Potentiation of quantal secretion by insulin-like growth factor-1 at developing motoneurons in Xenopus cell culture
- Corresponding author J.-C. Liou: Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan. Email: netliou{at}mail.nsysu.edu.tw
Abstract
Although evidence suggests that insulin-like growth factor (IGF) plays an important role in the development and growth of the nervous system, the effect of IGF-1 in the regulation of neurotransmitter release in the peripheral nervous system remains unknown. Here we show that acute application of IGF-1, a factor widely expressed in developing myocytes, dose-dependently enhances the spontaneous acetylcholine (ACh) secretion at developing neuromuscular synapses in Xenopus cell culture using whole-cell patch clamp recording. We studied the role of endogenously released IGF-1 by examining the effect of IGF-1 antibody on the frequency of spontaneous synaptic currents (SSCs) at high-activity synapses, and found SSC frequency was markedly reduced at these high-activity synapses. The IGF-1-induced synaptic potentiation was not abolished when Ca2+ was eliminated from the culture medium or there was bath-application of the pharmacological Ca2+ channel inhibitor Cd2+, indicating that Ca2+ influxes through voltage-activated Ca2+ channels are not required. Application of membrane-permeable inhibitors of inositol 1,4,5-trisphosphate (IP3) or ryanodine receptors effectively occluded the increase of SSC frequency elicited by IGF-I. Treating cells with the phosphoinositide-3 kinase (PI3-K) inhibitors wortmannin or LY294002, and with phospholipase Cγ (PLCγ) inhibitor U73122, but not the inhibitor of mitogen-activated protein (MAP) kinase PD98059, abolished IGF-1-induced synaptic potentiation. Taken collectively, these results suggest that endogenously released IGF-1 from myocytes elicits Ca2+ release from IP3- and/or ryanodine-sensitive intracellular Ca2+ stores of the presynaptic nerve terminal. This is done via PI3-K and PLCγ signalling cascades, leading to an enhancement of spontaneous transmitter release.
Successful synaptic transmission at the neuromuscular junction depends on the precise alignment of the nerve terminals with the postsynaptic specialization of the muscle fibre. The contact between presynaptic motoneurons and target muscle cells leading to the exchange of electrical signals and chemical factors serves to co-ordinate their spatial and temporal differentiation (Connor & Smith, 1994; Sanes & Lichtman, 1999). A rich history of research dating back to the time of Ramon y Cajal and Hamburger supports the observations that neuronal differentiation appears to be dependent on retrograde signals from the target, and many neurotrophic factors have been characterized and demonstrated to play important roles in the development of the neuron (Black, 1999; Bennet et al. 2002). During development, particular sets of genes are expressed at specific times and in specific contexts. The discovery that expression of IGF-1 in the developing skeletal muscle increases with the formation of differentiated skeletal muscle fibres and decreases to very low levels in the adult during the process of synapse elimination, paved the way for an expanding field of research that has focused on the role of IGF-1 in synapse formation (Ishii, 1989; Caroni, 1993).
Many experimental approaches have suggested a regulatory role for IGF-1 in the development of the nervous system: (a) both in vitro and in vivo, IGF-1 increases the rate of motor axon elongation, branching and synapse formation (Caroni & Grandes, 1990; Caroni et al. 1994); (b) blockade of synaptic activity increases the expression of IGF-1 and IGF-2 mRNA in skeletal muscle in vivo (Caroni, 1993); (c) IGF administration prevents motoneuron death and supports the re-establishment of synapses following nerve injury (Vergani et al. 1998; Lutz et al. 1999); (d) in vivo treatment of neuromuscularly blocked embryos with IGF-binding proteins (IGF-BPs) that interfere with the actions of endogenous IGFs reduce motoneuron survival, axon branching and synapse formation (Caroni et al. 1994; Pu et al. 1999). Besides being considered as a mitogen with long-term effects, IGF-1 has now also been demonstrated to be a rapid neuromodulator. It has been suggested that IGF-1 regulates ion channel currents and neuronal excitability (Blair & Marshall, 1997; Kar et al. 1997).
IGF-1 is a polypeptide hormone that is structurally similar to insulin and IGF-II. The diverse biological actions of insulin and IGF-1 are initiated by binding of the polypeptides to their respective cell surface receptors. IGF-1 interacts primarily with the heterotetrameric (β2β2) IGF-1 receptor, a transmembrane protein tyrosine kinase that is structurally related to the insulin receptor. Binding of IGF-1 to its receptor induces receptor autophosphorylation in the intracellular kinase domain of the β-subunit, which results in the activation of several cellular signal transduction cascades, including MAP kinase (Kim et al. 1997; Mehrhof et al. 2001), PI3-K (Blair et al. 1999; Leski et al. 2000; Mehrhof et al. 2001) and PLCγ (Foncea et al. 1997; Hong et al. 2001).
The activity of neuromuscular transmission at developing synapses is crucial in synaptic maturation and competition as well as in the differentiation of postsynaptic properties (Kidokoro & Saito, 1988; Lo & Poo, 1991; Dan & Poo, 1992; Balice-Gordon & Lichtman, 1993). The IGF-1 receptor is present in both developing and mature neurons (Kar et al. 1993) and the expression of IGF-1 in the developing skeletal muscle increases with the formation of differentiated skeletal muscle fibres before innervations. Although all the evidence to date supports the notion that IGF-1 is essential for neuronal growth and development, what is not as well understood is the role of IGF-1 in synaptogenesis. In the present study we examine the acute effect of IGF-1 on synaptic transmission, which provides insight into the related mechanisms in cultured Xenopus nerve-muscle preparation by virtue of its simplicity and easy accessibility. Cultures derived from embryos of the Xenopus offer several advantages in studying the early events of synaptogenesis. First, previous studies of neuromuscular synapses in Xenopus cell cultures have provided a detailed description of the morphological and physiological events associated with the timing of development. Second, Xenopus myoblasts do not fuse to form poly-nucleated myotubes in culture, remaining mono-nucleated as long as they survive. This provides us with good conditions for using whole-cell patch clamp to record the synaptic activity. Third, the cells remain viable for many hours in open air at room temperature on the microscope stage, which is ideal for electrophysiological recordings (Tabti & Poo, 1991). Our results suggest that IGF-1 released endogenously from myocytes may serve as a positive trophic factor at the developing neuromuscular junction; the underlying signalling mechanisms involved are also addressed.
METHODS
Cell culture
Xenopus nerve-muscle cultures were prepared as previously reported (Dan & Poo, 1992). Briefly, the neural tube and the associated myotomal tissue of 1-day-old (stage 20–22; Nieuwkoop & Faber, 1967) Xenopus embryos were dissected and dissociated in Ca2+- and Mg2+-free Ringer solution supplemented with 0.15 mm EDTA. The dissociated cells were plated and used for experiments after incubation at room temperature (20–25 °C) for 1 day. The culture medium consisted of 50 % (v/v) Ringer solution (mm: 115 NaCl, 2 CaCl2, 2.5 KCl, 10 Hepes, pH 7.6), 49 % L-15 Leibovitz medium (Sigma, St Louis, MO, USA), 1 % fetal bovine serum (Life Technologies, Gaithersburg, MD, USA) and antibiotics (100 u ml−1 penicillin and 100 μg ml−1 streptomycin sulfate). IGF-1 and various inhibitors were applied directly to the culture media at the time of recording.
One day following cell plating, functional synapses are rapidly established between cultured spinal neurons and embryonic muscle cells. The spontaneous synaptic activity of singly innervated myocytes was recorded in the present study. The frequency of spontaneous synaptic events during the first day of synaptogenesis was found to vary greatly from cell to cell, over two orders of magnitude, and the frequency of SSC events increased with the time that synapses developed (Evers et al. 1989; Fu & Huang, 1994). To test the potentiation effect induced by IGF-1 treatment in more simple conditions, our analyses were performed mostly in low-activity synapses (< 1.0 Hz) to mimic the early contact between motoneurons and myocytes.
Electrophysiology and data analysis
Gigaohm-seal whole-cell recording methods followed those described previously (Hamill et al. 1981). Patch pipettes (Hilgenberg, Malsfeld, Germany) were pulled with a two-stage electrode puller (PP-830, Narishige, Tokyo, Japan) and the tips were polished immediately before the experiment using a microforge (MF-830, Narishige, Tokyo, Japan). Spontaneous synaptic currents (SSCs) were detected from innervated myocytes by whole-cell recording in the voltage clamp mode. Recordings were made at room temperature in Ringer solution, and the solution inside the recording pipette contained (mm): 150 KCl, 1 NaCl, 1 MgCl2 and 10 Hepes (pH 7.2). In all recordings, the membrane currents were monitored by a patch clamp amplifier Axopatch 200A (Axon Instruments, Union City, CA, USA) and filtered at 10 kHz. The data were stored on a videotape recorder for later playback onto a storage oscilloscope or a polygraph (Gould RS3200, Valley View, OH, USA) and also for amplitude analysis using SCAN computer program (Dagan, Minneapolis, MN, USA). To quantitatively measure the changes in neurotransmitter release, a time course of SSC frequency was first constructed on a minute-to-minute basis. The SSC frequencies for a 6 min period immediately before drug application was averaged as control. The changes in SSC frequency were measured by averaging a 6 min period recording starting from the highest number after drug application (He et al. 2000) and the results were expressed as means ±s.e.m. The statistical significance was evaluated by Student's paired t test. For comparison of SSC amplitude distribution, the composite graph of cumulative frequency of all SSC events was constructed, and only the synapse with a total number of events exceeding 180 was used for analysis. The statistical difference between these graphs was tested by the Kolmogorov-Smirnov test.
Chemicals
The following chemicals were used: IGF-1 (PeproTech, London, UK), 8-(dethylamino) octyl 3,4,5-trimethoxybenzoate (TMB-8), thapsigargin, ruthenium red (Sigma, St Louis, MO, USA), PD98059, wortmannin, U73122, LY294002 (Tocris Cookson, Bristol, UK). Xestospongin C (XeC), 2-aminoethoxydiphenyl borate (2-APB) (Calbiochem, San Diego, CA, USA), and human anti-IGF-1 polyclonal antibody (RELIATech GmbH, Braunschweig, Germany) to neutralize biological activity of IGF-1 with neutralizing dose (ND)50 at ∼0.1-0.2 mg ml−1.
RESULTS
Potentiation of spontaneous ACh release by IGF-1
In Xenopus nerve-muscle cultures, functional synaptic transmission can be detected within minutes after nerve-muscle contact (Xie & Poo, 1986; Evers et al. 1989), although morphological maturation of the synapse requires many days to complete (Takahashi et al. 1987; Buchanan et al. 1989). SSCs are readily detectable from the innervated muscle cell with whole-cell voltage clamp recordings. These currents were caused by spontaneous ACh secretion from the neuron because they are abolished by bath application of d-tubocurarine and unaffected by tetrodotoxin, which blocks action potentials in neurons (Xie & Poo, 1986). Bath application of IGF-1 at 100 nm dramatically enhanced spontaneous transmitter release, as shown by a marked increase in the frequency of spontaneous synaptic events (Fig. 1A). The increase in SSC frequency produced by IGF-1 was rapid and reached a plateau within ∼15-25 min after bath-application of IGF-1, and the effect persisted for more than 20 min (Fig. 1B). On average, the frequency increased to 5.1 ± 0.8 (n = 17) times the control SSC frequency before the treatment (Fig. 1B and Fig. 4C). The effect of IGF-1 on spontaneous synaptic activity was concentration dependent with maximal potentiation effect at 100 nm (Fig. 2). For comparison, the synaptic potentiation effect of the structure-related polypeptide insulin was also tested. The change of SSC frequency was not significant (2.4 ± 1.0-fold of control, n = 5) after the application of insulin (100 nm; Fig. 2).
The culture medium was replaced with Ca2+-free Ringer solution (A) or addition of Cd2+ to block Ca2+ channels (B) ∼10–15 min prior to the experiment. The arrow marks the application of IGF-1. C, summary of the effects of IGF-1 on SSC frequency in normal culture medium, Ca2+-free medium and in medium containing the Cd2+ (n = ∼5–17). The SSC frequency from a single synapse was counted for a 6 min period in control and a 6 min period after IGF-1 application. The data were then averaged and normalized to control of the same synapse. For comparison, the horizontal dashed line, which defines basal activity as ‘1’, is shown. *P < 0.05 as compared with control SSC frequency of each set experiment before IGF-1 application (Student's paired t test).
A, the mean SSC frequency ∼15–25 min after the application of IGF-1 (•) or insulin (○) at different concentrations was normalized for each synapse by setting the mean SSC frequency to 1 before the addition of drugs. Each value represents the mean and the vertical line represents the s.e.m. of ∼5–17 determinations. B, changes in the frequency of SSC events in the presence of IGF-1 (100 nm) and insulin (100 nm). Data points represent mean SSC frequency, and the line connects data from the same cell before and after drug application.
A, the continuous trace depicts the membrane currents recorded from an innervated muscle cell in day 1 Xenopus cell culture, using the whole-cell recording method (holding potential (VH) = −70 mV, filtered at 10 kHz). Downward events are inward spontaneous synaptic currents (SSCs) resulting from quantal ACh secretion. Samples of current events are shown below at higher time resolution. B, changes in the SSC frequency (normalized to control frequency) with time after the addition of IGF-1 (mean ±s.e.m., n = 17). Synapses with SSC frequency lower than 1 Hz were chosen for these experiments. C, amplitude distribution of all SSC events before (○) and after (•) IGF-1 treatment. The cumulative frequency refers to the proportion of total events with amplitudes smaller than given amplitude. Each value represents the mean ±s.e.m. for 5 experiments. There was no significant difference between these two distributions (P > 0.05, Kolmogorov-Smirnov test).
Synaptic currents may be enhanced by an increased presynaptic release of neurotransmitter or by an increased postsynaptic sensitivity to the neurotransmitter. Increased postsynaptic ACh sensitivity could explain the increase in the SSC frequency, because previously undetected small ACh quanta may emerge after exposure to the factor. As shown in Fig. 1C, we found no detectable change in the amplitude distribution of the SSC amplitude (P > 0.05, Kolmogorov-Smirnov test), suggesting that it is unlikely that ACh sensitivity had been increased by the factor. The absence of any change in the rise time and the decay time of the SSC events after significant elevation of SSC frequency had occurred suggests that these factors did not affect the properties of postsynaptic ACh channels. The amplitude, rise time and decay time of SSC events after IGF-1 application were 101.4 ± 23.9, 108.7 ± 16.0 and 115.0 ± 10.9 % of those of the control (P > 0.05, Student's paired t test). Thus the primary action of IGF-1 at these synapses seems to be a presynaptic modulation of transmitter secretion mechanisms.
Effects of endogenously released IGF-1 on the spontaneous ACh release
In Xenopus nerve-muscle co-culture, the spontaneous ACh release from presynaptic motoneurons is capable of eliciting action potentials and contractions in muscle cells (Chow & Poo, 1985). In the present study, we found that bath-application of IGF-1 markedly increased the frequency of these spontaneous contractions. However, even without drug treatment, there exist some synapses in the culture that have a high frequency of spontaneous contractions resembling those induced by exogenous application of IGF-1. It seems likely that synapses with high-frequency events (high-activity synapse) are under the influence of endogenously released IGF-1. Taking advantage of the specificity and neutralizing activity of anti-IGF-1 antibody, we thus explored the possibility that IGF-1, which is capable of facilitating neurotransmitter release, can serve as an endogenous neuromodulator to regulate synaptic transmission in these Xenopus nerve- muscle cultures. Treatment synapses with spontaneous synaptic events at high-activity (> 3 Hz; Fu & Huang, 1994) with polyclonal IGF-1 antibody markedly reduced the SSC frequency. The inhibition of SSC frequency before and 15 min after antibody application was 59.5 ± 3.3 % (n = 5, Fig. 3), respectively. The amplitude, rise time and decay time of SSC events after IGF-1 antibody application were 110.9 ± 20.0, 95.1 ± 7.2 and 109.2 ± 12.2 % those of control (P > 0.05, Student's paired t test). These results provide evidence that endogenously released IGF-1 was responsible for the maintenance of high levels of spontaneous ACh release in these developing neuromuscular synapses.
The synapses with a frequency of spontaneous ACh release higher than 3 Hz were chosen for the following experiments. A, the SSC frequency of high-activity synapse was significantly reduced after IGF-1 antibody application. B, changes in the SSC frequency (normalized to control frequency) with time after the addition of IGF-1 antibody (mean ±s.e.m., n = 5.
Ca2+ influx is not involved in synaptic potentiation induced by IGF-1
How does IGF-1 enhance presynaptic efficacy? It is well known that the intracellular Ca2+([Ca2+]i) level in the nerve terminal exerts a dominant effect on the rate of spontaneous transmitter release (Miledi, 1973; Augustine et al. 1987). This increase in [Ca2+]i may be due to influx of Ca2+ from the extracellular fluid or release of Ca2+ from intracellular stores. We next examined the role of Ca2+ influx in the action of IGF-1. The Ca2+ was eliminated from the culture medium after several washes with the Ca2+-free Ringer solution. Treating the cells with IGF-1 elicited an increase in the SSC frequency even under the zero external Ca2+ condition (Fig. 4A and C). The SSC frequency was increased by 7.3 ± 2.3-fold (n = 8) under Ca2+-free conditions. To further examine the role of membrane Ca2+ channels, we blocked Ca2+ influx by bath-application of Cd2+ (0.5 mm), which competes with Ca2+ and blocks Ca2+ influx through Ca2+ channels. IGF-1 was capable of potentiating SSC frequency in the presence of Cd2+ (4.3 ± 1.1-fold of control, P > 0.05, Student's paired t test; Fig. 4B and C). Thus, IGF-1-induced potentiation of transmitter release does not require Ca2+ influx from extracellular fluid.
Role of intracellular Ca2+ stores
We further examined whether the Ca2+ released from intracellular stores is responsible for IGF-1-induced synaptic potentiation. To approach this problem, the Ca2+-ATPase inhibitor thapsigargin was initially used to deplete intracellular Ca2+ stores (He et al. 2000). The culture medium was also replaced with Ca2+-free Ringer solution to exclude the possibility of internal Ca2+ store depletion-induced Ca2+ entry through store-operated channels in the plasma membrane (Kanzaki et al. 1999; Tempia et al. 2001). Bath-application of thapsigargin (2 μm) elicited an increase in SSC frequency, which returned to control levels within 40–80 min (8.9 ± 1.1 times the control SSC frequency, n = 4. IGF-1 no longer elicited any changes in SSC frequency in thapsigargin-treated synapses (Fig. 5). These results suggest that Ca2+ released from intracellular stores was responsible for synaptic potentiation induced by IGF-1.
Representation of the potentiation of SSC frequency 8 min after thapsigargin (2 μm) application and the enhancement of spontaneous ACh release sustained for a long time (∼45 min in this case) due to the Ca2+ released from intracellular stores. Application of IGF-1 after SSC frequency returned to normal no longer increased the SSC frequency. Samples of synaptic currents are shown below the traces at higher time resolution.
Two major pathways are indicated for the release of Ca2+ from intracellular stores: the IP3-sensitive and the ryanodine-sensitive Ca2+ stores (Berridge, 1998). Pretreatment of the culture with membrane-permeable inhibitors of IP3-induced Ca2+ release 2-APB (75 μm) or XeC (1 μm) effectively occluded the increase of SSC frequency elicited by IGF-1 (Fig. 6). The synaptic potentiation of IGF-1 under the presence of 2-APB and XeC were 0.5 ± 0.1 (n = 5) and 1.8 ± 0.3-fold (n = 7) of control, respectively. The release of Ca2+ from IP3 receptors could further trigger Ca2+-induced Ca2+ release from ryanodine receptors (Berridge, 1998). Pretreatment of the cultures with ryanodine receptor antagonist TMB-8 (3 μm) or ruthenium red (10 μm) occluded the IGF-1 actions (1.9 ± 0.3-fold, n = 6 and 1.9 ± 0.1-fold, n = 5, of control, for TMB-8 and ruthenium red pretreatment, respectively; Fig. 6). Thus, Ca2+ released from both IP3- and ryanodine-sensitive pools is responsible for the IGF-1-induced synaptic potentiation.
Drugs that inhibit the IP3 receptor (Xestospongin C (XeC); 2-aminoethoxydiphenyl borate (2-APB)) or the ryanodine receptor (8-(dethylamino) octyl 3,4,5-trimethoxybenzoate (TMB-8); ruthenium red) were added to the culture medium 15–50 min before the experiment at the final concentration of: XeC, 2 μm; 2-APB, 75 μm; TMB-8 3 μm; and ruthenium red, 10 μm. The effect of IGF-1 on SSC frequency was then evaluated in the presence of XeC (A), 2-APB (B), TMB-8 (C) and ruthenium red (D). E, summary of the drug effects. The error bars refer to s.e.m. (n = ∼5–17). *P < 0.05 as compared with control SSC frequency of each set experiment before IGF-1 application (Student's paired t test).
Mechanisms of the IGF-1 action
Accumulated evidence suggests that IGF-1 signals through MAP kinase, PI3-K and PLCγ signal transduction pathways. Thus we next examined which signalling pathway is responsible for the action of IGF-1 in developing Xenopus neuromuscular synapses. The IGF-1-induced synaptic potentiation was abolished in the presence of PI3-K inhibitor wortmannin (100 nm; 1.2 ± 0.3-fold of control, n = 5; Fig. 7A). Pretreatment with another PI3-K inhibitor, LY294002 (5 μm), also prevented the IGF-1-induced increase in SSC frequency (0.8 ± 0.1-fold of control, n = 6; Fig. 7B). In contrast, the synaptic potentiation effect of IGF-1 was not hampered by the presence of MAP kinase inhibitor PD98059 (10 μm; 3.7 ± 1.0-fold of control, n = 5; Fig. 7C), suggesting that MAP kinase is not involved in the acute effect of IGF-1. Activation of PLCγ is another attractive candidate for mediation of synaptic potentiation because its activation would result in intracellular Ca2+ release via the second messenger IP3. We have shown the inhibition of IP3 receptor by 2-APB and XeC abolished the IGF-1 effect (Fig. 6). Consistent with this result, inhibition of PLCγ by U73122 (5 μm) completely prevents the IGF-1-induced increase in SSC frequency (1.3 ± 0.4-fold of control, n = 5; Fig. 7D). Thus, the synaptic potentiation induced by IGF-1 requires signalling pathways dependent on PI3-K and PLCγ, but not on MAP kinase.
The cultures were pretreated with various inhibitors for ∼20–50 min before IGF-1 challenge. A, wortamannin (100 nm); B, LY294002 (5 μm); C, U73122 (5 μm); and D, PD98059 (10 μm). E, summary of the drug effects. For each synapse, a time course of SSC frequencies was averaged from a 6 min recording period before IGF-1 application, and from a 6 min period starting from the highest number after IGF-1 application. The SSC frequencies after IGF-1 application were then normalized to those before IGF-1 application. For comparison, the horizontal dashed line, which defines basal activity as ‘1’, is shown. The error bars refer to s.e.m.n = ∼5–17. *P < 0.05 as compared with control SSC frequency of each set experiment before IGF-1 application (Student's paired t test).
DISCUSSION
Our studies demonstrate for the first time that IGF-1, under acute conditions, can increase spontaneous neurotransmitter release in the peripheral nervous system. It has been suggested that IGF-1 can decrease K+- as well as veratridine-evoked hippocampal ACh release. However, the finding that the inhibitory effect of IGF-1 was altered by a GABA antagonist suggests that the negative modulation of ACh release from hippocampus by IGF-1 is an indirect effect (Kar et al. 1997; Seto et al. 2002). Furthermore, this is the first evidence that muscle cell-derived IGF-1 is involved in the regulation of synaptic transmission at developing motoneurons. Several observations led to our present study on the role of endogenously released IGF-1. First, the frequency of spontaneous synaptic events was previously found to vary greatly from cell to cell, over two orders of magnitude (Evers et al. 1989; Fu & Huang, 1994). Second, synapses with high frequencies of spontaneous events resemble those induced by exogenous application of IGF-1 (Fig. 1). Third, focal application of neutralizing antibodies to IGF-1 reduces collateral axonal branching after peripheral nerve lesion (Streppel et al. 2002). Fourth, in vivo treatments of embryos with IGF-binding proteins (IGF-BPs), which are high affinity proteins that interfere with the actions of endogenous IGFs, reduce synapse formation in avian neuromuscular system (D'Costa et al. 1998). Fifth, subcutaneous administration of IGF-I can increase muscle endplate size in rats (Lewis et al. 1993). Taking advantage of the specificity and neutralizing activity of anti-IGF-1 antibody, we have shown the inhibitory effect of IGF-1 antibody on the SSC frequency of high-activity synapses indicating that endogenously released IGF-1 acts at synapses of higher activity in cell cultures. However, previously there has been no evidence concerning the expression of IGF-1 receptor at the nerve terminal in Xenopus. The possibility cannot be ruled out that paracrine and/orautocrine IGF-1, which is produced by motoneurons themselves or other cell types that happen to be in the culture other than myocytes, could be acting on the IGF-1 receptor on cell bodies.
It has been demonstrated that within seconds IGF-1 induces a PI3-K-dependent increase in N- and L-type Ca2+ channel activity in cerebellar granule neurons and might be involved in the control of Ca2+-dependent processes such as neurotransmitter release and survival (Blair & Marshall, 1997; Blair et al. 1999). In addition, many studies have shown that the activation of the IGF-1 receptor facilitates skeletal muscle L-type Ca2+ channel activity via a PKC-dependent phosphorylation mechanism, and that overexpression of IGF-1 exclusively in skeletal muscle increases the number of dihydropyridine receptors in adult transgenic mice (Delbono et al. 1997; Renganathan et al. 1998). Furthermore, a new mechanism for IGF-I-induced Ca2+ influx in cells was recently reported to involve the IGF-I-dependent translocation of a Ca2+-permeable channel to the plasma membrane through a PI3-K-dependent signal (Kanzaki et al. 1999). However, our experiments show that IGF-1 potentiates transmitter release either in Ca2+-free or Cd2+-containing medium, and that depletion of intracellular Ca2+ stores with thapsigargin prevented the IGF-1 effect, implying that IGF-1 modulates the machinery of transmitter release in a different way. Recently, transmitter release modulated by the release of Ca2+ from intracellular stores has been shown in a number of systems, such as the cholinergic synapse in Aplysia and sympathetic nerve terminals (Smith & Cunnane, 1996; Mothet et al. 1998). In the present study, IGF-1 induced Ca2+ release through IP3 receptors and subsequently triggered Ca2+-induced Ca2+ release from ryanodine receptors, leading to an increase in spontaneous transmitter release at the terminals of developing spinal neurons. The result that either IP3 or ryanodine receptor antagonist can effectively prevent synaptic potentiation induced by IGF-1 suggests that both pathways are necessary for the synaptic potentiation and the primary effect of IGF-1 is probably on the IP3 receptor.
Binding of IGF-1 to its receptor induces receptor autophosphorylation in the intracellular kinase domain of the β-subunit which resulted in the activation of several cellular signal transduction cascades, including MAP kinase (Kim et al. 1997; Mehrhof et al. 2001), PI3-K (Blair et al. 1999; Leski et al. 2000; Mehrhof et al. 2001) and PLCγ (Foncea et al. 1997; Hong et al. 2001). Activation of PLCγ is an attractive candidate for the mediation of synaptic potentiation because its activation would result in intracellular Ca2+ release via the second messenger IP3. There are two possible mechanisms that resulted in PLCγ activation. PLCγ is phosphorylated by diverse receptor tyrosine kinases and nonreceptor protein tyrosine kinases through a high affinity interaction with the SH2 (Src homology 2) domain of PLCγ (Rhee, 2001). Also it has been shown that the binding of the pleckstrin homology (PH) domain of PLCγ to phosphatidylinositol 3,4,5-trisphosphate (PI-3,4,5-P3) present in the membrane as a result of PI3-K activation leads to the activation of PLCγ (Bae et al. 1998; Falasca et al. 1998). It has been suggested that IGF-I is able to activate PLCγ via both tyrosine phosphorylation-dependent and PI3-K-dependent mechanisms (Hong et al. 2001; Chattopadhyay & Carpenter, 2002). Although in our experiments either the PI3-K inhibitor or the PLCγ inhibitor can effectively prevent the synaptic potentiation induced by IGF-1, suggesting that PLCγ activated from the PI3-K pathway appeared to play a more important role than tyrosine phosphorylation in IGF-I activation of PLCγ. However, we cannot exclude the possibility of direct activation of PLCγ via binding to phsophorylated tyrosine residues of the receptor tyrosine kinases.
It has been suggested that a large number of trophic factors are involved in the development of the neuromuscular junction. Furthermore, as expected, some of these are present in the skeletal muscle targets of presynaptic motoneurons (e.g. neurotrophic factor-3 (NT-3), NT-4 and IGF-1; Caroni, 1993; Funakoshi et al. 1995; Xie et al. 1997), whereas other factors are not (e.g. ciliary neurotrophic factor (CNTF)). Why are so many factors involved in the development of neuromuscular junction? One simple answer to this question is that motoneurons require multiple factors from diverse sources for optimal development. However, the more significant alternative might be that the spatial and/ortemporal expression of the trophic factors provides a consecutive programme during neuronal development and synapse formation. Indeed, there is good evidence suggesting that certain populations of neurons switch their survival requirements from one neurotrophin to another during an early stage in their development (Davies, 1997). During development, muscle IGF mRNA levels increase with the formation of differentiated skeletal muscle fibre, at the time prior to the contact of motor nerve growth cone (Ishii, 1989; Caroni, 1993) and it has also been suggested that the IGF-1 receptors are present in the spinal cord (Kar et al. 1993; Lewis et al. 1993). In the present study, our studies provide the first physiological evidence that endogenously released IGF-1 potentially enhances the spontaneous transmitter release at the developing neuromuscular synapse. What is the functional significance of potentiating ACh secretion by IGF-1 during the early phase of synaptogenesis? The activity of neuromuscular transmission at developing synapses is crucial in synaptic maturation and competition as well as in the differentiation of postsynaptic properties (Lo & Poo, 1991; Balice-Gordon & Lichtman, 1993). The potentiation of the spontaneous ACh release at developing neuromuscular synapses may have a profound developmental significance. Several studies have indicated that the gene expression and secretion of neurotrophic factors NT-3 and NT-4 in the neuromuscular junction was regulated by synaptic activity (Liou & Fu, 1997; Wang & Poo, 1997; Xie et al. 1997). It has also been suggested that activity-dependent secretion of neurotrophic factors is important in synaptic activity regulation and may be involved in Hebbian-type homosynaptic potentiation (Poo, 2001). Furthermore, SSCs at developing neuromuscular junctions in Xenopus cultures are capable of eliciting action potentials and spontaneous contractions in muscle cells (Chow & Poo, 1985). This frequent supra-threshold excitation produces a global influence on the development of contractile properties of the postsynaptic muscle cell (Kidokoro & Saito, 1988). In addition, spontaneous synaptic potentials are accompanied by a localized influx of ions at the subsynaptic site of the muscle, including Ca2+ (Decker & Dani, 1990). Local Ca2+ accumulation and the consequent Ca2+-dependent enzymatic reactions are likely to play an important role in regulating the development of postsynaptic structure. Overall, the conclusion drawn from these experiments is that endogenously released IGF-1 increases the spontaneous neurotransmitter release, thus perhaps having significant roles in initiating the consecutive and complex cross-interaction between presynaptic motoneurons and postsynaptic muscle cells that then lead to the maturation of the neuromuscular synapse.
Acknowledgments
The authors are most grateful for the continuous help provided by Professor W.-M. Fu and Y.-J. Chan. This work was supported by a grant from the National Science Council of Taiwan (NSC 89–2320-B-110-007).
Footnotes
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- Received July 9, 2003.
- Accepted September 25, 2003.
- © The Physiological Society 2003
