What is/are cross-striations?

Try this:Proc. Nati. Acad. Sci. USA

Vol. 85, pp. 1978-1982, March 1988

Neurobiology

Early cross-striation formation in twitching Xenopus myocytes

in culture

(spontaneous transmitter release/acetylcholine/trophic interaction)

YOSHIAKI KIDOKORO AND MITSUYOSHI SAITO

Jerry Lewis Neuromuscular Research Center, Department of Physiology, University of California, School of Medicine, Los Angeles, CA 90024

Communicated by Susumu Hagiwara, November 9, 1987

ABSTRACT Spontaneous release of neurotransmitter has

been demonstrated in various types of synapses. Its physiolog-

ical significance, however, is still unknown. In nerve-muscle

cultures of embryonic Xenopus laevis, we observed that acetyl-

choline, which is released spontaneously at the synaptic termi-

nal, caused frequent twitches of muscle cells. These muscle cells

developed cross-striations earlier than neighboring non-

twitching cells. This effect of innervation was unaffected by

tetrodotoxin but was blocked by a-bungarotoxin. Repeated

iontophoretic application of acetylcholine or KCI to muscle cells

caused twitches and also accelerated the formation of cross-

striations. Thus twitching apparently promotes lateral align-

ment of myofibrils. It is also known that myosin synthesis is

higher in twitching muscle cells. Therefore, successfully inner-

vated twitching muscle cells may have an advantage for faster

differentiation over neighboring non-twitching muscle cells. We

suggest that spontaneously released transmitter may serve as a

mediator for trophic interaction at forming synapses.

Since the discovery of miniature endplate potentials at the

neuromuscular junction (1), similar spontaneous release of

synaptic transmitter has been observed in many other chem-

ical synapses (2). Although the amplitude of miniature end-

plate potentials is small in adult muscle cells, at newly formed

neuromuscular junctions it is considerably larger due to the

higher input impedance of young myotubes (3). The amplitude

of synaptic potentials increases as acetylcholine (AcCho)

receptors accumulate at the postsynaptic membrane (4).

Spontaneous synaptic potentials are detected soon after a

growth cone contacts a muscle cell (5). In fact, the growth

cone releases AcCho even before contacting a muscle cell (6,

7). The physiological significance of spontaneous release of

transmitter is not yet known. AcCho thus released from

growing neurites may serve as a mediator for trophic interac-

tion.

MATERIALS AND METHODS

The dorsomedial portion of Xenopus embryos at stage 14 (8)

was dissected, and the covering skin was removed by using

a pair of needles. Neurons and myocytes were dissociated in

Ca2+- and Mg2+-free saline as described (9) and plated on

the collagen-coated cover glass in culture medium [0.6x

Dulbecco's modified Eagle's medium (buffered by Hepes to

pH 7.4, GIBCO) supplemented with 5% (vol/vol) horse

serum]. These cultures were maintained at room tempera-

ture (21-23°C) in the glass chamber.

Xenopus myocytes are mononucleated and constitute tail

musculature of the tadpole. Cultured myocytes are flat, and

cross-striation is clearly seen under a phase-contrast micro-

scope (40 x water-immersion objective, Zeiss). About 17 hr

after plating, cultures were rinsed three times with culture

medium to remove unattached cells. The great majority of

attached cells were myocytes, and some of them possessed an

adherent neuron. Typically a few thousand myocytes per dish

attached to the substrate, and 20-40 neurons adhered to the

myocytes.

AcCho and K+ were applied iontophoretically with a

current pulse 1.3 msec long and 100-600 nA in amplitude at

0.1-1 Hz. Electrodes were filled with 1 M AcChoCl dis-

solved in normal saline (120 mM NaCl; 3 mM KCl; 1 mM

CaCl2; 0.5 mM MgCl2; 8 mM Hepes'NaOH; 10 mM glucose;

pH 7.4) or with 3 M KCl dissolved in distilled water. For

AcCho application, a continuous braking current of 30-80

nA was applied to avoid desensitization of receptors.

All experiments were carried out at room temperature

(21-230C).

RESULTS

About two-thirds of myocytes contacted by neurons

twitched spontaneously after 24 hr of culture. These spon-

taneous twitches were blocked by 0.1 uM a-bungarotoxin,

indicating that they were the result of spontaneous release of

AcCho from neurons. Properties of spontaneous synaptic

potentials in Xenopus nerve-muscle cultures were studied

(4, 10). Their amplitudes were extremely variable, and some

spontaneous synaptic potentials were large enough to cause

twitches without muscle action potentials.

About 30 myocytes not contacted by a neuron and >10

myocytes contacted by a neuron in each culture dish were

examined closely under a phase-contrast microscope for

cross-striation formation, and the percentage of striated cells

was determined. A set of six cultures was thus examined at

various intervals after plating (Fig. 1A). Cross-striations in

myocytes not contacted by a neuron first developed =z23 hr

after plating. At first the number of striated bands was low,

and striation was confined to a small portion of the myocyte.

The percentage of myocytes with cross-striations as well as

the extent of striation in each myocyte increased with time.

Forty-four hours after plating virtually all myocytes were

cross-striated. The time for 50%o of myocytes not contacted

by a neuron to acquire cross-striation was measured (Fig.

1A) and found to be 28.5 + 1.0 hr (mean + SD) after

culturing at room temperature (for all four sets of cultures).

Myocytes, contacted by neurons and twitching, developed

cross-striations much earlier (Figs. 1A and 2B) than myo-

cytes not contacted by neurons (Fig. 2A). At 23 hr a majority

of twitching myocytes showed clear cross-striations. Some

myocytes contacted by neurons did not twitch during the

observation period of -5 min. Probably the neurons con-

tacting myocytes were not cholinergic or were cholinergic,

but released AcCho very infrequently. These myocytes were

not striated 23 hr after culturing (Fig. 1A and Fig. 2C),

Abbreviation: AcCho, acetylcholine.

1978

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payment. This article must therefore be hereby marked "advertisement"

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Proc. Natl. Acad. Sci. USA 85 (1988) 1979

A

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20 30 40 50

HOURS AFTER PLATING

B

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HOURS

FIG. 1. (A) Percentage of striated myocytes is plotted against hours after the myocytes were cultured. Filled circles, myocytes not

contacted by a neuron; open circles, myocytes contacted by a neuron and twitching; and open squares, myocytes contacted by a neuron but

not twitching. A smooth curve was drawn "by eye" to estimate the time when 50% of the myocytes not contacted by a neuron had acquired

striation. In this case it was 29 hr after plating (marked by a short vertical bar). (B) Frequency of spontaneous twitching (twitches, no. per min)

is plotted against time. The twitching was observed on the video screen, and twitches were registered with a photodiode placed on the monitor

screen, and the output was recorded on a chart recorder. Twitches were variable in amplitude, and obviously some small twitches were not

recorded. This myocyte had partial striation at the beginning of the recording, and the cross-striation developed further during the observation

period.

indicating that neuron contact by itself does not promote the

formation of cross-striation. Furthermore, when culture

medium contained 0.1 ,uM a-bungarotoxin, none of the

myocytes contacted by neurons twitched or developed

cross-striations ahead of myocytes not contacted by neu-

rons, indicating that activation of AcCho receptors is re-

quired for accelerated formation of cross-striation.

Another way to reduce spontaneous twitching is to in-

crease Mg2+ concentration in culture medium, which re-

duces the frequency of spontaneous transmitter release. In

medium with added 10 mM Mg2 +, muscle cells contacted by

neurons rarely twitched. Only one myocyte contacted by a

neuron twitched infrequently out of 26 examined, whereas in

control cultures 15 myocytes twitched out of 28 cells exam-

ined that were contacted by neurons. No accelerated forma-

tion of cross-striation was observed in high Mg2+ medium.

In contrast, 3 ,uM tetrodotoxin in culture medium neither

affected twitching nor blocked the facilitatory effect of

twitching on the formation of cross-striation, indicating that

Na+ action potentials are not involved in this phenomenon.

Therefore, we conclude that cholinergic neurons, by re-

leasing AcCho, often caused twitches in myocytes and

thereby accelerated the formation of cross-striation.

It has been reported in a rat skeletal muscle cell line that

high K+ medium (25 mM KCl added to culture medium)

promotes formation of cross-striations (11). In Xenopus

cultures, however, addition of KCl to culture medium to 10

or 20 mM did not increase the percentage of cross-striated

myocytes 23 and 31 hr after culturing compared with control.

Thus, maintained depolarization does not accelerate the

formation of cross-striations in Xenopus myocytes.

Myocytes contacted by a neuron twitched at various rates

between 0.1 and 108 twitches per min. In one culture 23 hr

after culturing, the average twitch frequency was 17 + 31

twitches per min (n = 11). The average twitch frequency in

each myocyte, however, increased gradually during the

period when cross-striations were forming (Fig. 1B). The

extent of cross-striation was generally greater in frequently

twitching myocytes than those twitching infrequen