Zinc Blocks Acetylcholine Release but not Vesicle Fusion at the Torpedo Nerve–Electroplate Junction

The combined effects of Zn²⁺ treatment and nerve stimulation were studied on cholinergic synapses of the Torpedo rnarmorafa electric organ. Incubation of small pieces of electric tissue in 250 μM ZnCI₂ for 2 h irreversibly blocked synaptic transmission by inhibiting the release of acetylcholine. This treatment, however, did not cause any significant fine structural alteration in the nerve-electroplate junctions. Preparations treated with Zn²⁺ were submitted to electrical stimulation. In spite of the fact that no transmitter was released, stimulation resulted in the accumulation of calcium in the tissue, and in marked ultrastructural changes. The density of synaptic vesicles was significantly reduced and many of the remaining vesicles were found in close proximity to the presynaptic membrane. Images of vesicles fused with the plasmalemma were abundant, indicating that numerous vesicles were caught in different phases of exocytosis or endocytosis. Freeze-fracture replicas made from quick-frozen or chemically fixed material showed a high number of vesicle openings (pits) in the presynaptic plasmalemma. No recovery occurred even after a prolonged period of rest, indicating that retrieval was impaired by zinc treatment. In conclusion, the present experimental paradigm created an unusual situation where fusion of synaptic vesicles to the plasma membrane could be activated independently from the release of transmitter.     

The ultrastructure of group Ia afferent fiber synapses in the lumbosacral spinal cord of the cat

Ia synapses in laminae VI and IX of the cat's spinal cord were examined in the electron microscope following iontophoretic injection of horseradish peroxidase (HRP) into single, identified, Ia afferent fibers from gastrocnemius muscles. Ia boutons contacting motoneuron dendrites in lamina IX contained spherical synaptic vesicles and generally contracted only one postsynaptic profile. The Ia boutons were often postsynaptic to smaller P-type axonal terminal. Consequently Ia boutons may be classified as S-boutons with axo-axonic contacts.     

The distribution of Ia antigen in the lesions of rat acute experimental allergic encephalomyelitis

Summary Ia antigen, encoded within the major histocompatibility complex, plays an important role in the activation of T lymphocytes. Since experimental allergic encephalitis is an essentially T cell-mediated disease, Ia antigen in the central nervous system (CNS) may be pathogenetically relevant. The occurrence of Ia antigen in the CNS of normal rats and of rats with experimental allergic encephalitis was studied by light and electron microscope immunocytochemistry using the monoclonal anti-Ia antibodies Ox 4 and Ox 6. In normal, unsensitized animals a distict population of stellate cells in the meninges and some perivascular mononuclear cells in the nervous tissue carried Ia antigen. In rats with experimental allergic encephalitis a dramatic increase of Ia-positive cells was found. In addition to the positive cells found in normal animals, monocytes, macrophages and many lymphocytes in the meningeal perivascular and parenchymal inflammatory infiltrates as well as activated microglia stained for Ia antigen. We did not find evidence for Ia expression on endothelial cells, astrocytes or other components of the CNS in either normal or diseased rats.     

The effects of naloxone, morphine and methionine enkephalinamide on Ia afferent terminations in the cat spinal cord

Naloxone, morphine, Met5-enkephalinamide (MENKA) and procaine were administered microelectrophoretically near extracellularly stimulated extensor muscle group Ia afferent fibres and terminations in the lumbar spinal cord of cats anaesthetized with pentobarbitone sodium. Observations were made of effects on the electrical threshold, on the depolarizing action of GABA or piperidine-4-sulphonate (P4S), and on bicuculline-sensitive primary afferent depolarization (PAD) generated by tetanic stimulation of flexor muscle low threshold afferents. All 4 agents reversibly elevated the threshold of Ia fibres in the dorsal column and Ia terminations in the ventral horn. The depolarizations of terminations by GABA or P4S were also reduced, an effect, which for all except MENKA, probably accounted for a concomitant reduction in PAD. In the absence of a consistent effect on either threshold or depolarization by GABAmimetics, MENKA reversibly diminished PAD, an action blocked by naloxone. Intravenously administered naloxone, in doses known to enhance spinal monosynaptic excitation in the cat, had no effect on GABAergic PAD and little or no effect on Ia termination threshold. The results are discussed in relation to a naloxone-sensitive effect of MENKA which reduces transmitter release from GABAergic axo-axonic synapses on Ia terminals, but which does not account for the enhancement of spinal reflexes by naloxone.     

The Effect of Barium on [3H]Noradrenalin Release from the Human Neuroblastoma SH-SY5Y

Replacement of Ca²⁺ with Ba²⁺ in HEPES-buffered saline stimulated [³H]noradrenalin release in the human neuroblastoma clone SH-SY5Y by up to 20% of the cell content in the absence of other secretory stimuli. The Ba²⁺-evoked release was inhibited by 85% by 3 μM tetrodotoxin and 95% by 5 μM nifedipine. Ba²⁺ also increased the potency of K⁺-evoked release of [³H]noradrenalin, as maximal release was observed with 60 mM K⁺ compared with the 100 mM K⁺ necessary to achieve maximal release in the presence of Ca²⁺. In contrast, replacing Ca²⁺ with Ba²⁺ had little effect on carbachol- and bradykinin-evoked release of [³H]noradrenalin. No evidence was obtained from studies on changes in [Ca²⁺]_i (in response to 100 pM carbachol) using fura-2 that Ba²⁺ could enter intracellular stores in SH-SY5Y cells. Whole-cell patch-clamp studies showed that Ba²⁺ depolarizes SH-SY5Y cells as well as enhancing inward Ca²⁺ channel currents and shifting their voltage dependence to more negative values. These results are discussed in terms of the hypothesis that Ba²⁺ blocks K⁺ channels, leading to depolarization followed by opening of voltage-sensitive Na⁺ channels. This in turn opens voltage-sensitive L-type Ca²⁺ channels, which are coupled to the release of [³H]noradrenalin in SH-SY5Y cells.