Impaired membrane traffic in defective ether lipid biosynthesis

The first steps of ether lipid biosynthesis are exclusively localized to peroxisomes and hence some peroxisomal disorders are characterized by a severe deficiency of plasmalogens, the main ether lipids in humans. Here we report on gene defects of plasmalogen biosynthesis, chromosomal localization of the corresponding genes and, as a consequence of plasmalogen deficiency, on structural alterations of caveolae, clathrin-coated pits, endoplasmic reticulum and Golgi cisternae, as well as on the reduced rate of transferrin receptor cycling. The data suggest that plasmalogens, analogous to cholesterol, are essential for correct membrane functioning and their deficiency results in impaired membrane trafficking.     

Norepinephrine uptake in aging adrenergic nerve terminals

Norepinephrine (NE) uptake has been studied in the adult (3-6 months) and aging (5 yr) chick iris, in which there is a discrete population of norepinephrine containing nerve terminals. although total accumulation of 3H-NE in the iris does not change with age, there is a decline in Na+-dependence, temperature-sensitivity, ouabain-and inhibitor -sensitivity of uptake. The results indicate either a loss of active, carrier-mediated NE uptake during aging, or a change in the biochemical and pharmacological characteristics of this uptake. changes in the composition of the organ and in the sites of accumulation of NE are considered.     

Anion permeability of motor nerve terminals

Motor nerve terminals in mouse and frog display behavior consistent with an appreciable permeability of the nerve terminal membrane to chloride. In mouse diaphragm, in the presence of 15 mM K⁺ and 2 mM or 8 mM Ca²⁺, replacement of Cl^– by NO ₃ ^– , Br^– or acetate causes a transient increase in the quantal release of acetylcholine, measured as the frequency of spontaneously occurring miniature end plate potentials (FMEPP); a rapid rise in FMEPP is followed by a slow decline, with a half-time of about 4 min, to an equilibration level close to the control level. After equilibration in a solution in which the Cl^– is replaced by another anion, return to Cl^–-containing solution causes a transient decrease in FMEPP with a subsequent slow recovery. The data are consistent with transient nerve terminal depolarization or hyperpolarization, reflecting a nerve terminal permeability to anions in the sequence Cl^–>Br^–>NO ₃ ^– >acetate. In 5 mM K⁺, changes in nerve terminal excitability, determined using focal stimulation, are also consistent with alteration of nerve terminal membrane potential as a consequence of anion substitution. The time course of relaxation of FMEPP after a change from Cl^– to an anion of lower permeability, or vice versa, is considerably slower than that expected if Cl^– permeability of nerve terminals is similar to that of skeletal muscle fibres, and if the nerve terminal behaves as a single compartment. In frog cutaneous pectoris, transient changes in FMEPP produced by substitution of anions in the bathing solution were similar to those produced in mouse diaphragm, but more rapid in time course.This work was supported by grants from the Muscular DystrophyAssociation of Canada and the Medical Research Council of Canada     

Electrotonic properties of motor nerve terminals

To obtain information about the electric membrane properties of frog motor nerve terminals we examined how depolarizing or hyperpolarizing current pulses of 2–8 ms duration to the preterminal, by electrotonic spread of potential, affected depolarization induced transmitter release. Sodium channels were blocked by tetrodotoxin. Under this condition a hyperpolarizing current pulse produced inhibition of release, followed by poten-tiation of release. Inhibition lasted more than 100 ms with a time constant of %I50 ms. When, in addition, potassium channels were blocked by 3,4-diaminopyridine or tetra-ethylammonium a depolarizing current pulse potentiated transmitter release for a period up to 50 ms. The results imply that inward currents in the nerve terminal are carried mainly by sodium and calcium ions and outward currents by potassium ions while “leak” conductances are negligible. A low “leak” conductance and therefore a high specific membrane resistance facilitates the spread of electrotonic potentials and thereby explains the long-lasting effects on transmitter release of brief current pulses to the preterminal.     

Functional analysis of neurotransmission at β2-laminin deficient terminals

Carotid body glomus cells release transmitters in response to hypoxia due to the increase of excitability resulting from inhibition of O₂ -regulated K⁺ channels. However, the mechanisms involved in the detection of changes of O₂ tension are unknown. We have studied the interaction between glomus cell O₂ sensitivity and inhibition of the mitochondrial electron transport chain (ETC) in a carotid body thin slice preparation in which catecholamine release from intact single glomus cells can be monitored by amperometry. Inhibition of the mitochondrial ETC at proximal and distal complexes induces external Ca²⁺-dependent catecholamine secretion. At saturating concentration of the ETC inhibitors, the cellular response to hypoxia is maintained. However, rotenone, a complex I blocker, selectively occludes the responsiveness to hypoxia of glomus cells in a dose-dependent manner. The effect of rotenone is mimicked by 1-methyl-4-phenylpyridinium ion (MPP⁺), an agent that binds to the same site as rotenone, but not by complex I inhibitors acting on different sites. In addition, the effect of rotenone is not prevented by incubation of the cells with succinate, a substrate of complex II. These data strongly suggest that sensitivity to hypoxia of carotid body glomus cells is not linked in a simple way to mitochondrial electron flow and that a rotenone (and MPP⁺)-sensitive molecule critically participates in acute oxygen sensing in the carotid body.     

Three potassium currents in mouse motor nerve terminals

A study of the K conductance of the presynaptic membrane has been performed in thetriangularis sterni muscle of the mouse. External currents generated in the presynaptic terminals upon invasion by action potentials have been recorded using microelectrodes inserted into the perineurium of preterminal nerve bundles. The voltage-dependent K current could be pharmacologically dissected into fast (IK_f) and slow (IK_s) components. While both are sensitive to 3,4-diaminopyridine (3,4-DAP), only IK_f is sensitive to tetraethylammonium (TEA). Uranyl (100–200 M) and guanidine (5–10 mM) produced a near complete block of IK_f and IK_s, which can explain their facilitatory effect upon transmitter release. The third K current of presynaptic terminals is Ca²⁺-dependent, but was activated also by Sr²⁺. This current could be suppressed by nanomolar doses of charybdotoxin; it is also sensitive to TEA but not to 3,4-DAP, uranyl or guanidine.     

Postnatal development of rat motor nerve terminals

Summary The nerve terminals of neuromuscular junctions in the rat diaphragm, extensor digitorum longus muscle and soleus muscle have been studied in animals between 3 weeks and 2.5 years of age using methylene blue stain and light microscopy. Dimensions, structure and organization of the nerve terminals were shown to change during life at various rates in different muscles and postnatal periods. The area and length of the terminals increase in all three muscles until young adult age. Later these dimensions continue to increase in the extensor digitorum longus and soleus muscles. In the diaphragm only the length increases, and this occurs late in adult life. The area also increases in relation to the diameter of the corresponding muscle fiber. Adult soleus terminals are more elongated than terminals in the diaphragm and extensor digitorum longus muscle. During adult life the extension of nerve terminals in relation to muscle fiber length increases in the extensor digitorum longus and soleus muscles, but is almost unchanged in the diaphragm. The nerve terminal branches are mainly coarse and irregular in young animals, but possess varying numbers of varicosities in adult animals. The number of varicosities is high in the extensor digitorum longus muscle and low in the diaphragm. In old animals the number of varicosities tends to be reduced. With increasing age the nerve terminal branches become organized in distinct groups with increasing distance between the groups. This is prominent in the soleus.     

Cholinergic receptors at sympathetic preganglionic nerve terminals

1. In the paravertebral sympathetic chain of bullfrogs, some part of the preganglionic nerve located within the ganglion was depolarized transiently when acetylcholine (ACh) was directly applied to the ganglion, particularly in the presence of an anticholinesterase (anti-ChE). The axonal part of the preganglionic nerve, on the other hand, showed no detectable depolarization following direct application of ACh (with anti-ChEs) to the interganglionic nerve trunk.2. The ACh depolarization was markedly depressed by nicotine, and less markedly by (+)-tubocurarine, whereas it was not affected by atropine. Nicotine, similar to ACh, transiently depolarized only the intraganglionic portion of the presynaptic fibres.3. The action potentials, recorded from the axonal as well as the terminal parts of the preganglionic nerve, showed spike potentials followed by a marked negative after-potential. The negative after-potential was followed by a positive after-potential which was markedly enhanced by repetitive nerve stimulation.4. A slow negative potential followed the positive after-potential in the repetitive responses of the terminal parts of the preganglionic nerve. The slow negative potential was enhanced by anti-ChEs, eliminated by ACh and nicotine, and unaffected by atropine.5. The amplitude of the action potentials of the terminal parts of the preganglionic nerve, and particularly that of the negative after-potential, was significantly depressed during the development of ACh depolarization as well as the slow negative potential, indicating that the two types of slow depolarization originated in the intraganglionic portion of the presynaptic fibres, presumably somewhere near or at the nerve terminals.6. ACh depolarization similar to that observed with bullfrog sympathetic ganglia was observed with rat superior cervical ganglia. The present experiments provide evidence that a certain part of the preganglionic nerve terminals is depolarized by the action of the transmitter released from their endings.     

Statistics of transmitter release at nerve terminals

This review presents an historical account of the developments of the statistical analysis of quantal transmission over the past half century and of the progress made in using this approach to reveal new properties of nerve terminals. In the early 1950s, Katz and his colleagues showed that evoked transmitter release occurred in quanta at the neuromuscular junction, opening up the study of transmitter release at nerve terminals to statistical analysis. In the subsequent two decades attempts were made to see if evoked quantal release could be described by binomial or compound binomial statistics, as originally suggested by Katz, and to relate the parameters of the statistic to various structures of the nerve terminal. During this period two hypotheses were enunciated, namely the 'vesicle hypothesis', which states that quanta arise as a consequence of the packaging of transmitter in vesicles; and the 'active zone hypothesis', which states that vesicles undergo exocytosis at discrete sites on the nerve terminal. Unsuccessful attempts were made to relate the binomial parameter n to the elements in these hypotheses, that is to the number of active zones possessed by the terminal or the number of vesicles available for release at these zones. This difficulty was part resolved in the late 1970s with the application of non-uniform binomial statistics to transmitter release from nerve terminals, in which n is the number of active zones each with their individual probabilities, p(j). Autocorrelation functions were subsequently introduced to detect if transmitter release is quantised at a particular nerve terminal. Statistical methods which would allow discrimination between different models of transmitter release over the active zones of a terminal were then developed. The introduction of maximum likelihood estimation procedures then allowed estimates to be made of the parameters in the statistical models of quantal release. The application of these procedures to experimental data from a variety of nerve terminals provided evidence for the concept that each synapse, taken as possessing a single active zone, possesses its own individual probability of secretion of a quantum by the exocytosis of a vesicle. In the late 1960s Stevens introduced the first stochastic approach to the analysis of the kinetics of the release of a quantum of transmitter at the neuromuscular junction following an impulse. In the subsequent decades this was developed into an explicit theory for the interaction of proteins involved in regulated exocytosis of a vesicle at an active zone. The parameters were the number of transition steps in the release process (k), each occurring at the same rate (alpha), with the possibility of each of these steps becoming blocked at the same rate (gamma). Maximum likelihood estimation procedures could then be used to obtain these parameter values. The discovery was made in the 1990s of the core proteins of the SNARE complex that govern regulated exocytosis. This offers the possibility in the near future of identifying the kinetic interaction of these proteins with the parameters of the stochastic process of exocytosis which confer a particular probability on individual synapses.     

Transitory inner medullary nerve terminals in the cat kidney

An indirect immunohistochemical method was used to visualize nerves immunoreactive for tyrosine hydroxylase (THI) and dopamine-beta-hydroxylase (DBHI) in kidney sections of cats 6 weeks and 2 and 3 months of age. THI and DBHI nerve terminals innervate the renal pelvis, interlobar veins and arterial tree including medullary vascular bundles of cats of each age studied. In kidneys of 6-week-old cats, THI and DBHI axons form elaborate plexuses that are distributed throughout much of the inner medulla, whereas some medullary axons appear to degenerate at 2 months and no inner medullary plexuses were visualized in 3-month-old cats. Transitory inner medullary nerves in the cat kidney may influence cellular development and play a role in salt and water balance.     

Fine structure of nerve terminals in the human gut

An electron microscopical investigation was made of neuronal terminals in operatively removed human duodenum and jejunum. No intraepithelial neuronal elements were found. The lamina propria mucosae, submucosa and muscular layer possessed, in addition to large bundles of unmyelinated nerve fibers, small ones consisting of three or four axons wrapped by infoldings of one Schwann cell. Some of these axons were light; others contained mitochondria and clear vesicles measuring 200–500 Å across. Still other axons had dense-cored vesicles with a diameter ～ 700 Å. The plasma membranes of vesiculated axons were not thickened and only in part invested by the Schwann cell, but were covered by a basement membrane. Such profiles were interpreted as nerve terminals, and were separated from fibers of the muscularis mucosae, bases of epithelial cells and submucous capillaries by interspaces measuring 5,000–6,800 Å. The gap between neuronal terminals and smooth muscle in the muscular wall was smaller (900 Å, more often 1,500–3,000 Å). The myenteric plexus contained nerve bundles consisting of the usual axons and of preterminals; some of the latter contained small clear vesicles, others predominantly larger granulated vesicles, and still others a mixture of both types of vesicles. The similarities and differences of neuronal terminals occurring in animals and in man were pointed out. The significance of the findings of this study was discussed in the light of current concepts of the physiology of autonomic nerve transmission.     

The storage of endogenous noradrenaline in sympathetic nerve terminals

1. The subcellular distribution of noradrenaline in sympathetic nerve terminals of rat vas deferens and cat spleen has been studied by cell fractionation methods combined with fluorescence and electronmicroscopic histochemical methods for noradrenaline.2. Pinched-off axon varicosities (synaptosomes) were isolated and identified by fluorescence and electronmicroscopy in the mitochondrial pellet.3. The proportion of large to small dense-cored vesicles in electronmicrographs of sympathetic nerve terminals varies in different organs. In rat vas deferens 4% and in cat spleen 20% are large vesicles.4. Density gradients of rat vas deferens have a single low density peak of noradrenaline at 0.6 M sucrose, whereas those of cat spleen have an additional peak of noradrenaline at 1.1 M sucrose.5. Small dense-cored vesicles were identified electronmicroscopically in the low density fractions and large dense-cored vesicles in the high density fractions from density gradients.6. We conclude that both small and large dense-cored vesicles store noradrenaline.     

Species specific morphology of mammalian motor nerve terminals

Summary Motor nerve terminals in the diaphragm, extensor digitorum longus and soleus muscles of young and adult rat, hamster and guinea pig were studied with the light microscope after staining with methylene blue. In adult animals the nerve terminals are smaller in the diaphragm than in the two other muscles. In the rat and hamster the extensor digitorum longus and soleus terminals are of similar area, but the terminals in extensor digitorum longus are shorter. In the guinea pig the terminals are smaller and shorter in soleus than in extensor digitorum longus. The density of nerve terminal varicosities is lowest in the diaphragm in all three species. In the rat and hamster the density is higher in extensor digitorum longus than in soleus. In the guinea pig the converse is found. In all three muscles the density of varicosities is higher in the rat than in the hamster and guinea pig. The nerve terminal branches in the diaphragm are mostly organized in one group. In the rat and hamster the soleus terminal branches are more separated in groups than the extensor digitorum longus terminal branches. In the guinea pig the number of groups is almost the same in the two muscles. These muscle and species-specific differences appear already in very young animals.     

Transmitter release from insect excitatory motor nerve terminals

1. Intracellular and extracellular electrodes were used to study spontaneous and impulse-linked release of transmitter at locust retractor unguis nerve-muscle synapses.2. At most extracellular recording sites the amplitude distributions of the excitatory post-synaptic potentials (e.p.s.p.s) were apparently non-Poisson. However, interpretation of these amplitude distributions was complicated by the effect on the extracellular recordings of the complex structural organization of the retractor unguis nerve terminal with its spatially distinct transmitter release sites extending over distances of 15-30 mum.3. The spontaneous miniature excitatory post-synaptic potentials (min e.p.s.p.s) did not occur at random intervals, bursts of min e.p.s.p.s being frequently recorded. As a result the spontaneous release of transmitter rarely approximated a Poisson process.4. For a period of at least 390 msec following a conditioning nerve impulse a test e.p.s.p. was facilitated and the probability of spontaneous transmitter release was enhanced. A large primary phase of facilitation of impulse-linked and spontaneous release was invariably followed by one or more secondary phases of smaller magnitude.