Response of Schwann Cells to Action Potentials in Development

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Science  24 Mar 2000:
Vol. 287, Issue 5461, pp. 2267-2271
DOI: 10.1126/science.287.5461.2267


Sensory axons become functional late in development when Schwann cells (SC) stop proliferating and differentiate into distinct phenotypes. We report that impulse activity in premyelinated axons can inhibit proliferation and differentiation of SCs. This neuron-glial signaling is mediated by adenosine triphosphate acting through P2 receptors on SCs and intracellular signaling pathways involving Ca2+, Ca2+/calmodulin kinase, mitogen-activated protein kinase, cyclic adenosine 3′,5′-monophosphate response element binding protein, and expression of c-fos andKrox-24. Adenosine triphosphate arrests maturation of SCs in an immature morphological stage and prevents expression of O4, myelin basic protein, and the formation of myelin. Through this mechanism, functional activity in the developing nervous system could delay terminal differentiation of SCs until exposure to appropriate axon-derived signals.

Neural impulse activity has a critical influence on development of the nervous system at late stages of prenatal development and early postnatal life. By regulating neuronal survival, outgrowth, synaptic organization, and gene expression, impulse activity in developing neural circuits helps tailor nervous system structure in accordance with functional requirements (1). Much less is known regarding possible activity-dependent regulation of nonneuronal cells (glia) during development. These cells provide essential structural and functional support for developing and adult neurons and undergo marked changes in proliferation, lineage progression, and differentiation during late stages of development when neural impulse activity could provide an instructive influence. The objective of the present study was to determine whether SCs can detect impulses from premyelinated axons and, if so, to identify the signaling pathways responsible and their functional consequences.

Time-lapse confocal microscopy was used to monitor changes in intracellular Ca2+ in SCs in response to electrical stimulation of dorsal root ganglion (DRG) neurons (2). Calcium imaging has been used to detect activity-dependent axon-SC communication in the adult nervous system at the nodes of Ranvier (3) and synaptic terminals (4) in association with K+ buffering and neurotransmitter secretion. However, it is not known whether SCs can detect impulse activity in extrasynaptic regions and in premyelinated axons before formation of nodes of Ranvier. This was investigated by culturing SCs (5) on DRG (6) axons in a preparation equipped with stimulating electrodes (7). Calcium levels increased immediately in neurons in response to action potential firing and activation of voltage-sensitive Ca2+ channels. Fifteen to 150 s after stimulation at 10 Hz (Fig. 1, A and B), intracellular Ca2+ increased to high levels in multiple SCs associated with the axons. The Ca2+ response in SCs varied proportionately with stimuli between 1 and 10 Hz and could be elicited repeatedly by electrical stimulation delivered several minutes after Ca2+ recovery to basal levels (Fig. 1B) (8).

Figure 1

Action potentials in premyelinated axons increase intracellular Ca2+ in SCs (2). (A) Scanning laser confocal microscopy was used to monitor changes in intracellular Ca2+ in SCs and DRG neurons in coculture, with the fluorescent Ca2+ indicator fluo-3/AM. Action potentials induced an immediate influx of Ca2+through voltage-sensitive Ca2+ channels in DRG neurons (arrow) (34 s). This was followed 15 to 150 s later by an increase in intracellular Ca2+ concentration in multiple SCs (*) (48 and 82 s). Bar, 20 μm. The Ca2+ responses for individual cells in (A) are plotted in (B) after stimulation at different frequencies (1 to 10 Hz). Ca2+ increased in DRG neurons (black traces) immediately upon stimulation (indicated by red bars), but responses in SCs (color traces) were delayed and proportionate to the stimulus frequencies, which were 1 , 3, and 10 Hz for (a), (b), and (c), respectively (8). (C) In SCs, the action potential-induced increase in intracellular Ca2+ (a) was blocked by electrical stimulation in the presence of apyrase (30 U/ml) (b), but Ca2+ responses in DRG neurons were not affected. Colors are as in (B). Stimulus frequency in (a) and (b) is 10 Hz; the duration of stimulation is indicated by the red bars. A time-lapse video of this figure may be viewed onScience Online

The delay between the neuronal and the SC response suggests involvement of a soluble signaling molecule released from nonsynaptic regions because synapses do not form in pure DRG cultures (9). The evidence suggests that the Ca2+ response of SCs is mediated by adenosine triphosphate (ATP) released from electrically active DRG neurons. SCs express P2Y-purinergic receptors, and ATP application has been shown to elicit Ca2+ responses in SCs of myelinated and unmyelinated nerves and in culture (10). Direct application of ATP (10 nM to 1 mM) to 2- to 3-day-old SC monocultures induced an immediate and large increase in intracellular Ca2+F/F 0 = 2.9 ± 0.168) followed by sustained low-frequency oscillations.

Activity-dependent release of ATP has been detected from synaptic terminals, but ATP release from nonsynaptic regions of axons has not previously been shown. To investigate this, we assayed culture medium from stimulated and unstimulated cultures of pure DRG neurons for ATP content (11). The measurements showed an activity-dependent increase in the concentration of ATP in conditioned medium (191% increase from control), which was dependent on activation of sodium-dependent action potentials (11). Electrical stimulation in the presence of apyrase (30 U/ml), an enzyme that rapidly degrades extracellular ATP (12), blocked the Ca2+ response in SCs but had no effect on the Ca2+ response in DRG neurons (Fig. 1C). Other factors may be released in response to impulse activity in DRG neurons; however, the blockade by apyrase is compelling evidence that ATP is the activity-dependent signaling molecule in these experiments.

Axon-derived signals have been shown to modulate several transcription factors related to the differentiation of SCs (13). We next investigated whether action potential-induced Ca2+ responses in SCs were of sufficient magnitude to activate signaling pathways regulating genes that control long-term adaptive responses of SCs. The cyclic adenosine 3′,5′-monophosphate (cAMP) response element binding protein (CREB) is an important transcription factor mediating Ca2+-dependent gene expression (14), but activation of CREB in SCs by ATP has not been shown. Immunocytochemical staining showed that CREB was activated by phosphorylation at Ser133 in SCs in response to electrical stimulation of DRG neurons or by direct application of ATP to SCs in monoculture (15) (Fig. 2). ATP-induced CREB phosphorylation was directly proportional to ATP concentration within the range of 10 nM to 1 mM, and no staining was seen when SCs were stimulated electrically in cultures without neurons (16). Electrical stimulation of co-cultures in the presence of apyrase, the purinergic receptor antagonist suramin, or tetrototoxin (TTX) blocked the activity-dependent phosphorylation of CREB in SCs (Fig. 2). Electrical stimulation in the presence of the Ca2+/calmodulin (CaM) kinase inhibitor KN62 (30 μM) or the MEK-1 inhibitor PD098059 (50 μM) significantly inhibited CREB phosphorylation in SCs (P < 0.0001) relative to controls stimulated at 10 Hz (60% reduction in KN62,n = 16; 100% reduction in PD098059; n= 14). These results implicate at least two Ca2+-dependent signaling pathways in the ATP-mediated phosphorylation of CREB.

Figure 2

The transcription factor CREB is phosphorylated in SCs by activity-dependent release of ATP from DRG neurons (15). (Inset) Levels of phosphorylated CREB (P-CREB) in the nucleus of SCs were increased on DRG axons stimulated for 30 min at 10 Hz to fire action potentials or in monocultures of SCs treated with 10 nM to 100 μM ATP for 15 min. The activity-dependent increase in P-CREB was blocked in the presence of 1 μM tetrototoxin (10 Hz + TTX), a 30 μM concentration of the P2 receptor antagonist suramin (10 Hz + surm), or apyrase (27 U/ml) (10 Hz + apy) . Levels of P-CREB returned to baseline 60 min after the 10 Hz stimulation (10 Hz + 60 min) stopped. Pulsed stimulation (phas) was applied for 0.5 s on intervals of 2 s. Error bars indicate SEM (P < 0.0001, one-way ANOVA;n = 58; *P < 0.001 versus control) (35).

Two immediate early genes, c-fos andKrox-24, have been implicated in adaptive responses of cells to extracellular stimulation. c-fos is regulated in part by CREB binding to a CRE element in the promoter (14). The zinc-finger transcription factor krox-24(Zif268, NGFI-A, Erg-1) is expressed in immature and nonmyelinating SCs, where it has been implicated in the control SC differentiation (17). Electrical stimulation also increased expression of both c-fos and Krox-24 in SCs; expression was blocked when stimulation was performed in the presence of apyrase. Direct application of ATP to purified SCs also induced c-fos and Krox-24 mRNA and protein expression in SC monoculture (Fig. 3), indicating a novel mechanism for inducing these genes.

Figure 3

Action potentials induce gene expression in SCs by the release of ATP (15). Immunocytochemical staining for c-Fos (black bars) and Krox-24 (gray bars) showed an increase in nuclear expression of these genes in SCs on axons stimulated for 30 min at 10 Hz (10 Hz) compared with unstimulated axons (0 Hz). Direct application of 100 μM ATP for 15 min stimulates expression of both genes (+ ATP), and electrical stimulation in the presence of apyrase (27 U/ml) blocks the response (10 Hz+apy). No increase in expression of a gene associated with myelinating phenotype, Krox-20, was detected in SCs in response to electrical stimulation or ATP application (16). Error bars indicate SEM. Krox-24,P = 0.000, one-way ANOVA; n = 28; *P < 0.005 versus control. c-Fos, P < 0.006, one-way ANOVA; n = 19; *P < 0.05 versus control (35). (Inset, right) Immunocytochemical staining for Krox-24 in SCs in control (0 Hz) and stimulated (10 Hz) co-cultures. (Inset, left), The ATP-induced increase in mRNA transcripts for both genes is shown by RT-PCR.

We then determined whether this activity-dependent axon-SC communication resulted in functional consequences that may be relevant to SC development. In the perinatal period, SCs undergo a sharp reduction in proliferation and differentiate into either myelinating or nonmyelinating phenotypes (18). This developmental stage coincides with the onset of active spontaneous and stimulus-evoked impulse activity in DRG axons (19). Therefore, SC proliferation was compared on axons firing at different frequencies. The rate of bromodeoxyuridine (BrdU) incorporation into mitotic nuclei was significantly reduced in SCs cultured on axons firing at 10 Hz (20) (Fig. 4), a frequency well within the normal physiological range of firing in DRG axons during the perinatal period (19) when proliferation of SCs declines precipitously (21). Moreover, 24 hours after direct application of 300 μM ATP, the proliferation rate of SCs was reduced significantly in co-culture (P < 0.0009,n = 76) or monoculture (P < 0.009,n = 22), and electrical stimulation in the presence of apyrase blocked the activity-dependent reduction in SC proliferation rate (Fig. 4).

Figure 4

Regulation of SC proliferation by activity-dependent release of ATP from DRG neurons (20). The proliferation rate of SCs was decreased on axons stimulated for 1 hour at 10 Hz (10 Hz) compared with SCs on unstimulated axons (0 Hz). Stimulation in the presence of apyrase (27 U/ml) (10 Hz+apy) prevented the reduction in proliferation rate, and direct application of 300 μM ATP for 24 hours significantly inhibited SC proliferation on unstimulated axons. Error bars indicate SEM (P < 0.0001, one-way ANOVA; n = 126; *P < 0.005 versus control) (35).

After SCs stop proliferating, they begin differentiating into myelinating and nonmyelinating phenotypes in vivo (18). SC differentiation can be initiated in culture by the addition of ascorbic acid (22), which results in marked changes in morphology and in gene expression associated with myelination. The normal maturation of SCs from a spindle-shaped to a more rounded and flattened morphology in vitro (23) was completely prevented by ATP treatment over 4 days (Fig. 5, A and C). In addition, expression of the O4 antigen, a marker of SC lineage progression, was strongly inhibited by ATP in a dose-dependent manner (Fig. 5, B and D) and by a 7- to 10-day phasic stimulation of neurons in co-culture (24). There were no differences in the total number of SCs, or evidence of apoptosis in these co-cultures as determined by cell counts or TUNEL (terminal deoxytransferase–mediated deoxyuridine triphosphate nick end labeling) assay after 1-hour or 7-day ATP treatment (25). In the rat sciatic nerve, O4 expression begins before differentiation into myelinating or nonmyelinating phenotype (26), suggesting that impulse activity could prevent or delay differentiation into either myelinating or nonmyelinating phenotypes.

Figure 5

ATP delays maturation and differentiation of SCs (31, 32). Chronic treatment of SCs with 300 μM ATP in co-culture with DRG neurons for 4 days prevented the normal development from spindle-shaped to rounded, flattened morphology (A and C) and prevented expression of the O4 antigen (B and D) just before differentiation into premyelinated or promyelinated phenotypes (18.2 ± 5.7 O4-positive cells per field control versus 0.03 ± 0.04 ATP;n = 24; t test, P < 0.004) (26). ATP treatment for 10 days prevented the close association of SCs with axons (E and G) and the formation of compact myelin and expression of MBP (F andH). Areas of the co-cultures containing no neuron cell bodies are shown (6). Bar in (H) indicates 20 μm; bar indicates 5 μm in (A) and (C), 10 μm in (B) and (D), and 20 μm in (E) through (F).

The developmental role of impulse activity in regulating glial responses such as myelination is controversial. Some studies suggest that impulse activity inhibits myelination (27) and others indicate that impulse activity promotes myelination (28) or has no effect (29). The present findings suggest that impulse activity could influence myelination by inhibiting the maturation and differentiation of SCs. Krox-20 has been associated with induction of genes characteristic of myelinating SCs (30). Expression of both Krox-20 (30) and galactocerebroside (Gal-C) (23), a myelin glycolipid, is reduced in SCs differentiating into the nonmyelinating phenotype. When long-term cultures were co-treated with ATP and ascorbic acid (31), the normal down-regulation of Krox-20 and Gal-C was inhibited, indicating failure to differentiate beyond the immature stage (89.7% versus 50% Krox-20+ cells, ATP versus control, P = 0.000, χ2 test,n = 1691 cells; and 7.2 ± 0.41 versus 0.5 ± 0.28 Gal-C positive profiles per field in ATP versus control,n = 8 cultures, P < 0.0001,t test). SCs remained spindle-shaped and were not aligned with axons even after 2 weeks of co-treatment with ascorbic acid and ATP (Fig. 5, E and G). Moreover, no compact myelin was detected in ATP-treated cultures, and myelin basic protein (MBP), a component of compact myelin, was not detected by immunocytochemistry (32) (Fig. 5, F and H).

The immunological and morphological evidence suggests that ATP arrests SC maturation before differentiation into either the myelinating or nonmyelinating phenotypes. Impulse activity may delay terminal differentiation of SCs until exposure to appropriate myelin-inducing signals. Many myelination signals are axon-specific and, like the caliber of the axon, are related to maturation of individual axons. Impulse activity may promote myelination by increasing the pool of SCs in a predifferentiated state available to respond to myelin-inducing signals.

SC proliferation, lineage progression, and differentiation are highly regulated by extrinsic and axonally derived factors. Firing frequency and pattern change with the developmental stage of the axon. Impulse activity may be one signal from the axon indicating the appropriate time for SCs to exit the cell cycle and become responsive to factors controlling differentiation into phenotypes necessary for SC functions related to neuronal excitability. A large number of factors contribute to regulation of SC development and proliferation. The present study indicates that electrical activity, acting through the release of ATP, can have a profound influence on SC development, proliferation, and gene expression.

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