Xenopus Cell Cultures Research Articles

Xenopus laevis as a model system to study cytoskeletal dynamics during axon pathfinding.

The model system, Xenopus laevis, has been used in innumerable research studies and has contributed to the understanding of multiple cytoskeletal components, including actin, microtubules, and neurofilaments, during axon pathfinding. Xenopus developmental stages have been widely characterized, and the Xenopus genome has been sequenced, allowing gene expression modifications through exogenous molecules. Xenopus cell cultures are ideal for long periods of live imaging because they are easily obtained and maintained, and they do not require special culture conditions. In addition, Xenopus have relatively large growth cones, compared to other vertebrates, thus providing a suitable system for imaging cytoskeletal components. Therefore, X. laevis is an ideal model organism in which to study cytoskeletal dynamics during axon pathfinding.

Mechanism of β-bungarotoxin in facilitating spontaneous transmitter release at neuromuscular synapse

The mechanism of the action of β-bungarotoxin (β-BuTx) in the facilitation of spontaneous transmitter release at neuromuscular synapse was investigated in Xenopus cell culture using whole-cell patch clamp recording. Exposure of the culture to β-BuTx dose-dependently enhances the frequency of spontaneous synaptic currents (SSCs). Buffering the rise of intracellular Ca 2+ with BAPTA-AM hampered the facilitation of SSC frequency induced by β-BuTx. The β-BuTx-enhanced SSC frequency was reduced when the pharmacological Ca 2+-ATPase inhibitor thapsigargin was used to deplete intracellular Ca 2+ store. Application of membrane-permeable inhibitors of inositol 1,4,5-trisphosphate (IP 3) but not ryanodine receptors effectively occluded the increase of SSC frequency elicited by β-BuTx. Treating cells with either wortmannin or LY294002, two structurally different inhibitors of phosphatidylinositol 3-kinase (PI3K) and with phospholipase C (PLC) inhibitor U73122, abolished the β-BuTx-induced facilitation of synaptic transmission. The β-BuTx-induced synaptic facilitation was completely abolished while there was presynaptic loading of the motoneuron with GDP βS, a non-hydrolyzable GDP analogue and inhibitor of G protein. Taken collectively, these results suggest that β-BuTx elicits Ca 2+ release from the IP 3 sensitive intracellular Ca 2+ stores of the presynaptic nerve terminal. This is done via PI3K/PLC signaling cascades and G protein activation, leading to an enhancement of spontaneous transmitter release.

Mechanism of β‐bungarotoxin in the facilitation of spontaneous transmitter release at developing neuromuscular synapse

The mechanism of action of β-bungarotoxin (β-BuTx) in the facilitation of spontaneous transmitter release at neuromuscular synapse was investigated in Xenopus cell culture using whole-cell patch clamp recording. Exposure of the culture to β-BuTx dose-dependently enhances the frequency of spontaneous synaptic currents (SSCs). Buffering the rise of intracellular Ca2+ with BAPTA-AM hampered the facilitation of SSC frequency induced by β-BuTx. The β-BuTx-enhanced SSC frequency was abolished when pharmacological Ca2+-ATPase inhibitor thapsigargin was used to deplete intracellular Ca2+ store. Application of membrane-permeable inhibitors of inositol 1,4,5-trisphosphate (IP3) but not ryanodine receptors effectively occluded the increase of SSC frequency elicited by β-BuTx. Treating cells with either wortmannin or LY294002, two structurally different inhibitors of phosphatidylinositol 3-kinase (PI3K) and with phospholipase C (PLC) inhibitor U73122, abolished β-BuTx-induced facilitation of synaptic transmission. The β-BuTx-induced synaptic facilitation was completely abolished while there was presynaptic loading of the motoneuron with GDPβS, a non-hydrolyzable GDP analogue and inhibitor of G protein. Taken collectively, these results suggest that β-BuTx elicits Ca2+ release from IP3 sensitive intracellular Ca2+ stores of the presynaptic nerve terminal. This is done via PI3Kγ/PLC signaling cascades and G protein activation, leading to an enhancement of spontaneous transmitter release.

A rapid, nongenomic pathway facilitates the synaptic transmission induced by retinoic acid at the developing synapse

We have previously shown that retinoic acid (RA), a factor highly expressed in spinal cord, rapidly and specifically enhances the spontaneous acetylcholine release at developing neuromuscular synapses in Xenopus cell culture, using whole-cell patch-clamp recording. We have now further investigated the underlying mechanisms that are involved in RA-induced facilitation on the frequency of spontaneous synaptic currents (SSCs). Buffering the rise of intracellular Ca2+ with BAPTA-AM hampered the facilitation of SSC frequency induced by RA. The prompt RA-enhanced SSC frequency 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+ influx through voltage-activated Ca2+ channels are not required. Application of membrane-permeable inhibitors of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] or ryanodine receptors effectively blocked the increase of SSC frequency elicited by RA. Treating cells with either wortmannin or LY294002, two structurally different inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase) and with the phospholipase Cgamma (PLCgamma) inhibitor U73122, abolished RA-induced facilitation of synaptic transmission. Preincubation of the cultures with pharmacological inhibitors, either genistein, a broad-spectrum tyrosine kinase inhibitor, or PP2, which predominantly inhibits the Src family of nonreceptor tyrosine kinase, completely abolished RA-induced synaptic facilitation. Taken collectively, these results suggest that RA elicits Ca2+ release from Ins1,4,5P3 and/or ryanodine-sensitive intracellular Ca2+ stores of the presynaptic nerve terminal. This is done via PLCgamma/PI 3-kinase signaling cascades and Src tyrosine kinase activation, leading to an enhancement of spontaneous transmitter release.

Non-genomic regulation of transmitter release by retinoic acid at developing motoneurons in Xenopus cell culture.

Although the long-term effects of all-trans retinoic acid (RA) on neuronal growth and differentiation have been intensively studied, nothing is known about its effect on synaptic transmission. Here we show that RA rapidly and specifically enhances the spontaneous acetylcholine release at developing neuromuscular synapses in Xenopus cell culture using whole-cell patch-clamp recording. Acute addition of RA dose-dependently and reversibly enhances the frequency of spontaneous synaptic currents (SSCs). Application of the lipophilic RA analogue all-trans retinol or RA metabolites produced by light-induced decomposition failed to provoke similar changes in SSC frequency, indicating the specificity of RA-induced facilitation of spontaneous transmitter release. Protein synthesis inhibitors anisomycin or cycloheximide had no effect on RA-induced SSC frequency facilitation. Treating cells with pan RA receptor (RAR) selective agonist or RARbeta-selective agonist, but not RARalpha-, RARgamma- or retinoid X receptor (RXR)-selective agonists, mimicked the action of RA. These results suggest that RA acts through the activation of RARbeta, to induce a rapid, non-genomic increase in the frequency of spontaneous transmitter release at developing neuromuscular synapses.

Potentiation of quantal secretion by insulin-like growth factor-1 at developing motoneurons in Xenopus cell culture.

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 Cgamma (PLCgamma) 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 PLCgamma signalling cascades, leading to an enhancement of spontaneous transmitter release.

Differential Regulation of Fibronectin Fibrillogenesis by Protein Kinases A and C

The assembly of fibronectin (Fn) matrix is a key event in regulating cell adhesion, migration and differentiation. To elucidate the regulatory role of protein kinases in the formation of fibrillar Fn matrix, we examined Fn fibril assembly from soluble bovine Fn underneath fibroblasts in Xenopus cell cultures and quantitated the resulting matrix by using a selectively cross-reactive antibody. The soluble form of bovine Fn was bath-applied to the cell cultures, and fibroblasts changed the soluble form of Fn into the fibrillar form in a time-dependent manner. Integrin antagonists, Arg-Gly-Asp-Ser peptide and Rhodostomin, inhibited the formation of Fn matrix from soluble Fn. Genistein, cytochalasin D, colchicine, H-7, Ro-31-8220 and forskolin exerted similar inhibitory action. However, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) significantly accelerated the formation of fibrillar Fn. The clustering of integrin and vinculin was enhanced and inhibited by TPA and forskolin, respectively. Following one day's incubation with soluble Fn to pre-organize fibrillar Fn matrix, bath application of these drugs caused the degradation of the Fn network, indicating that Fn fibrillogenesis is a dynamic state modulated by cytoskeleton and protein kinases. Aprotinin and leupeptin selectively antagonized the Fn degradative action of forskolin, but not that caused by H-7. These results suggest that the dynamic assembly of Fn matrix is integrin- and cell-dependent and that both PKA and PKC play important roles in regulating the dynamics of Fn matrix. PKC activation enhanced the assembly of Fn, whereas inhibition of PKC caused the disassembly of Fn matrix. On the other hand, activation of PKA inhibited Fn assembly and caused the degradation of Fn matrix mainly via the activation of multiple proteolytic enzymes. Serine and cysteinyl proteases play important roles in Fn disassembly caused by PKA activation, but not that caused by PKC inhibition.

Differential Regulation of Fibronectin Fibrillogenesis by Protein Kinases A and C

The assembly of fibronectin (Fn) matrix is a key event in regulating cell adhesion, migration and differentiation. To elucidate the regulatory role of protein kinases in the formation of fibrillar Fn matrix, we examined Fn fibril assembly from soluble bovine Fn underneath fibroblasts in Xenopus cell cultures and quantitated the resulting matrix by using a selectively cross-reactive antibody. The soluble form of bovine Fn was bath-applied to the cell cultures, and fibroblasts changed the soluble form of Fn into the fibrillar form in a time-dependent manner. Integrin antagonists, Arg-Gly-Asp-Ser peptide and Rhodostomin, inhibited the formation of Fn matrix from soluble Fn. Genistein, cytochalasin D, colchicine, H-7, Ro-31-8220 and forskolin exerted similar inhibitory action. However, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) significantly accelerated the formation of fibrillar Fn. The clustering of integrin and vinculin was enhanced and inhibited by TPA and forskolin, respectively. Following one day's incubation with soluble Fn to pre-organize fibrillar Fn matrix, bath application of these drugs caused the degradation of the Fn network, indicating that Fn fibrillogenesis is a dynamic state modulated by cytoskeleton and protein kinases. Aprotinin and leupeptin selectively antagonized the Fn degradative action of forskolin, but not that caused by H-7. These results suggest that the dynamic assembly of Fn matrix is integrin- and cell-dependent and that both PKA and PKC play important roles in regulating the dynamics of Fn matrix. PKC activation enhanced the assembly of Fn, whereas inhibition of PKC caused the disassembly of Fn matrix. On the other hand, activation of PKA inhibited Fn assembly and caused the degradation of Fn matrix mainly via the activation of multiple proteolytic enzymes. Serine and cysteinyl proteases play important roles in Fn disassembly caused by PKA activation, but not that caused by PKC inhibition.

Collaboration of fibronectin matrix and neurotrophin in regulating spontaneous transmitter release at developing neuromuscular synapses in Xenopus cell cultures

Integrins mediate cell-extracellular matrix connection and are particularly important during neuronal development. We here investigated the regulation of fibronectin (FN) matrix and neurotrophins on the embryonic synaptic transmission. Spontaneous synaptic currents (SSCs) were recorded from innervated myocytes of 1-day-old Xenopus cultures by whole-cell recordings. The SSC increasing action of α,β-methylene adenosine triphosphate was enhanced in neurons grown on FN substratum, which was further potentiated by chronic treatment with brain-derived neurotrophic factor (BDNF). The SSC increasing action of thapsigargin, carbonyl cyanide m-chlorophenylhydrazone and N-methyl- d-aspartate was also markedly potentiated in neurons grown on FN-coated glass coverslips and chronically treated with BDNF. FN matrix or BDNF alone only exerts slight potentiation on the SSC increasing action of these three drugs. Our results suggest that FN matrix can collaborate with neurotrophin in regulating synaptic transmission at developing motoneurons, which may play an important role in the maturation of embryonic neuromuscular junction.

Target-dependent regulation of acetylcholine secretion at developing motoneurons in Xenopus cell cultures.

1. Myocyte-dependent regulation of acetylcholine (ACh) quantal secretion from developing motoneurons was studied in day-3 Xenopus nerve-muscle co-cultures. Spontaneous synaptic currents (SSCs) were measured in manipulated synapses by using whole-cell voltage-clamped myocytes. Changes in SSC amplitude were assumed to reflect changes in the ACh content of secreted quantal packets. Compared with natural synapses, motoneurons without any contact with a myocyte (naive neurons) released ACh in smaller quantal packets. 2. Bipolar cultured motoneurons, which were in contact with a myocyte with one axon branch (contact-end) but remained free at another axon branch (free-end), were further used to examine quantal ACh secretion. The ACh quantal size recorded at free-end terminals was similar to that of naive neurons and was smaller than that at the contact-end, indicating that myocyte contact exerts differential regulation on quantal secretion in the same neuron. 3. Some of the neurons that formed a natural synapse with a myocyte continued to grow forward and ACh quantal secretion from the free growth cone was examined. The ACh quantal size recorded at free growth cones was inversely proportional to the distance to the natural synapse, implying localized regulation of quantal secretion by the myocyte. 4. Chronic treatment of day-1 cultures with veratridine and d-tubocurarine, respectively, increased and decreased the neurotrophic action of myocytes when assayed on day 3. 5. Taken together, these findings suggest that the myocyte is an important postsynaptic target in the regulation of quantal secretion and that the trophic action is spatially restricted to the neighbourhood of the neuromuscular junction.

Release of acetylcholine from embryonic myocytes in Xenopus cell cultures.

1. Acetylcholine (ACh) is important as the transmitter responsible for neuromuscular transmission. Here we report the non-quantal release of ACh from embryonic myocytes. 2. Co-cultures of spinal neurons and myotomal muscle cells were prepared from 1-day-old Xenopus embryos. Single channel currents were recorded in the non-innervated myocytes. When the patch pipette was filled with Ringer solution alone, spontaneous single channel currents occurred, which were inhibited by d-tubocurarine (d-Tc). 3. The channel conductance appearing in Ringer solution (37.3 pS) was similar to that of an embryonic-type ACh channel (36.9 pS), indicating that ACh is probably released from myocytes in normal Ringer solution. 4. When the patch pipette was filled with anticholinesterase alone to prevent hydrolysis of ACh released from myocytes, both physostigmine and neostigmine in a concentration-dependent manner increased channel open probability; it was reduced by d-Tc or alpha-bungarotoxin. 5. Vesamicol and quinacrine, vesicular transporter inhibitors, reduced the channel open probability caused by ACh released from myocytes in the presence of neostigmine or physostigmine. 6. Intracellular alkalinization with NH4Cl inhibited the ACh release from myocytes, whereas, extracellular alkalinization, brought about by replacing normal Ringer solution, with pH 8.6 Ringer solution enhanced ACh release. 7. The immunocytochemistry of choline acetyltransferase (ChAT) showed that ChAT exists in both myocytes and neuronal cells but not in fibroblasts. 8. These results suggest that embryonic myocytes are capable of synthesizing and releasing ACh in a non-quantal manner. Extracellular alkalinization enhanced and intracellular alkalinization inhibited ACh release from myocytes.

Regulation of acetylcholine release by intracellular acidification of developing motoneurons in Xenopus cell cultures.

1. The effects of intracellular pH changes on the acetylcholine (ACh) release and cytoplasmic Ca2+ concentration at developing neuromuscular synapses were studied in Xenopus nerve-muscle co-cultures. 2. Spontaneous and evoked ACh release of motoneurons was monitored by using whole-cell voltage-clamped myocytes. Intracellular alkalinization with 15 mM NH4Cl slightly reduced the frequency of spontaneous synaptic currents (SSCs). However, cytosolic acidification following withdrawal of extracellular NH4Cl caused a marked and transient increase in spontaneous ACh release. 3. Another method of cytosolic acidification was used in which NaCl in Ringer solution was replaced with weak organic acids. The increase in spontaneous ACh release paralleled the level of intracellular acidification resulting from addition of these organic acids. Acetate and propionate but not isethionate, methylsulphate and glucuronate, caused an increase in intracellular pH and a marked increase in spontaneous ACh release. 4. Impulse-evoked ACh release was slightly augmented by intracellular alkalinization and inhibited by cytosolic acidification. 5. Cytosolic acidification was accompanied by an elevation in the cytoplasmic Ca2+ concentration ([Ca2+]i), resulting from both external Ca2+ influx and intracellular Ca2+ mobilization. In contrast, the increase in [Ca2+]i induced by high K+ was inhibited by cytosolic acidification. 6. We conclude that cytosolic acidification regulates spontaneous and evoked ACh release differentially in Xenopus motoneurons, increasing spontaneous ACh release but inhibiting evoked ACh release.

Potentiation of transmitter release by ciliary neurotrophic factor requires somatic signaling.

Neurotrophic factors participate in the development and maintenance of the nervous system. Application of ciliary neurotrophic factor (CNTF), a protein that promotes survival of motor neurons, resulted in an immediate potentiation of spontaneous and impulse-evoked transmitter release at developing neuromuscular synapses in Xenopus cell cultures. When CNTF was applied at the synapse, the onset of the potentiation was slower than that produced by application at the cell body of the presynaptic neuron. The potentiation effect was abolished when the neurite shaft was severed from the cell body. Thus, transmitter secretion from the nerve terminals is under immediate somatic control and can be regulated by CNTF.

Heterosynaptic suppression of developing neuromuscular synapses in culture

Activity-dependent competition between two spinal neurons coinnervating an embryonic myocyte was studied in Xenopus cell culture. We have characterized in detail the phenomenon of heterosynaptic suppression by which tetanic stimulation of one neuron results in functional suppression of the synapse made by the untetanized neuron (Lo and Poo, 1991). Fluorescence labeling of the neurons using two different fluorophores revealed that the coinnervating nerve terminals on the spherical myocyte were in close proximity. Heterosynaptic suppression could be induced when the postsynaptic cell was held under either current-clamp or voltage-clamp conditions during the tetanic stimulation. This finding, together with the observation that repetitive postsynaptic depolarization of the myocyte by direct current injection was much less effective in inducing synaptic depression, suggests that postsynaptic ACh receptor activation plays a dominant role in the induction of heterosynaptic suppression. The heterosynaptic suppression appears to be mediated by a rise of Ca2+ levels in the postsynaptic cell, since it was not observed when the cytosolic Ca2+ concentration of the myocyte was buffered at a low level with intracellular loading of a Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA). The dependence of heterosynaptic suppression on the pattern of tetanic stimulation was also studied. At a stimulation frequency of 2 Hz, detectable heterosynaptic suppression could be induced after 20 repetitive stimuli were applied to one of the presynaptic neurons and the suppression was more effective with increasing number of stimuli. Over the range of 0.5-5 Hz, the extent of suppression was independent of the frequency of tetanic stimulation and, in some cells, detectable suppression could be induced at a frequency as low as 0.05 Hz. Except for a few cases, heterosynaptic suppression was found to last for as long as the recording was made after tetanus (up to 1 hr). The fact that the mean amplitude of spontaneous synaptic currents remained the same before and after the suppression while the evoked synaptic currents exhibited higher fluctuation after suppression suggests that the observed synaptic suppression involves a reduction of evoked ACh release from the nerve terminal, although postsynaptic changes have not been excluded. Finally we found that spontaneous synaptic activity may also contribute in part to the synaptic competition between coinnervating nerve terminals. Taken together, these findings provide a quantitative basis for further understanding of activity-dependent competition between developing neuromuscular synapses.

Potentiation by ATP of the postsynaptic acetylcholine response at developing neuromuscular synapses in Xenopus cell cultures.

1. Extracellular application of ATP to developing Xenopus neuromuscular synapses in culture resulted in a marked increase in the amplitude and frequency of spontaneous synaptic currents, using whole-cell recording. 2. The postsynaptic action of ATP was examined by studying the response of isolated muscle cells to iontophoretically applied acetylcholine (ACh). ATP enhanced the responses of the muscle membrane to ACh. The order of potency for various nucleotides (ATP = ADP >> AMP, adenosine, GTP) suggests that ATP acts through P2-purinoceptors. The effect of ATP on whole-cell currents was also abolished by the protein kinase inhibitor H-7. 3. Single-channel measurements indicate that ATP increased the mean open time of low-conductance ACh channels. No change in the conductance of ACh channels was observed. 4. Local application of ATP to one region of the elongated myocyte surface resulted in potentiated ACh responses only at the ATP-treated region, suggesting that the cytosolic second messengers were effectively confined within the muscle cytoplasm. 5. The results of the present study suggest that ATP released from the nerve terminals may potentiate the ACh response of developing muscle cells during the early phase of synaptogenesis, and that the action of ATP can be restricted to the subsynaptic region exposed to the secreted ATP.

Potentiation by endogenously released ATP of spontaneous transmitter secretion at developing neuromuscular synapses in Xenopus cell cultures.

1. Previously we have shown that extracellular application of ATP, a substance co-stored and co-released with acetylcholine (ACh) in the peripheral nervous system, markedly potentiated the frequency of spontaneous synaptic currents (SSCs) produced by ACh. In the present study, we have further characterized the purinoceptor which mediates the potentiation effect of ATP and the role of endogenously released ATP. 2. Pretreatment with a P2-purinoceptor antagonist, suramin (0.3 mM), but not a P1-purinoceptor antagonist, 8-phenyltheophylline (0.1 mM), prevented the potentiating effect of ATP. 3. We studied the role of endogenously released ATP by examining the effect of a specific P2-purinoceptor antagonist on the frequency of spontaneous synaptic events at high-activity synapses (> or = 3 Hz) and found that suramin, but not 8-phenyltheophylline markedly reduced the frequency of SSCs at these high-activity synapses. In addition, desensitizing the P2-purinoceptor with alpha,beta-methylene ATP also produced similar effects to suramin. 4. Extracellular application of the L-type Ca2+ channel blockers, verapamil, nifedipine or diltiazem (10 microM each) reduced SSC frequency of high-activity synapses, while the N-type Ca2+ channel blocker, omega-conotoxin had no appreciable effect. The potentiating effect of ATP was further prevented by pretreatment with the L-type Ca2+ channel blockers. On the other hand, Bay K 8644, which is a depolarization-dependent L-type Ca2+ channel agonist, potentiated SSC frequency at these high-activity synapses. 5. These results suggest that endogenous release of ATP at developing neuromuscular synapses is responsible for the maintenance of high levels of spontaneous ACh release, which is known to play a crucial role in regulating postsynaptic differentiation.

Activity-dependent modulation of developing neuromuscular synapses.

Spontaneous and impulse-evoked synaptic currents were observed immediately following nerve-muscle contact in Xenopus cell cultures. The functional significance of this early synaptic activity was examined. Stimulation of pre- and/or post-synaptic cells was found to exert immediate and persistent effects on the efficacy of synaptic transmission. Exogenous application of calcitonin gene-related peptide (CGRP) and neurotrophins, factors that may be coreleased with ACh in activity-dependent manner at the developing neuromuscular junctions, also modulate either the postsynaptic ACh response or presynaptic ACh release. These results underscore the plasticity of developing neuromuscular synapses and suggest a complex interplay between electrical activity and chemical factors during the formation and maturation of neuronal connections.

Pulsatile release of acetylcholine by nerve terminals (synaptosomes) isolated from Torpedo electric organ.

1. Electrophysiological detection of acetylcholine (ACh) release by synaptosomes from the electric organ of Torpedo was searched for by laying the isolated nerve terminals on a culture of Xenopus embryonic muscle cells (myocytes), and by recording the ACh-induced inward currents in the myocytes. 2. Whole-cell recording in one of the myocytes revealed rapid inward currents that where generated soon after synaptosome application. These pulsatile events strongly resembled those occurring normally during the early phase of synaptogenesis after nerve-muscle contact in Xenopus cell cultures. They were called spontaneous synaptic currents (SSCs). 3. The SSCs produced by the synaptosomes had a rapid time course, with mean time-to-peak and half-decay times of 2.6 +/- 0.4 ms and 6.0 +/- 1.1 ms, respectively. Most events had a falling phase that could be fitted with a single exponential. The mean time constant of decay was 6.2 +/- 1.1 ms. More than half of the SSCs (approximately 60%) constituted a rather homogenous population in which the time-to-peak versus amplitude showed a positive relationship, the smallest events displaying a shorter time course. The rest of the SSCs had a more variable and slower time course. Such events are also observed in young and mature junctions in situ. 4. The amplitudes of SSCs had a wide distribution which was skewed towards the smallest values. The mean amplitude was 65.2 +/- 16.1 pA. 5. During the minutes following an application of synaptosomes, the frequency of the SSCs tended to decrease, but their mean amplitude remained constant. Such behaviour could be reproduced during several successive additions of synaptosomes while recording in the same myocyte. 6. Just after synaptosome application, the SSCs were superposed to a noisy inward current that lasted for 20-60 s. Noise analysis of this current gave the values of 0.7 +/- 0.1 pA for the mean amplitude of the elementary event, and 4.7 +/- 0.2 ms for its mean duration, values that compare well with those reported for the activation of frog embryonic nicotinic receptor. This suggests that the noisy current was due to ACh molecules set free by synaptosomes which were either damaged or which released ACh at some distance. This view was strengthened by biochemical analysis of ACh release by synaptosomes in vitro. 7. Tubocurarine reversibly abolished the appearance of both the noise and the synaptosome-generated SSCs, showing that these currents were due to the action of ACh.(ABSTRACT TRUNCATED AT 400 WORDS)

Exogenous synapsin I promotes functional maturation of developing neuromuscular synapses

We have investigated the possible role of synapsin I, a nerve terminal-specific protein, in the maturation of neuromuscular synapses in Xenopus cell cultures. Purified synapsin I was loaded into embryonic spinal neurons by injection of the protein into one of the early blastomeres of a Xenopus embryo. At synapses made by synapsin I-loaded neurons, spontaneous synaptic currents occurred with higher frequency and amplitude, and the amplitude exhibited an earlier appearance of a bell-shaped distribution. These characteristics are indicative of more mature quantal secretion. Impulse-evoked synaptic currents also showed a significant increase in amplitude. Using cell manipulation techniques, enhanced transmitter release from synapsin I-loaded neurons was shown to occur at the onset of synaptogenesis, suggesting a presynaptic developmental action of synapsin I prior to synaptic contact. Taken together, these results suggest that endogenous synapsin 1 may participate in the functional maturation of synapses.

ATP potentiates spontaneous transmitter release at developing neuromuscular synapses

Extracellular application of ATP, a substance co-stored and co-released with acetylcholine in peripheral nervous systems, potentiates the spontaneous secretion of acetylcholine at developing neuromuscular synapses in Xenopus cell culture, as shown by a marked increase in the frequency of spontaneous synaptic currents recorded in the postsynaptic muscle cell. The effect of ATP is apparently mediated by the activation of cytosolic protein kinases and requires the influx of Ca 2+ through the plasma membrane. Since spontaneous acetylcholine release is known to regulate the development of contractile properties of the postsynaptic muscle cell, extracellular ATP may serve as a positive trophic factor at developing neuromuscular synapses.