Biomedical subjects

S W Kuffler

Publications and source records attributed to S W Kuffler.

6 recordsLinked to original sources

PubMed1979

A peptide as a possible transmitter in sympathetic ganglia of the frog.

A search was made in sympathetic ganglia of the bullfrog for a noncholinergic, nonaminergic transmitter that is released by a distinct group of preganglionic axons. These initiate a late slow excitatory postsynaptic potential which lasts for many minutes. The most promising candidate for the role of transmitter is a peptide that resembles luteinizing hormone-releasing hormone (LHRH; luliberin). The reasons are: (i) LHRH (1 muM) and some of its analogs cause a slow depolarization of ganglion cells. (ii) Radioimmunoassays established that 100-800 pg of a LHRH-like substance is contained in the lumbar chain of sympathetic ganglia. (iii) The LHRH-like material is specifically distributed in those spinal nerves that contain axons that initiate the slow noncholinergic synaptic responses. (iv) Five days after ipsilateral preganglionic axons are cut, 95% of the LHRH-like substance disappears from ganglia, while the LHRH immunoreactivity triples in the spinal nerves proximal to the cut region. (v) About 0.6% of the LHRH-like material within ganglia can be collected from the perfusate after 30 min of incubation in isotonic KC1; this release is Ca dependent. (vi) The candidate for transmitter has several chemical characteristics of a peptide and has a Mr near 1000.

Animals↗

PubMed1977

Synaptic excitation and inhibition resulting from direct action of acetylcholine on two types of chemoreceptors on individual amphibian parasympathetic neurones.

1. Synaptic transmission was studied in visually identified parasympathetic ganglion cells that modulate the heart beat of the mudpuppy Necturus maculosus).2. The brief pulse of acetylcholine (ACh) released from terminals of the vagus nerve after each impulse can produce two distinct post-synaptic responses in individual principal cells of the ganglion: (i) within a milli-second of release, ACh generates a rapid and strong excitatory post-synaptic potential (e.p.s.p.) that normally initiates a post-synaptic impulse; (ii) this excitation is usually followed by a slow hyperpolarizing inhibitory post-synaptic potential (i.p.s.p.) that lasts for several seconds. The magnitude and time course of the i.p.s.p. depends on the frequency and number of vagal stimuli. When the hydrolysis of ACh is inhibited by prostigmine, a train of nerve stimuli may be followed by an i.p.s.p. lasting half a minute or longer.3. The rapid e.p.s.p. and slow i.p.s.p. result from the direct action of ACh on two different types of chemoreceptors in the post-synaptic membrane of the principal cell. The e.p.s.p. can be preferentially blocked by the nicotinic antagonist dihydro-beta-erythroidine (5 x 10(-7)M), while the i.p.s.p. is selectively blocked by the muscarinic antagonist atropine (5 x 10(-9)M).4. Potentials resembling nerve-evoked e.p.s.p.s and i.p.s.p.s can be produced by iontophoretic release of ACh from micropipettes onto the post-synaptic membrane. Application of the muscarinic agonist bethanechol generates exclusively inhibitory responses.5. The reversal potential for the i.p.s.p. is about -105 mV, which is approximately the equilibrium potential for potassium (E(K)). When the external K(+) concentration is altered, the reversal potential for inhibition is shifted to the new value of E(K) as expected from the Nernst equation. Changes in the external Na(+) and Cl(-) concentrations have no appreciable effect on the reversal potential. Thus, the i.p.s.p. is the result of a conductance increase for K(+).6. The conductance change producing the i.p.s.p. is voltage sensitive. When the membrane potential is shifted from -40 to -60 mV, the i.p.s.p becomes larger and longer. Beyond -60 mV the inhibitory response decreases in proportion to the driving force on K(+) without any further change in time course.7. The inhibitory response produced by an iontophoretically applied pulse of bethanechol has a delayed onset of about 150 msec at 24 degrees C. The early portion of this response, including the delay, is proportional to t(3), where t is time. The proportionality factor (the apparent rate constant) decreases elevenfold when the temperature is lowered by 10 degrees C. This suggests that a multi-step process is involved in the activation of the conductance increase that leads to the inhibitory response. Inhibitory responses with similar kinetics were produced in heart muscles of the mudpuppy upon application of ACh.

Acetylcholine↗

PubMed1976

The number of acetylcholine molecules in a quantum and the interaction between quanta at the subsynaptic membrane of the skeletal neuromuscular synapse.

1. Postsynaptic responses to acetylcholine released from nerve terminals and from iontophoretic micropipettes were investigated in skeletal twitch-muscle fibers of the snake. The preparation consists of thin sheets of muscle fibers in which details of the end plate, including the outlines of individual synaptic boutons, are clearly seen in the living state. After treatment with collagenase, the motor nerve and its terminal boutons can be removed to expose the intact subsynaptic membrane to direct application of ACh by iontophoretic pipettes. 2. The number of ACh molecules in a quanta was estimated to be fewer than 10,000. This was done by developing a sensitive bioassay to measure the output of ACh from iontophoretic pipettes needed to produce synaptic responses closely resembling nerve-released miniature postsynaptic potentials. 3. Postsynaptic receptors are not saturated by the ACh in a quantum, since the peak of the quantal response produced by an appropriate background concentration of ACh from a pipette. 4. When acetylcholine esterase is inhibited, two or more quanta can act upon partially overlapping postsynaptic membrane areas and potentiate each other's effects. This potentiation reveals itself as a prolongation of the synaptic current. Postsynaptic potentiation is a consequence of the nonlinear dose-response characteristics of ACh receptors and can also be demonstrated in a model system in which ACh micropipettes substitute for quantal release from the nerve. 5. With AChE fully active, however, each quantum is functionally isolated from its neighbors and no postsynaptic potentiation is seen. 6. It is suggested that postsynaptic potentiation between quantum may play a role in signaling at synapses which have nonlinear dose-response characteristics and where transmitter is not so rapidly inactivated as at the neuromuscular synapse.

Acetylcholine↗

PubMed1975

The distribution of acetylcholine sensitivity at the post-synaptic membrane of vertebrate skeletal twitch muscles: iontophoretic mapping in the micron range.

1. The distribution of acetylcholine (ACh) sensitivity was mapped in skeletal twitch muscles of the snake, frog and mudpuppy with iontophoretic methods that provide a resolution in the mum range. 2. The preparations were thin sheets of muscle fibres that were viewed with Nomarski optics, giving sharp definition of cellular detail. The muscles in the snake were especially suitable. Their motor nerves terminate in a compact cluster of synaptic boutons that rest in distinct craters on the muscle surface. After treatment with collagenase the motor nerve and its terminal boutons can be removed, exposing the subsynaptic membrane in the craters. 3. The slopes of dose-response curves obtained by iontophoretic application of ACh were expressed in mV/nC and used as an index of ACh sensitivity. The areas of highest sensitivity, tested either with the terminals in place or removed, were those immediately under the presynaptic terminals. The greatest subsynaptic sensitivities were about 5000 mV/nC, and the time course of the potentials caused by ACh released iontophoretically closely matched that of synaptic potentials set up by ACh released by the nerve. 4. The sensitivity of the extrasynaptic surface less than 2 mum away was at least 50 times lower than that of the subsynaptic membrane. The low extrasynaptic sensitivity declined still further at greater distances. 5. Acetylcholinesterase was shown physiologically to be confined to subsynaptic areas. No activity of the enzyme was detected in extrasynaptic areas beyond about 2 mum from the edge of the synapse. 6. The confinement of high densities of receptors and of acetylcholinesterase to the subsynaptic membrane in muscles is also a feature in parasympathetic neurones. It is suggested that similar specialization may be a widespread property of neurones with chemical synapses.

Acetylcholine↗

PubMed1975

Post-synaptic potentiation: interaction between quanta of acetylcholine at the skeletal neuromuscular synapse.

1. Post-synaptic responses to acetylcholine (ACh) released from nerve terminals and from iontophoretic micropipettes were investigated in skeletal muscle fibres of the snake. Each fibre has a compact end-plate consisting of fifty to seventy synaptic boutons. The fibres were voltage clamped, and synaptic currents were recorded from visually identified end-plates. 2. When acetylcholinesterase (AChE) is inhibited, a potentiating interaction is observed between two or more quanta that are released close to each other from a synaptic bouton and act upon partially overlapping postsynaptic areas. The potentiation is expressed as a prolongation of the synaptic current. This potentiation also occurs under normal conditions of release when about 300 quanta are distributed over the entire end-plate, so thet the presynaptic release sites are separated by an average of 2 mum. An analogous potentiating interaction is observed when micropipettes, closely apposed to the subsynaptic membrane, substitute for quantal release sites. ACh from one pipette potentiates the response to ACh from another pipette less than 2 mum away. 3. In contrast, with AChE fully active no post-synaptic potentiation is seen when the normal complement of quanta is released over the entire end-plate. The time course of the synaptic currents in response to a single quantum or to 300 quanta is similar. It is concluded that functionally the quanta act independently of each other, because AChE isolates each quantum from its neighbours by limiting the lifetime of ACh and its lateral diffusion in the synaptic cleft. The estimated area over which a quantum normally acts is less than 2mum2. 4. Post-synaptic receptors are not saturated by the ACh in a quantum, since the peak of the quantal response adds linearly to the response produced by an appropriate background concentration of ACh from a pipette. This conclusion is supported by the observation that upon inhibition of AChE the peak amplitude of the quantal current response increases by about 20% with no change in its time to peak. 5. It is suggested that post-synaptic potentiation between quanta may play a role in signalling at synapses in which non-linear dose-response characteristics have been observed and where transmitter is not as repidly inactivated as the neuromuscular synapse.

Acetylcholine↗

PubMed1975

The number of transmitter molecules in a quantum: an estimate from iontophoretic application of acetylcholine at the neuromuscular synapse.

1. The sensitivity of the subsynaptic membrane of twitch muscles of the frog and snake to iontophoretically applied acetylcholine (ACh) was determined. Optimal placement of ACh micropipettes on to the postsynaptic membrane resulted in potentials that were similar, though not identical, to the miniature excitatory post-synaptic potentials (min e.p.s.p.s). A sensitive bio-assay was developed to measure the output of ACh from micropipettes; this allowed an estimate to be made of the upper limit of the number of ACh molecules in a quantum of transmitter that is released from the nerve to produce a min e.p.s.p. 2. The assay to calibrate the output of ACh from micropipettes used the end-plate of the snake muscle as an ACh concentration detector. The end-plate was situated within a few mum of an oil-water interface, and a 0-6 nl. droplet of Ringer solution containing a known concentration of ACh (1 muM or less) was formed in the oil phase. The droplet was brought to the interface and, upon touching it, discharged its contents into the Ringer phase immediately above the end-plate. This resulted in a membrane depolarization that was recorded with an intracellular microelectrode. By applying droplets containing various known ACh concentrations a standard curve was constructed. To measure the ACh output of micropipettes a 0-6 nl. droplet of Ringer solution was suspended in the oil. The ACh pipette tip was inserted into the droplet and several thousand pulses of ACh were then delivered. The ACh content of the test droplet was measured by comparing its effectiveness in depolarizing the end-plate with the standard curve. In this manner the number of ACh molecules released in a single pulse was determined as a function of charge passed through the pipette. The output of ACh was linear and an average of 30,000 molecules of ACh were released per pC. 3. The sensitivity of the subsynaptic membrane to iontophoretically applied ACh, using the linear slopes of dose-response curves, in preparations from frog and snake treated with anticholinesterases was usually about 5 mV/pC. It follows that 6000 molecules of ACh are sufficient to produce a depolarization of 1 mV in the subsynaptic membrane. 4. The mean min e.p.s.p.s of muscle fibres treated with anticholinesterase range from 1 to 3 mV. Since the ACh released from an iontophoretic pipette is less effective than the same amount released from the nerve, it is concluded that a quantum of transmitter consists of less than 10,000 molecules of ACh. 5. It is calculated that for each molecule of ACh released in a quantum there results a minimum net flow of 3000 univalent ions across the synaptic membrane.

Acetylcholine↗