Cholinergic stimulation of skeletal muscle cells induces rapid immediate early gene expression: role of intracellular calcium

MOLECULAR BRAIN RESEARCH ELSEVIER

Molecular Brain Research 26 (1994) 55-60

Research Report

Cholinergic stimulation of skeletal muscle cells induces rapid immediate early gene expression: role of intracellular calcium S.R. Abu-Shakra

a,., A . J . C o l e b, R . N . A d a m s

c, D . B . D r a c h m a n

c

a Department of Neurology, Wayne State University, School of Medicine, 4201 St. Antoine, 6E-UHC, Detroit, M148201, USA b Neurology Service, Massachusetts General Hospital, Boston, MA 02114, USA c Departments of Neurology and Netlroscience, Johns Hopkins University, School of Medicine, Baltimore, MD 21205, USA Accepted 26 April 1994

Abstract

Many properties of skeletal muscle are closely regulated by motor nerves. We have shown that nerve stimulation in vivo induced a rapid rise in mRNA for the immediate early gene (IEG) zif268 in stimulated muscle. However, the mechanisms leading to neural regulation of zif268 gene expression in muscle are not yet known. In this study, we used a mammalian skeletal muscle cell line (C2C12) to analyze the role of cholinergic transmission, and calcium flux, in the neural regulation of zif268. Treatment of the C2C12 muscle cells with carbachol, a cholinergic agonist, induced zif268 gene expression rapidly and transiently. This effect was blocked by a-bungarotoxin (a-BuTx), which specifically blocks nicotinic acetylcholine receptors. Treatment with ryanodine or dantrolene, which block the calcium release channel of the sarcoplasmic reticulum, inhibited the carbachol-induced zif268 response essentially completely. Calcium influx produced by A23187, a calcium ionophore, induced an increase of zif268 gene expression equivalent to the effect of carbachol stimulation. These results suggest that the effect of neural stimulation on zif268 may be attributable to cholinergic transmission, and the subsequent release of calcium from the sarcoplasmic reticulum.

Keywords: Skeletal muscle; Immediate early gene; Acetylcholine receptor; Calcium; Intracellular calcium store; Synaptic transmission

1. Introduction

One of the central issues in neurobiology involves the mechanisms by which neurons regulate the properties of the cells they innervate. Several properties of skeletal muscle ceils are closely regulated by motor nerves, including the synthesis and distribution of acetylcholine receptors (AChRs) and membrane ionic channels, and the profile of metabolic enzymes and contractile proteins [9,11,12]. Neuromuscular synaptic transmission plays a key role in mediating the neural regulation of many of these properties [20,21,24,30]. Several second messenger systems have also been shown to play a role in mediating these effects of

* Corresponding author. Fax: (1) (313) 577-7552. 0169-328X/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 9 - 3 2 8 X ( 9 4 ) 0 0 0 9 6 - W

neural activity [15]. However, little is known about the early events that occur in response to synaptic activity in muscle, leading to gene expression. In a previous study, we showed that the immediate early gene (lEG), zif268, which encodes a zinc finger protein [5] was rapidly and markedly induced in skeletal muscle after neural stimulation in vivo [1]. Zif268 belongs to the family of lEGs which encode transcription factors [18,19,22,29] and have now been shown in several systems to be responsive to synaptic activity [1,6,7,31]. The lEGs are thought to regulate programs of gene expression leading to long term phenotypic changes in cells [13,28]. In this study, we have focused on the effects of cholinergic stimulation and calcium flux on zif268 induction in muscle cells. For this purpose, we have used the mouse skeletal muscle cell line C 2 C12 [2,32] derived from myogenic cells, which fuse to form branch-

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S.R. Abu-Shakra ct aL / Molecular Brain Research 26 (1994) 55-00

ing myotubes and express surface AChRs, contractile proteins, and ionic channels. These features closely resemble those of adult non-innervated skeletal muscle. [3,16,23]

primer method. The labelled cDNA probe was added to the same above solution to obtain a specific activity of 1.5 2.0× 106 c p m / m l solution. Filters were hybridized at 42°C for 24-36 h. They were then washed with 2 exchanges of 2 × S S C , (/.1% SDS, followed by 2 exchanges of 0.2×SSC, 0.1% SDS, at 50°C for 15 min each. Filters were exposed to Kodak X A R film at - 7 0 ° C for l - 3 days for production of autoradiograms.

2. Materials and methods 2.5. Quantitation of autodiographic signal and statistical analysis 2.1. Muscle cultures The C2Ct2 myoblasts, obtained form the American Tissue Culture collection (ATCC C R L 1772) were first cultured in 'growth medium', consisting of Dulbecco's modified Eagle's m e d i u m ( D M E M ) and 10% fetal bovine serum (both from J R H Biosciences), to which antibiotics were added. Before the cells reached confluence in 2 - 3 days, they were switched to 'differentiation m e d i u m ' containing 7.5% horse serum (Hazleton Biologics) in D M E M . T h e cells were grown in 35 m m plastic dishes (95% air, 5% CO 2, at 37°C) for 4 - 5 days after the medium switch. The maturity of the cells was assessed by phase contrast microscopy, and by m e a s u r e m e n t of creatine kinase (CK) activity and surface acetylcholine receptors (AChRs).

2.2. Quantitation of surface AChRs and muscle creatine kinase At various days after switching to differentiation medium, the n u m b e r of surface A C h R s was measured using 125I-a-BuTx binding as previously described [8]. CK activity was measured in C2C12 culture homogenates using a standard spectrophotometric method (creatine kinase kit, Sigma).

2.3. Pharmacologic treatment of cultures All pharmacologic treatments were performed in serum-free medium to avoid the known stimulatory effect of serum on I E G expression. Differentiation medium was therefore replaced with serum-free medium one day before the scheduled treatment. In all experiments, 2 groups of 4 culture dishes each were used for each treatment. The drugs dissolved in H 2° (carbachol, a-BuTx), or 10% D M S O (all others), were added directly to the culture medium in 0.2 ml volumes of stock solutions. Control cultures were treated in parallel with the solvent vehicle (0.2 ml of H z O or 10% DMSO). In experiments where 2 drugs were added sequentially, the control group received the corresponding vehicle each time. Carbachol and dantrolene were obtained from Sigma. a-BuTx was obtained from the Miami Serpentarium and repurified by column chromatography. Ryanodine, A23187, and Actinomycin D were obtained from Calbiochem.

Signal intensity of the resultant autoradiograms was quantified using the Loats R densitometer which integrates the density within the area of the bands on the autoradiograms. Values obtained were then used for statistical analysis. For each treatment, the experiment was repeated at least three times using duplicate sets of samples, giving an n _> 6. The mean and standard error of the m e a n (S.E.M.) were calculated from the results. Paired t-test analysis was used to compare simultaneously run samples. To evaluate the relative effect of each drug on zif268 expression, the densitometry readings were normalized, using the simultaeouslyrun control culture readings as 100%. The results were expressed as percent of controls. We then determined the inhibitory effect of each of the drugs on carbachol-induced expression of zif286. For this purpose, we compared the carbachol-induced zif268 signal in cultures treated with carbachol alone, with the signal in cultures treated with drug plus carbachol. The results were given as percent inhibition of carbacholinduced zif268 expression.

3. Results

All experiments were performed 5 or 6 days after the C2C12 cultures were switched to differentiation medium. At this stage the cultures consisted mostly of branched fused myotubes, that expressed surface AChR and CK activity at high levels. At the time of the medium switch (day 0), there was only minimal expression of AChR (< 0.04 x 10 - 2 mol. per dish), and CK (0-3 IU per dish). By day 6, the AChR expression per culture dish was approximately 0.5 × 10 -12 mol, and CK levels rose to approximately 100 IU. Both of these parameters correspond closely to levels expressed by primary rat muscle cultures at the corresponding stage.

_28 S

2.4. Northern analysis" zif 268 The cultures were first washed with ice cold PBS, and total R N A was then extracted using the single step method [4]. Total R N A was separated by gel electrophoresis through 1% agarose, 5% formaldehyde gel, and then transferred to nitrocellulose filters, according to standard procedures [25]. After transfer, filters were photographed under U V light, showing the ethidium bromide-stained ribosomal R N A bands, to ensure equal loading of the wells. Filters were then baked and prehybridized at 56°C for 6 - 1 2 h in a solution containing 50% formamide, 4 x S S C , 4 × D e n h a r d t ' s solution, 0.1% SDS, 0.1% sodium pyrophosphate, and 100 /~g/ml salmon sperm DNA. The c D N A probe for the l E G zif268 [5] was a generous gift of D. Nathans. The probe was labelled with a-32p-dCTP, by the random

CC

_ 18 S

0

@01

A

3

;

10

pM

Fig. 1. Dose-dependent effect of carbachol on zif268 m R N A s shown by Northern blot analysis. Duplicate sets of 4 cultures each were treated with carbachol (CC) at the indicated concentrations, and total R N A was extracted at 90 min after addition of carbachol. North.ern blots were probed and processed as described. 100 ~ M carbachol induced a zif268 m R N A increase (not shown) similar to the 10 /zM effect. The 18S and 28S marks indicate the location of the ribosomal R N A bands.

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S.R. Abu-Shakra et al. / Molecular Brain Research 26 (1994) 55-60

a

b

c

d

I~Control Vehicle

a

b

c

II

Actinomycin D

dle

f

g

I

h

1

3

C

6 Fig. 2. Time course of zif268 mRNA response to carbachoi treatment; performed on parallel sets of cultures and run simultaneously, shown by northern blot analysis. Duplicate sets of 4 cultures each were treated with control vehicle (lanes a and b), or carbaehol (100 /zM; lanes c and d), and total RNA was extracted at indicated times. Northern blots were probed and processed as described. 10 /.~g of total RNA was loaded per lane.

T r e a t m e n t w i t h c a r b a c h o l , a c h o l i n e r g i c a g o n i s t , inc r e a s e d zif268 m R N A l e v e l s in a d o s e - d e p e n d e n t m a n n e r , r e a c h i n g a p l a t e a u at 1 0 - 1 0 0 / x M (Fig. 1). T h e r e f o r e , t h e c a r b a c h o l d o s e u s e d in all s u b s e q u e n t e x p e r i -

450 400 350 300

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.-'6

-

T

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t C

A CC

Fig. 4. Effect of actinomycin D on basal and carbachol-induced zif268 mRNA shown by northern analysis. Control vehicle (lanes a-d) or actinomycin D (4 ~tg/ml" lanes e-h) was added to duplicate sets of 4 cultures each at time = 0. Two hours later, carbachol (100 /~M) (CC) or control vehicle (C) was added to cultures as indicated. One hour later, RNA was extracted. 10 ~g of total RNA was loaded per lane. Northern blots were probed and processed as described. Note the marked inhibition of basal (lanes e and f) and CC-induced (lanes g and h) levels of zif268 mRNA in actinomycin-treated cultures.

ments was 100/~M. Zif268 mRNA levels rose within 1 h a f t e r t h e a d d i t i o n o f c a r b a c h o l , r e m a i n e d e l e v a t e d at 3 h, a n d r e t u r n e d t o b a s a l l e v e l s by 6 h (Fig. 2). T h e m e a n i n c r e a s e in zif268 m R N A signal a b o v e c o n t r o l s w a s 211 + 4 0 % ( S . E . M . ) , at 1 h (Fig. 3). T h e c a r b a c h o l induced IEG response involves increased gene trans c r i p t i o n s i n c e A c t i n o m y c i n D (4 / x g / m l ) , w h i c h inhibits t r a n s c r i p t i o n , d r a m a t i c a l l y r e d u c e d b o t h b a s a l a n d c a r b a c h o l - s t i m u l a t e d zif268 m R N A l e v e l s (Fig. 4). T r e a t m e n t w i t h a - B u T x (2 # g / m l ) , a s p e c i f i c n i c o tinic A C h R b l o c k e r , a b o l i s h e d this r e s p o n s e to c a r b a -

n-8

250 200

g=

150

28 S

n-8 100 50 0

l

z iF 268

18 S

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Fig. 3. Effects of various treatments on zif268 mRNA densitometric signals. CTL, control; carb, carbachol; t~-Butx, a-bungarotoxin; all shown 1 h after treatment. A23187 is shown at 1 and 3 h after treatment. ~ P < 0.01.

C

A

CC

A

Bu

A Ou+CC A

Fig. 5. Effect of a-bungarotoxin pretreatment on carbachol-induced zif268 mRNA response, shown by northern analysis. Duplicate sets of cultures received control vehicle (C), carbachol (CC), abungarotoxin (Bu), or a-bungarotoxin followed 2 h later by carbachol (Bu + CC). Total RNA was extracted 90 min after last treatment. 10 /zg of total RNA was loaded per lane. Northern blots w e r e probed

and processed as described.

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S.R. Abu-Shakra et al. / Molecular Brain Research 26 (1994) 55-60

chol virtually completely (Figs. 5 and 6). To determine whether the carbachol effect is dependent on calcium release from the sarcoplasmic reticulum (SR), we blocked the SR calcium release channels using ryanodine (30 IzM) or dantrolene (30 /xM), concentrations that are known to block calcium effiux [15,17]. Both agents blocked the carbachol-induced zif268 response significantly (Figs. 6 and 7). By contrast, treatment with PN 200-110, which blocks L-type voltage-sensitive calcium channels, did not attenuate the carbachol-induced zif268 response (Fig. 6). Treatment with the calcium ionophore A23187 (1 tzM) resulted in an increase in zif268 mRNA at 1 hr of 84% _+47% above controls, with a maximum increase, equivalent to that of carbachol after 3 h (201% _+63% [P < 0.01]) (Fig. 3).

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4. Discussion Fig. 6. Percent inhibition of carbachol induced zif268 m R N A response produced by the various pretreatments. Sets of cultures were treated at time = 0 with bungarotoxin, ryanodine, dantrolene, PN200-110, or control vehicle. Two hours later, carbachol (100 ~ M ) was added. Total R N A was extracted 90 min after carbachol treatment. Northern blots were analyzed by densitometry, as described. Carb, carbachol; a-Butx, a-bungarotoxin; Ryan., ryanodine; Dant., dantrolene; PN 200, PN 200-110. * P < 0.01, ** P < 0.05.

ryan. .I-

C

CC T

CC T

ZIF268

A

C

&

CC

dant. +

CC Fig. 7. Effect of ryanodine and dantrolene pre-treatment on carbachol-induced zif268 m R N A response, shown by northern blot analysis. T r e a t m e n t sequence as described in Fig. 5. Note marked inhibition of carbachol-induced levels of zif268 m R N A in cultures pretreated with ryanodine or dantrolene. C, control (vehicle); CC, carbachol; ryan., ryanodine; dant., dantrolene.

In a previous in vivo study we reported that a 15 min course of programmed electrical stimulation of the sciatic nerves of mice induced a rapid and marked increase of mRNA for the immediate early gene zif268 in the gastrocnemius muscle [1]. The mechanism by which synaptic activity induces zif268 gene expression in skeletal muscle is not yet known. In this study we focused on potential mechanisms by which neural acitivity could regulate early gene expression in muscle. In order to examine these mechanisms pharmacologically, we used the C2C12 skeletal muscle cell line in a culture system [2,32]. This myogenic cell line is known to fuse, forming myotubes, and to differentiate and express many features of relatively mature but non-innervated muscle. These features include surface AChRs, ionic channels, contractile proteins, and muscle specific enzymes [3,16,23]. The present studies show that cholinergic stimulation of these cells rapidly and markedly increased expression of zif268. This effect was attributable to the action of carbachol on the nicotinic AChR, since it was totally inhibited by the specific antagonist, a-BuTx. Cholinergic stimulation leads to the following sequence of events in skeletal muscle cells: Opening of the AChR channels allows influx of cations and focal depolarization. Upon reaching threshold, the voltagegated Na ÷ channels are activated causing propagated action potentials. When the action potential invades the T-tubules there is activation of the voltage-sensitive calcium channels (VSCC). Activation of L-type VSCC is coupled to calcium release from sarcoplasmic reticulum stores (via ryanodine-sensitive Ca 2+ release channels) leading to contraction. We have therefore examined the effect of calcium flux on zif268 expression. We found that zif268 induction by carbachol is highly dependent on calcium release from sarcoplasmic retie-

S.R. Abu-Shakra et al. / Molecular Brain Research 26 (1994) 55-60

ulum stores (SR), since it was blocked by ryanodine and dantrolene. Both of these agents are known to block Ca 2÷ efflux from the SR by acting on the ryanodine-sensitive Ca 2÷ release channels [15,17,27]. Treatment with A23187, a Ca 2÷ ionophore which leads to calcium influx from the extracellular compartment also resulted in a marked increase in zif268 expression, although the time course of this effect was slower than that of carbachol. This intracellular increase of Ca 2÷ due to Ca 2÷ influx from the extracellular compartment could have directly induced the zif268 response; alternatively the initial Ca 2÷ influx could have led to secondary release of calcium from SR (calcium induced calcium release, CICR), a well established characteristic of SR Ca 2÷ release channels [10,26]. In this study we have not distinguished between these possibilities, although the delayed onset of zif268 expression after A23187 treatment suggests that CICR may be involved in zif268 induction. Our findings show that cholinergic neurotransmission and the secondary release of intracellular Ca 2÷ stores lead to the induction of zif268. This is consistent with previous studies showing zif268 to be highly responsive to glutamatergic stimulation in hippocampus and to synaptic activity in visual cortex [6,31]. However, a unique feature of skeletal muscle is that lEG induction by the neurotransmitter involves Ca 2+ release from intracellular (SR) stores, in contrast to the requirement for calcium influx from an extracellular source in the PC12 cell system [14]. Since the release of SR calcium stores is also the main signal necessary for excitation-contraction coupling, this raises the possibility that zif268 may be involved in regulating the expression of genes that encode contractile proteins or enzymes involved in the contractile process. Our results suggest that cholinergic transmission plays an important role in mediating the l E G response to neural stimulation that has been observed in vivo. Furthermore, the release of intracellular Ca ÷2 stores is the main secondary signal involved in this response. This does not, however, exclude the possibility that other factors released from the axon terminal could play a role in lEG induction in skeletal muscle.

Acknowledgments This work was supported by grants from the Center for Alternatives to Animal Testing, the Muscular Dystrophy Association, the Myasthenia Gravis Foundation (SAS, DBD), NIH RO1 NS23719 (DBD), and NIH NS1370, Epilepsy Foundation of America, and the Klingenstein Foundation (AJC). We wish to thank C. Salemi and M. Bowman for secretarial support, Dr. D. Nathans for the zif268 cDNA, and Drs. P.F. Worley and J.M. Baraban for advice and suggestions.

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References [1] Abu-Shakra, S.R., Cole, A.J. and Drachman, D.B., Nerve stimulation and denervation induce differential patterns of immediate early gene mRNA expression in skeletal muscle, Mol. Brain Res., 18 (1993) 216-220. [2] Blau, H.M., Pavlath, G.K., Hardeman, E.C., Cjiu, C-P., Silberstein, L., Webster, S.G., Miller, S.C. and Webster, C., Plasticity of the differentiated state, Science, 230 (1985) 758-766. [3] Caffrey, J.M., Brown, A.M. and Schneider, M.D., Ca ÷2 and Na + currents in developing skeletal myoblasts are expressed in a sequential program: reversible suppression by transforming growth factor Beta-l, an inhibitor of the myogenic pathway, J. Neurosci., 9 (1989) 3443-3453 [4] Chomczynski, P. and Sacchi, N., Single step method of RNA isolation by acid guanidinium-thiocyanate-phenol-chloroform extraction, Anal. Biochem., 162 (1987) 156-159. [5] Christy, B.A., Lau, L.F. and Nathans, D., A gene activated in mouse 3T3 cells by serum growth factors encodes a protein with 'zinc finger' sequences, Proc. Natl. Acad. Sci. USA, 85 (1988) 7857-7861. [6] Cole, A.J., Saffen, D.W., Baraban, J.M. and Worley, P.F., Rapid increase of an immediate early gene messenger RNA in hippocampal neurons by synaptic NMDA receptor activation, Nature, 340 (1989) 474-476. [7] Cole, A.J., Abu-Shakra, S., Saffen, D.W., Baraban, J.M. and Worley, P.F., Rapid rise in transciption factor mRNAs in rat brain after electroshock-induced seizures, J. Neurochem., 55 (1990) 1920-1927. [8] Drachman, D.B., Angus, C.W., Adams, R.N. and Kao, I., Effect of myasthenic patients' immunoglobulin on acetylcholine receptor turnover: selectivity of the degradation process, Proc Natl. Acad. Sci. USA, 75 (1978) 3422-3426. [9] Drachman, D.B., Stanley, E.F. and Pestronk, A., Neural regulation of muscle properties. In G. Serratrice et al. (Eds.), Neuromuscular Diseases, Raven, New York, NY, 1984, pp. 415-424. [10] Endo, M., Tanaka, M. and Ogawa, Y., Calcium induced release of calcium from the sarcoplasmic reticulum of skinned skeletal muscle fibres, Nature, 228 (1970) 34-36. [11] Evered, D. and Whelan, J. (Eds.), Plasticity of the Neuromuscular System, Ciba Foundation Symposium, 1988, pp. 1-273. [12] Fambrough, D.M., Control of acetylcholine receptors in skeletal muscle, Physiol Rev., 59 (1979) 165-227. [131 Goelet, P., Castelluci, V.F., Schacher, S. and Kandel, E.R., The long and the short of long-term memory - - a molecular framework, Nature, 322 (1986) 419-422. [14] Greenberg, M.E. Ziff, E.B. and Greene, L.A., Stimulation of neuronal acetylcholine receptors induces rapid gene transcription, Science, 234 (1986) 80-83. [15] Klarsfeld, A., Laufer, R., Fontaine, B., Devillers-Thiery, A., Dubrenil, C. and Changeux, J.P., Regulation of muscle AChR a-subunit gene expression by electrical activity: involvement of protein kinase C and Ca +E, Neuron, 2 (1989) 1229-1239. [16] Kubo, Y., Comparison of initial stages of muscle differentiation in rat and mouse myoblastic and mouse mesodermal stem cell lines, J. Physiol., 442 (1991) 743-759. [17] Lai, F.A. and Meissner, G., The muscle ryanodine receptor and its Intrinsic Ca +2 channel activity. Mini-Review, J. Bioenerg. Biomembr., 21 (1989) 227-246. [18] Lau, L.F. and Nathans, D., Expression of a set of growth-related immediate early genes in Balbc/3T3 cells: coordinate regulation with c-fos and c-myc, Proc. Natl. Acad. Sci. USA, 84 (1987) 1182-1186. [19] Lemaire, P., Revelant O, Bravo, R. and Charnay, P., Two mouse genes encoding potential transcription factors with identical DNA-binding domains are activated by growth factors in cultured cells, Proc. Natl. Acad. Sci. USA, 85 (1988) 4691-4695

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[20] Lipsky, N.G., Drachman, D.B., Pestronk, A. and Shih, P.J.. Neural regulation of mRNA for the a-subunit of acetylcholine receptors: role of neuromuscular transmission, Exp. Neurol., 1(/5 (1989) 171-176. [21] Lomo, T. and Rosenthal, J., Control of acetylcholine sensitivity by muscle activity in the rat, J. Physiol., 221 (1972)493-513. [22] Milbrandt, J., A nerve growth factor-induced gene encodes a possible transcriptional regulatory factor, Science, 238 (1987) 797-799. [23] Miller, J.B., Regulation of acetylcholine receptors in the mouse muscle cell line C2, Exp. Cell Res., 154 (1984) 256-269. [24] Salpeter, M.M., Development and neural control of the neuromuscular junction and of the junctional acetylcholine receptor. In M.M. Salpeter (Ed.), Vertebrate Neuromuscular Juction, Liss, New York, NY, 1987, pp. 55-115 [25] Sambrook, J., Fritsch, E. and Maniatis, T., Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. [26] Smith, J.S., Coronado, R. and Meissner, G., Single channel measurements of the calcium release channel from skeletal muscle sarcoplasmic reticulum, activation by Ca +2 and ATP and modulation by Mg +2. J. Gen. Physiol., 88 (1986) 573-588.

[27] Smith, J.S., Imagawa, T., Ma, J., Fill, M., Campbell, K.P. and Coronado, P., Purified ryanodine receptor from rabbit skeletal muscle is the calcium release channel of sarcoplamic reticulum, J. Gen. Physiol., 92 (1988) 1-26. [28] Sonnenberg, J.L., Rauscher, F.J. Ill, Morgan, J.l. and Curran, T., Regulation of proenkephalin by Fos and Jun, Science, 246 (1989) 1622-1625. [29] Sukhatme, V.P., Cao, X.M., Chang, L.C., Tsai-Morris, C.H., Stamenkovich, D., Ferreira, P.C. Cohen, D.R., Edwards, S.A., Shows, T.B., Curran, T. et al., A zinc finger encoding gene coregulated with c-fos during growth and differentiation, and after cellular depolarization, Cell, 53 (1988) 37-43. [30] Witzemann, V., Brenner, H.R. and Sakmann, B., Neural factors regulate AChR subunit mRNAs at rat neuromuscular synapses, J. CellBiol., 114, (1991) 125-141. [31] Worley, P.F., Christy, B.A., Nakabeppu, Y., Bhat, R., Cole A.J. and Baraban, J.M., Constitutive expression of zif268 in neocortex is regulated by synaptic activity, Proc. Natl. Acad. Sci. USA, 88 (1991) 5106-5110. [32] Yaffe, D. and Saxel, O., Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle, Nature, 270 (1977) 725-727.