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The survival of cultured mouse cerebellar granule cells is not dependent on elevated potassium-ion concentration
~) Pergamon
Int. J. Devl Neuroscience, Vol. 12,No. 5, pp. 451-460, 1994
0736-5748(94)E0008-P
Elsevier ScienceLtd Copyright© 1994ISDN Printed in Great Britain. All rightsreserved 0736-5748/94$7.00+0.00
THE SURVIVAL OF C U L T U R E D MOUSE C E R E B E L L A R G R A N U L E CELLS IS NOT D E P E N D E N T ON E L E V A T E D POTASSIUM-ION CONCENTRATION HELLE S. MOGENSEN*~, NICOLA HACK~:, ROBERT BALAZS:~ and OLE STEEN JORGENSENt tLaboratory of Neuropsychiatry, Department of Pharmacology, University of Copenhagen, DK-2100 Copenhagen, Denmark :~Netherlands Institute for Brain Research, Amsterdam, The Netherlands (Received 7 October 1993; in revised form 7 January 1994; accepted 10 January 1994)
Abstract--The effects of K+-induced membrane depolarization were studied on the survival and biochemical parameters in mouse and rat cerebellar granule cells grown in micro-well cultures. Cell numbers were determined by estimating DNA content using the Hoechst 33258 fluorochrome binding assay. DNA from degenerated cells was removed by prior DNAase treatment. These DNA estimates of cell numbers were comparable with values obtained by direct counting of fluorescein diacetate-stalned viable cells. In agreement with previous studies, the survival of rat granule cells was promoted by increasing the concentration of K + in the medium from 5 to 25 mM throughout a 7-day culture period. In contrast, mouse granule cells survived in culture containing 'low' K + (5 or 10 mM), as well as in the presence of 'high' K + (25 mM). On the other hand, several biochemical parameters in mouse granule cells were markedly increased by cultivation in 'high' as compared with 'low' K +-containing media, demonstrated by increased fluorescein diacetate esterase activity, enhanced rate of NADPH-dependent tetrazolium reduction, augmented 2-deoxy-D-glucose accumulation and increased N-methyl-D-aspartate-evoked 45Ca2+ influx. It was concluded that although cultivation in 'high' K + promotes biochemical differentiation in mouse cerebellar granule cells, these cells differ from their rat counterparts in that they do not develop a survival requirement for K+-induced membrane depolarization. Key words: mouse, rat, cerebellar granule cells, K + concentration, maturation, survival.
W h e n g r o w n in culture, rat cerebellar granule cells develop b e t w e e n the s e c o n d and f o u r t h days in vitro, a survival r e q u i r e m e n t that can be satisfied either by elevating the m e d i u m K + c o n c e n t r a t i o n f r o m the n o r m a l 5 m M to > 2 0 m M 12 or by treating the cells with glutamate receptor agonists, N-methyl-D-aspartate ( N M D A ) being particularly effective. 5-7 If these r e q u i r e m e n t s are not met, a significant p r o p o r t i o n o f the cells g r o w n in serum-containing m e d i u m with ' l o w ' K + c o n c e n t r a t i o n (5 and 10 m M ) d e g e n e r a t e rather abruptly towards the e n d o f the first w e e k in culture. W e have suggested that the survival-promoting effect o f either 'high' K + c o n c e n t r a t i o n (25 mM; K25) or excitatory a m i n o acid t r e a t m e n t in vitro mimics the influence of the initial innervation received in v i v o o f the immediately p o s t - m i g r a t o r y granule cells by glutamatergic m o s s y fibre afferents. 5,13 In contrast to the observations in rat cells, P e n g et al. 21 have f o u n d that granule cells f r o m m o u s e cerebellum cultivated in m e d i u m containing ' l o w ' K + survive as well as in the presence of 'high' K +. Thus, with respect to survival r e q u i r e m e n t , m o u s e granule cells b e h a v e d in a serum-containing m e d i u m as rat granule cells did in serum-free, chemically defined m e d i u m . 17 P e n g et al. 21 explained the difference in the survival r e q u i r e m e n t of granule cells o b s e r v e d in their cultures c o m p a r e d with o t h e r studies (rat cells) by differences in plating density and the composition o f the culture m e d i u m . In o r d e r to characterize the differences better we studied m o u s e and rat granule cells g r o w n in s e r u m - c o n t a i n i n g m e d i u m u n d e r identical culture conditions. F o r estimating cell survival quantitatively, we modified the H o e c h s t 33258 f l u o r o c h r o m e binding m e t h o d 27 such that the n u m b e r of viable cells could be d e t e r m i n e d despite the p r e s e n c e of D N A fragments f r o m d e a d cells. W e f o u n d that, in contrast to rat granule cells, ' h i g h ' K + is n o t a survival *Author to whom correspondence should be addressed. Abbreviations: DIV, days in vitro; DOG, 2-deoxy-D-glucose;EDTA, ethylenediaminetetra-acetate; EGTA, ethylenegly-
coltetra-acetate; FDA, fluorescein diacetate; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid; MK-801, (+)-5-methyl-10.11-dihydro-5H-dibenzo-[a,d]cyclohepten-5,10-imine hydrogen maleate; NADPH, reduced nicotinamide adenine dinueleotide phosphate; NMDA, N-methyl-D-aspartate; PBS, phosphate-buffered saline; PI, propidium iodide. 451
452
H.S. Mogensen et a/
requirement for mouse granule cells. On the other hand, K t-induced membrane depolarization promotes the biochemical differentiation of both rat and mouse cerebellar granule cells in cultures. EXPERIMENTAL PROCEDURES Cell cultures Granule cell-enriched cultures were prepared from cerebella of 6-day-old mice and rats, as described previously.13.26 Briefly, cerebella were dissociated and cells were plated (in general 2.1 × 103 cells/ram2, 69 × 103 cells/well) in 96-well polystyrene micro-well culture plates (NUNC) that had been pre-coated with poly-L-lysine (10 txg/ml; Sigma). The culture medium consisted of 10% heat-inactivated fetal calf serum (Gibco) in basal Eagle's medium (Gibco) supplemented with 2 mM glutamine, and it contained 100 ixg/ml gentamicin. K + concentrations tested were: 5 mM (K5), 10 mM (K10) and 25 mM (K25). Cytosine arabinoside (10 IxM final concentration; Sigma) was added about 18 hr after seeding. Pre-treatment with DNAase For DNAase treatment, the culture medium was removed from the micro-wells using an eight-channel Octapette (Costar) and the cell monolayer gently washed once with 100 Ixl phosphate-buffered saline (PBS), pH 7.3. Cells were then covered with 50 ILl balanced salt solution (120 mM NaCI, 5 mM KCI, 1.0 mM CaC12, 0.5 mM NazHPO4, 5 mM glucose, 20 mM HEPES, 4.2 mM MgCI2, pH 7.4) supplemented with 0.3% (w/v) bovine serum albumin (fraction V; Sigma) and 0.32 mg DNAase/ml (EC 3.2.21.1; Sigma 05025). After 20 min at 20°C, the DNAase-containing solution was removed and the cells were gently washed once with 100 Ixl PBS. The cultures were then either immediately analysed or stored at -80°C until needed. Cell lysis and determination of DNA content DNA was released from the cells by alkaline lysis in 140 ixl basic EDTA solution (10 mM EDTA, adjusted to pH 12.3 with 5 M NaOH immediately prior to use) for 30 min at 37°C with moderate shaking.27 The lysates were transferred to 96-well black fluoroplates (Labsystems) using an Octapette, cooled on ice for 5-10 min and neutralized with about 10 ixl 1 M KH2PO4. Salmon testis DNA (Sigma), 0.05-0.50 txg/well added to neutralization buffer, was used as calibration standard. After a further 5-10 min on ice, the fluoroplates were analysed within 1 hr. Finally, 50 Ixl of Tris-NaC1 buffer (10 mM Tris-HC1, 100 mM NaC1, pH 7.0) containing 1.0 i~g/ml Hoechst 33258 (Sigma) were added to each well and fluorescence allowed to develop for 5 min. The fluorescence yield was measured at an excitation wavelength of 355 nm and an emission wavelength of 460 nm using a Fluoroscan II automatic microplate reader (Labsystems). Intravital staining of cultures with fluorescein diacetate and propidium iodide Standard fluorescein diacetate (FDA) and propidium iodide (PI) staining methods were adapted for estimation of cell viability in a simultaneous staining procedure, as described previously.15 After removing the culture medium, the cell monolayers were washed once with PBS and incubated for 3 min at 37°C with 50 Ixl PBS containing 15 ixg/ml FDA (Sigma) and 4.6 ixg/ml PI (Sigma). The monolayers were then washed once in PBS and examined with an inverted IMT2 fluorescence microscope equipped with a high pressure mercury burner. The combined use of a band pass 405 nm exciter filter, 455 nm dichroic mirror and 455 nm plus 515 nm barrier filters permitted the excitations of both the green and red dye and simultaneous observations of the red fluorescence from dead cells and the green fluorescence from viable cells. Photomicrographs were taken of two different areas in each micro-well and cells counted in five randomly distributed fields on each micrograph that together comprised 0.7% of the micro-well area, Biochemical parameters" NADPH tetrazolium reductase activity in unfixed cells was determined as described by Hope et aL 14 for NADPH-diaphorase, with modifications. Culture medium was removed from the
Survival of mouse cerebellar granule cells and potassium
453
micro-wells using an Octapette and the cell monolayers were gently washed once with 100 lxl PBS, pH 7.3. The cells were then incubated with 50 ixl Tris-HCl (50 mM, pH 8.0) containing 0.5 mM nitroblue tetrazolium (Sigma) and 1.0 mM N A D P H (Boehringer Mannheim) for 90 min at 37°C while shaking moderately. After washing twice with 100 Ixl PBS, the cells were disrupted and the formazan was dissolved in 200 ~1 alkaline dimethyl formamide, as described (Ref. 1; 1 part 0.2 M NaCI, 1 part 0.2 M glycine, 38 parts 0.5 M NaOH and 360 parts dimethyl formamide), and the concentration of formazan measured immediately using a TIM-200 automatic micro-well reader (Teknunc) at an absorbance wavelength of 415 nm and a reference wavelength of 600 nm. With respect to N A D P H concentration, the tetrazolium reductase activity followed Michaelis-Menten kinetics with a Km value of 1.0 mM. FDA esterase activity was estimated as described by Didier et al. 11 Briefly, cultures were incubated with F D A (15 ixg/ml) for 5 min and cells lysed in Tris-HCl (5 mM, pH 7.4) containing 1% sodium dodecyl sulphate. The lysates were transferred to 96-well black fluoroplates and fluoroscence measured using a Fluoroscan II automatic microplate reader (Labsystems) at an excitation wavelength of 485 nm and an emission wavelength of 538 nm. 2-Deoxy-D-glucose (DOG) accumulation was determined as described (JCrgensen, in preparation and Ref. 16). Briefly, cultures were exposed for 15 min at 20°C to 50 p~l [3H]DOG (NET-328, 5.4 txCi/ml, specific activity 8.00 Ci/mmol; DuPont/NEN) in balanced salt solution containing glutamine and pyruvate, pH 7.4. Cultures were washed three times at 2 min intervals and the 3H content of the cells determined by liquid scintillation counting. N M D A receptor expression The functional expression of NMDA receptors was assessed by estimating NMDA-induced
45Ca2+-influx, as described by Resink et al. 22 Briefly, cultures, plated in 35 mm dishes were washed twice with Mg2+-free buffered Locke's solution (154 mM NaC1, 5.6 mM KCI, 3.6 mM NaHCO3, 1.3 mM CaCI2, 5.6 mM D-glucose, 5 mM HEPES, pH 7.4) and then pre-incubated for 30 rain in the same solution at 35°C in 5% CO2 in air. The pre-incubation solution was replaced by the Locke's solution containing 1 p~Ci 45CAC12 (specificity activity 10-40 mCi/mg, Amersham, U.K.) and the cells incubated for 2 min at 20°C. Basal 45Ca2+ influx was determined in the presence of 1 ixM MK-801 (MK-801 hydrogen maleate, RBI, MA, U.S.A.). Responses to NMDA were elicited at 100 IxM (saturating concentration) in the presence of 10 IxM glycine. The reaction was stopped by washing the cells three times within 30 sec with 1 ml of a solution containing 154 mM choline chloride, 2 mM E G T A and 10 mM HEPES, pH 7.4. Finally, the cells were solubilized in i ml 0.1 M NaOH; half of the sample was used for 45Ca2+ counting and another aliquot for protein determination. 2° Statistical analysis Statistical analysis was conducted by one-way A N O V A followed by the Newman-Keuls test for multiple comparisons of means. RESULTS Modification of D N A estimation for cultures containing degenerating cells Because D N A of degenerating cells was not always broken down rapidly and D N A from the dead cells might stick to the poly-lysine coat of the micro-wells, the measurements of DNA content by Hoechst 33258-binding 27 could not be used without modification. Thus, D N A estimates were unchanged even when numerous dead cells could be detected by intravital staining with FDA/PI, for example after exposure to 5-10 p~M glutamate (Fig. 1). Furthermore, large red fluorescence spots indicating a diffuse halo of DNA around dead cells were often observed. In order to overcome the problem of D N A not associated with viable cells, the cultures were pre-treated with DNAase. D N A in healthy cells is not accessible to the extracellular enzyme, as also indicated by the observation here that DNAase treatment of cultures had no significant effect on either the phase-contrast appearance of the cells or the estimated D N A content (see Fig. 1, no added glutamate). Labarca and Paigen 18 have shown that the binding of Hoechst 33258 to DNA is
454
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Fig. 1. Effect of DNAase treatment on glutamate-treated mouse granule cell cultures that were nol supplemented with extra glucose. Glutamate was applied at 8 DIV at concentrations of up to 100 I~M and the number of surviving cells determined 24 hr later by the Hoechst 33258 binding assay with and without pre-treatment with DNAase (0.32 mg/ml for 20 min). Fluorescence yield is shown as a percentage of that in control cultures not pre-treated with DNAase arbitrarily normalized to 100. Closed circles represent cultures pre-treated with balanced salt solution only. Open circles, connected by the broken line, represent cultures pre-treated with DNAase in balanced salt solution. Values are means_+S.E.M. of replicates of 12 micro-well cultures obtained from two different cell preparations. DNAase pre-treatment clearly decreased fluorescence at the moderately toxic levels of glutamate (1-20 p.M).
considerably reduced by treating D N A with DNAase. We confirmed this finding: 20 min incubation of salmon testis D N A with 0.32 mg/ml DNAase halved the fluorescence yield obtained with Hoechst 33258 (Table 1). The importance of the DNAase pre-treatment for obtaining a valid estimate of live cells is indicated in Fig. 1, where granule cell response to glutamate was studied at 8 days in vitro (DIV). Granule cells become vulnerable to glutamate by about 8 DIV (Resink A. and Balfizs R., unpublished observations). However, monitoring the effect of 24 hr glutamate exposure by Hoechst 33258-binding, the extent of the toxic influence at low glutamate concentrations became only evident after the DNAase pre-treatment, which resulted in estimates in agreement with those obtained by counting viable cells with phase-contrast microscopy and after FDA/PI staining. In pilot studies, granule cell cultures were treated with various concentrations of DNAase at 37 and 20°C for various lengths of time. It was established that pre-treatment of the cultures with 0.32 mg/ml DNAase for 20 min at 20°C is the optimal procedure which resulted in the breakdown of 87% of a total of 0.14 ~g DNA derived from dead and lysed cells of a 12-day-old culture grown in the absence of glucose supplements (Table 1 ). Lower concentrations of DNAase were less effective; 0.03 mg DNAase/ml and 0.1 mg DNAase/ml reduced the fluorescence yield by only 39% and 63%, respectively (not shown). Table 1. The effect of DNAase pre-treatment on the fluorescence yield of the Hoechst 33258 DNA binding assay 0.14 p~gDNA (lysed granule cells) DNAase (%) 100_+15
+ DNAase (%) 13_+1
0.32 ~ g D N A (salmon testis) -
DNAase
(%) 100_+6
+
DNAase (%) 55_+5
The Hoechst 33258 fluorescence yield o b t a i n e d following DNAase pre-treatment (0.32 mg/ml for 20 min) is expressed as a percentage of that obtained without pre-treatment. Granule cell DNA was derived from dead and lysed cells of a 12-day-old culture which did not receive glucose s u p p l e m e n t a t i o n after 6 DIV; c o m p l e t e degeneration of the culture was confirmed by phasecontrast microscopy. Purified salmon testis DNA (Sigma) is shown for comparison.
Survival of mouse cerebellar granule cells and potassium 120
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Fig. 2. Survival of rat (A) and m o u s e (B) granule cells after 7 days growth in media containing 5, 10 or 25 m M K ÷. Cell survival was assessed using the Hoechst 33258 binding assay after D N A a s e pre-treatment. Data were normalized with respect to K25 cultures. Rat values are from one experiment performed in replicates of 12 micro-wells. Mouse values are from four different experiments, each performed in replicates of 10 micro-wells. M e a n s _ S . E . M . are shown. W h e r e a s the survival of rat granule cells was significantly enhanced when grown in 25 m M K +, that of m o u s e granule cells was not. Statistically significant differences from 25 m M K +. cultures are indicated: ***P<0.001.
The effect of DNAase treatment was not influenced by the simultaneous additiofi of trypsin inhibitor (0.5 mg/ml soy bean trypsin inhibitor; Sigma). The micro-well DNA assay using Hoechst 33258 was linear from 0.05 Ixg (detection limit) to more than 0.5 p~g D N A (not shown). Estimation of viable cell numbers in rat and mouse granule cells cultures
Figure 2 shows the influence of medium K + concentration on the number of DNAase-resistant and viable cells in rat and mouse granule cell cultures grown simultaneously under identical conditions for 7 days. The results are expressed as a percentage of the DNA content (i.e. cell number) in the mouse and rat K25 cultures. In rat cultures, only a fraction of the cells recovered in the K25 medium survived in the presence of"low" K + (about 60%, P<0.001; Fig. 2A). In contrast, in mouse cultures, granule cell survival was only slightly depressed in "low" K + media compared to "high" K + (Fig 2B). The depression in survival ranged between 0 and 15% and was occasionally statistically significant depending on the culture batch. The effect of plating density on thesurvival of mouse and rat granule cells was studied in K5 and K25 cultures (Table 2). There was no significant difference in the survival of mouse granule cells in the K5 and K25 media at low (1200 cells/mm 2) and normal (1500-2500 cells/mm 2) plating densities, whereas at high density (3500 cells/m2) survival was apparently slightly better in K5 than in K25 medium (P<0.05). In contrast, the survival of rat granule cells was compromised in the K5 compared with the K25 medium, irrespective of the plating densities studied. Thus, the survival requirement for elevated K + concentration shown by rat granule cells was not dependent on the plating density, the survival of granule cells in the K25 medium always being about twice that in the K5 medium. Table 2. T h e effects of elevated culture m e d i u m K + concentration on cell survival in 7-day-old rat and m o u s e granule cell cultures plated at different densities N u m b e r of cells plated/mm2: Medium: M o u s e (% survival) Rat (% survival)
1200
1500-2500
K5
K25
82±3 23-+2 ***
90±3 51---4
K5 81___3 22_+1 ***
K25 81-+3 48_+2
3500 K5 87-+3 * 22___1"**
K25 73±4 52±2
The cultures of m o u s e and rat cerebellar granule cells were grown in parallel in a m e d i u m containing either 5 m M or 25 m M K ÷ and cell survival (as a percentage of plated cells) determined after 7 D I V using the Hoechst 33258 binding assay after D N A a s e pre-treatment. Values are m e a n s ± S . E . M . Statistically significant differences between K5 and K25 are indicated: *P<0.05; ***P<0.001; n =25-68.
H.S. Mogensen et ,1.
45~
Table 2 also shows that mouse granule cells survivcd better than rat neurons during the 7-dax cultivation period.
Intravital staining o f mouse granule cell cultures Phase-contrast and fluorescence micrographs of K5 and K25 m o u s e granule cell cultures at 7 D I V are shown in Fig. 3. T h e m o r p h o l o g y of the two cultures was very similar with respect to the extent of neurite formation, but there was s o m e w h a t m o r e cell aggregation in the K5 cultures: however, the aggregation was never as p r o n o u n c e d as that usually seen in the rat cultures, lntravital staining with F D A / P I revealed that very few dead cells were present, as d e m o n s t r a t e d by the low n u m b e r of PI-stained red cells (Fig. 3, arrows). The majority of cells were viable, as indicated by the a b u n d a n c e of cells showing green fluorescence (an indication of F D A hydrolysis by intact cells). T h e n u m b e r of viable cells were c o m p a r e d in K5 and K25 m o u s e and rat granule cell cultures which were grown simultaneously (Table 3). Cell counting and D N A m e a s u r e m e n t gave c o m p a r a b l e estimates of viable cell numbers, thus validating the use of the modification of the H o e c h s t 33258 binding assay and d e m o n s t r a t i n g the methodological i n d e p e n d e n c e of the findings. Results of cell counting also c o n f i r m e d the observations r e p o r t e d in Table 2 using D N A estimation that the m e d i u m K + concentration does not significantly influence the survival of the m o u s e cells and that u n d e r the present culture conditions m o u s e granule cells survive better than rat cells.
Phase
Green
Red
K25
K5
Fig. 3. Phase-contrast and fluorescence micrographs of mouse granule cells grown for 7 days in medium containing 5 or 25 mM K +. Cells were intravitally stained by FDA/PI and examined by phase-contrast and fluorescence microscopy. The fluorescence micrographs were digitized and colour-separated to demonstrate viable cells (green window) and dead cells (red window). Dead cells stained red by propidium iodide. marked by arrows, gave almost no colour in the green window, whereas intense green cells also showed some colour in the red window. Horizontal bar=100 ixm. There was no evidence of any difference in survival in K5 and K25 cultures.
Survival of mouse cerebellar granule cells and potassium
457
Table 3. Cell survival determined by intravital staining with F D A / P I Cell n u m b e r Mouse
K5 K25
92_+9 100_+ 13
Rat
K5 K25
45_+5*** 74_+6**
Cultures of m o u s e and rat cerebellar granule cells were grown in parallel for 7 days in m e d i u m containing 5 m M or 25 m M K +. Cell n u m b e r is the average n u m b e r of viable cells f l u o r e s c i n g green in cultures incubated with F D A (n=10 microwells). Results are n o r m a l i z e d with r e s p e c t to m o u s e c u l t u r e s g r o w n in 25 m M K +. Significant differences in cell n u m b e r relative to m o u s e K25 cultures are indicated: **P<0.01; ***P<0.001. Table 4. Effects of K + concentration in the culture m e d i u m on biochemical parameters in m o u s e and rat cerebellar granule cell micro-well cultures Mouse
Rat
K5 N A D P H tetrazolium reductase (absorbance units) F D A esterase activity (fluorescence units) D O G (c.p.m.) Cell n u m b e r
K25
0.18_+0.02 ***
0.32_+0.01
21 _+1 *** 5900_+470** 55,100_+2300
34 +_2 8520+_750 54,800_+2300
K5 0.03_+0.01"** -1080_+75 *** 15,275_+950"***
K25 0.13+_0.01 -2640_+150 32,850_+1500
G r a n u l e cells were cultured for 7 days in m e d i u m containing 5 m M or 25 m M K + (plating density: 2000 cells/mm 2, i.e. 68,000 cells/well corresponding to 0.43 ~g D N A ) . Results are given in arbitrary units. For N A D P H tetrazolium reductase activity n = 1 0 - 3 2 , for F D A esterase n = 9 , for D O G accumulation n = 2 2 - 2 4 , and for cell n u m b e r (determined in micro-well cultures using the Hoecbst 33258 binding assay after pre-treatment with D N A a s e ) n=36-67. Values are means-+S.E.M. Statistically significant differences between K5 and K25 are indicated: **P<0.01; ***P<0.001; ****P<0.0001.
Granule cells and the extracellular K + concentration Although elevation of medium K + concentration did not influence the survival of mouse granule cells, it did influence the biochemical differentiation of these cells. Thus, at 7 DIV the rate of nitroblue tetrazolium reduction in K25 cultures was nearly double the value in K5 cultures, and significant differences were also detected in FDA esterase activity and DOG accumulation (Table 4). "High" K + also increased the value of these estimates in rat granule cell cultures. Even when corrected for the low number of cells in rat K5 cultures, the rate of tetrazolium reduction was significantly increased by a factor of three in "high" K +. The functional expression of NMDA receptors in mouse granule cells was measured as NMDA-evoked increase in 45Ca2+ influx under MgZ+-free conditions in order to avoid the Mg 2+ blockade of the NMDA receptor-linked channel and in the presence of 10 I~M glycine to ensure full activation of the receptor (see Ref. 22). NMDA receptor activity was approximately three-fold greater in mouse granule cells cultured in K25 than in K5 medium (Table 5).
DISCUSSION Quantitative estimation of viable cells in micro-well cultures DNAase pre-treatment of micro-well cultures resulted in the hydrolysis of DNA originating from degenerating cells. Since the enzyme does not penetrate membranes, it does not attack DNA contained in the viable cells. DNAase pre-treatment, as performed in this study, appeared to be
458
H.S. Mogensen et al. F a b l e 5. Effect of m e d i u m K ~ c o n c e n t r a t i o n on the N M D A r e c e p t o r activationi n d u c e d 45Cae ~ influx in m o u s e c e r e b e l l a r g r a n u l e cell c u l t u r e s K5 B a s a l (1 m M MK-801 ) 100 m M N M D A + 10 ~tM glycine N M D A - i n d u c e d 45Ca 2 + u p t a k e
K25
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NMDA receptor activitywas determined in granule cell cultures at 7 D1V, as the NMDA-evoked increase, in an Mg2+-free medium, in 45Ca2+ influx (nmol 45Ca2+ influx/mgprotein). Values are means-+S.E.M. (n=3 dishes). harmless to matrix-attached, intact granule cells at 7 DIV, even when the exposure was prolonged for up to 3 hr. However, the treatment dramatically decreased the fluorescence of Hoechst 33258 in cultures in which cells were forced to degenerate. The D N A content of viable cells could therefore be estimated in the micro-well cultures using the Hoechst 33258 fluorochrome binding method. 27 This method is based on the fact that the fluorescence yield of Hoechst 33258 at 450 nm is greatly enhanced upon binding to DNA; the enhancement increases linearly with the amount of DNA, and R N A does not interfere with the determination of DNA. 10,18,19 The effect o f elevated K + concentration on cell s u r v i v a l in rat a n d m o u s e granule cell cultures
After working for some time with rat cerebellar granule cell cultures, we were at first surprised to find that the mouse cells do not degenerate when grown in a serum-containing medium in the presence o f " l o w " K +. In order to exclude experimental variables, granule cells from rat and mouse cerebella were prepared, cultured and analysed simultaneously and under identical conditions. The observations confirmed the report of Peng et al. 21 and our initial findings by showing that mouse cerebellar granule cells have no survival requirement for K+-induced membrane depolarization. In contrast, the survival of the parallel grown rat granule cells was compromised, unless the culture medium contained "high" K +, as reported earlier (e.g. Ref. 13). The present findings therefore permit the exclusion of methodological differences, considered by Peng et al. 21 in connection with the discrepancy in the K + dependence of their mouse granule cell cultures and our rat cultures. Thus, we observed that different cell densities, suggested by Peng et al. as possibly the most important procedural difference, do not influence either the lack of K + dependence of the mouse cells or the survival requirement of the rat cells for "high" K +. Furthermore, we cultured the granule cells from the two species in the same medium, which contained fetal calf serum. Astrocytes are known to support the survival and maturation of nerve cells in culture. For example, in aggregated cell cultures of rat telencephalon, an effect of "high" K + on neuronal maturation was evident only after the depletion of glial cells. 23 The presence of a relatively high proportion of astrocytes in the mouse cultures might therefore have accounted for both the better survival of the mouse neurons and their lack of survival requirement for elevated K + concentration. Accordingly, we assessed astrocyte contribution to the cultures by estimating glial fibrillary acidic protein using E L I S A (data not shown). The amount of glial fibrillary acidic protein-positive material was low in both cultures, but it was 30-40% higher in the rat than in the mouse granule cell cultures, thus rendering the possibility that astrocytes might have accounted for the differences rather unlikely. We conclude, therefore, that the different survival requirements are genuine properties of the mouse and rat granule cells. These findings are apparently at variance with the report of Didier et al., 11 who assessed mouse granule cell survival by measuring fluorescein production from FDA. These authors considered that "high" K + promoted neuronal survival since it increased the conversion of F D A to fluorescein. However, we observed that after F D A loading the fluorescence of individual cells in K25 cultures was brighter compared with K5 cells. Peng et al. 21 have also commented on the paler fluorescence of cells in the "low" than in the "high" K+-containing cultures. Measurements of the fluorescein produced from F D A in the cells, together with the estimations of cell numbers in the K5 and K25 cultures, supported the microscopic impression and showed that fluorescein production/cell in K5 cultures was only 65% of that in K25.
Survival of mouse cerebellar granule cells and potassium
459
Effect of "high" K + on the differentiation of mouse granule cells It seems that cultivation in "high" K+-containing medium is not essential for the survival of mouse cerebellar granule cells. However, "high" K + has a powerful effect on the biochemical differentiation and the expression of NMDA receptors in these cells. All the biochemical parameters measured in this study showed higher values in the K25 than the K5 cultures. Thus, activities of NADPH tetrazolium reductase and FDA esterase, and rate of DOG accumulation were about 50% higher in K25 than in K5. Under the experimental conditions, the NADPH-diaphorase activity of nitric oxide synthase contributes to formazan formation 14 and this enzyme is greatly enriched in cerebellar granule cells. 25 The effect of chronic membrane depolarization was even more pronounced on the expression of phenotypically characteristic properties, compared with the above-mentioned household functions. The functional expression of the NMDA receptors was about three-fold higher in K25 than in K5 cultures, and in this respect the behaviour of mouse cells was comparable with rat granule cells. 8,22 It has been observed previously that stimulus-coupled glutamate release develops with the maturation of rat granule cells. 12 Comparable findings have been reported by Peng et al. 21 for mouse cells grown in K25 medium, but these authors failed to detect K+-evoked glutamate release in cultures grown in "low" K + medium. In this respect, the retarded maturation of mouse granule cells in "low" K + medium is even more pronounced than observed for rat cells in "low" K+. 13 Mouse granule cells are not unique among neurons in culture in exhibiting good survival in "low" K + medium. Most notably, hippocampal neurons 9 and GABAergic cerebral interneurons24 are routinely cultivated in "low" K + medium. Moreover, even rat granule cells fail to develop survival requirements for "high" K + under certain conditions. Cells grown in serum-flee, chemically defined medium survive as well in medium containing 5 mM K + as 25 mM K+. 17 The maturation of the serum-free, "low" K + cells is, however, markedly retarded compared with cells grown in serum-containing medium with "high" K + and it was proposed that serum-derived factors promote the maturation of rat granule cells to a stage at which survival becomes dependent on external stimulation, such as K +-induced membrane depolarization.4 Cultivation in "high" K + promotes the differentiation and the maturation of the rat granule cells grown in serum-free medium.3,4 The morphology of our serum-containing mouse cultures resembles that of serum-flee rat cultures expressing only minor neuronal aggregation, 17 thus indicating a degree of immaturity of the mouse granule cells. The present work shows that the biochemical differentiation and the maturation of mouse cerebellar granule cells is also promoted by cultivation in "high" K + containing medium, as indicated by increased FDA esterase activity, enhanced rate of NADPH-dependent tetrazolium reduction, augmented DOG accumulation and increased NMDA-evoked 45Ca2+ influx. During their development, both in vivo and in culture, nerve cells go through stages in differentiation and maturation in which their survival depends on the presence of various environmental factors.2 It seems that one such factor for cerebellar granule cells is K+-induced membrane depolarization, which in rat cells promotes both survival and differentiation, whereas in mouse cells it primarily promotes differentiation. The reason for this species-dependent difference in properties is not yet known. We are proposing here that mouse granule cells in culture are insensitive to the serum-derived factor(s) which promotes the differentiation and maturation of rat granule cells and therefore retain a less differentiated state in which their survival is not dependent on K+-induced membrane depolarization. Acknowledgements--This research was supported by grants from The Danish Medical Research Council, Ivan Nielsens Fond and the Royal Netherlands Academy of Science (KNAW). We are very grateful to Professor Niels Anker Thorn and Technical Assistant Birthe Linnerup Kristensen, Institute of Medical Physiology, University of Copenhagen, for access to and help with the Fluoroscan 11 instrument.
REFERENCES 1. Altman F. P. (1972) An Introduction to the Use o f Tetrazolium Salts in Quantitative Enzyme Cytochemistry, pp. 1-45. Koch-Light Laboratories Ltd, Colnbrook, Bucks, U.K. 2. Altman J. (1992) Programmed cell death: the paths to suicide. Trends Neurosci. 15, 278-280. 3. Bal~izs R., Gallo V., Kingsbury A., Thangnipon W., Smith R., Atterwill C. and Woodhams P. (1987) Factors affecting
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4. 5. 6. 7. 8.
9. 10. 11.
12. 13. 14. 15. 16.
17. 18. 19. 20. 21.
22. 23. 24. 25. 26. 27.
H . S . M o g e n s e n et al. the survival and maturation of nerve cells in culture. In GliaI-Neuronal Communicatum and Regeneration (eds Althous H. H. and Seifert W.), pp. 285-302. Springer, Berlin. Bal~izs R., Gallo V. and Kingsbury A. (1988) Effect of depolarization on the maturation of cerebellar granule cells ill culture. Brain Res. 468, 26%276. Bal~izs R., J0rgensen O. S. and Hack N. (1988) N-Methyt-D-aspartate promotes the survival of cerebellar granule cells in culture. Neuroscience 27, 437-451. Bal~izs R., Hack N., Jc~rgensen O. S. and Cotman C. W. (1989) N-Methyl-D-aspartate promotes the survival of cerebellar granule cells: pharmacological characterization. Neurosci. Lett. 10L 241-246. Bal~izs R., Hack N. and J0rgensen O. S. (1990) Selective stimulations of excitatory amino acid receptor subtypes and the survival of cerebellar granule cells in culture: effect of kainic acid. Neuroscience 37, 251-258. Bal~izs R.. Resink A., Hack N., Van der Valk J. B., Kumar K. N. and Michaelis E. (1992) N M D A treatment and K +-induced depolarization selectively promote the expression of an NMDA-preferring class of ionotropic glutamate receptors in cerebellar granule cells. Neurosci. Lett. 137, 109-113. Banker G. A. and Cowan W. M. (1979) Further observations on hippocampal neurons in dispersed cell culture. ,L comp. NeuroL 187, 469494. Cesarone C. F., Bolognesi C. and Santi L. (1979) Improved microfluoremetric DNA determination in biological material using 33258 Hoechst. Analyc Biochem. 100, 188-197. Didier M., Heaulme M., Soubrie P., Bockaert J. and Pin J.-P. (1990) Rapid, sensitive, and simple method for quantification of both neurotoxic and neurotrophic effects of N M D A on cultured cerebellar granule cells. J. Neurosci. Res. 27, 25-35. Gallo V., Ciotti M. T., Colletti A., Alosi F. and Levi G. (1982) Selective release of glutamate from cerebellar granule cells differentiating in culture. Proc. nam. Acad. Sci. U.S.A. 79, 7919--7923. Gal[o V., Kingsbury A., Bal~izs R. and J0rgensen O. S. (1987) The role of depolarization in the surviwd and differentiation of cerebellar granule cells in culture. J. Neurosci. 7, 220,3-2213. Hope T. B., Michael G. J., Knigge K. M. and Vincent S. R. ( 1991 ) Neuronal N A D P H diaphorase is a nitric oxide synth ase. Proc. natn. Acad. Sci. U.S.A. 88, 2811-2814. Jones K. H. and Senti J. A. (1985) An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium oidide. J. Histochem. Cvtochem. 33, 77-79. Jcrgcnsen O. S. and Bal~izs R. (1993) Plastic neuronal changes in Alzheimer's disease associated with activation of astrocytes and enhanced neurotrophic activity. In Alzheimer's Disease: Advances m Clinical and Basic Research (eds Corain B., Iqbal K., Nicolini M., Winblad B., Wisniewski H. and Zatta P.), pp. 18%198. John Wiley, New York. Kingsbury A., Gallo V., Woodhams P. L. and Balgzs R. (1985) Survival, morphology and adhesion properties of cerebellar interneurones cultured in chemically defined and serum-supplemented medium. Devl Brain Res. 17, 17-25. Labarca C. and Paigen K. (1980) A simple, rapid, and sensitive D N A assay procedure. Analyt Biochem. 102, 344-352. Latt S. A. (1973) Microfluoremetric detection of deoxyribonucleic acid replication in human metaphase chromosomes. Proc. natn. Acad. Sci. U.S.A. 70, 3395 3399. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. ( 1951 ) Protein measurements with the Folin phenol reagenl. ,I. biol. Chem. 193, 265-275. Peng L., Juurlink B. H. J. and Hertz L. (1991) Difference in transmitter release, morphology, and ischemia-induced cell injury between cerebellar granule cell cultures developing in the presence of and in the absence of a depolarizing potassium concentration. Devl Brain Res. 63, 1-12. Resink A., Boer G. J. and Belfizs R. (1992) Treatment with excitatory amino acids or high K ~ and N M D A receptors in cerebellar granule cells. NeuroReport 3, 75%760. Riederer B. M., Monnet-Tschudi F. and Honegger P. (1992) Development and maintenance of the neuronal cytoskeleton in aggregated cell cultures of fetal telencephalon and influence of elevated K + concentration. J. Nearochem. 58, 649~558. Schousboe A., Drejer J., Hansen G. H. and Meier E. (1985) Cultured neurons as model systems for biochemical and pharmacological studies on receptors for neurotransmitter amino acids. Devl Neurosci. 7, 252-262. Southam E., East S. J. and Garthwaite J. (1992) Sources and targets of nitric oxide in the cerebellum. Neurosct. Lett. 137, 241-244. Thangnipon W., Kingsbury A., Webb M. and B a l ~ s R. (1983) Observations on rat cerebellar cells in vitro: influence on substratum, potassium concentration and relationship between neurons and astrocytes. Devl Brain Res. 11, 177-189. West D. C., Sattar A. and Kumar S. (1985) A simplified in situ solubilization procedure for the determination of DNA and cell number in tissue cultured mammalian cells. Analyt. Biochem. 147, 289-295.
Int. J. Devl Neuroscience, Vol. 12,No. 5, pp. 451-460, 1994
0736-5748(94)E0008-P
Elsevier ScienceLtd Copyright© 1994ISDN Printed in Great Britain. All rightsreserved 0736-5748/94$7.00+0.00
THE SURVIVAL OF C U L T U R E D MOUSE C E R E B E L L A R G R A N U L E CELLS IS NOT D E P E N D E N T ON E L E V A T E D POTASSIUM-ION CONCENTRATION HELLE S. MOGENSEN*~, NICOLA HACK~:, ROBERT BALAZS:~ and OLE STEEN JORGENSENt tLaboratory of Neuropsychiatry, Department of Pharmacology, University of Copenhagen, DK-2100 Copenhagen, Denmark :~Netherlands Institute for Brain Research, Amsterdam, The Netherlands (Received 7 October 1993; in revised form 7 January 1994; accepted 10 January 1994)
Abstract--The effects of K+-induced membrane depolarization were studied on the survival and biochemical parameters in mouse and rat cerebellar granule cells grown in micro-well cultures. Cell numbers were determined by estimating DNA content using the Hoechst 33258 fluorochrome binding assay. DNA from degenerated cells was removed by prior DNAase treatment. These DNA estimates of cell numbers were comparable with values obtained by direct counting of fluorescein diacetate-stalned viable cells. In agreement with previous studies, the survival of rat granule cells was promoted by increasing the concentration of K + in the medium from 5 to 25 mM throughout a 7-day culture period. In contrast, mouse granule cells survived in culture containing 'low' K + (5 or 10 mM), as well as in the presence of 'high' K + (25 mM). On the other hand, several biochemical parameters in mouse granule cells were markedly increased by cultivation in 'high' as compared with 'low' K +-containing media, demonstrated by increased fluorescein diacetate esterase activity, enhanced rate of NADPH-dependent tetrazolium reduction, augmented 2-deoxy-D-glucose accumulation and increased N-methyl-D-aspartate-evoked 45Ca2+ influx. It was concluded that although cultivation in 'high' K + promotes biochemical differentiation in mouse cerebellar granule cells, these cells differ from their rat counterparts in that they do not develop a survival requirement for K+-induced membrane depolarization. Key words: mouse, rat, cerebellar granule cells, K + concentration, maturation, survival.
W h e n g r o w n in culture, rat cerebellar granule cells develop b e t w e e n the s e c o n d and f o u r t h days in vitro, a survival r e q u i r e m e n t that can be satisfied either by elevating the m e d i u m K + c o n c e n t r a t i o n f r o m the n o r m a l 5 m M to > 2 0 m M 12 or by treating the cells with glutamate receptor agonists, N-methyl-D-aspartate ( N M D A ) being particularly effective. 5-7 If these r e q u i r e m e n t s are not met, a significant p r o p o r t i o n o f the cells g r o w n in serum-containing m e d i u m with ' l o w ' K + c o n c e n t r a t i o n (5 and 10 m M ) d e g e n e r a t e rather abruptly towards the e n d o f the first w e e k in culture. W e have suggested that the survival-promoting effect o f either 'high' K + c o n c e n t r a t i o n (25 mM; K25) or excitatory a m i n o acid t r e a t m e n t in vitro mimics the influence of the initial innervation received in v i v o o f the immediately p o s t - m i g r a t o r y granule cells by glutamatergic m o s s y fibre afferents. 5,13 In contrast to the observations in rat cells, P e n g et al. 21 have f o u n d that granule cells f r o m m o u s e cerebellum cultivated in m e d i u m containing ' l o w ' K + survive as well as in the presence of 'high' K +. Thus, with respect to survival r e q u i r e m e n t , m o u s e granule cells b e h a v e d in a serum-containing m e d i u m as rat granule cells did in serum-free, chemically defined m e d i u m . 17 P e n g et al. 21 explained the difference in the survival r e q u i r e m e n t of granule cells o b s e r v e d in their cultures c o m p a r e d with o t h e r studies (rat cells) by differences in plating density and the composition o f the culture m e d i u m . In o r d e r to characterize the differences better we studied m o u s e and rat granule cells g r o w n in s e r u m - c o n t a i n i n g m e d i u m u n d e r identical culture conditions. F o r estimating cell survival quantitatively, we modified the H o e c h s t 33258 f l u o r o c h r o m e binding m e t h o d 27 such that the n u m b e r of viable cells could be d e t e r m i n e d despite the p r e s e n c e of D N A fragments f r o m d e a d cells. W e f o u n d that, in contrast to rat granule cells, ' h i g h ' K + is n o t a survival *Author to whom correspondence should be addressed. Abbreviations: DIV, days in vitro; DOG, 2-deoxy-D-glucose;EDTA, ethylenediaminetetra-acetate; EGTA, ethylenegly-
coltetra-acetate; FDA, fluorescein diacetate; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid; MK-801, (+)-5-methyl-10.11-dihydro-5H-dibenzo-[a,d]cyclohepten-5,10-imine hydrogen maleate; NADPH, reduced nicotinamide adenine dinueleotide phosphate; NMDA, N-methyl-D-aspartate; PBS, phosphate-buffered saline; PI, propidium iodide. 451
452
H.S. Mogensen et a/
requirement for mouse granule cells. On the other hand, K t-induced membrane depolarization promotes the biochemical differentiation of both rat and mouse cerebellar granule cells in cultures. EXPERIMENTAL PROCEDURES Cell cultures Granule cell-enriched cultures were prepared from cerebella of 6-day-old mice and rats, as described previously.13.26 Briefly, cerebella were dissociated and cells were plated (in general 2.1 × 103 cells/ram2, 69 × 103 cells/well) in 96-well polystyrene micro-well culture plates (NUNC) that had been pre-coated with poly-L-lysine (10 txg/ml; Sigma). The culture medium consisted of 10% heat-inactivated fetal calf serum (Gibco) in basal Eagle's medium (Gibco) supplemented with 2 mM glutamine, and it contained 100 ixg/ml gentamicin. K + concentrations tested were: 5 mM (K5), 10 mM (K10) and 25 mM (K25). Cytosine arabinoside (10 IxM final concentration; Sigma) was added about 18 hr after seeding. Pre-treatment with DNAase For DNAase treatment, the culture medium was removed from the micro-wells using an eight-channel Octapette (Costar) and the cell monolayer gently washed once with 100 Ixl phosphate-buffered saline (PBS), pH 7.3. Cells were then covered with 50 ILl balanced salt solution (120 mM NaCI, 5 mM KCI, 1.0 mM CaC12, 0.5 mM NazHPO4, 5 mM glucose, 20 mM HEPES, 4.2 mM MgCI2, pH 7.4) supplemented with 0.3% (w/v) bovine serum albumin (fraction V; Sigma) and 0.32 mg DNAase/ml (EC 3.2.21.1; Sigma 05025). After 20 min at 20°C, the DNAase-containing solution was removed and the cells were gently washed once with 100 Ixl PBS. The cultures were then either immediately analysed or stored at -80°C until needed. Cell lysis and determination of DNA content DNA was released from the cells by alkaline lysis in 140 ixl basic EDTA solution (10 mM EDTA, adjusted to pH 12.3 with 5 M NaOH immediately prior to use) for 30 min at 37°C with moderate shaking.27 The lysates were transferred to 96-well black fluoroplates (Labsystems) using an Octapette, cooled on ice for 5-10 min and neutralized with about 10 ixl 1 M KH2PO4. Salmon testis DNA (Sigma), 0.05-0.50 txg/well added to neutralization buffer, was used as calibration standard. After a further 5-10 min on ice, the fluoroplates were analysed within 1 hr. Finally, 50 Ixl of Tris-NaC1 buffer (10 mM Tris-HC1, 100 mM NaC1, pH 7.0) containing 1.0 i~g/ml Hoechst 33258 (Sigma) were added to each well and fluorescence allowed to develop for 5 min. The fluorescence yield was measured at an excitation wavelength of 355 nm and an emission wavelength of 460 nm using a Fluoroscan II automatic microplate reader (Labsystems). Intravital staining of cultures with fluorescein diacetate and propidium iodide Standard fluorescein diacetate (FDA) and propidium iodide (PI) staining methods were adapted for estimation of cell viability in a simultaneous staining procedure, as described previously.15 After removing the culture medium, the cell monolayers were washed once with PBS and incubated for 3 min at 37°C with 50 Ixl PBS containing 15 ixg/ml FDA (Sigma) and 4.6 ixg/ml PI (Sigma). The monolayers were then washed once in PBS and examined with an inverted IMT2 fluorescence microscope equipped with a high pressure mercury burner. The combined use of a band pass 405 nm exciter filter, 455 nm dichroic mirror and 455 nm plus 515 nm barrier filters permitted the excitations of both the green and red dye and simultaneous observations of the red fluorescence from dead cells and the green fluorescence from viable cells. Photomicrographs were taken of two different areas in each micro-well and cells counted in five randomly distributed fields on each micrograph that together comprised 0.7% of the micro-well area, Biochemical parameters" NADPH tetrazolium reductase activity in unfixed cells was determined as described by Hope et aL 14 for NADPH-diaphorase, with modifications. Culture medium was removed from the
Survival of mouse cerebellar granule cells and potassium
453
micro-wells using an Octapette and the cell monolayers were gently washed once with 100 lxl PBS, pH 7.3. The cells were then incubated with 50 ixl Tris-HCl (50 mM, pH 8.0) containing 0.5 mM nitroblue tetrazolium (Sigma) and 1.0 mM N A D P H (Boehringer Mannheim) for 90 min at 37°C while shaking moderately. After washing twice with 100 Ixl PBS, the cells were disrupted and the formazan was dissolved in 200 ~1 alkaline dimethyl formamide, as described (Ref. 1; 1 part 0.2 M NaCI, 1 part 0.2 M glycine, 38 parts 0.5 M NaOH and 360 parts dimethyl formamide), and the concentration of formazan measured immediately using a TIM-200 automatic micro-well reader (Teknunc) at an absorbance wavelength of 415 nm and a reference wavelength of 600 nm. With respect to N A D P H concentration, the tetrazolium reductase activity followed Michaelis-Menten kinetics with a Km value of 1.0 mM. FDA esterase activity was estimated as described by Didier et al. 11 Briefly, cultures were incubated with F D A (15 ixg/ml) for 5 min and cells lysed in Tris-HCl (5 mM, pH 7.4) containing 1% sodium dodecyl sulphate. The lysates were transferred to 96-well black fluoroplates and fluoroscence measured using a Fluoroscan II automatic microplate reader (Labsystems) at an excitation wavelength of 485 nm and an emission wavelength of 538 nm. 2-Deoxy-D-glucose (DOG) accumulation was determined as described (JCrgensen, in preparation and Ref. 16). Briefly, cultures were exposed for 15 min at 20°C to 50 p~l [3H]DOG (NET-328, 5.4 txCi/ml, specific activity 8.00 Ci/mmol; DuPont/NEN) in balanced salt solution containing glutamine and pyruvate, pH 7.4. Cultures were washed three times at 2 min intervals and the 3H content of the cells determined by liquid scintillation counting. N M D A receptor expression The functional expression of NMDA receptors was assessed by estimating NMDA-induced
45Ca2+-influx, as described by Resink et al. 22 Briefly, cultures, plated in 35 mm dishes were washed twice with Mg2+-free buffered Locke's solution (154 mM NaC1, 5.6 mM KCI, 3.6 mM NaHCO3, 1.3 mM CaCI2, 5.6 mM D-glucose, 5 mM HEPES, pH 7.4) and then pre-incubated for 30 rain in the same solution at 35°C in 5% CO2 in air. The pre-incubation solution was replaced by the Locke's solution containing 1 p~Ci 45CAC12 (specificity activity 10-40 mCi/mg, Amersham, U.K.) and the cells incubated for 2 min at 20°C. Basal 45Ca2+ influx was determined in the presence of 1 ixM MK-801 (MK-801 hydrogen maleate, RBI, MA, U.S.A.). Responses to NMDA were elicited at 100 IxM (saturating concentration) in the presence of 10 IxM glycine. The reaction was stopped by washing the cells three times within 30 sec with 1 ml of a solution containing 154 mM choline chloride, 2 mM E G T A and 10 mM HEPES, pH 7.4. Finally, the cells were solubilized in i ml 0.1 M NaOH; half of the sample was used for 45Ca2+ counting and another aliquot for protein determination. 2° Statistical analysis Statistical analysis was conducted by one-way A N O V A followed by the Newman-Keuls test for multiple comparisons of means. RESULTS Modification of D N A estimation for cultures containing degenerating cells Because D N A of degenerating cells was not always broken down rapidly and D N A from the dead cells might stick to the poly-lysine coat of the micro-wells, the measurements of DNA content by Hoechst 33258-binding 27 could not be used without modification. Thus, D N A estimates were unchanged even when numerous dead cells could be detected by intravital staining with FDA/PI, for example after exposure to 5-10 p~M glutamate (Fig. 1). Furthermore, large red fluorescence spots indicating a diffuse halo of DNA around dead cells were often observed. In order to overcome the problem of D N A not associated with viable cells, the cultures were pre-treated with DNAase. D N A in healthy cells is not accessible to the extracellular enzyme, as also indicated by the observation here that DNAase treatment of cultures had no significant effect on either the phase-contrast appearance of the cells or the estimated D N A content (see Fig. 1, no added glutamate). Labarca and Paigen 18 have shown that the binding of Hoechst 33258 to DNA is
454
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Fig. 1. Effect of DNAase treatment on glutamate-treated mouse granule cell cultures that were nol supplemented with extra glucose. Glutamate was applied at 8 DIV at concentrations of up to 100 I~M and the number of surviving cells determined 24 hr later by the Hoechst 33258 binding assay with and without pre-treatment with DNAase (0.32 mg/ml for 20 min). Fluorescence yield is shown as a percentage of that in control cultures not pre-treated with DNAase arbitrarily normalized to 100. Closed circles represent cultures pre-treated with balanced salt solution only. Open circles, connected by the broken line, represent cultures pre-treated with DNAase in balanced salt solution. Values are means_+S.E.M. of replicates of 12 micro-well cultures obtained from two different cell preparations. DNAase pre-treatment clearly decreased fluorescence at the moderately toxic levels of glutamate (1-20 p.M).
considerably reduced by treating D N A with DNAase. We confirmed this finding: 20 min incubation of salmon testis D N A with 0.32 mg/ml DNAase halved the fluorescence yield obtained with Hoechst 33258 (Table 1). The importance of the DNAase pre-treatment for obtaining a valid estimate of live cells is indicated in Fig. 1, where granule cell response to glutamate was studied at 8 days in vitro (DIV). Granule cells become vulnerable to glutamate by about 8 DIV (Resink A. and Balfizs R., unpublished observations). However, monitoring the effect of 24 hr glutamate exposure by Hoechst 33258-binding, the extent of the toxic influence at low glutamate concentrations became only evident after the DNAase pre-treatment, which resulted in estimates in agreement with those obtained by counting viable cells with phase-contrast microscopy and after FDA/PI staining. In pilot studies, granule cell cultures were treated with various concentrations of DNAase at 37 and 20°C for various lengths of time. It was established that pre-treatment of the cultures with 0.32 mg/ml DNAase for 20 min at 20°C is the optimal procedure which resulted in the breakdown of 87% of a total of 0.14 ~g DNA derived from dead and lysed cells of a 12-day-old culture grown in the absence of glucose supplements (Table 1 ). Lower concentrations of DNAase were less effective; 0.03 mg DNAase/ml and 0.1 mg DNAase/ml reduced the fluorescence yield by only 39% and 63%, respectively (not shown). Table 1. The effect of DNAase pre-treatment on the fluorescence yield of the Hoechst 33258 DNA binding assay 0.14 p~gDNA (lysed granule cells) DNAase (%) 100_+15
+ DNAase (%) 13_+1
0.32 ~ g D N A (salmon testis) -
DNAase
(%) 100_+6
+
DNAase (%) 55_+5
The Hoechst 33258 fluorescence yield o b t a i n e d following DNAase pre-treatment (0.32 mg/ml for 20 min) is expressed as a percentage of that obtained without pre-treatment. Granule cell DNA was derived from dead and lysed cells of a 12-day-old culture which did not receive glucose s u p p l e m e n t a t i o n after 6 DIV; c o m p l e t e degeneration of the culture was confirmed by phasecontrast microscopy. Purified salmon testis DNA (Sigma) is shown for comparison.
Survival of mouse cerebellar granule cells and potassium 120
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Fig. 2. Survival of rat (A) and m o u s e (B) granule cells after 7 days growth in media containing 5, 10 or 25 m M K ÷. Cell survival was assessed using the Hoechst 33258 binding assay after D N A a s e pre-treatment. Data were normalized with respect to K25 cultures. Rat values are from one experiment performed in replicates of 12 micro-wells. Mouse values are from four different experiments, each performed in replicates of 10 micro-wells. M e a n s _ S . E . M . are shown. W h e r e a s the survival of rat granule cells was significantly enhanced when grown in 25 m M K +, that of m o u s e granule cells was not. Statistically significant differences from 25 m M K +. cultures are indicated: ***P<0.001.
The effect of DNAase treatment was not influenced by the simultaneous additiofi of trypsin inhibitor (0.5 mg/ml soy bean trypsin inhibitor; Sigma). The micro-well DNA assay using Hoechst 33258 was linear from 0.05 Ixg (detection limit) to more than 0.5 p~g D N A (not shown). Estimation of viable cell numbers in rat and mouse granule cells cultures
Figure 2 shows the influence of medium K + concentration on the number of DNAase-resistant and viable cells in rat and mouse granule cell cultures grown simultaneously under identical conditions for 7 days. The results are expressed as a percentage of the DNA content (i.e. cell number) in the mouse and rat K25 cultures. In rat cultures, only a fraction of the cells recovered in the K25 medium survived in the presence of"low" K + (about 60%, P<0.001; Fig. 2A). In contrast, in mouse cultures, granule cell survival was only slightly depressed in "low" K + media compared to "high" K + (Fig 2B). The depression in survival ranged between 0 and 15% and was occasionally statistically significant depending on the culture batch. The effect of plating density on thesurvival of mouse and rat granule cells was studied in K5 and K25 cultures (Table 2). There was no significant difference in the survival of mouse granule cells in the K5 and K25 media at low (1200 cells/mm 2) and normal (1500-2500 cells/mm 2) plating densities, whereas at high density (3500 cells/m2) survival was apparently slightly better in K5 than in K25 medium (P<0.05). In contrast, the survival of rat granule cells was compromised in the K5 compared with the K25 medium, irrespective of the plating densities studied. Thus, the survival requirement for elevated K + concentration shown by rat granule cells was not dependent on the plating density, the survival of granule cells in the K25 medium always being about twice that in the K5 medium. Table 2. T h e effects of elevated culture m e d i u m K + concentration on cell survival in 7-day-old rat and m o u s e granule cell cultures plated at different densities N u m b e r of cells plated/mm2: Medium: M o u s e (% survival) Rat (% survival)
1200
1500-2500
K5
K25
82±3 23-+2 ***
90±3 51---4
K5 81___3 22_+1 ***
K25 81-+3 48_+2
3500 K5 87-+3 * 22___1"**
K25 73±4 52±2
The cultures of m o u s e and rat cerebellar granule cells were grown in parallel in a m e d i u m containing either 5 m M or 25 m M K ÷ and cell survival (as a percentage of plated cells) determined after 7 D I V using the Hoechst 33258 binding assay after D N A a s e pre-treatment. Values are m e a n s ± S . E . M . Statistically significant differences between K5 and K25 are indicated: *P<0.05; ***P<0.001; n =25-68.
H.S. Mogensen et ,1.
45~
Table 2 also shows that mouse granule cells survivcd better than rat neurons during the 7-dax cultivation period.
Intravital staining o f mouse granule cell cultures Phase-contrast and fluorescence micrographs of K5 and K25 m o u s e granule cell cultures at 7 D I V are shown in Fig. 3. T h e m o r p h o l o g y of the two cultures was very similar with respect to the extent of neurite formation, but there was s o m e w h a t m o r e cell aggregation in the K5 cultures: however, the aggregation was never as p r o n o u n c e d as that usually seen in the rat cultures, lntravital staining with F D A / P I revealed that very few dead cells were present, as d e m o n s t r a t e d by the low n u m b e r of PI-stained red cells (Fig. 3, arrows). The majority of cells were viable, as indicated by the a b u n d a n c e of cells showing green fluorescence (an indication of F D A hydrolysis by intact cells). T h e n u m b e r of viable cells were c o m p a r e d in K5 and K25 m o u s e and rat granule cell cultures which were grown simultaneously (Table 3). Cell counting and D N A m e a s u r e m e n t gave c o m p a r a b l e estimates of viable cell numbers, thus validating the use of the modification of the H o e c h s t 33258 binding assay and d e m o n s t r a t i n g the methodological i n d e p e n d e n c e of the findings. Results of cell counting also c o n f i r m e d the observations r e p o r t e d in Table 2 using D N A estimation that the m e d i u m K + concentration does not significantly influence the survival of the m o u s e cells and that u n d e r the present culture conditions m o u s e granule cells survive better than rat cells.
Phase
Green
Red
K25
K5
Fig. 3. Phase-contrast and fluorescence micrographs of mouse granule cells grown for 7 days in medium containing 5 or 25 mM K +. Cells were intravitally stained by FDA/PI and examined by phase-contrast and fluorescence microscopy. The fluorescence micrographs were digitized and colour-separated to demonstrate viable cells (green window) and dead cells (red window). Dead cells stained red by propidium iodide. marked by arrows, gave almost no colour in the green window, whereas intense green cells also showed some colour in the red window. Horizontal bar=100 ixm. There was no evidence of any difference in survival in K5 and K25 cultures.
Survival of mouse cerebellar granule cells and potassium
457
Table 3. Cell survival determined by intravital staining with F D A / P I Cell n u m b e r Mouse
K5 K25
92_+9 100_+ 13
Rat
K5 K25
45_+5*** 74_+6**
Cultures of m o u s e and rat cerebellar granule cells were grown in parallel for 7 days in m e d i u m containing 5 m M or 25 m M K +. Cell n u m b e r is the average n u m b e r of viable cells f l u o r e s c i n g green in cultures incubated with F D A (n=10 microwells). Results are n o r m a l i z e d with r e s p e c t to m o u s e c u l t u r e s g r o w n in 25 m M K +. Significant differences in cell n u m b e r relative to m o u s e K25 cultures are indicated: **P<0.01; ***P<0.001. Table 4. Effects of K + concentration in the culture m e d i u m on biochemical parameters in m o u s e and rat cerebellar granule cell micro-well cultures Mouse
Rat
K5 N A D P H tetrazolium reductase (absorbance units) F D A esterase activity (fluorescence units) D O G (c.p.m.) Cell n u m b e r
K25
0.18_+0.02 ***
0.32_+0.01
21 _+1 *** 5900_+470** 55,100_+2300
34 +_2 8520+_750 54,800_+2300
K5 0.03_+0.01"** -1080_+75 *** 15,275_+950"***
K25 0.13+_0.01 -2640_+150 32,850_+1500
G r a n u l e cells were cultured for 7 days in m e d i u m containing 5 m M or 25 m M K + (plating density: 2000 cells/mm 2, i.e. 68,000 cells/well corresponding to 0.43 ~g D N A ) . Results are given in arbitrary units. For N A D P H tetrazolium reductase activity n = 1 0 - 3 2 , for F D A esterase n = 9 , for D O G accumulation n = 2 2 - 2 4 , and for cell n u m b e r (determined in micro-well cultures using the Hoecbst 33258 binding assay after pre-treatment with D N A a s e ) n=36-67. Values are means-+S.E.M. Statistically significant differences between K5 and K25 are indicated: **P<0.01; ***P<0.001; ****P<0.0001.
Granule cells and the extracellular K + concentration Although elevation of medium K + concentration did not influence the survival of mouse granule cells, it did influence the biochemical differentiation of these cells. Thus, at 7 DIV the rate of nitroblue tetrazolium reduction in K25 cultures was nearly double the value in K5 cultures, and significant differences were also detected in FDA esterase activity and DOG accumulation (Table 4). "High" K + also increased the value of these estimates in rat granule cell cultures. Even when corrected for the low number of cells in rat K5 cultures, the rate of tetrazolium reduction was significantly increased by a factor of three in "high" K +. The functional expression of NMDA receptors in mouse granule cells was measured as NMDA-evoked increase in 45Ca2+ influx under MgZ+-free conditions in order to avoid the Mg 2+ blockade of the NMDA receptor-linked channel and in the presence of 10 I~M glycine to ensure full activation of the receptor (see Ref. 22). NMDA receptor activity was approximately three-fold greater in mouse granule cells cultured in K25 than in K5 medium (Table 5).
DISCUSSION Quantitative estimation of viable cells in micro-well cultures DNAase pre-treatment of micro-well cultures resulted in the hydrolysis of DNA originating from degenerating cells. Since the enzyme does not penetrate membranes, it does not attack DNA contained in the viable cells. DNAase pre-treatment, as performed in this study, appeared to be
458
H.S. Mogensen et al. F a b l e 5. Effect of m e d i u m K ~ c o n c e n t r a t i o n on the N M D A r e c e p t o r activationi n d u c e d 45Cae ~ influx in m o u s e c e r e b e l l a r g r a n u l e cell c u l t u r e s K5 B a s a l (1 m M MK-801 ) 100 m M N M D A + 10 ~tM glycine N M D A - i n d u c e d 45Ca 2 + u p t a k e
K25
9.1±t).8
(~.7-q).(~
~ 4 +1.1 13.3 + 1.1
47 ..... ~.2 40.5 -: 2.2
NMDA receptor activitywas determined in granule cell cultures at 7 D1V, as the NMDA-evoked increase, in an Mg2+-free medium, in 45Ca2+ influx (nmol 45Ca2+ influx/mgprotein). Values are means-+S.E.M. (n=3 dishes). harmless to matrix-attached, intact granule cells at 7 DIV, even when the exposure was prolonged for up to 3 hr. However, the treatment dramatically decreased the fluorescence of Hoechst 33258 in cultures in which cells were forced to degenerate. The D N A content of viable cells could therefore be estimated in the micro-well cultures using the Hoechst 33258 fluorochrome binding method. 27 This method is based on the fact that the fluorescence yield of Hoechst 33258 at 450 nm is greatly enhanced upon binding to DNA; the enhancement increases linearly with the amount of DNA, and R N A does not interfere with the determination of DNA. 10,18,19 The effect o f elevated K + concentration on cell s u r v i v a l in rat a n d m o u s e granule cell cultures
After working for some time with rat cerebellar granule cell cultures, we were at first surprised to find that the mouse cells do not degenerate when grown in a serum-containing medium in the presence o f " l o w " K +. In order to exclude experimental variables, granule cells from rat and mouse cerebella were prepared, cultured and analysed simultaneously and under identical conditions. The observations confirmed the report of Peng et al. 21 and our initial findings by showing that mouse cerebellar granule cells have no survival requirement for K+-induced membrane depolarization. In contrast, the survival of the parallel grown rat granule cells was compromised, unless the culture medium contained "high" K +, as reported earlier (e.g. Ref. 13). The present findings therefore permit the exclusion of methodological differences, considered by Peng et al. 21 in connection with the discrepancy in the K + dependence of their mouse granule cell cultures and our rat cultures. Thus, we observed that different cell densities, suggested by Peng et al. as possibly the most important procedural difference, do not influence either the lack of K + dependence of the mouse cells or the survival requirement of the rat cells for "high" K +. Furthermore, we cultured the granule cells from the two species in the same medium, which contained fetal calf serum. Astrocytes are known to support the survival and maturation of nerve cells in culture. For example, in aggregated cell cultures of rat telencephalon, an effect of "high" K + on neuronal maturation was evident only after the depletion of glial cells. 23 The presence of a relatively high proportion of astrocytes in the mouse cultures might therefore have accounted for both the better survival of the mouse neurons and their lack of survival requirement for elevated K + concentration. Accordingly, we assessed astrocyte contribution to the cultures by estimating glial fibrillary acidic protein using E L I S A (data not shown). The amount of glial fibrillary acidic protein-positive material was low in both cultures, but it was 30-40% higher in the rat than in the mouse granule cell cultures, thus rendering the possibility that astrocytes might have accounted for the differences rather unlikely. We conclude, therefore, that the different survival requirements are genuine properties of the mouse and rat granule cells. These findings are apparently at variance with the report of Didier et al., 11 who assessed mouse granule cell survival by measuring fluorescein production from FDA. These authors considered that "high" K + promoted neuronal survival since it increased the conversion of F D A to fluorescein. However, we observed that after F D A loading the fluorescence of individual cells in K25 cultures was brighter compared with K5 cells. Peng et al. 21 have also commented on the paler fluorescence of cells in the "low" than in the "high" K+-containing cultures. Measurements of the fluorescein produced from F D A in the cells, together with the estimations of cell numbers in the K5 and K25 cultures, supported the microscopic impression and showed that fluorescein production/cell in K5 cultures was only 65% of that in K25.
Survival of mouse cerebellar granule cells and potassium
459
Effect of "high" K + on the differentiation of mouse granule cells It seems that cultivation in "high" K+-containing medium is not essential for the survival of mouse cerebellar granule cells. However, "high" K + has a powerful effect on the biochemical differentiation and the expression of NMDA receptors in these cells. All the biochemical parameters measured in this study showed higher values in the K25 than the K5 cultures. Thus, activities of NADPH tetrazolium reductase and FDA esterase, and rate of DOG accumulation were about 50% higher in K25 than in K5. Under the experimental conditions, the NADPH-diaphorase activity of nitric oxide synthase contributes to formazan formation 14 and this enzyme is greatly enriched in cerebellar granule cells. 25 The effect of chronic membrane depolarization was even more pronounced on the expression of phenotypically characteristic properties, compared with the above-mentioned household functions. The functional expression of the NMDA receptors was about three-fold higher in K25 than in K5 cultures, and in this respect the behaviour of mouse cells was comparable with rat granule cells. 8,22 It has been observed previously that stimulus-coupled glutamate release develops with the maturation of rat granule cells. 12 Comparable findings have been reported by Peng et al. 21 for mouse cells grown in K25 medium, but these authors failed to detect K+-evoked glutamate release in cultures grown in "low" K + medium. In this respect, the retarded maturation of mouse granule cells in "low" K + medium is even more pronounced than observed for rat cells in "low" K+. 13 Mouse granule cells are not unique among neurons in culture in exhibiting good survival in "low" K + medium. Most notably, hippocampal neurons 9 and GABAergic cerebral interneurons24 are routinely cultivated in "low" K + medium. Moreover, even rat granule cells fail to develop survival requirements for "high" K + under certain conditions. Cells grown in serum-flee, chemically defined medium survive as well in medium containing 5 mM K + as 25 mM K+. 17 The maturation of the serum-free, "low" K + cells is, however, markedly retarded compared with cells grown in serum-containing medium with "high" K + and it was proposed that serum-derived factors promote the maturation of rat granule cells to a stage at which survival becomes dependent on external stimulation, such as K +-induced membrane depolarization.4 Cultivation in "high" K + promotes the differentiation and the maturation of the rat granule cells grown in serum-free medium.3,4 The morphology of our serum-containing mouse cultures resembles that of serum-flee rat cultures expressing only minor neuronal aggregation, 17 thus indicating a degree of immaturity of the mouse granule cells. The present work shows that the biochemical differentiation and the maturation of mouse cerebellar granule cells is also promoted by cultivation in "high" K + containing medium, as indicated by increased FDA esterase activity, enhanced rate of NADPH-dependent tetrazolium reduction, augmented DOG accumulation and increased NMDA-evoked 45Ca2+ influx. During their development, both in vivo and in culture, nerve cells go through stages in differentiation and maturation in which their survival depends on the presence of various environmental factors.2 It seems that one such factor for cerebellar granule cells is K+-induced membrane depolarization, which in rat cells promotes both survival and differentiation, whereas in mouse cells it primarily promotes differentiation. The reason for this species-dependent difference in properties is not yet known. We are proposing here that mouse granule cells in culture are insensitive to the serum-derived factor(s) which promotes the differentiation and maturation of rat granule cells and therefore retain a less differentiated state in which their survival is not dependent on K+-induced membrane depolarization. Acknowledgements--This research was supported by grants from The Danish Medical Research Council, Ivan Nielsens Fond and the Royal Netherlands Academy of Science (KNAW). We are very grateful to Professor Niels Anker Thorn and Technical Assistant Birthe Linnerup Kristensen, Institute of Medical Physiology, University of Copenhagen, for access to and help with the Fluoroscan 11 instrument.
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