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Biomedical subjects

R Miledi

Publications and source records attributed to R Miledi.

At least 217 records · Page 12Linked to original sources
PubMed1980

Visualization of satellite cells in living muscle fibres of the frog.

Satellite cells were visualized in living muscle fibres of the frog. Single fibres or bundles consisting of a few fibres were isolated after treatment with collagenase, and viewed under the light microscope. Subsequent electron microscopy of identified cells confirmed that they were satellite muscle cells. Under the light microscope, satellite cells appear as fusiform cells, tapering into long fine processes usually orientated parallel to the muscle fibre axis. Horseradish peroxidase injected into the muscle fibre was not transferred to the satellite cells.

Animals↗
PubMed1980

Intracellular calcium and desensitization of acetylcholine receptors.

Acetylcholine (ACh) was applied iontophoretically to voltage-clamped endplates in frog muscle. The current induced by prolonged application of ACh decreases progressively as the membrane becomes desensitized. Desensitization was sharply localized, and at a distance of 15 micrometer or less the ACh sensitivity of the membrane remained normal. Desensitization still occurred in muscles exposed to Ca2+-free media for several hours. In these conditions the rate of desensitization was not greatly affected by altering the membrane potential. In normal Ringer (1.8 mM Ca2+) desensitization was more pronounced and ACh application was frequently accompanied by localized contraction of the muscle fibre. Both the desensitization and the contraction were reduced after intracellular injection of EGTA, probably because this opposes the rise in internal Ca2+ normally caused by ACh action.

Acetylcholine↗
PubMed1980

Electrical synapses between motoneurons in the spinal cord of the newborn rat.

Ventral roots of the newborn rat spinal cord were stimulated while recording intracellularly from motoneurons. In many cells, stimulation subthreshold for an antidromic action potential in the impaled cell produced a small, short-latency depolarization, which was unaffected by membrane polarization. This response (antidromic synaptic potential, a.s.p.) was also seen, in some cells, on stimulating the ventral root of an adjacent segment. Replacement of Ca2+ (2 mM) with Mn2+ (3 mM) or Mg2+ (10 mM) completely abolished orthodromic synaptic potentials, but the a.s.p. persisted. These results strongly suggest that the a.s.p. is produced by an electrical interaction between motoneurons.

Animals↗
PubMed1980

On the purification of beta-bungarotoxin.

It has recently been claimed that our beta-bungarotoxin preparation contained three contaminants, including a postsynaptic toxin. We have extended our purification procedure and found no evidence of such contaminants.

Bungarotoxins↗
PubMed1980

Effects of a Dendroaspis neurotoxin on synaptic transmission in the spinal cord and the neuromuscular junction of the frog.

A neurotoxin from the venom of Dendroaspis jamesoni was tested at the neuromuscular junction and at a cholinergic pathway in the isolated spinal cord of the frog. The toxin reduced the amplitude and time constant of decay of miniature endplate currents in the presence of prostigmine, indicating a curare-like action. In the spinal cord it selectively blocked transmission in the cholinergic pathway and increased spontaneous activity. Partial protection against toxin action in the spinal cord was provided by atropine or carbachol. The results suggest that the toxin acts on cholinergic receptors at both sites and also provide further evidence that the pharmacology of the two sites is different.

Animals↗
PubMed1980

Calcium transients in mammalian muscles.

Contraction of vertebrate skeletal muscle is caused by calcium ions released from the sarcoplasmic reticulum (see refs 1, 2 for reviews). The ensuing transient change in the intracellular level of ionised calcium has been monitored using various Ca2+ indicators, sich as murexide, aequorin, and arsenazo III. So far, most of what is known about these calcium transient derives from experiments on barnacle or frog muscles fibres, and it is desirable to extend such studies to mammalian muscle. We report here that the photoprotein aequorin can be used to monitor calcium transients in rat and human muscles, and that the transients decay more quickly in fast contracting muscle fibres.

Aequorin↗
PubMed1980

Induction of action potentials in cultured slow muscle fibres of the frog.

1. Slow muscle fibres of the frog were maintained in a simple organ culture system for up to 28 days at 18 degrees C. Slow fibres cultured for one week resembled innervated fibres in their lack of ability to generate an action potential. 2. By 10 days a few fibres had acquired the action potential mechanism and in muscles cultured for 28 days all slow fibres examined were capable of generating action potentials most of which reached or exceeded 0 mV membrane potential. 3. Slow muscle fibres which were denervated for two weeks in vivo retained their ability to generate overshooting action potentials after 4 weeks of culture. Thus, in the culture system described, slow muscle fibres are capable of developing action potentials and the action potential mechanism in these fibres remains for at least one month in culture.

Action Potentials↗
PubMed1980

On the release of transmitter at normal, myasthenia gravis and myasthenic syndrome affected human end-plates.

1. Transmitter release has been studied at normal, myasthenia gravis (m.g.) and myasthenic syndrome (m.s.) affected human end-plates. At normal and diseased end-plates evoked transmitter release is Poisson for a mean quantal content, m less than ten. 2. The relation between log m and log [Ca]o, at normal and m.g. end-plates is linear, with a slope of 3.3-3.4. The value of m at m.g. end-plates is about five times larger than normal, below Ca 0.7 mM (Mg, 2mM). This difference in m is reduced at higher Ca levels. 3. The slope of the relation between log m.e.p.p. frequency and log [K]o is similar at normal and m.g. end-plates. Over its linear portion the relationship has a slope of approximately 6. 4. Fluctuations in the latency of evoked transmitter release were compared at normal and m.g. nerve terminals. At normal end-plates the probability of release reaches a peak about 0.3-0.4 msec after unitary e.p.p.s of the shortest latency and returns to zero about 1.0 msec after the peak. At m.g. end-plates the distribution of latencies shows less uniformity. 5. At m.s. end-plates m is approximately 5 in normal Ringer solution (2 mM-Ca, 1 mM-Mg). The relation between log m and log [Ca]o is linear, with a slope of 1.0-1.5. The K dependence of m.e.p.p. frequency appears reduced at m.s. end-plates. 6. Assuming a co-operative mechanism for transmitter release at normal human motor nerve terminals, the dissociation constant for the Ca complex is about 1.6X10(-3) M and the dissociation constant for the Mg complex is about 1.0X10(-3) M. 7. It is concluded that the presynaptic changes, at m.g. end-plates, are not the primary cause of the defect in nerve muscle transmission. At m.s. end-plates the presynaptic changes are sufficient to account for failure in transmission. Possible mechanisms for the abnormalities in transmitter release are considered.

Action Potentials↗
PubMed1980

Transmitter induced calcium entry across the post-synaptic membrane at frog end-plates measured using arsenazo III.

1. The Ca2+ influx occurring across the post-synaptic membrane during transmitter action was studied at the frog neuromuscular junction, using the Ca sensitive dye arsenazo III to monitor the resulting changes in free myoplasmic Ca2+ concentration. 2. Calibration experiments showed a linear relationship between the amount of Ca2+ injected by ionophoresis into a muscle fibre, and the peak size of the arsenazo light absorbance record. 3. Ionophoretic application of acetylcholine (ACh) to voltage clamped end-plates gave rise to an arsenazo signal. The size of this response varied with the Ca2+ concentration in the bathing solution. 4. The arsenazo light response increased in size steeply, and non-linearly, with hyperpolarization of the end-plate membrane, even when the end-plate current increased approximately linearly with hyperpolarization. The voltage dependence of the light response could be fitted well by an exponential with a voltage constant of 28 mV. Changes in Ca2+ concentration of the bathing medium had little effect on this relationship. 5. At end-plates bathed in isotonic CaCl2 solution the voltage dependence of both the arsenazo light response, and the end-plate current showed a closely similar, non-linear relationship. 6. Addition of 12 mM-Co2+ to a bathing solution initially containing 12 mM-Ca2+ substantially reduced the size of the arsenazo light response, and the voltage dependence of this response became more linear. 7. Arsenazo light responses were also recorded in response to transmitter release evoked by nerve stimulation. The size of the nerve evoked light response showed a non-linear voltage dependence, whilst the end-plate current was a linear function of membrane potential.

Acetylcholine↗
PubMed1980

Effects of strontium ions on end-plate channel properties.

1. Changes in end-plate channel properties resulting from substitution of Sr2+ for Ca2+ in the Ringer solution have been analysed at the voltage clamped frog end-plate, by recording m.e.p.c.s and ACh induced noise variance. 2. In 2 mM-Sr2+--Ringer the peak size of m.e.p.c.s showed a very small increase, and the time constant of the decay phase (tau m.e.p.c.), at any given voltage, was increased by a factor of about two compared to control Ringer. The voltage dependence of tau m.e.p.c. was the same in both solutions. 3. Addition of increasing amounts of CaCl2 to 2 mM-Sr2+--Ringer produced a progressive shortening of tau m.e.p.c., with no change in voltage dependence. 4. Estimates of single channel properties from noise analysis showed that the elementary conductance appeared to be slightly increased in 2 mM-Sr2+--Ringer, whilst the mean channel life-time was prolonged by a factor of about two. These changes in single channel properties are sufficient to account for the observed changes in m.e.p.c.s. 5. Following inhibition of cholinesterase activity by neostigmine, similar effects on m.e.p.c.s and single channel properties were still observed on changing to 2 mM-Sr2+--Ringer. The shapes of m.e.p.c.s in Sr2+ + neostigmine Ringer were often altered, and showed flat 'plateaus'. 6. The observed effects of Sr2+--Ringer on channel life-time cannot be explained on the basis of changes in surface charge density on the membrane, and suggest that divalent cations have an additional, and more direct, influence on receptor channel properties.

Acetylcholine↗
PubMed1980

Voltage sensitive calcium entry in frog motoneurones.

1. The electrical properties of motoneurone membrane were investigated in the isolated and hemisected spinal cord of frogs, using intracellular recording techniques. 2. TTX (1 x 10(-6) g/ml.) blocked action potentials produced either by intracellular depolarizing current pulses or ventral root stimuli. Voltage--current relations from these cells showed a diminishing slope for depolarizing current pulses of increasing intensity. 3. If TEA (5--10 mM) was added to the media containing TTX, intracellular depolarizing pulses elicited prolonged regenerative depolarizations characterized by a peak of variable amplitude and a repolarizing phase preceded by a prolonged plateau of variable duration. 4. During the plateau of the response, the membrane conductance was increased above its resting value. 5. The response was shortened during repetitive stimulation and could be curtailed by applying a hyperpolarizing pulse during the plateau. 6. The response depended on the presence of external Ca2+ and increased in size and duration with increasing Ca2+ concentration. Sr2+ substituted effectively for Ca2+. Sr2+-dependent responses were considerably longer than the Ca2+-dependent ones. Ca2+ or Sr2+ dependent responses persisted in Na+-free media containing isotonic TEA, and were abolished by addition of Co2+. 7. Ca2+ or Sr2+-dependent regenerative responses were followed by a hyperpolarization which could last several seconds. The current responsible for this after-hyperpolarization was TTX and TEA resistant. 8. It is concluded that the TTX-resistant regenerative response is probably generated in the soma-dendritic membrane, and is due to influx of Ca2+ or Sr2+ through voltage sensitive channels different to those through which Na+ permeates during generation of 'normal' action potentials. In addition it is shown that the hyperpolarization following 'Ca spikes', and which might be due to an increase in K+ conductance can also be triggered by Sr2+.

Animals↗
PubMed1980

The effect of lanthanum ions on acetylcholine in frog muscle.

1. Frog sartorius muscles were treated with an irreversible cholinesterase inhibitor and then incubated in Ringer with 2 mM-LaCl3. The amounts of ACh in the tissue and medium were assayed by mass fragmentography, miniature end-plate potentials (min. e.p.p.s) were recorded and the end-plate was investigated by electron microscopy. 2. Addition of La3+ caused in normal, but not in denervated, muscles a discharge of both min. e.p.p.s and chemically detectable ACh. After 30 min both min. e.p.p.s and ACh release decreased. Between 4 and 5 hr after the addition of La3+ min. e.p.p.s had practically ceased and the rate of ACh release was almost back to that in the absence of La3+. 3. La3+ caused a 50% reduction in the ACh content of the tissue within the first 30 min; thereafter ACh gradually increased to 110% by 5 hr. At this time synaptic vesicles were practically absent in most terminals. The ACh was predominantly located in the end-plate regions of the muscles, before as well as after the incubation with La3+. ACh in end-plate free parts of the muscles was unchanged by La3+. 4. Hemicholinium-3 inhibited the synthesis of ACh in the muscles, but it had almost no influence on La3+-induced ACh release. 5. From these and other results, it is concluded that the ACh released by La3+ originates exclusively from the nerve terminals, that most likely this ACh is released via exocytosis from synaptic vesicles, and that the synthesis of ACh following the release of ACh takes place in the nerve terminals. The results further indicate that in freshly excised muscle the greater part (80-90%) of the ACh contained in the nerve terminals is located in the vesicles.

Acetylcholine↗
PubMed1979

Estimates of quantal content during 'chemical potentiation' of transmitter release.

The number of quantal transmitter packets (m), released from motor nerve terminals in response to a single stimulus, has been estimated from the ratio of the amplitudes of endplate currents (e.p.c.) to spontaneous miniature endplate currents (m.e.p.c.), in voltage-clamped endplates of the frog. At 6 degrees C, the average value of m at normal nerve-muscle junctions was about 300. If allowance is made for the temporal dispersion of quantal transmitter release during the e.p.c., this value is increased by about 30%. After treatment with diaminopyridine or tetraethylammonium, transmitter release in response to a nerve stimulus is greatly enhanced and values of m exceeding 10(4) are frequently found. Moreover, the duration of the e.p.c. becomes much longer than that of the m.e.p.cs. The number of packets then liberated during the e.p.c. is much larger than the number of 'active zones' of the endplate and may even exceed the total number of vesicles lined up in twin-files adjacent to the presynaptic membrane.

Animals↗