Nerve-muscle Cultures Research Articles

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.

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.

Synapsin IIa accelerates functional development of neuromuscular synapses.

We have investigated the possible involvement of the synaptic vesicle protein synapsin IIa in synapse development. Synapsin IIa was introduced into Xenopus embryonic spinal neurons by early blastomere injection, and nerve-muscle cultures were prepared. Synaptic currents were measured by comparing synapses in which the presynaptic neuron either contained [syn IIa (+)] or lacked (control) exogenous synapsin IIa. Syn IIa (+) synapses had a 3.6-fold increase in the frequency and a 2.1-fold increase in the amplitude of spontaneous synaptic currents, compared to controls, after 2 days in culture. Synapsin IIa also increased the amplitude of evoked synaptic currents by 2.3-fold in 2-day cultures. The evoked synaptic current amplitudes of syn IIa (+) synapses had a lower coefficient of variation indicating a more stable evoked response. These enhanced synaptic activities were independent of the presence or absence of the protein in the postsynaptic muscle cell. The findings indicate a role for synapsin IIa in synapse maturation.

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.

Effect of myasthenic patient sera on the number and distribution of acetylcholine receptors in muscle and nerve-muscle cultures from rat: Correlations with clinical state

We studied the functional activities (FA) of sera obtained from 83 myasthenic patients on rat muscle cultures. Using the same sets of cultures, two parameters were evaluated after exposure to sera: residual fraction (RF) of acetylcholine receptors (AChR) coupled to 125I-labelled α-bungarotoxin (αBgt) (81 sera) and the number of rhodamine labelled clusters (56 sera). Two types of culture were assayed: muscle alone and nerve-muscle cocultures (12 cases). In all combinations (fluorescence, radio-labelling, muscle alone and nerve-muscle cocultures), we found a significant correlation between FA and antibody (Ab) titre, and no correlation between FA and clinical severity: only sera with a high or intermediate Ab titre were effective, whatever the clinical severity of disease. With active sera, AChR loss was about 50% whereas the disappearance of AChR clusters was quite complete, which suggests AChR redistribution induced by MG sera.

Early cross-striation formation in twitching Xenopus myocytes in culture.

Spontaneous release of neurotransmitter has been demonstrated in various types of synapses. Its physiological significance, however, is still unknown. In nerve-muscle cultures of embryonic Xenopus laevis, we observed that acetylcholine, which is released spontaneously at the synaptic terminal, caused frequent twitches of muscle cells. These muscle cells developed cross-striations earlier than neighboring non-twitching cells. This effect of innervation was unaffected by tetrodotoxin but was blocked by alpha-bungarotoxin. Repeated iontophoretic application of acetylcholine or KCl to muscle cells caused twitches and also accelerated the formation of cross-striations. Thus twitching apparently promotes lateral alignment of myofibrils. It is also known that myosin synthesis is higher in twitching muscle cells. Therefore, successfully innervated twitching muscle cells may have an advantage for faster differentiation over neighboring non-twitching muscle cells. We suggest that spontaneously released transmitter may serve as a mediator for trophic interaction at forming synapses.

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)

Trophic influence of sympathetic neurons on the cardiac alpha-adrenergic response requires close nerve-muscle association.

In tissue culture, sympathetic neurons exert a trophic effect on myocardial cells to alter the alpha-adrenergic response from positive to negative chronotropism. We hypothesized that this neurotrophic effect might be a result of the release of a humoral substance into the bulk phase of the culture medium by the neurons. The negative chronotropic response to alpha stimulation persisted in nerve-muscle cultures pretreated with the muscarinic antagonist atropine, indicating that acetylcholine is not the trophic agent. Further, variations in the concentration of norepinephrine, epinephrine or dopamine in the nerve-muscle culture media do not account for the presence of a negative chronotropic response to alpha stimulation. Finally, muscle cells grown in the same petri dish with innervated muscle cells, to allow conditioning of the muscle cell environment by the neurons, do not acquire a negative chronotropic response to alpha stimulation. The results of this study clearly demonstrate that this trophic effect is not due to the release of a humoral substance into the bulk phase of the culture medium, but requires close nerve-muscle association. This suggests that the trophic effect may involve localized release and transsynaptic transfer of a chemical substance other than a neurotransmitter.

Presynaptic or postsynaptic origin of acetylcholinesterase at neuromuscular junctions? An immunological study in heterologous nerve-muscle cultures

Numerous studies have shown that the acetylcholine receptor (AChR) is inserted in the plasma membrane of the mnuscle fiber, and that it is focalized at the site of neuromuscular junctions, as an effect of neural influence. In contrast, acetylcholinesterase (AChE) may be presynaptic or anchored in the basal lamina, as well as postsynaptic at neuromuscular junctions. We investigated the origin of the junctional enzyme, particularly the collagen-tailed asymmetric A 12 forms, by studying the AChE contents of heterologous rat and chicken neuromuscular cocultures by immunohistochemical and biochemical methods. We found that the overall content of AChE, in the neuromuscular cocultures, including the A 12 form, was essentially identical to the sum of the contents of separate myotube and motoneuron cultures. The sedimentation coefficients of the rat and chicken asymmetric forms are sufficiently different to clearly differentiate these enzymes in sucrose gradients: 16 S for rat, 20 S for chicken A 12 AChE. Sedimentation analyses of AChE in cocultures thus showed that the A 12 form was of muscular origin. In the case of aneural cultures of myotubes, histochemical staining of AChE activity or immunohistochemical staining with specific antibodies showed only very scarce, faint concentrations of enzyme. Some patches of acetylcholine receptor (AChR) were, however, visible in these cultures. Neuromuscular contacts are readily established in cocultures of myotubes with embryonic motoneurons from spinal cords. In the presence of motoneurons, the myotubes presented a larger number of AChR patches. The most remarkable feature of neuromuscular cocultures was the presence of numerous intense AChE patches which always coincided with AChR clusters. By specifically staining nerve terminals with tetanus toxin, we could show an excellent correlation between neuromuscular contacts and the presence of AChE-AChR patches. We found that the AChE patches in heterologous cocultures could be stained exclusively by the anti-myotube AChE antiserum. The focalized enzyme is therefore exclusively, or very predominantly, provided by the myotube.

Tissue culture studies of muscle disorders: Part 2. Biochemical studies, nerve-muscle culture, metabolic myopathies, and animal models.

This review continues with studies of protein, lipid, and purine metabolism of Duchenne muscular dystrophy (DMD) cells in vitro and of muscle cells in combined culture with nerve cells. In vitro studies of human metabolic myopathies are tabulated. Results using the hamster, chicken, and mouse (dy25, dy, mdg, and mdx) myopathies are discussed. Interesting findings include suggestions of altered collagen synthesis by DMD cells. Analysis of cell proteins by two-dimensional gel electrophoresis and the use of combined nerve-muscle cultures remain important areas of development. It is disappointing that so few attempts have been made to repeat significant findings in this field, and when a number of laboratories have examined the same phenomenon, the results are often contradictory. It remains to be shown how these various abnormalities found in cells in vitro are related to each other and to those pathologic features of diseased muscle observed in vivo.

Acquisition by innervated cardiac myocytes of a pertussis toxin-specific regulatory protein linked to the alpha 1-receptor.

During development, the chronotropic response of rat ventricular myocardium to alpha 1-adrenergic stimulation changes from positive to negative. The alpha 1-agonist phenylephrine increases the rate of contraction of neonatal rat myocytes cultured alone but decreases the rate of contraction when the myocytes are cultured with functional sympathetic neurons. The developmental induction of the inhibitory myocardial response to alpha 1-adrenergic stimulation in intact ventricle and in cultured myocytes was shown to coincide with the functional acquisition of a substrate for pertussis toxin. A 41-kilodalton protein from myocytes cultured with sympathetic neurons and from adult rat myocardium showed, respectively, 2.2- and 16-fold increases in pertussis toxin-associated ADP-ribosylation (ADP, adenosine diphosphate) as compared to controls. In nerve-muscle cultures, inhibition of the actions of this protein by pertussis toxin-specific ADP-ribosylation reversed the mature inhibitory alpha 1-adrenergic response to an immature stimulatory pattern. The results suggest that innervation is associated with the appearance of a functional pertussis toxin substrate by which the alpha 1-adrenergic response becomes linked to a decrease in automaticity.

Formation of acetylcholine receptor clusters at neuromuscular junction in Xenopus cultures

The formation of acetylcholine receptor (AChR) clusters at the neuromuscular junction was investigated by observing the sequential changes in AChR cluster distribution on cultured Xenopus muscle cells. AChRs were labeled with tetramethylrhodamine-conjugated α-bungarotoxin (TMR-αBT). Before innervation AChRs were distributed over the entire surface of muscle cells with occasional spots of high density (hot spots). When the nerve contacted the muscle cell, the large existing hot spots disappeared and small AChR clusters (less than 1 μm in diameter) initially emerged from the background along the area of nerve contact. They grew in size, increased in number, and fused to form larger clusters over a period of 1 or 2 days. Receptor clusters did not migrate as a whole as observed during “cap” formation in B lymphocytes. The rate of recruitment of AChRs at the nerve-muscle junction varied from less than 50 binding sites to 1000 sites/hr for αBT. In this study the diffusion-trap mechanism was tested for the nerve-induced receptor accumulation. The diffusion coefficient of diffusely distributed AChRs was measured using the fluorescence photobleaching recovery method and found to be 2.45 × 10 −10 cm 2/sec at 22°C. There was no significant difference in these values among the muscle cells cultured without nerve, the non-nerve-contacted muscle cells in nerve-muscle cultures, and the nerve-contacted muscle cells. It was found that the diffusion of receptors in the membrane is not rate-limiting for AChR accumulation.

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.