- Email: [email protected]
Regulation of intracellular free calcium in normal and dystrophic mouse cerebellar neurons
Brain Research, 578 (1992) 49-54 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
49
BRES 17619
Regulation of intracellular free calcium in normal and dystrophic mouse cerebellar neurons F. Woodward Hopf and Richard A. Steinhardt Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720 (USA) (Accepted 26 November 1991) Key words: Cerebellum; Granule neuron; Calcium; Muscular dystrophy; Fura-2 We measured free intracellular calcium ([Ca2+]i) in cultured cerebellar granule cells from normal and mdx mice. Resting levels of ([Ca2+]i) were 24% higher in the dystrophic neurons (normal: 61.2 + 1.5 nM calcium, n = 104; dystrophic: 76.1 + 2.4 nM calcium, n = 136, P <0.01). Distrophic neurons showed a significantly greater increase in ([Ca2÷]i) in the presence of elevated (18 mM) extracellular calcium levels. Resting sodium levels ([Na+]i), however, were found to be similar in normal and dystrophic granule neurons. In addition, sodium influx rates after ouabain inhibition of the Na+/K + ATPase were also identical. Therefore, the increased permeability of granule neurons was specific to calcium, and did not result from a non-selective cation-permeable conductance. Unlike granule cells, astrocytes do not express dystrophin. Glial cells from normal and dystrophic mice showed no difference in their resting free calcium levels or their response to a high calcium load. Thus, cerebellar granule neurons from mdx mice show a calcium-specific regulatory defect similar to that found in dystrophic muscle fibers 15'47'48, while cerebellar glial cells, which do not normally express dystrophin, have no such defect. INTRODUCTION Duchenne Muscular Dystrophy (DMD) is an X-linked genetic disease in humans characterized by skeletal muscle fiber degeneration, and replacement with connective tissue. In addition, 30% of the D M D cases express a mild, non-progressive mental retardation 35, although no consistent histopathological alterations have been found 26'35. Patients with D M D have a deletion in the gene encoding for the 400 kDa protein dystrophin, and thus do not express functional dystrophin 23. D M D can be studied using a mouse model, the m d x mouse, which has genetic, biochemical, and physiological defects similar to those observed in D M D individuals 4. In normal muscle, dystrophin is located primarily on the inner surface of the sarcolemma 44'5°. Dystrophin has some sequence homology with a-actinin and spectrin, and is bound to several integral membrane glycoproteins 7"39. The function of dystrophin is unknown. Whole brain homogenates express the dystrophin protein 24'31 and m R N A 8'9"24'37. However, brain dystrophin m R N A is spliced differently than that found in muscle 14'38 and is transcribed from a different p r o m o t e r 3. In the CNS, dystrophin has been found in neurons of the cerebellum and cerebrum, but not in astrocytes in any location 24'31. Early observations that total muscle calcium content is elevated in D M D patients 2 suggested a role for calcium in the etiology of dystrophy. In the mouse, the
resting level of free intracellular cytosolic calcium ([Ca2+]i) is higher in mdx (92 + 9.8 nM) than in normal (40 + 2.8 nM) isolated skeletal muscle fibers 47. Furthermore, after a fiber is subjected to an ten-fold increase in extracellular calcium (to 18 mM), ([Ca2+]i) rises, but to a significantly greater level in dystrophic fibers 47. This is also observed in mouse and human cultured myotubes 15. The calcium regulatory defect might be due to an increased ion influx across the sarcolemma, or an impaired efflux. Studies of calcium resequestration rates after electrical stimulation demonstrate that calcium efflux pathways function normally in dystrophic muscle fibers, suggesting that any regulatory impairment must be an ion influx 48. This influx might be due to a specific calcium leak or a non-specific leak admitting other cations. When the Na+/K+-ATPase is inhibited with ouabain, intracellular sodium ([Na+]i) rises due to an inward sodium leak. No difference in the initial rate of rise of [Na÷]i is found between normal and dystrophic muscle cells, arguing in favor of a calcium-specific sarcolemmal leak 48. Using the patch-clamp technique, cultured mouse and human myotubes are found to have a calcium leak channel which shows altered behavior in dystrophic cells 15. Compared to normal myotubes, the mean channel closed time of dystrophic cells is greatly reduced, resulting in an increased open probability. Other channel properties (mean open time, single channel conductance, ion selec-
Correspondence: R.A. Steinhardt, Dept. of Molecular and Cell Biology, Division of Cell and Developmental Biology, University of California at Berkeley, Berkeley, CA 94720, USA.
50 tivity, and b e h a v i o r in the p r e s e n c e of p h a r m a c o l o g i c a l agents) are similar b e t w e e n the two fiber types. T h e inc r e a s e d o p e n p r o b a b i l i t y of t h e c a l c i u m l e a k c h a n n e l m a y be r e s p o n s i b l e for the o b s e r v e d e l e v a t e d influx of calcium into d y s t r o p h i c cells. A c a l c i u m l e a k c h a n n e l has b e e n f o u n d in c e r e b e l l a r g r a n u l e n e u r o n s ; l e a k c h a n n e l s
tifacts associated with single intensity measurements, which may be effected by dye leakage or changes in cell thickness TM. Some experiments included astrocytes from 4-week-old cultures, to allow measurement without the interference of associated granule cells, which have died by this time. Measurements from 4-week glial cells were not different from 3-day-old cultures. The approximate actual ([Ca2])i can be calculated from the following formula18:
f r o m d y s t r o p h i c n e u r o n s possess a l t e r e d c h a n n e l b e h a v [Ca 2+] = b × K d (R
ior similar to that f o u n d in d y s t r o p i c m y o t u b e s 2°. To investigate w h e t h e r g r a n u l e n e u r o n s f r o m dystrophic animals h a v e c a l c i u m r e g u l a t o r y defects similar to those
observed
in
dystrophic
muscle,
we
examined
[Ca2+]i in n e u r o n s at rest and after c h a l l e n g e with a high e x t r a c e l l u l a r calcium load. F u r t h e r m o r e , we e x a m i n e d the calcium-specificity of the r e g u l a t o r y
defect(s)
by
studying c h a n g e s in [Na+]i after o u a b a i n inhibition o f the Na+/K+-ATPase. MATERIALS AND METHODS Isolation and maintenance o f cultured cells (modified from re.[. 25) All experiments in this study used the C57 mdx mouse, or the corresponding normal mouse strain. The cerebelli of a litter of postnatal day 7 mice were removed, pooled, and minced. The tissue was incubated on a shaker at 37°C for 35-40 min in 20 U/ml papain (Worthington) in a modified Earle's balanced salt solution (BSS; Gibco) containing (in raM) NaCI 117, KCI 5.3, MgSO4 0.8, CaC12 1.8, NaH2PO4.H20 1.0, cysteine 1.0, EDTA 0.5, HEPES 10, NaHCO 3 4.0, glucose 25, pH 7.4. The tissue was removed and triturated 5 or 6 times with a fire polished pasteur pipette in DMEM (Gibco) supplemented with the following components: 10 mM HEPES, 26 mM NaHCO3, 25 mM glucose, 0.5 mM glutamine, 8% fetal calf serum (Hyclone), 100 U/ml penicillin, 0.1 mg/ml streptomycin, 1.5 mg/ml each of BSA (Sigma) and ovomucoid (Sigma), pH 7.4. After trituration 200-500 /tl of the cell suspension was plated onto polylysine-laminin (50/~g/ml of each) coated glass coverslips. The cells were allowed to settle for 3-6 h in a humid incubator at 37°C under a 5% CO2/95% air atmosphere. After settling, 2 ml of medium (DMEM, modified as above, except without BSA and ovomucoid, and with a total of 25 m M [K+] 6'22"29"4"1)was added to each dish. After 2-5 days recovery, the cells were used for microfluorimetry experiments. Just before an experiment, the cells were placed in a normal [K ÷] (5 mM) medium for 15-30 min 11. Experiments included litters from 4 mdx mice and 4 normal mice. Calcium fluorescence measurements We used the AM ester form of fura°2 (Molecular Probes), a membrane-permeant, fluorescent calcium-chelating dye 46. Fura-2 stocks (10 mM) were prepared in dimethyl sulfoxide (DMSO) and stored at -20°C. Prior to cell loading, an aliquot of the fura-2 stock was mixed 1:1 with 25% w/v Pluronic F-127 (Molecular Probes). A dish of cells was loaded for 35-45 min at 20°C in the presence of 10/zM fura-2. After loading, the dish was washed 3 times with fresh Ringer's (BSS plus 26 mM NaHCO3), and placed onto the stage of an inverted Nikon Diaphot-TMD microscope (using a 40× bright field quartz objective). The cells were maintained at 32°C under a 5% CO2/95% air atmosphere. Healthy neuronal cells were selected based on size (6-8 #M) and morphology (bipolar with round or eliptical soma, smooth gray appearance) 21"32. As observed by other investigators 1~, the amount of dye loaded into granule cells is variable. Fura-2 was excited alternately at 350 nm and 385 nm wavelengths. For each, the emission intensity was measured at 510 nm by a photomultiplier tube. The ratio of the intensities at 350 nm over 385 nm was calculated. Ratio measurements eliminate the ar-
-
Rmin)/(Rmax
-
R),
where R is the measured ratio, and Rmin and Rmax are the ratios of fura-2 in CaZ+-free and saturated-Ca 2+ calibration solutions. The K a of fura-2 for calcium at 32°C is 185 nM, and b is the ratio of the 385-nm excitation efficiency in the absence of calcium to the 385-nm excitation efficiency at saturating calcium concentrations. Intracellular Rmm and Rma x are somewhat different from those in calibration buffers, because of differences in intracellular viscosity, ionic strength, or dye binding 4°'46. Therefore, a correction factor was calculated 43. Granule cells were placed in 5/~M ionomycin, a calcium ionophore, and intracellular Rmin and Rn~ax were determined by placing the cells in a nominally Ca2+-free buffer (plus 3 mM EGTA) and a 20 mM Ca 2+ buffer, respectively. Rmin was 0.343, R~,ax was 7.426 and b was 10.57. The correction factor for granule neurons was found to be 0.62, from two experiments. A separate correction factor was not calculated for astrocytes. The cell-permeant fura-2 am-ester is known to enter intracellular compartments, where it contributes to fluorescent emissions, but is not responsive to changes in [Ca2+]i, leading to spurious results ~. To correct for this possibility, cells were placed in digitonin, which permeabilizes the plasma membrane, and leads to rapid loss of cytoplasmic, but not compartmentalized, signal 27. At the end of a normal one hour recording period, digitonin permeabilization (75 /~M; n = 3) led to loss of all but 7% of the fluorescence signal, showing that very little of the dye is compartmentalized. Sodium fluorescence measurements Sodium experiments were similar to calcium as described above, except that intracellular free sodium levels were measured with the sodium chelating dye, sodium-binding benzofuran isopthalate (SBFI, Molecular Probes) 33. Cells were loaded for 60 min using a final concentration of 20/~M SBFI AM ester (mixed with an equal volume Pluronic F-127, in DMSO), then washed with BSS 3 times prior to experiments. Excitation and emission wavelengths were the same as for fura-2. The rate of sodium leak was examined after 3.3 mM ouabain was added to inhibit the Na+/K +- ATPase 19"36. Intracellular dye calibrations were performed by permeabilizing the cells with a mixture of 15/~M monensin and 15/~M nigericin. External sodium concentration was varied as previously described 36, and the fluorescence ratio at each sodium concentration was monitored. All statistics were calculated using a one- or two-tailed Student t-test. All values are expressed as the mean + S.E.M.
RESULTS I s o l a t e d cell cultures w e r e m o r p h o l o g i c a l l y similar to t h o s e s e e n by o t h e r investigators 28'32'45. H o w e v e r , to assure that the cells studied w e r e g r a n u l e cells, kainic acid ( K A ) n e u r o t o x i c i t y was tested. W h e n c u l t u r e d in 100/~M K A , all n e u r o n a l types e x c e p t g r a n u l e n e u r o n s die within 48 h 42. A f t e r 4 days in K A , 70% of the n e u r o n s in o u r cultures s u r v i v e d (n = 3). A s t r o c y t e s w e r e u n a f f e c t e d by the t r e a t m e n t . M o r e o v e r , o t h e r c e r e b e l l a r n e u r o n a l types ( P u r k i n j e , G o l g i , basket, stellate) do n o t survive in significant n u m b e r s w h e n t a k e n f r o m p o s t n a t a l animals 22'
51 GLU APV Hi [K+I
130' 120
500
A
,
110' 100"
400
90" 80"
300 "~
70-
200
60'
100
50" 40
0
I
0
I
1000
I
I
2000
I
I
3000
I
)
i
i
)
5
15
25
35
time (rain)
I
4000
seconds Fig. 1. Example of a fura-2 experiment, showing some characteristic responses of a granule neuron. This trace shows the response of a normal granule cell to 2 pulses of 100 #M glutamate (GLU; the endogenous neurotransmitter), and to a 25 mM [K+] depolarizing pulse. At the end of the second GLU pulse, 200 #M APV (an NMDA receptor agonist) was added to demonstrate the presence of NMDA receptors on these cells.
28,32,45; those that do survive are distinguishable by larger size (15/~M) and morphology (generally stellate as opposed to bipolar). Fibroblasts and endothelial cells compose less than 1% of the non-neuronal cells in these cultures 32'45, especially in the presence of serum 28. Fig. 1 shows a typical response of a granule neuron to rapid bath applications of 100/~M glutamate (Glu), the endogenous neutrotransmitter 6'16, and 25 mM [K+]. When Glu (n = 5) was applied, [Ca2+]i r o s e rapidly, and remained at an elevated plateau level. In response to a depolarizing [K ÷] application (n = 6), [Ca2+]i r o s e sharply, then dropped rapidly to a plateau above the resting [Ca2÷]i. Both Glu and high [K ÷] responses were fully reversible; when either agonist was replaced by normal Ringer's, [Ca2+]i returned to baseline. These responses are consistent with those seen by other investigators 5,n. N-Methyl-o-aspartate ( N M D A ) receptors are present in granule neurons at t h i s a g e 5'13'17'41. Application of DL-2-amino-5-phosphonovaleric acid (APV, 200 #M, n = 4), an N M D A receptor antagonist 34, partially reduced the Glu-induced [Ca2+]i rise, confirming the presence of N M D A receptors in this cell preparation. The difference in resting [Ca2+]i o f mdx and normal granule neurons was small but significant (normal: 61.2 + 1.5 riM, n = 104; dystrophic: 76.1 + 2.4 nM, n = 136, P < 0.01). These resting values are within the range observed by other investigators 5,n. Astrocytes were chosen for comparison with neurons because they do not express detectable levels of dystrophin 24'31. Astrocytes from normal and dystrophic mice showed no difference in their resting [Ca2+]i (normal: 66.6 + 3.4 nM, n = 28;
120" 110
B
100
E E
90 80
¢3
70 60 50 -5
i
i
!
5
15
25
3'5
time (rain) Fig. 2. [Ca2+]ichanges in normal and dystrophic cerebellar granule neurons (A) and astrocytes (B) in response to a high calcium stress. At time zero, the normal medium containing 1.8 mM calcium was replaced by a high calcium (18 raM) medium. Calcium values at time zero are corrected to the total population means. [Ca2+]i of dystrophic neurons (A; n = 26) was significantly higher than normal (O; n = 21) at all time points ( , ; P < 0.01). [Ca2÷]i of dystrophic (A; n = 10) and normal (O; n = 11) astrocytes was not significantly different at any time point (P > 0.25). Cells not shifted to a high calcium levels showed no change in [Ca2÷]i (0; granule neurons: n = 16 for normal, n = 20 for dystrophic; astrocytes: n = 5 for normal, n = 9 for dystrophic; only normal shown for both cell types).
dystrophic: 65.8 + 3.5 nM, n = 49, P > 0.25). In the absence of perturbations, both granule cells and astrocytes show stable resting [Ca2+]i for at least 30 min. To investigate possible defects in calcium homeostatic regulation across the plasma membrane, the cells were subjected to an external calcium load (Fig. 2). At time zero, calcium was added to a dish to give a total external calcium concentration of 18 mM (10 times the normal calcium concentration of 1.8 mM). Compared to normal granule neurons, mdx neurons showed a significantly elevated [CaZ+]i, at all time points (P < 0.01) (Fig. 2A). Astrocytes from normal and dystrophic mice showed no difference in their response to high calcium levels at any time point (P > 0.25) (Fig. 2B). These data suggest that dystrophic neurons are defective in their regulation of calcium fluxes across the plasma membrane.
52 1.6-
1.2 -
creased ion permeability across the plasma membrane of dystrophic granule neurons was specific to calcium, and did not result from a non-specific increase in cation permeability.
1.0-
DISCUSSION
0.8-
The results show that postnatal day 11-13 cerebellar granule neurons lacking dystrophin possess a calciumspecific regulatory defect similar to that observed in dystrophic skeletal muscle cells. This is consistent with the observation 2° that dystrophic granule neurons possess a defect in the regulation of a calcium leak channel similar to that observed in muscle cells of dystrophic origin 15. Astrocytes, which do not express dystrophin, show no differences in calcium regulation. However, the difference in resting [Ca2÷]i b e t w e e n mdx and normal granule neurons is not as great as that observed in muscle cells. Dystrophic mouse muscle fibers 47 and cultured myotubes ~5 show an elevation of 50 nM and 28 nM calcium, respectively, over normal levels. Dystrophic and normal granule neurons only showed a difference of 15.1 nM. However, the regulatory defect in granule neurons in response to high calcium levels is similar to that found in muscle c e l l s 15'47.
1'4. O
"*~
0.6 5
, 15
2'5
, 35
sodium (mM) Fig. 3. Calibration curve for SBFI in granule neurons (n = 5). Intracellular calibration of SBFI fluorescence signals, as described in Materials and Methods, allows approximations of internal sodium values. Note that the dye response is approximately linear in the 0-30 mM [Na÷] range, as previously observed 36. The S.E.M. at 10 mM [Na ÷] is smaller than the symbol used. All cells had ratios in the range corresponding to 0-10 mM sodium. Therefore, the following formula was derived to calculate [Na+]i from a measured ratio: [Na÷] = ((Ratio - 0.79)/0.03).
To test whether the regulatory defect in dystrophic neurons was a non-specific cation leak, or specific to calcium ions, sodium influx in the absence of regulatory mechanisms was measured. The major regulatory mechanism for sodium is the Na+/K÷-ATPase. Therefore, we inhibited this pathway with ouabain (3.3 mM), and measured the initial, maximal rate of passive sodium influx. The resting [Na+]i and sodium influx rate after ouabain addition were estimated from the SBFI calibration curve (Fig. 3). Resting [Na+]i levels were similar in normal and dystrophic neurons. Furthermore, sodium influx rates after inhibition of the Na+/K+-ATPase were also similar (Table I). Two experiments were conducted in very low (61 nM) calcium to control for possible regulatory effects of the Na+/Ca 2+ exchanger. The rate of sodium rise after ouabain addition was similar to that observed in normal (1.8 mM) calcium (data not shown). In summary, there was no difference between normal and dystrophic neurons in either resting [Na+]i or sodium influx after inhibition of the Na+/K+-ATPase. Therefore, the in-
TABLE I
lntracellular free sodium values of granule neurons at rest, and the initial rate of rise of sodium after ouabain inhibition of the Na+/K + pump Cell type
Level at rest (raM Na ÷)
Rate of rise (mM Na+/min)
Normal
5.4 + 0.4, n = 64 5.8 + 0.6, n = 81
1.15 + 0.21, n = 9 1.12 + 0.13, n = 8
mdx
There is no significant difference between normal and m d x granule neurons, either in the resting levels (P > 0.25), or the initial rate of rise following addition of ouabain (P > 0.25).
This more pronounced difference in resting [Ca2+]i observed in muscle cells may be a reflection of the wide distribution of dystrophin under the sarcolemma 44's°. In contrast, in adult mouse brain slices, dystrophin is located on the postsynaptic regions of axodendritic and axosomatic synapses of cerebellar Purkinje neurons, and pyramidal neurons in certain regions of the cerebral cortices 3t. Thus, in neurally derived preparations, dystrophin is localized to a restricted subset of membrane domains. Therefore, the effects of a calcium regulatory impairment might not be evident at resting [Ca2+]i . However, the precise subcellular distribution of dystrophin in neonatal neurons of the stage studied here is unknown. Hence, the effect of dystrophin location on [Ca2+]i in these cells is not clear. It should be noted that Lidov et al. 3~ did not find dystrophin expression in cerebellar granule neurons from normal adult mice in slice. We did not study adult cells. However, our results, and those of Haws et al. 2°, which demonstrate a regulatory defect in neonatal granule neurons from mdx mice, suggest the precense of dystrophin in normal neonatal neurons. Resting [Na+]i does not differ between normal and dystrophic granule neurons. However, tight intracellular sodium regulation by various Nat-related transporters and ATPases may mask any alterations in sodium leak. Inhibition of these regulatory mechanisms would reveal any such increases in sodium influx. However, sodium
53 influx after inhibition of the Na+/K+-ATPase with oua-
stricted neuronal distribution of dystrophin, together
bain was not different between normal and dystrophic
with the t r e m e n d o u s ability of the developing nervous
granule neurons, arguing against the possibility of a dys-
system to compensate for abberations, may contribute to the variability and mildness of the observed mental retardation in dystrophic individuals.
trophy-related sodium leak. The sodium resting levels reported here are lower than those reported using SBFI in other cell types, which are in the range of 8-10 mM [Na+]i (refs. 19, 36, 48). Despite the lower resting levels, the observed rate of rise after the addition of ouabain is similar to that seen by other observers ( - 1 . 0 mM/min (ref. 48), - 3 . 0 mM/min (ref. 36), - 0 . 5 mM/ rain (ref. 19). It is not clear how aberrations in the cerebellum might
In conclusion, dystrophic granule neurons display an impairment in calcium regulation which is similar to that observed in dystrophic muscle 15'47'48. Elevated intracellular calcium accumulation leads to excitotoxic damage and neuronal death u n d e r hypoxic and ishemic conditions 1°. Thus, the defect in calcium regulation reported here may lead to cellular damage in granule neurons in
lead to a cognitive deficit. No clear cerebellar patholog-
a similar m a n n e r , and hence may contribute to the men-
ical syndromes (such as tremors and ataxia) are associated with D M D , although they are seen in older m d x mice 31. However, recent observations of a possible as-
tal retardation sometimes associated with D M D .
sociation between autism and disorganization of the cerebellar vermis ~2 suggest that cerebellar damage may play a role in cognitive dysfunctions3°,49. The normally re-
Acknowledgements. This work was supported by private donations and the Muscular Dystrophy Association. We would like to thank Dr. Peying Fong and Dr. Paul Turner for comments and suggestions on the manuscript.
REFERENCES
bellar granule neurones, J, Physiol., 400 (1988) 189-122. 14 Feener, C.A., Koenig, M. and Kunkel, L.M., Alternative splicing of human dystrophin mRNA generates isoforms at the carboxy terminus, Nature, 338 (1989) 509-511. 15 Fong, P., Turner, P.R., Denetclaw, W.E and Steinhardt, R.A., Increased activity of calcium leak channels in myotubes of Duchenne human and mdx mouse origin, Science, 250 (1990) 673-676. 16 Gallo, V., Ciotti, M.T., Coletti, A., Aloisi, E and Levi, G., Selective release of glutamate from cerebellar granule cells differentiating in culture, Proc. Natl. Acad. Sci. USA, 79 (1982) 7919-7923. 17 Garthwaite, G., Yamini Jr., B. and Garthwaite, J., Selective loss of Purkinje and granule cell responsiveness to N-Methyl-Daspartate in rat cerebellum during development, Dev. Brain Res., 36 (1987) 288-292. 18 Grynkiewicz, G., Poeni, M. and Tsien, R.Y., A new generation of Caz* indicators with greatly improved fluorescence properties, J. Biol. Chem., 260 (1985) 3440-3450. 19 Harootunian, A.T., Kao, J.P., Eckert, B.K. and Tsien, R.Y., Fluorescence ratio imaging of cytosolic free Na ÷ in individual fibroblasts and lymphocytes, J. Biol. Chem., 264 (1989) 1945819467. 20 Haws, C.M. and Lansman, J.B., Calcium-permeable ion channels in cerebellar neurons from mdx mice, Proc. Roy. Soc. Lond. B, 244 (1991) 185-189. 21 Hockberger, P.E., Tseng, H.-Y. and Connor, J.A., Immunocytochemical and electrophysiological differentiation of rat cerebellar granule cells in explant cultures, J. Neurosci., 7 (1987) 1370-1383. 22 Hockberger, P.E., Tseng, H.-Y. and Connor, J.A., Development of rat cerebellar Purkinje cells: electrophysiological properties following acute isolation and in long-term culture, J. Neurosci., 9 (1989) 2258-2271. 23 Hoffman, E.P., Brown Jr., R.H. and Kunkel, L.M. Dystrophin: the protein product of the Duchenne muscular dystrophy locus, Cell, 51 (1987) 919- 928. 24 Hoffman, E.P., Hudecki, M.S., Rosenberg, P.A., Pollina, C.M. and Kunkel, L.M., Cell and fiber-type distribution of dystrophin, Neuron, 1 (1988) 411-420. 25 Huettner, J.E. and Baughman, R.W., Primary culture of identified neurons from the visual cortex of postnatal rats, J. Neurosci., 6 (1986) 3044-3060.
1 Almers, W. and Neher, E., The Ca signal from fura-2 loaded mast cells depends strongly on the method of dye-loading, FEBS Lett., 192 (1985) 13-18. 2 Bertorini, T.E., Bhattacharya, S.K., Palmieri, G.M.A., Chesney, C.M., Pifer, D. and Baker, B., Muscle calcium and magnesium content in Duchenne muscular dystrophy, Neurology, 32 (1982) 1088-1092. 3 Boyce, EM., Beggs, A.H., Feener, C. and Kunkel, L.M., Dystrophin in brain is transcribed from a distant upstream promotor, Proc. Natl. Acad. Sci. U.S.A., 88 (1991) 1276-1280. 4 Bulfield, G., Siller, W.G., Wight, P.A.L. and Moore, K.J., X chromosome linked muscular dystrophy (mdx) in the mouse, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 1189-1192. 5 Burgoyne, R.D., Pearce, I.A. and Cambray-Deakin, M., N-Methyl-D-aspartate raises cytosolic calcium concentration in rat cerebellar granule cells in culture, Neurosci. Lett., 91 (1988) 47-52. 6 Burgoyne, R.D. and Cambray-Deakin, M., The cellular neurobiology of neuronal development: the cerebellar granule cell, Brain Res. Rev., 13 (1988) 77-101. 7 Campbell, K.P. and Kahl, S.D., Assocation of dystrophin and an integral membrane glycoprotein, Nature, 338 (1989) 259262. 8 Chamberlain, J.S., Pearlman, J.A., Muzny, D.M., Gibbs, R.A., Ranier, J.E., Reeves, A.A. and Caskey, C.T., Expression of the murine Duchenne muscular dystrophy gene in muscle and brain, Science, 239 (1988) 1416-1420. 9 Chelly, J., Kaplan, J.-C., Maire, P., Gautron, S. and Kahn, A., Transcription of the dystrophin gene in human muscle and nonmuscle tissue, Nature, 333 (1988) 858-860. 10 Choi, D.W., Calcium mediated neurotoxicity: relationship to specific channel types and role in ishemic damage, Trends Neurosci., 11 (1988) 465-469. 11 Connor, J.A., Tseng, H.-Y. and Hockberger, P.E., Depolarization- and transmitter-induced changes in intracellular Ca2+ of rat cerebellar granule cells in explant cultures, J. Neurosci., 7 (1987) 1384-1400. 12 Courchesne, E., Neuroanatomic imaging in autism, Pediatrics, 87 (1991) 781-790. 13 Cull-Candy, S.G., Howe, J.R. and Ogden, D.C., Noise and single channels activated by excitatory amino acids in rat cere-
54 26 Jagadha, V. and Becket, L.E., Brain morphology in Duchenne muscular dystrophy: a Golgi study, Pediat. Neurol., 4 (1988) 87-92. 27 Kao, J.P., Harootunian, A.T. and Tsien, R.Y., Photochemically generated calcium pulses and their detection by fluo-3, J. Biol. Chem., 264 (1989) 8179-8184. 28 Kingsbury, A.E., Callo, V., Woodhams, P.L. and Balazs, R., Survival, morphology and adhesion properties of cerebellar interneurones cultured in chemically defined and serum-supplemented medium, Dev. Brain Res., 17 (1985) 17-25. 29 Lasher, R.S. and Zagon, I.S., The effect on potassium of neuronal differentiation in cultures of dissociated newborn rat cerebellum, Brain Res., 41 (1972) 482-488. 30 Leiner, H.C., Leiner, A.L. and Dow, R.S., Reappraising the cerebellum: what does the hindbrain contribute to the forebrain?, Behav. Neurosci., 103 (1989) 998-1008. 31 Lidov, H.G.W., Byers, T.J., Watkins, S.C. and Kunkel, L.M., Localization of dystrophin to postsynaptic regions of central nervous system cortical neurons, Nature, 348 (1990) 725-728. 32 Messer, A., The maintenance and identification of mouse cerebellar granule cells in monolayer culture, Brain Res., 130 (1977) 1-12. 33 Minta, A. and Tsien, R.Y., Fluorescent indicators for cytosolic sodium, J. Biol. Chem., 264 (1989) 19449-19457. 34 Monaghan, D.T., Bridges, R.J. and Cotman, C.W., The excitatory amino acid receptors: their classes, pharmacology, and distinct properties in the function of the central nervous system, Annu. Rev. Pharmacol. Toxicol., 29 (1989) 365-402. 35 Moser, H., Duchenne muscular dystrophy: pathogenic aspects and genetic prevention, Hum. Genet., 66 (1984) 17-40. 36 Negulescu, P.A., Harootunian, A., Tsien, R.Y. and Machen, T.E., Fluorescence measurements of cytosolic free Na concentration, influx and efflux in gastric cells, Cell. Regul., 1 (1990) 259-268. 37 Nudel, U., Robzyk, K. and Yaffe, D., Expression of the putative Duchenne muscular dystrophy gene in differentiated myogenic cultures and the brain, Nature, 331 (1988) 635-638. 38 Nudel, U., Zuk, D., Einat, P., Zeelon, E., Levy, Z., Neuman, S. and Yaffe, D., Duchenne muscular dystrophy gene product is not identical in muscle and brain, Nature, 337 (1989) 76-78. 39 Ohlendieck, K., Ervasti, J.M., Snook, J.B. and Campbell, K.P., Dystrophin-glycoprotein complex is highly enriched in isolated skeletal muscle sarcolemma, J. Cell Biol., 112 (1991) 135-148.
40 Poenie, M., Alderton, J., Steinhardt, R. and Tsien, R., Calcium rises abruptly and briefly throughout the cell at the onset of anaphase, Science, 233 (1986) 886-889. 41 Sciancalepore, M., Forti, L. and Moran, O., Changes of N-methyl-n-aspartate activated channels of cerebellar granule cells with days in culture, Biochem. Biophys. Res. Commun., 165 (1989) 481-487. 42 Sell, F.J., Blank, N.K. and Leiman, A.L., Toxic effects of kainic acid on mouse cerebellum in tissue culture, Brain Res., 161 (1979) 253-265. 43 Sorimachi, M., Morita, Y. and Nakamura, H., Possible regulation of the cytosolic-free calcium concentration by Na ÷ spikes in immature cerebellar Purkinje cells, Neurosci. Left., 111 (1990) 133. 44 Sugita, H., Arahata, K., Ishiguro, T., Suhara, Y., Tsukahara, T.. Ishiura, S., Eguchi, C., Nonaki, I. and Ozawa, E., Negative immunostaining of Duchenne muscular dystrophy and muscle surface membrane with antibody against synthetic peptide fragment predicted from DMD cDNA, Proc. Jpn. Acad., 64B (1987) 37-39. 45 Thangnipon, W., Kingsbury, A., Webb, M. and Balazs, R., Observations on rat cerebellar cells in vitro: influence of substratum, potassium concentration and relationship between neurons and astrocytes, Dev. Brain Res., 11 (1983) 177-189. 46 Tsien, R.Y., Rink, T.J. and Poenie, M., Measurement of cytosolic free Ca 2+ in individual small cells using fluorescence microscopy with dual excitation wavelengths, Cell Calcium, 6 (1985) 145-157. 47 Turner, ER., Westwood, T., Regan, C.M. and Steinhardt, R.A., Increased protein degradation results from elevated free calcium levels found in muscle from mdx mice, Nature, 335 (1988) 735-738. 48 Turner, ER., Fong, P., Denetclaw, W.E and Steinhardt, R.A., Increased calcium influx in dystrophic muscle, J. Cell Biol., 115 (1991) 1701-1712. 49 Wallesch, C.-W. and Horn, A., Long-term effects of cerebellar pathology on cognitive functions, Brain Cogn., 14 (1990) 1925. 50 Zubryzycka-Gaarn, E.E., Bulman, D.E., Karpati, G., Burghes, A.H.M., Belfall, B., Klamut, H.J., Talbot, J., Hodges, R.S., Ray, P.N. and Worton, R.G., The Duchenne muscular dystrophy gene product is localized in the sarcolemma of human skeletal muscle, Nature, 333 (1988) 466-469.
49
BRES 17619
Regulation of intracellular free calcium in normal and dystrophic mouse cerebellar neurons F. Woodward Hopf and Richard A. Steinhardt Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720 (USA) (Accepted 26 November 1991) Key words: Cerebellum; Granule neuron; Calcium; Muscular dystrophy; Fura-2 We measured free intracellular calcium ([Ca2+]i) in cultured cerebellar granule cells from normal and mdx mice. Resting levels of ([Ca2+]i) were 24% higher in the dystrophic neurons (normal: 61.2 + 1.5 nM calcium, n = 104; dystrophic: 76.1 + 2.4 nM calcium, n = 136, P <0.01). Distrophic neurons showed a significantly greater increase in ([Ca2÷]i) in the presence of elevated (18 mM) extracellular calcium levels. Resting sodium levels ([Na+]i), however, were found to be similar in normal and dystrophic granule neurons. In addition, sodium influx rates after ouabain inhibition of the Na+/K + ATPase were also identical. Therefore, the increased permeability of granule neurons was specific to calcium, and did not result from a non-selective cation-permeable conductance. Unlike granule cells, astrocytes do not express dystrophin. Glial cells from normal and dystrophic mice showed no difference in their resting free calcium levels or their response to a high calcium load. Thus, cerebellar granule neurons from mdx mice show a calcium-specific regulatory defect similar to that found in dystrophic muscle fibers 15'47'48, while cerebellar glial cells, which do not normally express dystrophin, have no such defect. INTRODUCTION Duchenne Muscular Dystrophy (DMD) is an X-linked genetic disease in humans characterized by skeletal muscle fiber degeneration, and replacement with connective tissue. In addition, 30% of the D M D cases express a mild, non-progressive mental retardation 35, although no consistent histopathological alterations have been found 26'35. Patients with D M D have a deletion in the gene encoding for the 400 kDa protein dystrophin, and thus do not express functional dystrophin 23. D M D can be studied using a mouse model, the m d x mouse, which has genetic, biochemical, and physiological defects similar to those observed in D M D individuals 4. In normal muscle, dystrophin is located primarily on the inner surface of the sarcolemma 44'5°. Dystrophin has some sequence homology with a-actinin and spectrin, and is bound to several integral membrane glycoproteins 7"39. The function of dystrophin is unknown. Whole brain homogenates express the dystrophin protein 24'31 and m R N A 8'9"24'37. However, brain dystrophin m R N A is spliced differently than that found in muscle 14'38 and is transcribed from a different p r o m o t e r 3. In the CNS, dystrophin has been found in neurons of the cerebellum and cerebrum, but not in astrocytes in any location 24'31. Early observations that total muscle calcium content is elevated in D M D patients 2 suggested a role for calcium in the etiology of dystrophy. In the mouse, the
resting level of free intracellular cytosolic calcium ([Ca2+]i) is higher in mdx (92 + 9.8 nM) than in normal (40 + 2.8 nM) isolated skeletal muscle fibers 47. Furthermore, after a fiber is subjected to an ten-fold increase in extracellular calcium (to 18 mM), ([Ca2+]i) rises, but to a significantly greater level in dystrophic fibers 47. This is also observed in mouse and human cultured myotubes 15. The calcium regulatory defect might be due to an increased ion influx across the sarcolemma, or an impaired efflux. Studies of calcium resequestration rates after electrical stimulation demonstrate that calcium efflux pathways function normally in dystrophic muscle fibers, suggesting that any regulatory impairment must be an ion influx 48. This influx might be due to a specific calcium leak or a non-specific leak admitting other cations. When the Na+/K+-ATPase is inhibited with ouabain, intracellular sodium ([Na+]i) rises due to an inward sodium leak. No difference in the initial rate of rise of [Na÷]i is found between normal and dystrophic muscle cells, arguing in favor of a calcium-specific sarcolemmal leak 48. Using the patch-clamp technique, cultured mouse and human myotubes are found to have a calcium leak channel which shows altered behavior in dystrophic cells 15. Compared to normal myotubes, the mean channel closed time of dystrophic cells is greatly reduced, resulting in an increased open probability. Other channel properties (mean open time, single channel conductance, ion selec-
Correspondence: R.A. Steinhardt, Dept. of Molecular and Cell Biology, Division of Cell and Developmental Biology, University of California at Berkeley, Berkeley, CA 94720, USA.
50 tivity, and b e h a v i o r in the p r e s e n c e of p h a r m a c o l o g i c a l agents) are similar b e t w e e n the two fiber types. T h e inc r e a s e d o p e n p r o b a b i l i t y of t h e c a l c i u m l e a k c h a n n e l m a y be r e s p o n s i b l e for the o b s e r v e d e l e v a t e d influx of calcium into d y s t r o p h i c cells. A c a l c i u m l e a k c h a n n e l has b e e n f o u n d in c e r e b e l l a r g r a n u l e n e u r o n s ; l e a k c h a n n e l s
tifacts associated with single intensity measurements, which may be effected by dye leakage or changes in cell thickness TM. Some experiments included astrocytes from 4-week-old cultures, to allow measurement without the interference of associated granule cells, which have died by this time. Measurements from 4-week glial cells were not different from 3-day-old cultures. The approximate actual ([Ca2])i can be calculated from the following formula18:
f r o m d y s t r o p h i c n e u r o n s possess a l t e r e d c h a n n e l b e h a v [Ca 2+] = b × K d (R
ior similar to that f o u n d in d y s t r o p i c m y o t u b e s 2°. To investigate w h e t h e r g r a n u l e n e u r o n s f r o m dystrophic animals h a v e c a l c i u m r e g u l a t o r y defects similar to those
observed
in
dystrophic
muscle,
we
examined
[Ca2+]i in n e u r o n s at rest and after c h a l l e n g e with a high e x t r a c e l l u l a r calcium load. F u r t h e r m o r e , we e x a m i n e d the calcium-specificity of the r e g u l a t o r y
defect(s)
by
studying c h a n g e s in [Na+]i after o u a b a i n inhibition o f the Na+/K+-ATPase. MATERIALS AND METHODS Isolation and maintenance o f cultured cells (modified from re.[. 25) All experiments in this study used the C57 mdx mouse, or the corresponding normal mouse strain. The cerebelli of a litter of postnatal day 7 mice were removed, pooled, and minced. The tissue was incubated on a shaker at 37°C for 35-40 min in 20 U/ml papain (Worthington) in a modified Earle's balanced salt solution (BSS; Gibco) containing (in raM) NaCI 117, KCI 5.3, MgSO4 0.8, CaC12 1.8, NaH2PO4.H20 1.0, cysteine 1.0, EDTA 0.5, HEPES 10, NaHCO 3 4.0, glucose 25, pH 7.4. The tissue was removed and triturated 5 or 6 times with a fire polished pasteur pipette in DMEM (Gibco) supplemented with the following components: 10 mM HEPES, 26 mM NaHCO3, 25 mM glucose, 0.5 mM glutamine, 8% fetal calf serum (Hyclone), 100 U/ml penicillin, 0.1 mg/ml streptomycin, 1.5 mg/ml each of BSA (Sigma) and ovomucoid (Sigma), pH 7.4. After trituration 200-500 /tl of the cell suspension was plated onto polylysine-laminin (50/~g/ml of each) coated glass coverslips. The cells were allowed to settle for 3-6 h in a humid incubator at 37°C under a 5% CO2/95% air atmosphere. After settling, 2 ml of medium (DMEM, modified as above, except without BSA and ovomucoid, and with a total of 25 m M [K+] 6'22"29"4"1)was added to each dish. After 2-5 days recovery, the cells were used for microfluorimetry experiments. Just before an experiment, the cells were placed in a normal [K ÷] (5 mM) medium for 15-30 min 11. Experiments included litters from 4 mdx mice and 4 normal mice. Calcium fluorescence measurements We used the AM ester form of fura°2 (Molecular Probes), a membrane-permeant, fluorescent calcium-chelating dye 46. Fura-2 stocks (10 mM) were prepared in dimethyl sulfoxide (DMSO) and stored at -20°C. Prior to cell loading, an aliquot of the fura-2 stock was mixed 1:1 with 25% w/v Pluronic F-127 (Molecular Probes). A dish of cells was loaded for 35-45 min at 20°C in the presence of 10/zM fura-2. After loading, the dish was washed 3 times with fresh Ringer's (BSS plus 26 mM NaHCO3), and placed onto the stage of an inverted Nikon Diaphot-TMD microscope (using a 40× bright field quartz objective). The cells were maintained at 32°C under a 5% CO2/95% air atmosphere. Healthy neuronal cells were selected based on size (6-8 #M) and morphology (bipolar with round or eliptical soma, smooth gray appearance) 21"32. As observed by other investigators 1~, the amount of dye loaded into granule cells is variable. Fura-2 was excited alternately at 350 nm and 385 nm wavelengths. For each, the emission intensity was measured at 510 nm by a photomultiplier tube. The ratio of the intensities at 350 nm over 385 nm was calculated. Ratio measurements eliminate the ar-
-
Rmin)/(Rmax
-
R),
where R is the measured ratio, and Rmin and Rmax are the ratios of fura-2 in CaZ+-free and saturated-Ca 2+ calibration solutions. The K a of fura-2 for calcium at 32°C is 185 nM, and b is the ratio of the 385-nm excitation efficiency in the absence of calcium to the 385-nm excitation efficiency at saturating calcium concentrations. Intracellular Rmm and Rma x are somewhat different from those in calibration buffers, because of differences in intracellular viscosity, ionic strength, or dye binding 4°'46. Therefore, a correction factor was calculated 43. Granule cells were placed in 5/~M ionomycin, a calcium ionophore, and intracellular Rmin and Rn~ax were determined by placing the cells in a nominally Ca2+-free buffer (plus 3 mM EGTA) and a 20 mM Ca 2+ buffer, respectively. Rmin was 0.343, R~,ax was 7.426 and b was 10.57. The correction factor for granule neurons was found to be 0.62, from two experiments. A separate correction factor was not calculated for astrocytes. The cell-permeant fura-2 am-ester is known to enter intracellular compartments, where it contributes to fluorescent emissions, but is not responsive to changes in [Ca2+]i, leading to spurious results ~. To correct for this possibility, cells were placed in digitonin, which permeabilizes the plasma membrane, and leads to rapid loss of cytoplasmic, but not compartmentalized, signal 27. At the end of a normal one hour recording period, digitonin permeabilization (75 /~M; n = 3) led to loss of all but 7% of the fluorescence signal, showing that very little of the dye is compartmentalized. Sodium fluorescence measurements Sodium experiments were similar to calcium as described above, except that intracellular free sodium levels were measured with the sodium chelating dye, sodium-binding benzofuran isopthalate (SBFI, Molecular Probes) 33. Cells were loaded for 60 min using a final concentration of 20/~M SBFI AM ester (mixed with an equal volume Pluronic F-127, in DMSO), then washed with BSS 3 times prior to experiments. Excitation and emission wavelengths were the same as for fura-2. The rate of sodium leak was examined after 3.3 mM ouabain was added to inhibit the Na+/K +- ATPase 19"36. Intracellular dye calibrations were performed by permeabilizing the cells with a mixture of 15/~M monensin and 15/~M nigericin. External sodium concentration was varied as previously described 36, and the fluorescence ratio at each sodium concentration was monitored. All statistics were calculated using a one- or two-tailed Student t-test. All values are expressed as the mean + S.E.M.
RESULTS I s o l a t e d cell cultures w e r e m o r p h o l o g i c a l l y similar to t h o s e s e e n by o t h e r investigators 28'32'45. H o w e v e r , to assure that the cells studied w e r e g r a n u l e cells, kainic acid ( K A ) n e u r o t o x i c i t y was tested. W h e n c u l t u r e d in 100/~M K A , all n e u r o n a l types e x c e p t g r a n u l e n e u r o n s die within 48 h 42. A f t e r 4 days in K A , 70% of the n e u r o n s in o u r cultures s u r v i v e d (n = 3). A s t r o c y t e s w e r e u n a f f e c t e d by the t r e a t m e n t . M o r e o v e r , o t h e r c e r e b e l l a r n e u r o n a l types ( P u r k i n j e , G o l g i , basket, stellate) do n o t survive in significant n u m b e r s w h e n t a k e n f r o m p o s t n a t a l animals 22'
51 GLU APV Hi [K+I
130' 120
500
A
,
110' 100"
400
90" 80"
300 "~
70-
200
60'
100
50" 40
0
I
0
I
1000
I
I
2000
I
I
3000
I
)
i
i
)
5
15
25
35
time (rain)
I
4000
seconds Fig. 1. Example of a fura-2 experiment, showing some characteristic responses of a granule neuron. This trace shows the response of a normal granule cell to 2 pulses of 100 #M glutamate (GLU; the endogenous neurotransmitter), and to a 25 mM [K+] depolarizing pulse. At the end of the second GLU pulse, 200 #M APV (an NMDA receptor agonist) was added to demonstrate the presence of NMDA receptors on these cells.
28,32,45; those that do survive are distinguishable by larger size (15/~M) and morphology (generally stellate as opposed to bipolar). Fibroblasts and endothelial cells compose less than 1% of the non-neuronal cells in these cultures 32'45, especially in the presence of serum 28. Fig. 1 shows a typical response of a granule neuron to rapid bath applications of 100/~M glutamate (Glu), the endogenous neutrotransmitter 6'16, and 25 mM [K+]. When Glu (n = 5) was applied, [Ca2+]i r o s e rapidly, and remained at an elevated plateau level. In response to a depolarizing [K ÷] application (n = 6), [Ca2+]i r o s e sharply, then dropped rapidly to a plateau above the resting [Ca2÷]i. Both Glu and high [K ÷] responses were fully reversible; when either agonist was replaced by normal Ringer's, [Ca2+]i returned to baseline. These responses are consistent with those seen by other investigators 5,n. N-Methyl-o-aspartate ( N M D A ) receptors are present in granule neurons at t h i s a g e 5'13'17'41. Application of DL-2-amino-5-phosphonovaleric acid (APV, 200 #M, n = 4), an N M D A receptor antagonist 34, partially reduced the Glu-induced [Ca2+]i rise, confirming the presence of N M D A receptors in this cell preparation. The difference in resting [Ca2+]i o f mdx and normal granule neurons was small but significant (normal: 61.2 + 1.5 riM, n = 104; dystrophic: 76.1 + 2.4 nM, n = 136, P < 0.01). These resting values are within the range observed by other investigators 5,n. Astrocytes were chosen for comparison with neurons because they do not express detectable levels of dystrophin 24'31. Astrocytes from normal and dystrophic mice showed no difference in their resting [Ca2+]i (normal: 66.6 + 3.4 nM, n = 28;
120" 110
B
100
E E
90 80
¢3
70 60 50 -5
i
i
!
5
15
25
3'5
time (rain) Fig. 2. [Ca2+]ichanges in normal and dystrophic cerebellar granule neurons (A) and astrocytes (B) in response to a high calcium stress. At time zero, the normal medium containing 1.8 mM calcium was replaced by a high calcium (18 raM) medium. Calcium values at time zero are corrected to the total population means. [Ca2+]i of dystrophic neurons (A; n = 26) was significantly higher than normal (O; n = 21) at all time points ( , ; P < 0.01). [Ca2÷]i of dystrophic (A; n = 10) and normal (O; n = 11) astrocytes was not significantly different at any time point (P > 0.25). Cells not shifted to a high calcium levels showed no change in [Ca2÷]i (0; granule neurons: n = 16 for normal, n = 20 for dystrophic; astrocytes: n = 5 for normal, n = 9 for dystrophic; only normal shown for both cell types).
dystrophic: 65.8 + 3.5 nM, n = 49, P > 0.25). In the absence of perturbations, both granule cells and astrocytes show stable resting [Ca2+]i for at least 30 min. To investigate possible defects in calcium homeostatic regulation across the plasma membrane, the cells were subjected to an external calcium load (Fig. 2). At time zero, calcium was added to a dish to give a total external calcium concentration of 18 mM (10 times the normal calcium concentration of 1.8 mM). Compared to normal granule neurons, mdx neurons showed a significantly elevated [CaZ+]i, at all time points (P < 0.01) (Fig. 2A). Astrocytes from normal and dystrophic mice showed no difference in their response to high calcium levels at any time point (P > 0.25) (Fig. 2B). These data suggest that dystrophic neurons are defective in their regulation of calcium fluxes across the plasma membrane.
52 1.6-
1.2 -
creased ion permeability across the plasma membrane of dystrophic granule neurons was specific to calcium, and did not result from a non-specific increase in cation permeability.
1.0-
DISCUSSION
0.8-
The results show that postnatal day 11-13 cerebellar granule neurons lacking dystrophin possess a calciumspecific regulatory defect similar to that observed in dystrophic skeletal muscle cells. This is consistent with the observation 2° that dystrophic granule neurons possess a defect in the regulation of a calcium leak channel similar to that observed in muscle cells of dystrophic origin 15. Astrocytes, which do not express dystrophin, show no differences in calcium regulation. However, the difference in resting [Ca2÷]i b e t w e e n mdx and normal granule neurons is not as great as that observed in muscle cells. Dystrophic mouse muscle fibers 47 and cultured myotubes ~5 show an elevation of 50 nM and 28 nM calcium, respectively, over normal levels. Dystrophic and normal granule neurons only showed a difference of 15.1 nM. However, the regulatory defect in granule neurons in response to high calcium levels is similar to that found in muscle c e l l s 15'47.
1'4. O
"*~
0.6 5
, 15
2'5
, 35
sodium (mM) Fig. 3. Calibration curve for SBFI in granule neurons (n = 5). Intracellular calibration of SBFI fluorescence signals, as described in Materials and Methods, allows approximations of internal sodium values. Note that the dye response is approximately linear in the 0-30 mM [Na÷] range, as previously observed 36. The S.E.M. at 10 mM [Na ÷] is smaller than the symbol used. All cells had ratios in the range corresponding to 0-10 mM sodium. Therefore, the following formula was derived to calculate [Na+]i from a measured ratio: [Na÷] = ((Ratio - 0.79)/0.03).
To test whether the regulatory defect in dystrophic neurons was a non-specific cation leak, or specific to calcium ions, sodium influx in the absence of regulatory mechanisms was measured. The major regulatory mechanism for sodium is the Na+/K÷-ATPase. Therefore, we inhibited this pathway with ouabain (3.3 mM), and measured the initial, maximal rate of passive sodium influx. The resting [Na+]i and sodium influx rate after ouabain addition were estimated from the SBFI calibration curve (Fig. 3). Resting [Na+]i levels were similar in normal and dystrophic neurons. Furthermore, sodium influx rates after inhibition of the Na+/K+-ATPase were also similar (Table I). Two experiments were conducted in very low (61 nM) calcium to control for possible regulatory effects of the Na+/Ca 2+ exchanger. The rate of sodium rise after ouabain addition was similar to that observed in normal (1.8 mM) calcium (data not shown). In summary, there was no difference between normal and dystrophic neurons in either resting [Na+]i or sodium influx after inhibition of the Na+/K+-ATPase. Therefore, the in-
TABLE I
lntracellular free sodium values of granule neurons at rest, and the initial rate of rise of sodium after ouabain inhibition of the Na+/K + pump Cell type
Level at rest (raM Na ÷)
Rate of rise (mM Na+/min)
Normal
5.4 + 0.4, n = 64 5.8 + 0.6, n = 81
1.15 + 0.21, n = 9 1.12 + 0.13, n = 8
mdx
There is no significant difference between normal and m d x granule neurons, either in the resting levels (P > 0.25), or the initial rate of rise following addition of ouabain (P > 0.25).
This more pronounced difference in resting [Ca2+]i observed in muscle cells may be a reflection of the wide distribution of dystrophin under the sarcolemma 44's°. In contrast, in adult mouse brain slices, dystrophin is located on the postsynaptic regions of axodendritic and axosomatic synapses of cerebellar Purkinje neurons, and pyramidal neurons in certain regions of the cerebral cortices 3t. Thus, in neurally derived preparations, dystrophin is localized to a restricted subset of membrane domains. Therefore, the effects of a calcium regulatory impairment might not be evident at resting [Ca2+]i . However, the precise subcellular distribution of dystrophin in neonatal neurons of the stage studied here is unknown. Hence, the effect of dystrophin location on [Ca2+]i in these cells is not clear. It should be noted that Lidov et al. 3~ did not find dystrophin expression in cerebellar granule neurons from normal adult mice in slice. We did not study adult cells. However, our results, and those of Haws et al. 2°, which demonstrate a regulatory defect in neonatal granule neurons from mdx mice, suggest the precense of dystrophin in normal neonatal neurons. Resting [Na+]i does not differ between normal and dystrophic granule neurons. However, tight intracellular sodium regulation by various Nat-related transporters and ATPases may mask any alterations in sodium leak. Inhibition of these regulatory mechanisms would reveal any such increases in sodium influx. However, sodium
53 influx after inhibition of the Na+/K+-ATPase with oua-
stricted neuronal distribution of dystrophin, together
bain was not different between normal and dystrophic
with the t r e m e n d o u s ability of the developing nervous
granule neurons, arguing against the possibility of a dys-
system to compensate for abberations, may contribute to the variability and mildness of the observed mental retardation in dystrophic individuals.
trophy-related sodium leak. The sodium resting levels reported here are lower than those reported using SBFI in other cell types, which are in the range of 8-10 mM [Na+]i (refs. 19, 36, 48). Despite the lower resting levels, the observed rate of rise after the addition of ouabain is similar to that seen by other observers ( - 1 . 0 mM/min (ref. 48), - 3 . 0 mM/min (ref. 36), - 0 . 5 mM/ rain (ref. 19). It is not clear how aberrations in the cerebellum might
In conclusion, dystrophic granule neurons display an impairment in calcium regulation which is similar to that observed in dystrophic muscle 15'47'48. Elevated intracellular calcium accumulation leads to excitotoxic damage and neuronal death u n d e r hypoxic and ishemic conditions 1°. Thus, the defect in calcium regulation reported here may lead to cellular damage in granule neurons in
lead to a cognitive deficit. No clear cerebellar patholog-
a similar m a n n e r , and hence may contribute to the men-
ical syndromes (such as tremors and ataxia) are associated with D M D , although they are seen in older m d x mice 31. However, recent observations of a possible as-
tal retardation sometimes associated with D M D .
sociation between autism and disorganization of the cerebellar vermis ~2 suggest that cerebellar damage may play a role in cognitive dysfunctions3°,49. The normally re-
Acknowledgements. This work was supported by private donations and the Muscular Dystrophy Association. We would like to thank Dr. Peying Fong and Dr. Paul Turner for comments and suggestions on the manuscript.
REFERENCES
bellar granule neurones, J, Physiol., 400 (1988) 189-122. 14 Feener, C.A., Koenig, M. and Kunkel, L.M., Alternative splicing of human dystrophin mRNA generates isoforms at the carboxy terminus, Nature, 338 (1989) 509-511. 15 Fong, P., Turner, P.R., Denetclaw, W.E and Steinhardt, R.A., Increased activity of calcium leak channels in myotubes of Duchenne human and mdx mouse origin, Science, 250 (1990) 673-676. 16 Gallo, V., Ciotti, M.T., Coletti, A., Aloisi, E and Levi, G., Selective release of glutamate from cerebellar granule cells differentiating in culture, Proc. Natl. Acad. Sci. USA, 79 (1982) 7919-7923. 17 Garthwaite, G., Yamini Jr., B. and Garthwaite, J., Selective loss of Purkinje and granule cell responsiveness to N-Methyl-Daspartate in rat cerebellum during development, Dev. Brain Res., 36 (1987) 288-292. 18 Grynkiewicz, G., Poeni, M. and Tsien, R.Y., A new generation of Caz* indicators with greatly improved fluorescence properties, J. Biol. Chem., 260 (1985) 3440-3450. 19 Harootunian, A.T., Kao, J.P., Eckert, B.K. and Tsien, R.Y., Fluorescence ratio imaging of cytosolic free Na ÷ in individual fibroblasts and lymphocytes, J. Biol. Chem., 264 (1989) 1945819467. 20 Haws, C.M. and Lansman, J.B., Calcium-permeable ion channels in cerebellar neurons from mdx mice, Proc. Roy. Soc. Lond. B, 244 (1991) 185-189. 21 Hockberger, P.E., Tseng, H.-Y. and Connor, J.A., Immunocytochemical and electrophysiological differentiation of rat cerebellar granule cells in explant cultures, J. Neurosci., 7 (1987) 1370-1383. 22 Hockberger, P.E., Tseng, H.-Y. and Connor, J.A., Development of rat cerebellar Purkinje cells: electrophysiological properties following acute isolation and in long-term culture, J. Neurosci., 9 (1989) 2258-2271. 23 Hoffman, E.P., Brown Jr., R.H. and Kunkel, L.M. Dystrophin: the protein product of the Duchenne muscular dystrophy locus, Cell, 51 (1987) 919- 928. 24 Hoffman, E.P., Hudecki, M.S., Rosenberg, P.A., Pollina, C.M. and Kunkel, L.M., Cell and fiber-type distribution of dystrophin, Neuron, 1 (1988) 411-420. 25 Huettner, J.E. and Baughman, R.W., Primary culture of identified neurons from the visual cortex of postnatal rats, J. Neurosci., 6 (1986) 3044-3060.
1 Almers, W. and Neher, E., The Ca signal from fura-2 loaded mast cells depends strongly on the method of dye-loading, FEBS Lett., 192 (1985) 13-18. 2 Bertorini, T.E., Bhattacharya, S.K., Palmieri, G.M.A., Chesney, C.M., Pifer, D. and Baker, B., Muscle calcium and magnesium content in Duchenne muscular dystrophy, Neurology, 32 (1982) 1088-1092. 3 Boyce, EM., Beggs, A.H., Feener, C. and Kunkel, L.M., Dystrophin in brain is transcribed from a distant upstream promotor, Proc. Natl. Acad. Sci. U.S.A., 88 (1991) 1276-1280. 4 Bulfield, G., Siller, W.G., Wight, P.A.L. and Moore, K.J., X chromosome linked muscular dystrophy (mdx) in the mouse, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 1189-1192. 5 Burgoyne, R.D., Pearce, I.A. and Cambray-Deakin, M., N-Methyl-D-aspartate raises cytosolic calcium concentration in rat cerebellar granule cells in culture, Neurosci. Lett., 91 (1988) 47-52. 6 Burgoyne, R.D. and Cambray-Deakin, M., The cellular neurobiology of neuronal development: the cerebellar granule cell, Brain Res. Rev., 13 (1988) 77-101. 7 Campbell, K.P. and Kahl, S.D., Assocation of dystrophin and an integral membrane glycoprotein, Nature, 338 (1989) 259262. 8 Chamberlain, J.S., Pearlman, J.A., Muzny, D.M., Gibbs, R.A., Ranier, J.E., Reeves, A.A. and Caskey, C.T., Expression of the murine Duchenne muscular dystrophy gene in muscle and brain, Science, 239 (1988) 1416-1420. 9 Chelly, J., Kaplan, J.-C., Maire, P., Gautron, S. and Kahn, A., Transcription of the dystrophin gene in human muscle and nonmuscle tissue, Nature, 333 (1988) 858-860. 10 Choi, D.W., Calcium mediated neurotoxicity: relationship to specific channel types and role in ishemic damage, Trends Neurosci., 11 (1988) 465-469. 11 Connor, J.A., Tseng, H.-Y. and Hockberger, P.E., Depolarization- and transmitter-induced changes in intracellular Ca2+ of rat cerebellar granule cells in explant cultures, J. Neurosci., 7 (1987) 1384-1400. 12 Courchesne, E., Neuroanatomic imaging in autism, Pediatrics, 87 (1991) 781-790. 13 Cull-Candy, S.G., Howe, J.R. and Ogden, D.C., Noise and single channels activated by excitatory amino acids in rat cere-
54 26 Jagadha, V. and Becket, L.E., Brain morphology in Duchenne muscular dystrophy: a Golgi study, Pediat. Neurol., 4 (1988) 87-92. 27 Kao, J.P., Harootunian, A.T. and Tsien, R.Y., Photochemically generated calcium pulses and their detection by fluo-3, J. Biol. Chem., 264 (1989) 8179-8184. 28 Kingsbury, A.E., Callo, V., Woodhams, P.L. and Balazs, R., Survival, morphology and adhesion properties of cerebellar interneurones cultured in chemically defined and serum-supplemented medium, Dev. Brain Res., 17 (1985) 17-25. 29 Lasher, R.S. and Zagon, I.S., The effect on potassium of neuronal differentiation in cultures of dissociated newborn rat cerebellum, Brain Res., 41 (1972) 482-488. 30 Leiner, H.C., Leiner, A.L. and Dow, R.S., Reappraising the cerebellum: what does the hindbrain contribute to the forebrain?, Behav. Neurosci., 103 (1989) 998-1008. 31 Lidov, H.G.W., Byers, T.J., Watkins, S.C. and Kunkel, L.M., Localization of dystrophin to postsynaptic regions of central nervous system cortical neurons, Nature, 348 (1990) 725-728. 32 Messer, A., The maintenance and identification of mouse cerebellar granule cells in monolayer culture, Brain Res., 130 (1977) 1-12. 33 Minta, A. and Tsien, R.Y., Fluorescent indicators for cytosolic sodium, J. Biol. Chem., 264 (1989) 19449-19457. 34 Monaghan, D.T., Bridges, R.J. and Cotman, C.W., The excitatory amino acid receptors: their classes, pharmacology, and distinct properties in the function of the central nervous system, Annu. Rev. Pharmacol. Toxicol., 29 (1989) 365-402. 35 Moser, H., Duchenne muscular dystrophy: pathogenic aspects and genetic prevention, Hum. Genet., 66 (1984) 17-40. 36 Negulescu, P.A., Harootunian, A., Tsien, R.Y. and Machen, T.E., Fluorescence measurements of cytosolic free Na concentration, influx and efflux in gastric cells, Cell. Regul., 1 (1990) 259-268. 37 Nudel, U., Robzyk, K. and Yaffe, D., Expression of the putative Duchenne muscular dystrophy gene in differentiated myogenic cultures and the brain, Nature, 331 (1988) 635-638. 38 Nudel, U., Zuk, D., Einat, P., Zeelon, E., Levy, Z., Neuman, S. and Yaffe, D., Duchenne muscular dystrophy gene product is not identical in muscle and brain, Nature, 337 (1989) 76-78. 39 Ohlendieck, K., Ervasti, J.M., Snook, J.B. and Campbell, K.P., Dystrophin-glycoprotein complex is highly enriched in isolated skeletal muscle sarcolemma, J. Cell Biol., 112 (1991) 135-148.
40 Poenie, M., Alderton, J., Steinhardt, R. and Tsien, R., Calcium rises abruptly and briefly throughout the cell at the onset of anaphase, Science, 233 (1986) 886-889. 41 Sciancalepore, M., Forti, L. and Moran, O., Changes of N-methyl-n-aspartate activated channels of cerebellar granule cells with days in culture, Biochem. Biophys. Res. Commun., 165 (1989) 481-487. 42 Sell, F.J., Blank, N.K. and Leiman, A.L., Toxic effects of kainic acid on mouse cerebellum in tissue culture, Brain Res., 161 (1979) 253-265. 43 Sorimachi, M., Morita, Y. and Nakamura, H., Possible regulation of the cytosolic-free calcium concentration by Na ÷ spikes in immature cerebellar Purkinje cells, Neurosci. Left., 111 (1990) 133. 44 Sugita, H., Arahata, K., Ishiguro, T., Suhara, Y., Tsukahara, T.. Ishiura, S., Eguchi, C., Nonaki, I. and Ozawa, E., Negative immunostaining of Duchenne muscular dystrophy and muscle surface membrane with antibody against synthetic peptide fragment predicted from DMD cDNA, Proc. Jpn. Acad., 64B (1987) 37-39. 45 Thangnipon, W., Kingsbury, A., Webb, M. and Balazs, R., Observations on rat cerebellar cells in vitro: influence of substratum, potassium concentration and relationship between neurons and astrocytes, Dev. Brain Res., 11 (1983) 177-189. 46 Tsien, R.Y., Rink, T.J. and Poenie, M., Measurement of cytosolic free Ca 2+ in individual small cells using fluorescence microscopy with dual excitation wavelengths, Cell Calcium, 6 (1985) 145-157. 47 Turner, ER., Westwood, T., Regan, C.M. and Steinhardt, R.A., Increased protein degradation results from elevated free calcium levels found in muscle from mdx mice, Nature, 335 (1988) 735-738. 48 Turner, ER., Fong, P., Denetclaw, W.E and Steinhardt, R.A., Increased calcium influx in dystrophic muscle, J. Cell Biol., 115 (1991) 1701-1712. 49 Wallesch, C.-W. and Horn, A., Long-term effects of cerebellar pathology on cognitive functions, Brain Cogn., 14 (1990) 1925. 50 Zubryzycka-Gaarn, E.E., Bulman, D.E., Karpati, G., Burghes, A.H.M., Belfall, B., Klamut, H.J., Talbot, J., Hodges, R.S., Ray, P.N. and Worton, R.G., The Duchenne muscular dystrophy gene product is localized in the sarcolemma of human skeletal muscle, Nature, 333 (1988) 466-469.