Xenopus Nerve-muscle Cultures Research Articles

Both A chain and B chain of β-bungarotoxin are functionally involved in the facilitation of spontaneous transmitter release in Xenopus nerve–muscle cultures

In the present study, Xenopus nerve–muscle cultures were used to explore the functional roles of A chain (a phospholipase A 2 subunit) and B chain (a non-phospholipase A 2 subunit) of Bungarus multicinctus β-bungarotoxin. It was found that β-bungarotoxin induced an increment of the frequency of spontaneous synaptic currents (SSCs) in the nerve–muscle cultures. Modification of β-bungarotoxin with pyridoxal-5′-phosphate or substitution of Ca 2+ with Ba 2+ in buffer abolished the phospholipase A 2 activity of β-bungarotoxin and the facilitatory phase of SSC as well. Antibodies that were directed specifically against A chain or B chain effectively inhibited phospholipase A 2 activity, and as a consequence the SSC frequency was not greatly different from the control rate. These results suggest that both A and B chains are indispensable parts of β-bungarotoxin for inducing the facilitation of SSC frequency with Xenopus nerve–muscle cultures.

Localized Synaptic Potentiation by BDNF Requires Local Protein Synthesis in the Developing Axon

Brain-derived neurotrophic factor (BDNF) is known to promote neuronal survival, guide axonal pathfinding, and participate in activity-dependent synaptic plasticity. In Xenopus nerve-muscle cultures, localized contact of a single BDNF-coated bead with the presynaptic axon resulted in potentiation of transmitter secretion at the developing synapses, but only when the bead was placed within 60 μm from the synapse. The localized potentiation induced by BDNF is accompanied by a persistent local elevation of [Ca 2+] i in the axon and requires constitutive presynaptic protein translation, even for axons severed from the cell body. Thus, presynaptic local TrkB signaling and protein synthesis allow a localized source of BDNF to potentiate transmitter secretion from nearby synapses, a property suited for spatially restricted synaptic modification by neurotrophins.

Tracking presynaptic Ca2+ dynamics during neurotransmitter release with Ca2+-activated K+ channels.

Neurotransmitter release during action potentials is thought to require transient, localized [Ca2+]i as high as hundreds of micromolar near presynaptic release sites. Most experimental attempts to characterize the magnitude and time course of these Ca2+ domains involve optical methods that sample large volumes, require washout of endogenous buffers and often affect Ca2+ kinetics and transmitter release. Endogenous calcium-activated potassium (KCa) channels colocalize with presynaptic Ca2+ channels in Xenopus nerve-muscle cultures. We used these channels to quantify the rapid, dynamic changes in [Ca2+]i at active zones during synaptic activity. Confirming Ca2+-domain predictions, these KCa channels revealed [Ca2+]i over 100 microM during synaptic activity and much faster buildup and decay of Ca2+ domains than shown using other techniques.

Activity-induced potentiation of developing neuromuscular synapses.

Electrical activity plays a critical role in shaping the structure and function of synaptic connections in the nervous system. In Xenopus nerve-muscle cultures, a brief burst of action potentials in the presynaptic neuron induced a persistent potentiation of neuromuscular synapses that exhibit immature synaptic functions. Induction of potentiation required an elevation of postsynaptic Ca2+ and expression of potentiation appeared to involve an increased probability of transmitter secretion from the presynaptic nerve terminal. Thus, activity-dependent persistent synaptic enhancement may reflect properties characteristic of immature synaptic connections, and bursting activity in developing spinal neurons may promote functional maturation of the neuromuscular synapse.

Overexpression of synaptotagmin modulates short-term synaptic plasticity at developing neuromuscular junctions

The level of synaptotagmin I or II in developing spinal neurons was increased by injection of synaptotagmin messenger RNA into early blastomeres of Xenopus embryos. The effect of overexpression of synaptotagmin on synaptic function was assayed in Xenopus nerve–muscle cultures within two days after injection. At neuromuscular synapses made by synaptotagmin-overexpressing neurons, the frequency of miniature postsynaptic currents was markedly reduced, while their mean amplitude was unchanged, as compared to those of control neurons in the same culture. The amplitude of evoked postsynaptic currents elicited by low-frequency test stimuli was not affected by overexpression. However, synapses made by synaptotagmin-overexpressing neurons exhibited significantly higher paired-pulse facilitation and reduced tetanus-induced depression of the synaptic response, and there was also an increased number of synaptic vesicles at regions 100–300nm from the plasmalemma at such synapses.These results show that synaptotagmins can exert an inhibitory action on the spontaneous exocytosis of synaptic vesicles. The effects on short-term plasticity suggest that synaptotagmin may facilitate vesicular supply for the evoked release during higher frequency transmission.

Potentiation of Developing Synapses by Postsynaptic Release of Neurotrophin-4

The hypothesis that synaptic functions can be regulated by neurotrophins secreted from the postsynaptic cell was examined in Xenopus nerve-muscle cultures. Neuromuscular synapses formed on myocytes overexpressing neurotrophin-4 (M+ synapses) exhibited a higher level of spontaneous synaptic activity and enhanced evoked synaptic transmission as compared to those formed on normal control myocytes (M− synapses). The NT-4 effects involve a potentiation of presynaptic transmitter secretion as well as a lengthening of the mean burst duration of postsynaptic low conductance acetylcholine channels. Repetitive stimulation of either the presynaptic neuron or the postsynaptic myocyte led to a potentiation of synaptic transmission at M+ synapses. All potentiation effects of NT-4 overexpression were abolished by the extracellular presence of TrkB-IgG but not by the presence of TrkA-IgG, indicating that postsynaptic secretion of NT-4 was responsible for the synaptic modification.

Spread of Synaptic Depression Mediated by Presynaptic Cytoplasmic Signaling

Postsynaptic activity may modulate presynaptic functions by transsynaptic retrograde signals. At developing neuromuscular synapses in Xenopus nerve-muscle cultures, a brief increase in the cytosolic calcium ion (Ca2+) concentration in postsynaptic myocytes induced persistent depression of presynaptic transmitter secretion. This depression spread to distant synapses formed by the same neuron. Clearance of extracellular fluid did not prevent the spread of depression, and depression could not be induced by increasing the Ca2+ concentration in a nearby myocyte not in contact with the presynaptic neuron. Thus, the spread of depression is mediated by signaling in the presynaptic cytoplasm, rather than by a retrograde factor in the extracellular space.

Effects of cytochalasin treatment on short-term synaptic plasticity at developing neuromuscular junctions in frogs.

1. The role of actin microfilaments in synaptic transmission was tested by monitoring spontaneous and evoked transmitter release from developing neuromuscular synapses in Xenopus nerve-muscle cultures, using whole-cell recording of synaptic currents in the absence and presence of microfilament-disrupting agents cytochalasins B and D. 2. Treatment with cytochalasins resulted in disruption of microfilament networks in the growth cone and the presynaptic nerve terminal of spinal neurons in Xenopus nerve-muscle cultures, as revealed by rhodamine-phalloidin staining. 3. The same cytochalasin treatment did not significantly affect the spontaneous or evoked synaptic currents during low-frequency stimulation at 0.05 Hz in these Xenopus cultures. Synaptic depression induced by high-frequency (5 Hz) stimulation, however, was reduced by this treatment. Paired-pulse facilitation for short interpulse intervals was also increased by the treatment. 4. These results indicate that disruption of microfilaments alters short-term changes in transmitter release induced by repetitive activity, without affecting normal synaptic transmission at low frequency. 5. Our results support the notion that actin microfilaments impose a barrier for mobilization of synaptic vesicles from the reserve pool, but do not affect the exocytosis of immediately available synaptic vesicles at the active zone.

Depression of developing neuromuscular synapses induced by repetitive postsynaptic depolarizations

Effect of postsynaptic activity on the synaptic efficacy was studied in Xenopus nerve-muscle cultures. Repetitive postsynaptic depolarizations induced by injection of current pulses into singly innervated myocytes resulted in significant reduction in the frequency of spontaneous synaptic currents and the amplitude of nerve-evoked synaptic currents at the majority of synapses that showed immature synaptic properties. Repetitive hyperpolarizations and steady depolarizations of similar duration were without effect. The depolarization-induced synaptic depression appeared to result predominantly from a reduced ACh secretion from the presynaptic nerve terminal. Buffering the myocyte cytosolic Ca2+ at a low level with intracellular loading of a Ca2+ buffer, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA), significantly reduced the effect of the depolarizations. Thus postsynaptic electrical activity can regulate the synaptic efficacy of the developing neuromuscular synapases and the regulation may be mediated by retrograde transsynaptic interactions.

L-type Ca 2+ channel is involved in the regulation of spontaneous transmitter release at developing neuromuscular synapses

Involvement of an L-type Ca 2+ channel in the regulation of spontaneous transmitter release was studied in Xenopus nerve-muscle cultures. The frequency of spontaneous synaptic currents, which reflects impulse-independent acetylcholine release from the nerve terminals, showed a marked increase in high-K + medium or after treatment with a phorbol ester, 12- O-tetradecanoyl-phorbol 13-acetate, a drug that activates protein kinase C and depolarizes the presynaptic neuron. The potentiation effect of high K + and 12- O-tetradecanoyl-phorbol 13-acetate requires Ca 2+ influx through the L-type Ca 2+ channel in the plasma membrane, since it was significantly reduced by the presence of nifedipine, verapamil or diltiazem and enhanced by Bay K 8644, an L-type Ca 2+ channel agonist. It was shown recently that adenosine 5'-triphosphate markedly potentiates the spontaneous acetylcholine release at these synapses through the binding of P 2-purinoceptors and the activation of protein kinase C. We found in the present study that potentiation effects of adenosine 5'-triphosphate are inhibited by L-type Ca 2+ channel blockers, suggesting that the L-type Ca 2+ channel is responsible for the positive regulation of spontaneous acetylcholine secretion by adenosine 5'-triphosphate at the developing neuromuscular synapses. Our data suggest that modulation of the L-type Ca 2+ channel in embryonic motor nerve terminals is important for the regulation of spontaneous transmitter release.

Heparin and heparan sulfate partially inhibit induction of acetylcholine receptor accumulation by nerve in Xenopus culture

It has been demonstrated that ACh receptors in Xenopus nerve-muscle cultures migrate in the membrane to the nerve contact area during junction formation (Anderson et al., 1977) and that "diffusion trapping" is the major mechanism for nerve-induced receptor accumulation (Kidokoro and Brass, 1985; Kuromi et al., 1985; Kidokoro et al., 1986). A crucial remaining question is how the nerve induces the trap for randomly diffusing ACh receptors. In this study we examined the effect of various glycosaminoglycans in the culture medium on the nerve-induced receptor accumulation and found that heparin and heparan sulfate partially inhibited nerve-induced receptor accumulation, but similar molecules, chondroitin sulfate type A and type C, did not. By chemical modification of heparin we also showed that N-sulfate residues and a large-molecular-weight molecule are essential for this inhibitory effect. Heparin did not affect ACh receptor clustering (hot-spot formation) in myocytes cultured without nerve. By changing the time and duration of heparin application, we found that heparin was effective in inhibiting nerve-induced receptor accumulation only when it was present in the culture medium during the period that neurites are actively forming contact with muscle membrane.

Action potentials and sodium inward currents of developing neurons in Xenopus nerve-muscle cultures

Action potentials and voltage-gated Na+ inward currents from cultured embryonic neurons of Xenopus laevis were recorded using the patch-clamp technique in the whole cell configuration. Neurons together with muscle cells were dissociated from embryos shortly after completion of gastrulation. Under the voltage-clamp condition the voltage-gated Na+ inward current was isolated from other currents by pharmacological means and by ion substitution. A small Na+ current was observed in round cells without neurites (presumptive neurons). The mean amplitude of the peak Na+ current was 2.5 times larger in neurons with short processes than in presumptive neurons. As they developed further by extending longer processes, the maximum amplitude of the Na+ inward current recorded at the soma decreased. In varicosities, the Na+ inward current density was greater than that at the soma of neurons with extended neurites but kinetic properties and voltage-dependency were similar.

Distribution of synaptic specializations along isolated motor units formed in Xenopus nerve-muscle cultures

The capacity of individual muscle cells and neurons to establish synaptic specializations along the entire extent of their neurite-muscle contacts was assessed in culture. Spinal cord neurons derived from embryos of Xenopus laevis were plated at low density in cultures of Xenopus myotomal muscle cells in order to obtain isolated motor units whose neuron and muscle cells were not contacted by any other neuron. These isolated motor units were examined for localization of acetylcholine receptors (AChRs) after staining with fluorescent alpha-bungarotoxin and, in some cases, for localization of a synaptic vesicle antigen by immunofluorescence. The neurite-muscle contacts formed by competent neurons exhibited discontinuous sites of AChR localization occupying about 25% of the contact length as compared with 2% for incompetent neurons. Competent neurons, unlike incompetent ones, established these neurite-associated receptor patches (NARPs) on virtually all the muscle cells they contacted and functionally innervated them. These and other observations on the distribution of NARPs throughout the isolated motor units indicate that the capacity of competent neurons to establish NARPs extends to the limits of growth of most if not all of their neurites, that this capacity is least in the most proximal portions of initial neuritic segments, and that the overall capacity of muscle cells to generate NARPs can be saturated by long lengths of neurite-muscle contact. The results also suggest that even in the absence of competitive interactions between neurons there are spatial discontinuities in neuritic action and/or muscle response during the establishment of NARPs. Synaptic vesicle antigen patches (SVAPs) occurred along the neuritic arbor of all neurons, but their distribution in competent neurons (those which established NARPs) and in incompetent ones differed. For competent neurons the percentage of neurite length occupied by SVAPs was 4.8-fold greater on muscle cells than off, whereas the corresponding value for incompetent neurons was only 1.5-fold. This large preferential localization of SVAPs along neurite-muscle contacts of competent neurons was further associated with a colocalization of SVAPs and NARPs that was greater than predicted by chance. These results suggest that muscle cells are much more effective in influencing the distribution of synaptic vesicles along the neuritic arbor of competent neurons than along the arbor of incompetent neurons and that this influence is greatest at sites of postsynaptic differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)

Release of acetylcholine from embryonic neurons upon contact with muscle cell

When a spherical muscle cell (myoball) was manipulated into contact with either the soma or the neurite of an isolated neuron in 2-day-old Xenopus nerve-muscle cultures, depolarizations similar to miniature endplate potentials (MEPPs) were frequently detected in the muscle cell. These depolarizations occurred within minutes after myoball-soma contact and within seconds after myoball-neurite contact. They had time course and amplitude distribution similar to those of the MEPPs recorded from naturally occurring neuromuscular synapses between neurites and muscle cells found in the same cultures, but they occurred at a lower frequency and had smaller average amplitudes. These depolarizations were induced by acetylcholine (ACh) since they were reversibly blocked by addition of d-tubocurarine into the culture, and they were abolished in muscle cells pretreated with alpha-bungarotoxin before contact with the neuron. Greater than 60% of the neuronal population in these cultures released ACh upon this direct muscle contact. The appearance of MEPP-like potentials in the myoball upon contact with an isolated neuron suggests that the cellular machinery responsible for ACh release is present throughout the neuron and that packages of ACh molecules are available for release prior to nerve-muscle synapse formation. We also found that neurons which had previously made synapse with other muscle cells in the culture all failed to release ACh from the soma and showed reduced release capability at the neurite for the first 30 min to 1 hr of contact with a myoball. This finding suggests that, during synapatogenesis, there is a depletion of ACh molecules and/or substances responsible for the triggering of their release in the extrasynaptic regions of the neuron.

Two types of miniature endplate potentials in Xenopus nerve-muscle cultures

The frequency distribution of miniature endplate potential (mepp) amplitudes was examined in Xenopus nerve-muscle cultures. In the early days of nerve-muscle co-culture (1-2 days) the mepp amplitude histogram was skewed in the majority of muscle cells and had a peak approximately at 0.6 mV. Innervated muscle cells in older cultures (3-4 days), however, often had two peaks. The distribution at the smaller amplitude was skewed and the peak was approximately at 0.6 mV, whereas the peak at the larger amplitude was more-or-less symmetrical and about 18 times greater than the small one. The time-to-peak value of mepps in the peak at the smaller amplitude was slightly smaller than those in the peak at the larger amplitude. Small mepps were not due to electrotonic spread of mepps generated at distant sites nor due to focal development of acetylcholinesterase. Two peaks were also observed in the amplitude histogram of extracellularly recorded miniature endplate currents. Therefore, these two types of mepps must be generated practically at the same site. Mepps in the peak at the smaller amplitude had similar properties as sub-miniature endplate potentials (s-mepps) described in the adult animal. The second symmetrical peak in older cultures may correspond to classical mepps (c-mepps). The age-dependent shift from the amplitude distribution with one peak to that with two peaks has also been reported in developing and regenerating neuromuscular junctions. Therefore, I suggest that during development in culture, mepps appear at first as s-mepps and subsequently c-mepps develop, forming the second peak in the amplitude histogram.

Initial synaptic transmission at the growth cone in Xenopus nerve-muscle cultures.

The excellent visibility of cultured cells allows the early events during formation of the neuromuscular junction to be suitably studied. It has been shown in various culture systems that synaptic transmission occurs early after nerve-muscle contact. Early synaptic potentials are small in amplitude and slow in time course reflecting a low acetylcholine receptor density at the site of nerve contact. Acetylcholine receptors accumulate later at the contact region. We have examined initial synaptic transmission at the growth cone-muscle contact in Xenopus nerve-muscle cultures. The approaching growth cone was observed under a phase-contrast microscope while the membrane potential of its target muscle cell was continuously monitored by using an intracellular microelectrode. The innervating neuron was stimulated extracellularly at the cell body. No synaptic potential was evoked when the growth cone was contacting the muscle only at the tip of filopodia. However, as soon as the main portion of the growth cone contacted the muscle membrane, nerve-evoked synaptic potentials were detected after stimulation of the nerve. This immediate appearance of synaptic potentials raises the possibility that acetylcholine could be released at the growth cone even prior to contact with muscle cells. As the area of contact enlarged during the observation period the amplitude of end-plate potentials also increased. Spontaneous synaptic potentials (miniature end-plate potentials) were rarely observed in these early growth cone-muscle contacts. Although there were several inherent difficulties, quantal analysis of the end-plate potentials was attempted by using binomial statistics. This analysis suggests that nerve-evoked transmitter release at the growth cone occurs in a quantal fashion.