Presynaptic effects of 4-aminopyridine and changes following a single giant impulse at the Torpedo nerve-electroplaque junction

The presynaptic changes caused by 4-aminopyridine were studied in the electric organ of Torpedo marmorata, in the resting state and during the period following transmission of a single giant discharge. Incubation with 4-aminopyridine provoked a 30-40% decrease in the density of synaptic vesicles in nerve terminals, and a similar decrease in the content of vesicular and free acetylcholine. These changes were not observed when 4-aminopyridine was applied in a low-calcium, high-magnesium solution. In the standard medium, 4-aminopyridine treated junctions were able to generate a giant electrical discharge of long duration in response to a single stimulus. During the seconds and minutes following the giant discharge, the number of synaptic vesicles was not found to be significantly altered in the whole population of nerve terminals. However, new membranous structures--looking like sacs with double membranes encircling a part of cytoplasm--were seen in approximately 25% of nerve endings; in those terminals, the number of synaptic vesicles was significantly decreased. At this stage, the junctions had not recovered their capability to generate a second giant discharge of full size and the yield of acetylcholine, adenosine 5'-triphosphate (ATP) and creatine phosphate was diminished. Thirty minutes after the single discharge, the functional recovery was achieved and the membranous sacs had disappeared; but the levels of acetylcholine, ATP and creatine phosphate were still not restored.     

Endo-exocytotic images and changes in synaptic transmission induced by diamide at a cholinergic junction

Small tissue fragments excised from the electric organ of Torpedo marmorata were treated with diamide, a penetrating thiol oxidizing agent, until synaptic transmission was blocked. At this stage, we found an unexpected number of exo-endocytotic images in the presynaptic plasmalemma. Omega-shaped profiles, some of them coated, were seen in thin sections of fixed tissue and pits opened in the P-face of the presynaptic membrane in freeze-fracture replicas from rapidly-frozen preparations. Diamide-treated specimens were frozen at 1 ms time intervals before, during and after a single electrical stimulus. This stimulation did not result in a further increase in the density of presynaptic pits, not in any change affecting the density or size distribution of intramembrane particles. This result is in contrast with what is observed in untreated specimens where transmission of a nerve impulse is accompanied by a momentary rise in the number of large particles. The density of synaptic vesicles--especially that of a subpopulation of small size vesicles--transiently increased within the first 2 h of diamide treatment. During the first stages of intoxication, diamide prolonged the time course of postsynaptic potentials--both spontaneous and evoked--probably by altering the gating properties of receptors (acetyl-cholinesterase activity was not impaired). Later on, all evoked responses were blocked. The spontaneous transmitter release greatly increased, first in the form of quantal miniature potentials. These then subsided whereas a class of very small potentials was generated at a high frequency. Also under the action of diamide, calcium progressively accumulated in the tissue but the number of synaptic vesicles containing calcium deposits was reduced. It is concluded that diamide causes a marked increase in the number of exo-endocytotic images in the presynaptic membrane, suppresses quantal but not subquantal release, and interferes with calcium sequestration in and extrusion from terminals.     

Absence of sterol-specific complexes at active zones of degenerating and regenerating frog neuromuscular junctions

Summary Freeze-fracture combined with filipin treatment has been used as a cytochemical probe for membrane cholesterol. As previously shown at the frog neuromuscular junction, distinctive sterol-specific complexes were formed on the presynaptic membrane after filipin treatment, except at active zones. The absence of sterol-specific complexes from active zones was confirmed using two other cytochemical agents — digitonin and saponin. We also studied the maintenance and differentiation of the presynaptic membrane heterogeneity revealed by membrane cholesterol probes at degenerating and regenerating neuromuscular junctions. During degeneration, active zones in frog nerve terminals were disorganized, but still lacked sterol-specific complexes. After engulfing the degenerating nerve terminals, Schwann cells occupied the synaptic gutters and displayed a uniform distribution of sterol-specific complexes. Schwann cell ridges opposite the postjunctional folds also had prominent sterol-specific complexes in regions formerly occupied by active zones. By 2 weeks after nerve crush, nerve terminals reinvaded the endplate region and active zones began to regenerate. While the intramembrane particles of the early regenerating active zones were not arranged in the normal double-rowed organization, filipin-sterol complexes were nevertheless excluded from these primitive active zones. Areas of nerve terminal membrane opposite to junctional folds but lacking active zones were covered with filipin-sterol complexes. These results show that the normal double-rowed organization is not required for the expression of the membrane heterogeneity associated with the active zone. In addition, the absence of sterol-specific complexes is closely associated with the active zone particles and not simply the membrane regions opposite to the postjunctional folds. The membrane heterogeneity does not seem to be directly linked with the functional state of the active zone since it is still associated with degenerating active zones after transmission failure has occurred.     

Structure and distribution of neuromuscular junctions on slow muscle fibers in the frog

Neuromuscular junctions on slow fibers in frog cruralis muscle have been examined with light and electron microscopy. The slow fiber bundle of this muscle is approximately half slow fibers and half fast twitch fibers. Each slow fiber has two-five clusters of nerve terminals in its central region. In contrast, fast fibers in the same bundle have single anastomosing neuromuscular junctions. The average length of terminal clusters on slow fibers is half that of the terminals on fast fibers. Less cholinesterase activity is associated with nerve terminals on slow muscle fibers. The two types of muscle fiber were identified in freeze-fracture replicas by characteristic patterns of the sarcolemmal caveolae and square arrays. Presynaptic membranes of terminals on fast fibers have long, paired double rows of intramembrane particles which lie along the sides of ridges aligned above each fold in the muscle. Ridges are less prominent or absent in terminals on slow fibers and the associated membrane particles are more often in single rows. The lengths of the particle rows also tend to be shorter and rows tend to branch and to lie at various orientations with respect to the longitudinal axis of the terminal. The average length of the particle rows per unit length of nerve terminal on slow fiber terminals is half that of particle rows on fast fiber terminals. It is concluded that the total length as well as the concentration of these active zone specializations is less at terminals on slow fibers. The lack of parallel orientation of the presynaptic active zones at slow fiber terminals corresponds to a lack of postsynaptic folds. Postsynaptic specializations seen in thin sections through slow fibers are either on flat expanses of sarcolemma or on low sarcolemmal bulges. Patches of large intramembrane particles, similar to those on fast fibers, mark regions of postsynaptic specialization in freeze-fracture replicas of slow fibers.     

Synaptic transmission in the guinea-pig vas deferens: the role of nerve action potentials

To determine how transmitter release is related to presynaptic nerve activities, pre- and postsynaptic electrical events of the vas deferens in the guinea-pig were recorded with a suction electrode. Stimulation of the hypogastric nerve elicited excitatory junction currents and nerve action potentials. Intermittence of excitatory junction currents was observed. In some instances, this was related to the absence of nerve action potentials, suggesting failure of impulse propagation into the nerve terminals. Facilitation of both the nerve action potentials and the excitatory junction currents was also observed. Internal perfusion of the recording electrode with tetrodotoxin blocked the nerve impulse, and the polarity of the excitatory junction current became positive. Similar effects on the polarity of the excitatory junction current were observed with alpha, beta-methylene ATP. Perfusion of the suction pipette with 4-aminopyridine or tetraethylammonium increased the amplitude of the excitatory junction currents and prolonged the nerve action potential duration. These experiments show that: (1) transmission failure in some cases can be related to conduction block into the terminal region: (2) facilitation of excitatory junction currents may be related to facilitation of the nerve action potentials; (3) enhancement of transmitter release by potassium channel blockers may be related to prolongation of the duration of the nerve action potential. It is concluded that transmitter release is intimately related to presynaptic nerve activities.     

Spontaneous synaptic potentials and quantal release of transmitter in the stellate ganglion of the squid

1. Several kinds of synapses have been studied in the stellate ganglion of the squid.2. A small electric coupling was found between giant fibres in different stellar nerves.3. Post-synaptic potentials recorded from the cells of small axons are composite, indicating that there are converging inputs from several pre-ganglionic fibres.4. Spontaneous miniature synaptic potentials were recorded from all types of synapses. Miniature potentials in the cells of small axons had a slower time course than those in the giant fibre system.5. Tetrodotoxin abolished nerve impulses in the ganglion but did not prevent the spontaneous quantal release of transmitter from the terminals, or its action on the post-synaptic membrane; nor did it prevent the increase in rate of release produced by depolarization of the presynaptic fibre.6. Glutamate depolarized the giant fibre when applied iontophoretically to the synaptic region. Similar doses applied intracellularly were without effect.     

A freeze-fracture study of photoreceptor presynaptic membranes during ribbon synapse formation

Summary The outer plexiform layer (OPL) of the developing chick retina from 11 embryonic days to 11/2 weeks posthatching has been studied by freeze-fracture to characterize changes in the membrane structure of photoreceptor terminals during synaptogenesis. At early stages, the undifferentiated photoreceptor synaptic base is characterized by a sparse distribution of intramembrane particles on the inner leaflet (P-face). Later, as the synaptic base begins to differentiate by extending filopodia into the OPL, numerous small aggregates of large particles appear between and on filopodial surfaces. Many of the aggregates occupy crater-like depressions, which are seen in cross-fractures through the underlying cytoplasm to be associated with vesicular invaginations of the presynaptic membrane. Corresponding thin sections through these regions at this time reveal immature arciform densities and coated vesicles fusing with the presynaptic membrane adjacent to these densities. At later stages, many of the particle aggregates on the photoreceptor membrane appear to have coalesced into longer arrays overlying ridges surrounded by numerous vesicle fusion sites. These intramembrane changes correlate with the formation of the mature arciform density-synaptic ribbon specialization in the photoreceptor presynaptic terminal and with physiological maturation of the chick retina.     

Histamine H2 receptor mediates postsynaptic excitation and presynaptic inhibition in submucous plexus neurons of the guinea-pig

Intracellular recordings were made from submucous plexus neurons of the guinea-pig cecum maintained in vitro. Histamine (0.3-10 microM) produced a dose-dependent membrane depolarization (congruent to 13 mV with 3 microM) in about 28% of the cells tested; most of these cells showed a prominent calcium-activated potassium conductance (AH cells). The depolarization was due primarily to an inactivation of potassium conductance which is available at the resting membrane potential of -60 mV. Peak amplitude of the fast excitatory postsynaptic potential was depressed by histamine (0.1-10 microM) in a dose-dependent manner (congruent to 62% depression with 1 microM). This was observed even in those cells in which histamine did not produce any membrane depolarizations (mostly S cells). The depression of the fast excitatory postsynaptic potential resulted from the presynaptic inhibition of acetylcholine release. Histamine also reduced the amplitude of the non-cholinergic, presumably peptidergic, slow excitatory postsynaptic potential by suppressing peptide release from presynaptic nerve terminals. Peak amplitude of the adrenergic inhibitory synaptic potential was not depressed by histamine suggesting that histamine receptors are not present on presynaptic terminals of sympathetic nerve fibres. Both postsynaptic and presynaptic actions of histamine were blocked by cimetidine or ranitidine but not by pyrilamine implying that H2 receptors are involved.     

Facilitation of synaptic activity in the frog spinal cord produced by 4-aminopyridine

Reflex activity in the isolated frog spinal cord is increased after adding 4-aminopyridine (4-AP) to the bath. Depression of synaptic transmission by increased magnesium or EGTA is effectively antagonized by 4-AP. It is suggested that 4-AP facilitates synaptic transmission in the central nervous system by interacting with the calcium mechanism involved in transmitter release from presynaptic nerve terminals.     

4-Ammopyridine-induced ultrastructural alterations of pinched-off nerve terminals from rat cerebral cortex

Summary Pinched-off nerve terminals (synaptosomes) from rat cerebral cortex were depolarized with 60 mM KCl and treated with 20 mM 4-aminopyridine in order to evaluate ultrastructural alterations. The empty presynaptic terminals were counted and their number was given as a percentage of the normal terminals. The proportion of empty terminals increased from 10.47±1.56% to 32.45±1.88% (P < 0.001) following treatment with 20 mM 4-aminopyridine. This effect of 4-aminopyridine depended on the presence of Ca⁺⁺ in the incubation medium. The results are discussed in terms of facilitation by 4-aminopyridine of exocytotic transmitter release. We think that the increase of the empty synaptosomes was due to the exhaustion or inhibition of the synaptic vesicle recycling mechanism.The work was supported by the Hungarian Ministry of Health (Grant No. 05/4-01/449) and the Hungarian Academy of Sciences (Grant No. 375/82/3.2)     

Quantal transmitter release by glioma cells: quantification of intramembrane particle changes

Glial cells in situ are able to release neurotransmitters such as glutamate or acetylcholine (ACh). Glioma C6BU-1 cells were used to determine whether the mechanisms of ACh release by a glial cell line are similar or not to quantal release from neurones. Individual C6BU-1 cells, pre-filled with ACh, were moved into contact with a Xenopus myocyte that was used as a real-time ACh detector. Upon electrical stimulation, C6BU-1 cells generated evoked ACh impulses which were Ca(2+)-dependent and quantal (quantal steps of ca. 100 pA). Changes in plasma membrane ultrastructure were investigated by using a freeze-fracture technique designed for obtaining large and flat replicas from monolayer cell cultures. A transient increase in the density of medium and large size intramembrane particles--and a corresponding decrease of small particles--occurred in the plasma membrane of C6BU-1 cells stimulated for ACh release. Changes in interaction forces between adjacent medium and large particles were investigated by computing the radial distribution function and the interaction potential. In resting cells, the radial distribution function revealed a significant increase in the probability to find two particles separated by an interval of 24 nm; the interaction potential suggested repulsive forces for intervals shorter than 24 nm and attractive forces between 24 and 26 nm. In stimulated cells, this interaction was displaced to 21 nm and made weaker, despite of the fact that the overall particle density increased. The nature of this transient change in intramembrane particles is discussed, particularly with regard to the mediatophore proteolipid which is abundant in the membranes C6-BU-1 like in those of cholinergic neurones. In conclusion, evoked ACh release from pre-filled C6-BU-1 glioma cells is quantal and Ca(2+)-dependent. It is accompanied by a transient changes in the size distribution and the organisation of intramembrane particles in the plasma membrane. Thus, for the release characteristics, glioma cells do not differ fundamentally from neurones.     

Mechanisms underlying the riluzole inhibition of glutamate release from rat cerebral cortex nerve terminals (synaptosomes)

We have examined the effect of riluzole, a neuroprotective agent with anticonvulsant properties, on the release of endogenous glutamate from rat cerebrocortical synaptosomes using an on-line enzyme-coupled fluorometric assay. Riluzole inhibited the calcium-dependent release of glutamate that was evoked by exposing cerebrocortical synaptosomes to the potassium channel blocker 4-aminopyridine, and this presynaptic inhibition was concentration-dependent. Riluzole did not alter either 4-aminopyridine-evoked depolarization of the synaptosomal membrane potential or ionomycin-mediated glutamate release, indicating that riluzole-mediated inhibition of glutamate release is not due to a decrease in synaptosomal excitability or a direct effect on the exocytotic machinery. Examination of the effect of riluzole on Ca2+ influx revealed that the diminution of glutamate release could be attributed to a reduction in cytosolic calcium. A possible effect of riluzole on synaptosomal calcium channels was confirmed in experiments where synaptosomes pretreated with P/Q-type calcium channel blocker omega-agatoxin IVA, which abolished the riluzole-mediated inhibition of glutamate release. In addition, pretreatment of synaptosomes with either the Gi/Go protein inhibitor pertussis toxin or the GABAB receptor agonist baclofen, completely prevented the inhibitory effect of riluzole on 4-aminopyridine-evoked glutamate release. It is concluded that riluzole exerts their presynaptic inhibition, likely through a reduction in the calcium influx mediated by P/Q-type calcium channels, and thereby inhibits the release of glutamate from rat cerebrocortical nerve terminals. This release inhibition may involve a pertussis toxin-sensitive G protein signalling pathway. This finding provides further support that presynaptic calcium channel blockade concomitant with inhibition of glutamate release could be an important mechanism underlying the therapeutic actions of this drug.     

Ion channels involved in the presynaptic hyperexcitability induced by herpes virus suis in rat superior cervical ganglion.

Rat superior cervical ganglia infected with herpes virus suis (pseudorabies virus) display a spontaneous bursting activity of still unknown origin. Previous intracellular recordings from the ganglionic neurons combined with pharmacological studies showed that the postganglionic action potentials are induced by acetylcholine release spontaneously from the preganglionic nerve. In this study we investigated whether the acetylcholine release is caused by mechanisms which are dependent on action potentials spontaneously generated on the preganglionic nerve or by mechanisms which occur without any changes in the excitability of presynaptic fibers. Simultaneous intra- and extracellular recordings from the ganglion cells and from the preganglionic nerve, respectively, were performed 32-38 h after the inoculation of herpes virus suis (strain Aujeszky) into the anterior chamber of one eye of the rat. Tetrodotoxin, well known to prevent the generation of action potentials by blocking the fast sodium channels, completely and reversibly abolished, whereas the potassium channel blockers 4-aminopyridine and apamin, enhanced the spontaneous, bursting activity at pre- and postsynaptic levels. The nicotinic receptor antagonist hexamethonium abolished the postsynaptic discharges and reduced the preganglionic activity by 50%. Pre- and postsynaptic electrical activities were suppressed in low calcium Krebs' solution, demonstrating that extracellular calcium is required not only for acetylcholine release but also for triggering the presynaptic action potentials. It is concluded that in the infected ganglia the spontaneous acetylcholine release is due to the generation of action potentials in the preganglionic nerve. Voltage-gated sodium and calcium channels contribute to the presynaptic electrogenesis, while the latter appears to be damped by the activation of voltage- and calcium-dependent potassium channels. Possible factors as well as mechanisms inducing such an increase in excitability are discussed.     

The action of calcium on neuronal synapses in the squid

1. The isolated stellate ganglion of the squid (L. pealii) was studied with intracellular and extracellular micro-electrodes. Three or four nerve fibres in the preganglionic nerve establish synaptic relations with the giant axon in the last stellar nerve. Accordingly, 1-3 small presynaptic spikes (< 1 mV) could be recorded from within the post-synaptic axon.2. A micro-electrode was inserted in the presynaptic fibre and used to polarize and record simultaneously. In the distal (giant) synapse, hyperpolarization of the ending produced an increase in the size of the presynaptic action potential and post-synaptic potential (PSP). Depolarization had the opposite effect. These effects of polarization took more than 10 sec to develop fully, and declined with a similar time course at the end of polarization. Analogous results were obtained with two other preganglionic fibres, which make contacts in the proximal synaptic region.3. The second of a pair of preganglionic impulses evoked a PSP larger than the first. This facilitation of PSP was sometimes accompanied by a small increase in the size of the second action potential in the presynaptic axon. At some shorter intervals, the second presynaptic action potential was reduced in amplitude, but the PSP was still increased. Hyperpolarization of the presynaptic terminal increased the size of both PSPs in a pair and abolished the facilitation. With stronger hyperpolarization the second PSP was even smaller than the first.4. Removing or reducing the Ca in the bathing fluid reversibly abolished the post-synaptic response. The small presynaptic spikes remained practically unaffected. In these conditions a nerve impulse still invaded the ending and normal action potentials could be recorded from the pre-synaptic terminal. This shows that electrical coupling between pre- and post-synaptic axons is insufficient to account for synaptic transmission.5. In low-Ca solution synaptic transmission could be restored locally by extracellular ionophoretic application of Ca to a small portion of the synapse. At sensitive spots a post-synaptic current (recorded with the Ca pipette) and PSP could be detected earlier than 1 sec after commencing the application of Ca.6. Ca was ineffective when injected intracellularly into the presynaptic fibre at a spot where extracellular ionophoresis of Ca restored the PSP.7. The results indicate that synaptic transmission in the squid stellate ganglion is not electrical but due to the release of an unidentified transmitter. Release of this transmitter by the presynaptic nerve impulse requires the presence of Ca in the external medium. During the impulse Ca would combine with a ;Ca-receptor' in the membrane and initiate the reactions which lead to transmitter release. It appears that the ;Ca-receptor' is only accessible from the outside of the membrane.     

Effects of iontophoretically applied drugs on spinal interneurons of the lamprey

1. Intracellular records were obtained from giant interneurones in the isolated spinal cord of the sea lamprey. The cells had a mean resting potential of about 75 mV and action potentials with overshoots of about 35 mV. Their input resistances, measured by passing polarizing currents through the recording pipette, were in the range 3-7 MOmega.2. Iontophoretic ejection of gamma-aminobutyric acid (GABA) from a micropipette placed near the surface of a cell resulted in a slight hyperpolarization, accompanied by a marked reduction in input resistance. The reversal point for the potential change was about 5 mV greater than the resting membrane potential.3. Iontophoretic application of L-glutamate to the cells produced a depolarization with a decrease in input resistance much smaller than that accompanying a GABA potential of similar amplitude. The action potential amplitude was reduced by L-glutamate application. The reversal potential could not be determined accurately but appeared to be near zero membrane potential.4. Glutamate application produced, in addition, a burst of inhibitory synaptic potentials in the cell, presumably by depolarizing either inhibitory presynaptic nerve terminals or nearby inhibitory cell bodies.5. Acetylcholine (ACh) produced no detectable change in membrane resistance or potential.6. Application of the three drugs to first-order sensory cells in the spinal cord had no effect on their membrane properties.     

On the presynaptic acetylcholine receptors in sympathetic ganglia of the frog

1. Action potentials have been recorded extracellularly from individual preganglionic nerve terminals in the lumbar sympathetic chain of frogs (cf. Koketsu & Nishi, 1968).2. The action potentials were unaffected by changes in external Mg(2+) or Ca(2+) concentration, but were attenuated or even abolished by acetylcholine or carbachol. The effect on the presynaptic action potential was transient, in contrast to the blockade of synaptic transmission caused by these drugs, which as is well known persists indefinitely in their presence.3. In the presence of tubocurarine (5 x 10(-5)M), acetylcholine and carbachol had no effect on the presynaptic action potential.4. The action potentials were reduced by an increase in external K(+) concentration and abolished at between 9 and 16 mM K(+). Synaptic transmission persisted until the amplitude of the action potential was reduced to less than one third of its control value.5. It is concluded that although the presynaptic nerve is endowed with acetylcholine receptors, they are not the source of the long lasting blockade of synaptic transmission caused by cholinergic substances.     

KCNQ5 channels control resting properties and release probability of a synapse

Little is known about which ion channels determine the resting electrical properties of presynaptic membranes. In recordings made from the rat calyx of Held, a giant mammalian terminal, we found resting potential to be controlled by KCNQ (Kv7) K(+) channels, most probably KCNQ5 (Kv7.5) homomers. Unlike most KCNQ channels, which are activated only by depolarizing stimuli, the presynaptic channels began to activate just below the resting potential. As a result, blockers and activators of KCNQ5 depolarized or hyperpolarized nerve terminals, respectively, markedly altering resting conductance. Moreover, the background conductance set by KCNQ5 channels, together with Na(+) and hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels, determined the size and time course of the response to subthreshold stimuli. Signaling pathways known to directly affect exocytic machinery also regulated KCNQ5 channels, and increase or decrease of KCNQ5 channel activity controlled release probability through alterations in resting potential. Thus, ion channel determinants of presynaptic resting potential also control synaptic strength.     

Analysis of hyperpolarizations induced by glutamate and acetylcholine on Onchidium neurones

1. Four giant neurones, designated G-H cells, in the right pleural ganglion of the marine pulmonate mollusc, Onchidium verruculatum, showed characteristic membrane hyperpolarization during applications of either acetylcholine (ACh) or L-glutamate. In the presence of ACh the membrane was hyperpolarized only transiently, while in the presence of glutamate the response was maintained. Significant increases in membrane conductance accompanied the changes in membrane potential.2. In excess potassium sea water, a slight hyperpolarization occurred when the normal concentration was increased between one- and twofold. However, depolarization usually occurred when the concentration was increased tenfold except on a few occasions when a slight but definite hyperpolarization occurred. These changes were all accompanied by a substantial increase in the membrane conductance. This hyperpolarization was in all probability the result of an increase in chloride ion permeability caused by the release of an ACh-like transmitter from depolarized presynaptic nerve terminals.3. The reversal levels for glutamate- and ACh-induced hyperpolarization respectively were approximately - 20 and - 17 mV with respect to the resting membrane potential.4. By changing the external ion composition, glutamate- and ACh-induced hyperpolarization were shown to be the result of an increased permeability of the subsynaptic membrane to potassium and chloride ions respectively. It appears therefore that inhibition in the same G-H cells can be activated by two different transmitter substances and that each of them activates a change in the membrane permeability to a different ion.5. The relationship between the concentration of glutamate and the membrane conductance change was suggestive of two glutamate molecules reacting with a single receptor site.     

Muscarinic inhibition of quantal transmitter release from the magnesium-paralysed frog sartorius muscle

The existence of presynaptic muscarinic acetylcholine receptors on motor nerve terminals of the isolated frog sartorius muscle was investigated. The modulatory role of these receptors was studied by observing the effects of muscarinic ligands on the frequency of miniature endplate potentials and on the quantal content of endplate potentials. The agonist oxotremorine reduced in concentration-dependent fashion the frequency of spontaneous potentials and the amplitude of evoked potentials. Also, high concentrations of oxotremorine depolarized the postsynaptic membrane and reduced the amplitude of the miniature endplate potentials. The depolarizing action of the drug was blocked byd-tubocurarine. The muscarinic antagonist atropine attenuated agonist-induced reductions in endplate potential amplitude and miniature endplate potential frequency but did not affect the depression in amplitude of the spontaneous potentials evoked by oxotremorine. It is concluded that activation of presynaptic muscarinic receptors inhibits the release of acetylcholine from motor nerve terminals.

Atropine itself had no effect on the quantal content of evoked potentials or on the frequency of spontaneous potentials suggesting that the nerve terminal is not affected by non-quantal acetylcholine.     

A freeze-fracture study of synaptic junction development in the superficial layers of the chick optic tectum

Summary Synaptogenesis in the superficial layers of the rostral pole of the chick optic tectum has been studied using freeze-fracture techniques. The developmental sequence of intramembrane organization at synaptic junctions involves the accumulation and assembly of intramembrane particles into aggregates characteristic of the mature junctions.By embryonic day seven, areas of loosely-arranged clusters of medium-sized particles are observed on the cytoplasmic membrane leaflets (P-faces) of developing neurites. These clusters are characteristic of the intramembrane organization at presynaptic active zones. At later stages, small pits, characteristic of vesicle fusion sites, are observed interspersed among such P-face particle clusters. Complementary intramembrane specializations are also present on the external leaflets (E-faces) of presynaptic membranes at the active zones.Small solitary aggregates of large-sized particles on the E-faces of neurite plasma membranes are also seen at early embryonic stages. As development progresses, these aggregates increase in size and packing density and occupy large oval domains in postsynaptic membranes. These intramembrane specializations may represent the postsynaptic active zones of asymmetric synapses. Another type of intramembrane specialization, observed during the third week of incubation, is characterized by aggregates of small- and medium-sized particles on the P-face of postsynaptic membranes and is often seen directly apposed to the E-face of a presynaptic terminal. This type of intramembrane specialization may represent the postsynaptic active zone region at symmetrical synaptic contacts.