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Biomedical subjects

M M Poo

Publications and source records attributed to M M Poo.

At least 19 recordsLinked to original sources
PubMed1992

Quantal transmitter secretion from myocytes loaded with acetylcholine.

It is well known that transmitter secretion requires specialized secretory organelles, the synaptic vesicles, for the packaging, storage and exocytotic release of the transmitter. Here we report that when acetylcholine (ACh) is loaded into an isolated Xenopus myocyte, there is spontaneous quantal release of ACh from the myocyte which results in activation of its own surface ACh channels and the appearance of membrane currents resembling miniature endplate currents. This myocyte secretion probably reflects Ca(2+)-regulated exocytosis of ACh-filled cytoplasmic compartments. Furthermore, step depolarization of the myocyte membrane triggers evoked ACh release from the myocyte with a weak excitation-secretion coupling. These findings suggest that quantal transmitter secretion does not require secretory pathways unique to neurons and that the essence of presynaptic differentiation may reside in the provision of transmitter supply and modification of the preexisting secretion pathway.

Acetylcholine↗
PubMed1992

Calcium-dependent transmitter secretion reconstituted in Xenopus oocytes: requirement for synaptophysin.

Calcium-dependent glutamate secretion was reconstituted in Xenopus oocytes by injecting the oocyte with total rat cerebellar messenger RNA (mRNA). Co-injection of total mRNA with antisense oligonucleotides to synaptophysin message decreased the expression of synaptophysin in the oocyte and reduced the calcium-dependent secretion. A similar effect on secretion was observed for oocytes injected with total mRNA together with an antibody to rat synaptophysin. These results indicate that synaptophysin is necessary for transmitter secretion and that the oocyte expression system may be useful for dissecting the molecular events associated with the secretory process.

Animals↗
PubMed1992

Hebbian depression of isolated neuromuscular synapses in vitro.

Modulation of synaptic efficacy may depend on the temporal correlation between pre- and postsynaptic activities. At isolated neuromuscular synapses in culture, repetitive postsynaptic application of acetylcholine pulses alone or in the presence of asynchronous presynaptic activity resulted in immediate and persistent synaptic depression, whereas synchronous pre- and postsynaptic coactivation had no effect. This synaptic depression was a result of a reduction of evoked transmitter release, but induction of the depression requires a rise in postsynaptic cytosolic calcium concentration. Thus, Hebbian modulation operates at isolated peripheral synapses in vitro, and transsynaptic retrograde interaction appears to be an underlying mechanism.

Animals↗
PubMed1992

Retrograde modulation at developing neuromuscular synapses: involvement of G protein and arachidonic acid cascade.

Intracellular loading of nonhydrolyzable GTP analogs into innervated muscle cells in Xenopus cultures led to a marked increase in the frequency of spontaneous synaptic currents (SSCs), while extracellular application of the drugs at the same concentration was without effect. The increase in SSC frequency appeared to be unrelated to changes in the muscle membrane sensitivity toward acetylcholine (ACh), but resulted from an elevated spontaneous ACh secretion from the presynaptic nerve terminal. Postsynaptic loading of arachidonic acid (AA) produced a similar effect as the GTP analogs, and the potentiation effect of both GTP analogs and AA was reversed by an inhibitor of AA metabolism, AA861. Further studies indicate that a lipoxygenase metabolite, 5-HPETE, appears to be a likely candidate for the retrograde factor involved in modulating ACh secretion. These results suggest that G protein activation of the AA cascade in the postsynaptic cell could produce a retrograde signal to modulate transmitter secretion from the presynaptic nerve terminal at developing synapses.

Acetylcholine↗
PubMed1992

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 I may participate in the functional maturation of synapses.

Acetylcholine↗
PubMed1992

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)

Acetylcholine↗
PubMed1992

Diffusional transport of macromolecules in developing nerve processes.

Passive transport of macromolecules in growing nerve processes was analyzed quantitatively by measuring the rate of diffusion of fluorescently labeled molecules injected into the soma of cultured Xenopus neurons. We found that the diffusion of globular proteins in the neurite's cytoplasm was about five times slower than that in aqueous solution, a rate considerably higher than those inferred from previous studies on cultured non-neuronal cells. The dependence of the diffusion coefficient, D, on the size of diffusing molecules was examined by measuring the diffusional spread of fluorescently labeled dextrans over a wide range of molecular weights. We found that the size dependence of D deviates considerably from that expected for diffusion in a viscous aqueous medium: larger dextrans encounter disproportionately higher viscous resistance. Treatment of the neuron with the microfilament-disrupting agent cytochalasin B, or pre-loading of the cells with dephospho-synapsin I, a molecule that induces bundling of actin filaments, significantly increased the diffusion rate for large dextrans without affecting that of small dextrans. Taken together, these results provide a quantitative basis for assessing diffusion as a potential transport mechanism along nerve processes, and suggest that the microfilament meshwork imposes a selective constraint on the diffusion of large macromolecular components within the neuronal cytoplasm.

Animals↗
PubMed1992

Asymmetric modulation of cytosolic cAMP activity induces growth cone turning.

The possible role of cyclic nucleotides as second messengers mediating growth cone turning was studied by producing an asymmetric distribution of cyclic nucleotides across the growth cone. A repetitive pulse application method was developed to produce microscopic chemical gradients near the growth cone of embryonic Xenopus neurons in cell culture. When picoliters of a solution containing 20 mM dibutyryl cAMP (dB-cAMP), a membrane-permeable analog of cAMP, were repetitively ejected from a micropipette near the growth cone, neurite growth was consistently directed toward the pipette. Theoretical analysis of the diffusion gradient showed that the neurite is capable of detecting a 10% difference in dB-cAMP concentration across the growth cone. Similar responses were also observed using gradients of the phosphodiesterase inhibitor isobutylmethylxanthine, or of forskolin, which activates adenylate cyclase. Dibutyryl cGMP, however, produced no significant turning. These results suggest that a cytoplasmic gradient of cAMP across the growth cone is sufficient to initiate its turning response, and that cAMP in the growth cone could serve as a second messenger in mediating the action of extracellular guidance cues.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Activity-dependent synaptic competition in vitro: heterosynaptic suppression of developing synapses.

The development and stability of synaptic connections in the nervous system are influenced by the pattern of electrical activity and the competitive interaction between the adjacent nerve terminals. To investigate this influence, a culture system of nerve and muscle cells has been developed in which a single embryonic muscle cell is coinnervated by two spinal neurons. The effect of electrical activity on the synaptic efficacy was examined after repetitive electrical stimulation was applied to one or both neurons. Brief tetanic stimulation of one neuron resulted in immediate functional suppression of the synapse made by the unstimulated neuron innervating the same muscle cell. This heterosynaptic suppression was largely absent when the tetanic stimulation was applied concurrently to both neurons. This result demonstrates that activity-dependent synaptic competition can be studied in vitro at a cellular level.

Animals↗
PubMed1991

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 Ca2+ 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.

Acetylcholine↗
PubMed1991

Repetitive impulse activity potentiates spontaneous acetylcholine secretion at developing neuromuscular synapses.

The effects of presynaptic impulse activity on the transmitter secretion at developing neuromuscular junctions were examined in Xenopus nerve-muscle cultures. Repetitive suprathreshold stimulation of the presynaptic neuron results in marked potentiation of spontaneous synaptic activity, as shown by whole-cell voltage-clamp recording of synaptic currents in the postsynaptic muscle cell. Our results are consistent with the notion that synaptic efficacy of the developing synapse is potentiated by the presence of electrical activity. Such activity-dependent synaptic modulation enables the early neuronal activity to play a regulatory role during the maturation of synaptic connections.

Acetylcholine↗
PubMed1990

Diffusional and electrokinetic redistribution at the synapse: a physicochemical basis of synaptic competition.

Coinnervating nerve terminals may compete for "stabilizing factors" confined within the postsynaptic cell. The competition could be achieved through a diffusion-mediated trapping the factor, facilitated by an activity-dependent electrokinetic migration of the factor toward the synaptic site. We have examined the evidence for diffusional and electrokinetic motions of cell surface and cytoplasmic components, the profile and magnitude of the electric field produced by the synaptic current, and the plausibility that these motions underlie the process of synaptic competition.

Animals↗
PubMed1990

Synaptic contact between embryonic neurons and acetylcholine receptor-fibroblast.

1. Mouse fibroblast cell lines were established that stably express Torpedo californica acetylcholine receptors (AChR) on their cell surface in quantities sufficient for biochemical and pharmacological analyses, as well as electrophysiological analysis at the single channel level. 2. Surface-expressed AChRs were shown to be assembled into proper alpha 2 beta gamma delta pentamers. 3. The distribution of surface-AChRs was uniform and identical in every cell. 4. We were able to successfully coculture AChR-fibroblasts with 1-day old Xenopus laevis embryonal neurons and maintain expression of cell surface AChRs. 5. Using the voltage-clamp technique, miniature end-plate currents were recorded from AChR-fibroblasts which were contacted by neurons. The current amplitudes of these AChRs were approximately 10-fold smaller than those observed in Xenopus myocytes, and the rise-times were slower.

Action Potentials↗
PubMed1989

In vitro analysis of position- and lineage-dependent selectivity in the formation of neuromuscular synapses.

The hypotheses that selective formation of nerve-muscle connections depends upon intrinsic cellular properties, endowed either by the cell's rostral-caudal position in the embryo or its lineage, were tested directly in Xenopus embryonic cell cultures. The position or the lineage of embryonic cells was traced in vitro by previous injection of fluorophore-conjugated dextran molecules into individual blastomeres. Synaptic efficacy was assayed by recording synaptic currents from neurite-contacted muscle cells in the culture, and the physical affinity of neurites for muscle cells of different positional or clonal origins was assayed by counting the frequency of association between the neurites' growth cones and the muscle cells. Both assays showed no apparent preference between nerve and muscle cells of similar rostral-caudal positions or clonal origins, suggesting that there is little position- or lineage-dependent selectivity in the initial nerve-muscle interactions.

Animals↗
PubMed1989

Studies of nerve-muscle interactions in Xenopus cell culture: analysis of early synaptic currents.

We have studied the spontaneous and nerve-evoked synaptic currents during the initial period of nerve-muscle contact in Xenopus cell cultures. The precise timing of the contact was achieved by physically manipulating embryonic muscle cells into contact with co-cultured spinal neurons. Previous studies have shown that physical contact of the muscle membrane induces pulsatile release of acetylcholine (ACh) from the growth cone of these neurons, resulting in spontaneous synaptic currents (SSCs) in the muscle cell within seconds following the contact. In the present work, we first showed that these SSCs at the manipulated nerve-muscle contacts are similar to those observed at naturally occurring synapses. We then examined the possible cellular mechanisms responsible for the marked variation in SSC amplitude and showed that it most likely results from differences in either the amount of ACh contained in each release event or the extent of close membrane apposition near the release sites. During the first 20 min following the nerve-muscle contact, there was an increase in the frequency and mean amplitude of the SSCs. During a similar period, the evoked synaptic currents (ESCs), which were induced by suprathreshold electrical stimulation of the neuronal soma, also showed an increase in the mean amplitude and a reduction in the delay of onset following the stimulus. These postcontact changes in the efficacy of synaptic transmission may be related to an increase in the total area of close membrane apposition between the nerve and muscle cells. This was suggested by the finding that neurite-muscle adhesion increases over a similar postcontact period. The transition from low- to high-efficacy transmission during the early phase of contact may reflect the process of selective adhesion between the cells, and thus signify the formation of specific synapse. Analysis of the fluctuation in the ESC amplitude at the early nerve-muscle contact suggests that evoked release of ACh occurs as multiples of a quantal unit. However, this unit is apparently related to only a small subpopulation of SSCs of relatively high amplitudes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Studies of nerve-muscle interactions in Xenopus cell culture: fine structure of early functional contacts.

We have studied the fine structure of nerve-muscle contacts during the first few hours of synaptogenesis in embryonic Xenopus cell cultures. The precise timing of contact was achieved by manipulating isolated spherical myocytes (myoballs) into contact with growth cones or neurites of co-cultured spinal neurons. The contacts were shown to be functional by whole-cell voltage-clamp recording of nerve-evoked synaptic currents in the muscle cell. The ultrastructure of these functional contacts was examined by thin-section electron microscopy. In total, 20 nerve-muscle pairs were studied with contact periods ranging from 20 min to 12 hr, during which time a substantial increase in the amplitude of synaptic currents occurred. The structure of noncontacting cells and of nerve-muscle contacts formed between the cells by natural encounters in 1-d-old cultures were also examined in order to identify the features and the time course of morphological differentiation of early functional contacts. Prominent features of the contact area during the first few hours included: close apposition of the nerve and muscle membranes, greater frequency of coated pits and vesicles, and thickening of postsynaptic muscle membrane. Occasionally, clusters of clear vesicles occurred near presynaptic membrane, but no further sign of active zone differentiation was observed. In comparison, definitive active zone structure, well-formed extracellular basal lamina, and widened cleft were seen in natural contacts less than 24 hr old. This study of the identified functional contacts may help us to understand the structural basis for early nerve-muscle interaction and the functional significance of various synaptic specializations.

Animals↗
PubMed1988

In vitro analysis of specificity during nerve-muscle synaptogenesis.

The early phase of synapse formation was studied in cultures of Xenopus laevis spinal neurons and myotomal muscle cells. Two early events are described: the pulsatile secretion of acetylcholine from the nerve terminal in response to myocytic or neuronal contacts, and the development of nerve-myocyte adhesion during the first few minutes of contact. The specificity in these early events in synaptogenesis was assessed with respect to the positional and clonal relationships of the neurons and myocytes. Axial position and lineage were determined by injecting embryos with a fluorescent dye, such that dissociated cells could subsequently be identified in culture. We examined the efficacy of spontaneous synaptic currents, and the relative preponderance of growth cone-myocyte associations, for neurite-myocyte pairs of the same or dissimilar origin. Neither of these two assays revealed a dependence on the axial position or the lineage of the cells. Although these studies indicate that early nerve-muscle interactions show little positional or clonal selectivity, myocytes clearly influence the onset of synaptic function.

Acetylcholine↗