in the RU486 treated cells when square current pulses of increasing amplitude (180 ms in duration) are injected. This behavior is observed either at resting potential (left) or at –70 mV (right). C) Relationship between resting potential and the number of spikes generated by depolarizing pulses. RU486 cells (black spots) were more depolarized and fireless than control cells (open spots).

The action potential firing properties of Purkinje cells treated with mifepristone were then compared to untreated cells. In the absence of mifepristone, 9 of 14 Purkinje cells (64%) displayed spontaneous action potentials when no current was injected, as described (20 21) . In contrast, only 1 of the 18 mifepristone-treated Purkinje cells (5%) fired spontaneously at resting potential.

To test the spiking properties of Purkinje cells in response to depolarization, square current pulses 180 ms in duration of increasing amplitude (+10 up to +500 pA) were injected across the Purkinje cell membrane through the patch pipette. Cells were first left at their resting potential (Fig. 3A-C , left panels). In control cells a threshold current stimulus produced repetitive firing in 9 of 12 cells (Fig. 3B ). In cells treated with mifepristone, similar depolarizing current pulses induced no spike in 5 cells, one spike in 9 and more than one spike in 3 of the 17 cells tested (Fig. 3A-C , left panel). Thus, when left at their resting potential, Purkinje cells treated with mifepristone display either no or very few spikes in response to depolarization. As expected and as illustrated in Fig. 3C , there was a strong correlation between the membrane potential and the number of action potentials induced by the depolarizing pulses. Indeed, when Purkinje cells are depolarized, they fire fewer action potentials.

We next tested whether maintaining mifepristone-treated Purkinje cells at –70 mV by a continuous injection of current could restore their discharge capabilities by allowing voltage-dependent channels to recover from inactivation. In this condition, Purkinje cells treated with mifepristone still displayed no or low spiking activity, similar to Purkinje cells left at their resting potential (Fig. 3A , right panel). Indeed, the depolarizing current pulses induced no spike in six cells or a maximum of one spike in eight cells. Only 3 of the 17 cells tested displayed more than one spike on depolarizing pulses. Thus, mifepristone significantly depolarizes Purkinje cells from P3 slices and prevents action potential firing.

Depolarizing agents promote Purkinje cell survival
To further test the role of depolarization in preventing the apoptosis of Purkinje cells, we treated P3 cerebellar slices with different depolarizing agents, including high K+, the K+ channel blocker TEA and the Na+ channel activator veratridine. Treatment of the slices during 5 DIV with high K+ (30 mM) significantly increased the number of surviving Purkinje cells: ~70-fold more Purkinje cells survived than in slices cultured in the presence of standard concentrations of K+ (5 mM) (Fig. 4 C). Thus, high K+ allows the rescue of some of Purkinje neurons in cerebellar slice cultures of P3 rat cerebellum. Purkinje cell survival could also be increased by treating slices during 5 DIV with TEA (2 mM, Fig. 4B, C ) (~300-fold increase in Purkinje cell survival when compared to untreated slices, Fig. 4A ). This neuroprotective effect of TEA was dose dependent. Even at a concentration as low as 0.5 mM, TEA resulted in an ~100-fold increase in Purkinje cell survival compared with control (data not shown).


Figure 4
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  		Figure 4. Purkinje cell death in organotypic slice cultures of postnatal rat cerebellum is mainly rescued by depolarizing stimulus. To confirm the effect of depolarization on Purkinje cell survival, depolarizing agents such as high K+ (30 mM), TEA, and veratridine were used in this study. A) Untreated P3 control slices (Ctr). B) Slices treated with 2 mM TEA. C) Quantitative analysis of Purkinje cell survival after treatment with 20 µM RU486 or with depolarizing agents KCl (30 mM) or with TEA (2 mM). D) Slices treated with 2 µM veratridine. E) The veratridine effect was abolished when cultures were simultaneously treated by veratridine (2 µM) and by 2.5 µM tetrodotoxin (TTX), a noncompetitive antagonist of voltage-gated sodium channels. F) Quantitative analysis of Purkinje cell survival after treatment with 20 µM RU486 or with depolarizing agent veratridine (2 µM). Note that TTX abolished the effect of veratridine but not that of RU486, suggesting that RU486 does not act via the voltage-gated sodium channels. Scale bar is 200 µm.

Treatment of P3 cerebellar slices with 0.5–5 µM of veratridine, another depolarizing agent, also increased Purkinje cell survival in a dose-dependent manner (a significant increase in Purkinje cell survival could already be observed at 2 µM) (Fig. 4D-F ). To confirm that veratridine increases Purkinje cell survival through its effect on voltage-sensitive Na+ channels, slices were simultaneously treated with veratridine and with tetrodotoxin (TTX), a noncompetitive antagonist of Na+ channels. The neuroprotective effect of veratridine was indeed abolished by 2.5 µM TTX (Fig. 4E, F ). This observation suggests that Purkinje neurons may be dependent on sodium ion influx for their survival. However, TTX only partially blocked the neuroprotective effects of mifepristone or TEA (data not shown), suggesting different direct targets.

Mifepristone-induced Purkinje cell survival and Ca2+ influx
Neuronal membrane depolarization can drive Ca2+ influx, for instance, through voltage-dependent Ca2+ channels, and Ca2+ has been widely implicated in cell death and survival. In P3 cerebellar slices, Purkinje cell survival supported by veratridine could be completely blocked by the coapplication of the low voltage-activated T-type calcium channel inhibitor flunarizine (5 µM). In contrast, the L-type calcium channel antagonist nifedipine (10 µM) did not affect the survival effect of veratridine (Fig. 5 C). These results demonstrate that the depolarization of Purkinje cells with veratridine promotes their survival by increasing the influx of Ca2+ through T-type calcium channels.


Figure 5
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  		Figure 5. Treatment by RU486 is followed by a moderate increase in Ca2+ influx. A) Representative whole slice fluorescence of Ca2+ release in response to application of 20 µM RU486, as determined by quantifying the calcium Fluo-4 staining density. B) Neither T-type nor L-type voltage-gated Ca2+ channels [blocked respectively by flunarizine (Fluna, 5 µM) and nifedipine (Nif, 10 µM)] was necessary for the effect of RU486 on Purkinje cell survival. In addition, the extracellular Ca2+ chelator EGTA (1 mM) did not block the effect of RU486. C) The depolarizing agent, veratridine, induced Purkinje cell survival in cerebellar slice cultures by Ca2+ influx through T-type voltage-gated Ca2+ channels, as it was blocked only by flunarizine.

We then examined whether treatment by mifepristone also causes an increase in Ca2+ influx. Our electrophysiological results showed that after 1 h in culture, mifepristone depolarized Purkinje cells in cerebellar slices. This depolarization was accompanied by a 1.4-fold increase in the cytoplasmic concentrations of Ca2+ as determined by quantifying calcium Fluo-4 staining density (Fig. 5A ). However, as shown in Fig. 4 , neither flunarizine nor nifedipine significantly reduced the effects of mifepristone on Purkinje cell survival. In agreement with this observation, the extracellular Ca2+ chelator EGTA (1 mM) was also ineffective (Fig. 5B ). Thus, voltage-gated Ca2+ channels seem not to be involved in the neuroprotective effects of mifepristone, but the release of Ca2+ from internal stores (ER) may play a role.

Purkinje cells survive in inferior olivo-cerebellar cocultures
Inferior olivary neurons, through their olivo-cerebellar projections, dynamically regulate the maturation and functions of Purkinje neurons. However, in organotypic cerebellar slice cultures, Purkinje cells are deprived of these glutamatergic excitatory synapses, which mainly derive from climbing fibers (22 23) . To test the hypothesis that the lack of excitatory inputs from inferior olivary neurons may contribute to the death of Purkinje cells in cerebellar slice cultures at P3, we performed an olivo-cerebellar organotypic slice coculture.

After 5 DIV, we counted 2.3-fold more surviving Purkinje cells in olivo-cerebellar slice cultures than in cerebellar slices cultured alone (Fig. 6 B). In many of these olivo-cerebellar slices, the surviving Purkinje cells were localized at the boundary in proximity of the olivary slices (Fig. 6A ). Moreover, VGluT2 (vesicular glutamate transporter) immunoreactive fibers were observed to enter the cerebellar slices and in close apposition with the Purkinje cell soma and dendrites (Figure 6C, D ). At that age, three types of VGluT2 immunoreactive fibers have been described in the rodent cerebellum: climbing, mossy, and parallel fibers (17 18) . However, in cerebellar slices, cultured alone, an extremely low labeling with VGluT2 was detected (Fig. 6E-J ), indicating that in the coculture experiments, the VGluT2 positive fibers originate from the olivary slices. Furthermore, the fact that these fibers terminate on Purkinje cell soma and dendrites suggests that they are more likely climbing fibers than mossy fibers. Indeed, mossy fibers normally do not contact Purkinje cells directly. These results strongly suggest that olivo-cerebellar connections may play a role in Purkinje cell survival during postnatal development.


Figure 6
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  		Figure 6. Purkinje cell survival in inferior olivo-cerebellar cocultures. A) P3 cerebellar slices were cocultured with the inferior olivary slices for 5DIV and double immunolabeling with VGluT2 (red) and CaBP (green) to visualize respectively glutamatergic fibers and Purkinje cells. The dashed lines represent the borderlines between inferior olivary and cerebellum slices in the cocultures. B) Quantification of surviving Purkinje cells in the olivo-cerebellar coculture. C) Apposition between glutamatergic fibers and Purkinje neurons. D) Higher magnification of panel C. Double immunostaining for VGluT2 and CaBP are pointed in Purkinje cell somata and dendritic shafts by the arrows. Scale bar: 250 µm (A), 60 µm (C), 20 µm (D). E, F) Slices of P3 inferior olivary neurons were separately cultured and respectively immunostained with CaBP and with VGluT2. G) A merger of panels E and F. H, I) Slice cultures of P3 cerebellum were respectively immunostained with CaBP and with VGluT2. J) A merger of panels H and I. Note that VGluT2 immunostaining was high in inferior olivary slices but extremely low in cerebellum. In inferior olivary slice cultures, some neurons were CaBP-positive (green). Scale bar: 200 µm in panels E–I.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We previously demonstrated that mifepristone, well known as an antagonist of the glucocorticosteroid and progesterone receptors (7) , exerts a powerful neuroprotective action on postnatal Purkinje cells in organotypic slice cultures of postnatal rat and mouse cerebellum. This mifepristone effect involves a novel mechanism, since neither classical intracellular steroid receptors nor the antioxidant properties of the steroid analog were found to be involved (8) . Here, we demonstrate that mifepristone promotes survival of these Purkinje cells by causing their persistent depolarization. Further, results strongly suggest that this could result from inhibiting the increase in Na+/K+-ATPase {alpha}3 subunit expression and activity. Thus, a certain degree of excitation may be necessary for Purkinje cells to survive during the early postnatal period, corresponding to a developmental stage when intense cerebellar remodeling and synaptogenesis take place (4 5 6) . Depolarizing agents such as high K+, TEA, and veratridine also promoted Purkinje cell survival in organotypic culture. However, the neuroprotective effect of mifepristone involved the Na+/K+ pump and bypassed the voltage-gated ion channels, as it could be mimicked by low concentrations of the Na+/K+-ATPase inhibitor ouabain and was not inhibited by the sodium channel blocker TTX, by Ca2+ channel blockers flunarizine and nifedipine, or by the extracellular Ca2+ chelator EGTA. The current data reveal another interesting feature of the developing Purkinje neurons: after being placed in culture, they respond with an increase in Na+/K+-ATPase expression and activity, which is restored to levels comparable to those observed in vivo by mifepristone or ouabain treatment of the slices. This contrasts with the generally observed decrease in neuronal Na+/K+-ATPase activity and {alpha}3 subunit expression in response to injury (24 25 26) . The Na+/K+-ATPase increase, most likely resulting in Purkinje cell hyperpolarization (12) , turns out to be fatal for the immature neurons relying on excitatory innervation for their survival, provided by climbing fibers. Indeed, coculturing cerebellar slices with slices containing glutamatergic excitatory inferior olivary neurons allowed rescue of part of the Purkinje cells.

Regulation of the Na+/K+-ATPase and Purkinje cell survival
Down-regulation of the Na+/K+-ATPase {alpha}3 subunit in the presence of mifepristone revealed by microarray analysis was confirmed by measuring protein levels and pump activity. The inhibition of increased Na+/K+-ATPase expression could already be observed after 1 h of treatment, started immediately at the beginning of the culture, and protein levels then slightly increased after 9 h. These results indicate that a transient action of mifepristone may be sufficient for its neuroprotective effects. Indeed, treating cerebellar slices with mifepristone for only 12 h was sufficient to rescue a large number of Purkinje cells, which normally die at P3 by apoptosis. We have shown that the caspase-3 pathway is already activated in Purkinje cells 3 h after being placed in culture (2) .

Decreasing Na+/K+-ATPase activity by a low concentration of ouabain also strongly enhanced Purkinje cell survival, suggesting that down-regulation of the pump may account for the neuroprotective effects of mifepristone. A reduction in Na+/K+-ATPase activity is generally associated with neuron death (26) , but it has also been proposed to be involved in adaptative responses of brain cells to hypoxia or ischemia (27) . Furthermore, down-regulation of the Na+/K+-ATPase has been shown to protect neurons in culture against hypoxia, glutamate, or low extracellular K+ (28 29 30) . Down-regulation of the Na+/K+-ATPase could also provide a pro-survival signal for neurons and stimulate DNA and protein synthesis (31 32 33 34) . Thus, depending on the pathophysiological context, a transient decrease in Na+/K+-ATPase activity may exacerbate or protect against neuronal death. Here, we show that decreasing the Na+/K+-ATPase by mifepristone prevents the age-dependent Purkinje cell death in organotypic cultures. The classical steroid receptors seem not to be involved in the effect of mifepristone on Na+/K+ATPase, as neither progesterone nor corticosterone inhibited this effect (data not shown) This is consistent with our previous finding that mifepristone protects Purkinje cells in slices of progesterone and glucocorticoid receptor knockout mice. It would be of interest to elucidate the novel mechanism by which mifepristone could regulate Na/K-ATPase expression.

Neuroprotective effects of mifepristone and depolarization of Purkinje cells
Each cycle of the Na+/K+ pump extrudes three Na+ ions from the cell and moves 2 K+ ions into the cell. Thus, blocking its activity has two consequences for Purkinje cells: their rapid depolarization due to the removal of the associated hyperpolarizing current and, at longer time scales, a modification of the Na+ and K+ membrane gradients (13) . As a consequence, transmembrane electrochemical gradients of these ions are changed and Purkinje cells are expected to display reduced spiking capabilities even when maintained at –70 mV. This is indeed what we observed here. Thus, the inhibition of spike discharge in Purkinje cells treated with mifepristone is not simply a consequence of their persistent depolarization, but most likely also reflects a shift in Na+ and K+ concentrations, expected to disrupt the driving force of these ions and to block action potential discharge. Indeed, Purkinje cells treated with mifepristone were continuously depolarized, displayed either little or no action potential firings, and, as could be expected, neither TTX nor flunarizine blocked the neuroprotective effects of mifepristone. This would favor the possibility that there are at least additional mechanisms of mifepristone action.

The activity of the Na+/K+ pump was increased in P3 Purkinje cells following the culture process and their deprivation of main afferents. Thus, under these conditions the response of the neurons at P3 appears to be inappropriate, as it rapidly leads to their death. As we demonstrate here, preventing induction of the Na+/K+ pump with mifepristone protects P3 Purkinje cells from apoptosis in culture and depolarizing procedures promotes their survival. McKay and Turner recently reported that Purkinje cells at P0 are depolarized at rest (–34±3 mV) and cannot fire sodium spikes even when hyperpolarized (35) . This electrophysiological status of P0 Purkinje cells closely resembles that of P3 Purkinje cells treated by RU486. This could be one reason why P0 Purkinje neurons survive well in culture. In this view, one could consider that mifepristone treatment at P3 brings the cells back to a less mature status, thereby protecting them from death in culture.

The age-dependent death of Purkinje cells in organotypic cultures
In organotypic cultures of rat cerebellum, Purkinje cell apoptosis is age dependent (1) . Most of these cells degenerate when cerebellar slices are taken between P1 and P5, but they survive before or after this period. This critical period of Purkinje cell vulnerability corresponds to a time window when Purkinje cells are engaged in intense synaptogenesis, dendritic remodeling, and cell death (4 5 6) . We previously showed that neurotrophic factors, known to play a crucial role in the development and survival of nerve cells namely, brain-derived neurotrophic factor, neurotrophin 3, and insulin-like growth factor I have only marginal effects on the survival of P3 Purkinje cells (3) . Membrane depolarization by the Na+/K+-ATPase blocker (ouabain), high potassium chloride (KCl), or other depolarizing agents such as TEA and verataridine treatment has been shown to prevent the death of many neuronal cell types (29 , 30 , 36 37 38 39) . Our present results suggest for the first time that depolarization could be one of the mechanisms by which the steroid mifepristone allows neuron survival. Indeed, we show here that all the compounds that maintain or induce a depolarization (either down-regulation of Na+/K+-ATPase by mifepristone or the use of depolarizing agents such as high K+, TEA, or veratridine) induce Purkinje cell survival.

At the first postnatal week of rat cerebellum development, glutamatergic excitatory synapses on Purkinje neurons are mainly derived from climbing fibers originating in the inferior olive nucleus, whereas the excitatory actions of parallel fibers only appear by the end of the first postnatal week (40 41 42 43) . Postnatal Purkinje cells receive multiple innervation by climbing fibers, which is maximal between P3 and P7, in contrast to the one-to-one relationship characteristic of the adult stage (44 , 45) . Our present data suggest that the strong excitation arising from the transient multiple innervation by climbing fibers may play an important role in the survival of immature Purkinje cells. Indeed, removal of this excitatory innervation by the culturing process may explain the age-dependent death of Purkinje cells between P3 and P7 in cerebellar slice preparations. This conclusion is supported by the observations that 1) coculturing cerebellar slices with inferior olivary slices promotes Purkinje cell survival; and 2) climbing fibers originating from the olivary neurons, as documented by our results and as reported by Audinat et al. (18) , form contacts with the surviving Purkinje cells.

Thus, neuron depolarization is necessary for Purkinje cell survival in cerebellar slice culture and it represents a novel function of the steroid receptor antagonist mifepristone. Treatment of the cerebellar slices by mifepristone may allow restoration of the physiological environment and maintenance of the resting potential at a value permitting neuron survival. These observations will be of interest for neuroprotective strategies in other brain regions after injury or under pathological conditions.


   ACKNOWLEDGMENTS
 
We thank Philippe Leclerc for image analysis. This work was supported in part by an Artemis-Fondation Nationale de Gerontologie grant to E.E.B. and by the EXELGYN company.

Received for publication February 6, 2006. Accepted for publication February 27, 2006.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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