行政院國家科學委員會專題研究計畫 成果報告
在胚胎發育過程中 Insulin-like growth factor-1 調節神
經-肌傳導作用之研究
計畫類別： 個別型計畫 計畫編號： NSC91-2320-B-110-016- 執行期間： 91 年 08 月 01 日至 92 年 07 月 31 日 執行單位： 國立中山大學生物科學系(所) 計畫主持人： 劉昭成 計畫參與人員： 蔡鳳如 報告類型： 精簡報告 處理方式： 本計畫涉及專利或其他智慧財產權，2 年後可公開查詢中 華 民 國 92 年 10 月 22 日
中文摘要
神經細胞與肌細胞形成突觸的過程非常複雜，這過程中除了細胞彼此間藉著 細胞黏著因子的直接接觸產生交互作用之外，許多訊號分子如 ATP、NO、HPETE 的出現及一些神經滋養因子的產生會進一步穩定整個突觸結構。在本論文中我們 將探討 Insulin-like growth factor-I (IGF-I) 在胚胎時期對神經細胞與肌細胞突觸 形成過程所扮演的角色。本實驗利用非洲爪蟾的神經細胞與肌細胞的混合培養 ( Xenopus nerve-muscle co-culture )，以 whole-cell patch clamp 的電生理記錄的方 式來探討 IGF-I 在胚胎發育早期突觸形成過程中所扮演的角色以及這中間可能的 訊息傳遞路徑。藉由在肌細胞記錄神經所釋放的 ACh 之後，ACh 打開肌細胞上 ACh receptor 而造成自發性電流，我們可以清楚的觀察到神經細胞的活性，當我 們在培養皿中加入 IGF-I 之後約經 15 分鐘後我們發現自發性神經傳導物質釋放 的頻率有顯著的增加。由於神經細胞釋放神經傳導物質和神經末梢內鈣離子的濃 度有很大的關係，因此我們設計一系列實驗來釐清鈣離子的來源。實驗結果顯示 在 Ca2+
free Ringer 以及鈣離子通道阻斷劑 Cd2+的存在下 IGF-I 對神經活性的促進 作用依然存在，顯示 IGF-I 對神經活性的促進作用所需的鈣離子來源不是來自細 胞外，而是由細胞內的鈣離子儲存池所提供。此外，為了更進一步證實 IGF-I 的
作用和鈣離子儲存池的關係，我們利用鈣離子儲存池上的鈣離子通道阻斷劑：IP3
receptor inhibitor (XeC, 2-APB) 及 ryanodine receptor inhibitor (TMB-8) 或是用鈣 離子儲存池的排空劑 thapsigargin 以阻斷細胞內鈣離子的來源，實驗結果一致顯 示在沒有細胞內的鈣離子來源的情況下 IGF-I 便無法再促進神經活性。至於 IGF-I 的那些訊息傳遞路徑和促進神經活性有關？目前已知 IGF-I 有三條訊息傳遞路徑 ─PI 3-kinase、PLCγ和 MAP kinase。這次的實驗結果發現，IGF-I 的作用會因 PI
3-kinase 和 PLCγ的活性被抑制而消失，但對於 MAP kinase 的抑制作用沒有明顯
的影響。
當神經末梢內鈣離子濃度升高時，Ca2+會透過和 calmoldulin 形成複合物而將
Ca2+/calmoldulin-dependent protein kinase (CaMK II) 活化，使得原本藉著 synapsin I 而束縛在細胞骨架上的突觸小泡( synaptic vesicle )因 synapsin I 被 CaMK II 磷酸 化而釋放出來，增加了突觸小泡被釋放的機率。實驗結果發現在 CaMK II 抑制 劑的處理下，IGF-I 的作用明顯被抑制，顯示 IGF-I 確實能提高細胞內 Ca2+而活 化 CaMK II 來增加神經傳導物質釋放的頻率。 綜合以上結果，我們認為IGF-I能夠藉由PI 3-kinase及PLCγ路徑的活化而打開 IP3及ryanodine sensitive的鈣離子儲存池，使神經末梢內鈣離子濃度升高後，鈣離 子經由活化CaMK II pathway而增加神經傳遞物質的釋放。
英文摘要
Although evidence suggests that insulin-like growth factor plays an 1
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 insulin-like growth factor-1 (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 calcium was eliminated from the culture medium or there was bath application of the pharmacological calcium channel inhibitor cadmium, indicating that calcium influxes through voltage-activated calcium 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 inhibitor of mitogens-activated protein (MAP) kinase PD98059 abolished IGF-1-induced synaptic potentiation. Taken collectively, these results suggest that endogenously released IGF-1 from myocytes elicits calcium release from IP3 and/or ryanodine sensitive
intracellular calcium stores of the presynaptic nerve terminal. This is done via PI3 kinase and PLC-γ signaling cascades, leading to an enhancement of spontaneous transmitter release.
關鍵詞
Insulin-like growth factor, Motoneuron, Synaptic activity, Acetylcholine
INTRODUCTION
Successful synaptic transmission at the neuromuscular junction depends on the precise alignment of the nerve terminals with the postsynaptic specialization of the muscle fiber. 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 appear to be dependent on retrograde signals from the target, and enormous neurotrophic
factors have been characterized and demonstrated to play important roles in the development of the neuron (Bennet et al. 2002; Black, 1999). During development, particular sets of genes are expressed at specific times and in specific context. The discoveries that expression of IGF-1 in the developing skeletal muscle increase with the formation of differentiated skeletal muscle fibers and decrease to very low adult levels during the process of synapse elimination, paved the way for an expanding field of research that focuses on the role of IGF-1 in synapse formation (Caroni, 1993; Ishii, 1989).
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 increase 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) IGFs administration prevent motoneuron death and support the reestablishment of synapses following nerve injury (Lutz et al. 1999; Vergani et al. 1998); (d) in vivo treatment of paralyzed 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 mitogens with long-term effects, IGF-1 has now also been demonstrated to be rapid neuromodulators. It has been suggested that IGF-1 regulates ion channel currents and neuronal excitability (Blair & Marshall, 1997; Kar et al. 1997; Kendra et
al. 2000).
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 (α2β2) 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 (Balice-Gordon & Lichtman, 1993; Dan & Poo, 1992; Kidokoro & Saito, 1988; Lo & Poo, 1991). The IGF-1 receptors 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 fibers before innervations. Although all the evidences to date support 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 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 endogenously released IGF-1 from myocytes may serve as a positive trophic factor at developing neuromuscular junction, and the underlying signaling mechanism involved were 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 the Ca2+ and Mg2+-free Ringer supplemented with 0.15 mM EDTA. The dissociated cells were plated and used for experiments after incubation at room temperature (20-25oC) for 1 day. The culture medium consisted of 50% (vol/vol) Ringer solution (115 mM NaCl, 2 mM CaCl2, 2.5 mM KCl, 10 mM HEPES [pH 7.6]),
49% L-15 Leibovitz medium (Sigma, St Louis, MO, USA), and 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 present study utilized synapses that myocytes were innervated by single co-cultured spinal neurons. 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 increase with time that synapses developed (Evers et al. 1989; Fu & Huang, 1993). 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.
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 (Evers et al. 1989; Xie & Poo, 1986), although morphological maturation of the synapse requires many days to complete (Buchanan et al. 1989; Takahashi et al. 1987). SSCs are readily detectable from the innervated muscle cell with the 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 evidenced 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 minutes after bath application of IGF-1, and the effect persisted for more than 20 minutes (Fig. 1B). On average, the frequency increased to 5.1±0.8 (n=17) times the control SSC frequency before the treatment (Fig. 1B & 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 folds of control, n=5) after the application of insulin (100 nM; Fig. 2).
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 undetectable small ACh quanta may emerge after exposure to the factor. As shown in Figure 1C, we found no detectable change in the amplitude distribution of the SSC amplitude (P>0.05, Kolmogorov-Simirnov 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% those of control (P>0.05, Student 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 motoneuron 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 endogenous neuromodulators to regulate synaptic transmission in these Xenopus nerve-muscle cultures. Treatment synapses with spontaneous synaptic events at high-activity (> 3 Hz; Fu & Huang, 1993) with polyclonal IGF-1 antibody markedly reduced the SSC frequency. The inhibition of SSC frequency before and 15 minutes 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 paired t test). This result provides evidence that endogenously released IGF-1 was responsible for the maintenance of high levels of spontaneous ACh release in these developing neuromuscular synapses.
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 calcium ([Ca2+]i) level in the nerve terminal exerts a dominant effect on
the rate of spontaneous transmitter release (Augustine et al. 1987; Miledi, 1973). 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. Treating the cells with IGF-1 still elicited an increase in the SSC frequency under the zero external Ca2+ condition (Fig. 4A & C). The SSC frequency was increased by 7.3±2.3 folds (n=8) under Ca2+-free condition. 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 block Ca2+ influx
through Ca2+ channels.IGF-1 was still capable of potentiating SSC frequency in the presence of Cd2+ (4.3±1.1 folds of control, P>0.05, Student paired t test; Fig. 4B & C). Thus, IGF-1-induced potentiation of transmitter release does not require Ca2+ influx from extracellular fluid.
Role of intracellular calcium stores
We further examine if the Ca2+ released from intracellular stores is responsible for IGF-1-induced synaptic potentiation. To approach this problem, 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 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.
Two major pathways are indicated for the release of Ca2+ from intracellular stores: the IP3-sensitive and the ryanodine-sensitive calcium stores (Berridge, 1998).
Pretreatment of the culture with membrane-permeable inhibitors of IP3-induced Ca2+
release 2-aminoethoxydiphenyl borate (2-APB, 75 µM) or Xestospongin C (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 (n=7) folds 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 8-(dethylamino) octyl 3, 4, 5-trimethoxybenzoate (TMB-8; 3 µM) or ruthenium red (10 µM) occluded the IGF-1 actions (1.9±0.3, n=6, and 1.9±0.1, n=5, folds 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.
Mechanisms of the IGF-1 action
Accumulated evidences suggest that IGF-1 signals through MAP kinase, PI3 kinase and PLCγ signal transduction pathways. Thus we next examined which signaling 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 folds of control, n=5; Fig. 7A). Pretreatment with another PI3 kinase inhibitor, LY294002 (5 µM), also prevented the IGF-1 induced increase in SSC frequency (0.8±0.1 folds 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 folds 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 folds of control, n=5; Fig. 7D). Thus, the synaptic potentiation induced by IGF-1 requires signaling pathways dependent on PI3 kinase and PLCγ, but not on MAP kinase.
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 inhibitory effect of IGF-1 could be altered by GABA antagonist suggest 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, 1993). 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 rule out that paracrine/autocrine 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 IGF-1 receptor on cell bodies.
It has been demonstrated that within seconds IGF-1 induces a PI3-kinase-dependent increase in N and L type calcium channel activity in cerebellar granule neurons and might involve 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 toinvolve the IGF-I-dependent translocation of a calcium-permeablechannel to the plasma membrane through a PI-3-kinase-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 (Mothet et al. 1998; Smith & Cunnane, 1996). In the present study, the 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.
It has been suggest that a large number of trophic factors are involved in the development of neuromuscular junction. Furthermore, as expected, some of these are present in the skeletal muscle targets of presynaptic motoneurons (e.g., NT-3, NT-4, and IGF-1; Caroni, 1993; Funakoshi et al. 1995; Xie et al. 1997), whereas other factors are not (e.g., 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/or temporal expression of the
trophic factors provides a consecutive program during the 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 fiber, at the time prior to the contact of motor nerve growth cone (Caroni, 1993; Ishii, 1989) 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 (Balice-Gordon & Lichtman, 1993; Lo & Poo, 1991). 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 are 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.
