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M M Poo

Publications and source records attributed to M M Poo.

At least 73 records · Page 4Linked to original sources
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↗
PubMed1987

Evoked release of acetylcholine from the growing embryonic neuron.

An excised patch of embryonic muscle membrane was used as a probe for measuring the release of acetylcholine (AcCho) from growing spinal neurons in Xenopus cell culture. The neuron was stimulated extracellularly at the soma, and the evoked AcCho release was monitored at the growth cone, along the neurite, and near the soma. For a majority of the neurons studied, a brief suprathreshold stimulation of the soma triggered a pulse of AcCho release from the growth cone. This release showed many of the characteristics reminiscent of the transmitter release at the nerve terminal of a mature neuromuscular synapse: it occurs within a few ms following the stimulation, depends on extracellular Ca2+ concentration, and exhibits depression and potentiation during and after high-frequency stimulation, respectively. Similar evoked release was also observed only at selected points along the neurite, and prolonged suprathreshold stimulus was required to induce release from the soma. These results indicate that some of the growing spinal neurons have acquired a substantial number of AcCho molecules as well as an efficient mechanism for excitation-secretion coupling at the growth cone, ready for establishing functional contact with the target muscle cell. This notion was further supported by the finding that the evoked AcCho release is capable of inducing suprathreshold excitation of the muscle cell within the first minute following neurite-muscle contact.

Acetylcholine↗
PubMed1986

Initial events in the formation of neuromuscular synapse: rapid induction of acetylcholine release from embryonic neuron.

We have studied the electrical events during the initial phase of nerve-muscle contact in embryonic Xenopus culture. Using a G omega-seal, whole-cell recording method, we monitored the membrane current of a muscle cell continuously while it was manipulated into close proximity of the growth cone of a cocultured spinal neuron. We found a rapid appearance of pulsatile inward currents at the muscle cell after the neurite-muscle contact. These currents were abolished by d-tubocurarine and alpha-bungarotoxin but were unaffected by tetrodotoxin. Both the drug sensitivity and the time course of these currents are similar to that of the spontaneous miniature end-plate currents (MEPCs) resulting from spontaneous release of pulses of acetylcholine (AcCho) from the nerve terminal. Unlike the MEPCs at the mature neuromuscular synapse, these early MEPCs varied greatly in their amplitudes, and there was a gradual increase in the frequency of the MEPCs of larger amplitudes during the first 20 min after the contact. Independent measurement of AcCho concentration near the growth cone by an excised patch of AcCho-sensitive muscle membrane showed that very little AcCho is released from the isolated growth cone, and marked release can be triggered by the contact with a muscle cell or with the excised membrane itself. The induction of release is relatively specific: contact with a neuron or the tip of a clean glass pipette was capable of inducing only a transient release, while persistent release was induced by contacts made with muscle membrane. This contact-dependent AcCho release may be responsible for an early induction of muscle activity and serve as a signal for the establishment of synaptic contact.

Acetylcholine↗
PubMed1986

Contact-induced redistribution of specific membrane components: local accumulation and development of adhesion.

We have used a model system to explore the importance of long-range lateral diffusion of membrane proteins in specific membrane-membrane adhesion. Single, cell-size phospholipid vesicles containing a dinitrophenyl (DNP)-lipid hapten were maneuvered into contact with rat basophilic leukemia (RBL) cells carrying fluorescent anti-DNP IgE in their cell-surface Fc epsilon receptors. Upon cell-vesicle contact the antibody molecules underwent a marked lateral redistribution, accumulating at the site of contact and becoming significantly depleted from noncontacting membrane. As assayed with a micropipette suction method, there was a time-dependent increase in the strength of cell-vesicle adhesion. This development of adhesion paralleled the kinetics of accumulation of the adhesion-mediating antibody molecules at the zone of membrane-membrane contact. Both adhesion and redistribution were absolutely dependent upon a specific interaction of the IgE with the hapten: No redistribution occurred when vesicles lacking the DNP hapten were pushed against IgE-armed RBL cells, and on cells bearing a 1:1 mixture of nonimmune rat IgE and anti-DNP mouse IgE, only the latter underwent redistribution. Vesicles containing DNP-lipids bound to RBL cells carrying anti-DNP IgE but not to cells carrying nonimmune rat IgE. Measurable nonspecific binding did not develop even after 15 min of pushing DNP-bearing vesicles against RBL cells sensitized with nonimmune IgE. Neither redistribution nor adhesion was blocked by metabolic poisons such as NaN3 and NaF. Both redistribution and adhesion occurred in plasma membrane blebs previously shown to lack cytoskeletal filaments. The above observations are consistent with contact-induced redistribution of the IgE being a result of passive diffusion-mediated trapping rather than active cellular responses. Thus, long-range diffusion of specific proteins can in some cases contribute to the formation of stable adhesion between membranes.

Animals↗
PubMed1985

Three types of transmitter release from embryonic neurons.

Prior to the contact with their target muscle cells in culture, growth cones of many isolated Xenopus embryonic neurons release acetylcholine (ACh) spontaneously. Using patch clamp techniques, this release can be detected by an outside-out patch of muscle membrane placed near the growth cone. Intracellular recording from innervated muscle cells showed spontaneous miniature endplate potentials (MEPPs) of varying amplitudes. Amplitude histograms showed a skewed distribution with multiple peaks, suggesting the existence of subunits in either the quantal packages of ACh released by the nerve terminal or in the postsynaptic muscle response. In addition to the quantal ACh release reflected by MEPPs, nerve terminal also release a large amount of ACh in a non-quantal fashion. This non-quantal ACh release is revealed by the hyperpolarization of the muscle membrane following extracellular application of curare or alpha-bungarotoxin, as well as by denervation of the muscle cell.

Acetylcholine↗
PubMed1985

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.

Acetylcholine↗
PubMed1985

Non-quantal release of acetylcholine at a developing neuromuscular synapse in culture.

Local, pulsed application of d-tubocurarine at neuromuscular synapses in embryonic Xenopus nerve-muscle culture resulted in a transient hyperpolarization of muscle membrane potential. Miniature endplate potentials (MEPPs) were abolished during the hyperpolarization and recovered after the return of resting membrane potential. The magnitude of hyperpolarization was independent of the frequency of MEPPs before curarization, and it had an average peak value of 4.3 mV in medium containing physiological levels of Ca2+. Prolonged application of curare or alpha-bungarotoxin led to sustained hyperpolarizations up to 8 mV in magnitude. Denervation produced by mechanically removing the neurite from the muscle cell also produced similar hyperpolarization, and curarization after denervation was without significant hyperpolarizing effect. Increasing the extracellular Ca2+ concentration to about 8 mM abolished the curare-induced hyperpolarization response, in sharp contrast to its effect in elevating the frequency of MEPPs. Taken together, our results indicate that innervated embryonic muscle cells were maintained at a depolarized state relative to that of uninnervated muscle cells by a steady, spontaneous release of acetylcholine (ACh) from the innervating neurite. The cellular mechanism underlying this mode of ACh release appears to be different from that of the quantal ACh release responsible for MEPPs.

Acetylcholine↗
PubMed1984

Migration of cell surface concanavalin A receptors in pulsed electric fields.

Concanavalin A (con A) receptors on the surface of cultured Xenopus myoblasts redistributed in response to monopolar, pulsed electric fields. The prefield uniform distribution of the receptors became asymmetrical, and was polarized toward the cathodal pole, in the same way as in DC fields. The extent of asymmetry depended on the duration of field exposure, pulse width (or alternatively, interpulse interval), frequency, and intensity. This relationship was most conveniently expressed by using duty cycle, a quantity determined by both pulse width and frequency. Pulses of average intensity 1.5 V/cm induced detectable asymmetry within 5 min. At the lowest average field intensity used, 0.8 V/cm, significant asymmetry was detected at 150 min. For pulses of high duty cycle (greater than 25%), steady state was reached after 30 min exposure and the steady state asymmetry was dependent on average field intensity. For low duty cycle fields, the time required to reach steady state was prolonged (greater than 50 min). Before reaching a steady state, effectiveness of the pulses, as compared with DC fields of equivalent intensity, was a function of duty cycle. A low duty cycle field (fixed number of pulses at low frequency or long interpulse interval) was less effective than high duty cycle fields or DC.

Animals↗