The optically determined size of exo/endo cycling vesicle pool correlates with the quantal content at the neuromuscular junction of Drosophila larvae.
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According to the current theory of synaptic transmission, the amplitude of evoked synaptic potentials correlates with the number of synaptic vesicles released at the presynaptic terminals. Synaptic vesicles in presynaptic boutons constitute two distinct pools, namely, exo/endo cycling and reserve pools (). We defined the vesicles that were endocytosed and exocytosed during high K+ stimulation as the exo/endo cycling vesicle pool. To determine the role of exo/endo cycling vesicle pool in synaptic transmission, we estimated the quantal content electrophysiologically, whereas the pool size was determined optically using fluorescent dye FM1-43. We then manipulated the size of the pool with following treatments. First, to change the state of boutons of nerve terminals, motoneuronal axons were severed. With this treatment, the size of exo/endo cycling vesicle pool decreased together with the quantal content. Second, we promoted the FM1-43 uptake using cyclosporin A, which inhibits calcineurin activities and enhances endocytosis. Cyclosporin A increased the total uptake of FM1-43, but neither the size of exo/endo cycling vesicle pool nor the quantal content changed. Third, we increased the size of exo/endo cycling vesicle pool by forskolin, which enhances synaptic transmission. The forskolin treatment increased both the size of exo/endo cycling vesicle pool and the quantal content. Thus, we found that the quantal content was closely correlated with the size of exo/endo cycling vesicle pool but not necessarily with the total uptake of FM1-43 fluorescence by boutons. The results suggest that vesicles in the exo/endo cycling pool primarily participate in evoked exocytosis of vesicles. (+info)
Cellular sites for dynorphin activation of kappa-opioid receptors in the rat nucleus accumbens shell.
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The nucleus accumbens (Acb) is prominently involved in the aversive behavioral aspects of kappa-opioid receptor (KOR) agonists, including its endogenous ligand dynorphin (Dyn). We examined the ultrastructural immunoperoxidase localization of KOR and immunogold labeling of Dyn to determine the major cellular sites for KOR activation in this region. Of 851 KOR-labeled structures sampled from a total area of 10,457 microm2, 63% were small axons and morphologically heterogenous axon terminals, 31% of which apposed Dyn-labeled terminals or also contained Dyn. Sixty-eight percent of the KOR-containing axon terminals formed punctate-symmetric or appositional contacts with unlabeled dendrites and spines, many of which received convergent input from terminals that formed asymmetric synapses. Excitatory-type terminals that formed asymmetric synapses with dendritic spines comprised 21% of the KOR-immunoreactive profiles. Dendritic spines within the neuropil were the major nonaxonal structures that contained KOR immunoreactivity. These spines also received excitatory-type synapses from unlabeled terminals and were apposed by Dyn-containing terminals. These results provide ultrastructural evidence that in the Acb shell (AcbSh), KOR agonists play a primary role in regulating the presynaptic release of Dyn and other neuromodulators that influence the output of spiny neurons via changes in the presynaptic release of or the postsynaptic responses to excitatory amino acids. The cellular distribution of KOR complements those described previously for the reward-associated mu- and delta-opioid receptors in the Acb shell. (+info)
Augmentation is a potentiation of the exocytotic process.
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Short-term synaptic enhancement is caused by an increase in the probability with which synaptic terminals release transmitter in response to presynaptic action potentials. Since exocytosed vesicles are drawn from a readily releasable pool of packaged transmitter, enhancement must result either from an increase in the size of the pool or an elevation in the fraction of releasable vesicles that undergoes exocytosis with each action potential. We show here that at least one major component of enhancement, augmentation, is not caused by an increase in the size of the readily releasable pool but is instead associated with an increase in the efficiency with which action potentials induce the exocytosis of readily releasable vesicles. (+info)
Nerve terminal damage by beta-bungarotoxin: its clinical significance.
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We report here original data on the biological basis of prolonged neuromuscular paralysis caused by the toxic phospholipase A2 beta-bungarotoxin. Electron microscopy and immunocytochemical labeling with anti-synaptophysin and anti-neurofilament have been used to show that the early onset of paralysis is associated with the depletion of synaptic vesicles from the motor nerve terminals of skeletal muscle and that this is followed by the destruction of the motor nerve terminal and the degeneration of the cytoskeleton of the intramuscular axons. The postjunctional architecture of the junctions were unaffected and the binding of fluorescein-isothiocyanate-conjugated alpha-bungarotoxin to acetylcholine receptor was not apparently affected by exposure to beta-bungarotoxin. The re-innervation of the muscle fiber was associated by extensive pre- and post-terminal sprouting at 3 to 5 days but was stable by 7 days. Extensive collateral innervation of adjacent muscle fibers was a significant feature of the re-innervated neuromuscular junctions. These findings suggest that the prolonged and severe paralysis seen in victims of envenoming bites by kraits (elapid snakes of the genus Bungarus) and other related snakes of the family Elapidae is caused by the depletion of synaptic vesicles from motor nerve terminals and the degeneration of the motor nerve terminal and intramuscular axons. (+info)
Presence of the vesicular inhibitory amino acid transporter in GABAergic and glycinergic synaptic terminal boutons.
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The characterization of the Caenorhabditis elegans unc-47 gene recently allowed the identification of a mammalian (gamma)-amino butyric acid (GABA) transporter, presumed to be located in the synaptic vesicle membrane. In situ hybridization data in rat brain suggested that it might also take up glycine and thus represent a general Vesicular Inhibitory Amino Acid Transporter (VIAAT). In the present study, we have investigated the localization of VIAAT in neurons by using a polyclonal antibody raised against the hydrophilic N-terminal domain of the protein. Light microscopy and immunocytochemistry in primary cultures or tissue sections of the rat spinal cord revealed that VIAAT was localized in a subset (63-65%) of synaptophysin-immunoreactive terminal boutons; among the VIAAT-positive terminals around motoneuronal somata, 32.9% of them were also immunoreactive for GAD65, a marker of GABAergic presynaptic endings. Labelling was also found apposed to clusters positive for the glycine receptor or for its associated protein gephyrin. At the ultrastructural level, VIAAT immunoreactivity was restricted to presynaptic boutons exhibiting classical inhibitory features and, within the boutons, concentrated over synaptic vesicle clusters. Pre-embedding detection of VIAAT followed by post-embedding detection of GABA or glycine on serial sections of the spinal cord or cerebellar cortex indicated that VIAAT was present in glycine-, GABA- or GABA- and glycine-containing boutons. Taken together, these data further support the view of a common vesicular transporter for these two inhibitory transmitters, which would be responsible for their costorage in the same synaptic vesicle and subsequent corelease at mixed GABA-and-glycine synapses. (+info)
Role of mitochondrial dysfunction in the Ca2+-induced decline of transmitter release at K+-depolarized motor neuron terminals.
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The present study tested whether a Ca2+-induced disruption of mitochondrial function was responsible for the decline in miniature endplate current (MEPC) frequency that occurs with nerve-muscle preparations maintained in a 35 mM potassium propionate (35 mM KP) solution containing elevated calcium. When the 35 mM KP contained control Ca2+ (1 mM), the MEPC frequency increased and remained elevated for many hours, and the mitochondria within twitch motor neuron terminals were similar in appearance to those in unstimulated terminals. All nerve terminals accumulated FM1-43 when the dye was present for the final 6 min of a 300-min exposure to 35 mM KP with control Ca2+. In contrast, when Ca2+ was increased to 3.6 mM in the 35 mM KP solution, the MEPC frequency initially reached frequencies >350 s-1 but then gradually fell approaching frequencies <50 s-1. A progressive swelling and eventual distortion of mitochondria within the twitch motor neuron terminals occurred during prolonged exposure to 35 mM KP with elevated Ca2+. After approximately 300 min in 35 mM KP with elevated Ca2+, only 58% of the twitch terminals accumulated FM1-43. The decline in MEPC frequency in 35 mM KP with elevated Ca2+ was less when 15 mM glucose was present or when preparations were pretreated with 10 microM oligomycin and then bathed in the 35 mM KP with glucose. When glucose was present, with or without oligomycin pretreatment, a greater percentage of twitch terminals accumulated FM1-43. However, the mitochondria in these preparations were still greatly swollen and distorted. We propose that prolonged depolarization of twitch motor neuron terminals by 35 mM KP with elevated Ca2+ produced a Ca2+-induced decrease in mitochondrial ATP production. Under these conditions, the cytosolic ATP/ADP ratio was decreased thereby compromising both transmitter release and refilling of recycled synaptic vesicles. The addition of glucose stimulated glycolysis which contributed to the maintenance of required ATP levels. (+info)
Simultaneous measurement of evoked release and [Ca2+]i in a crayfish release bouton reveals high affinity of release to Ca2+.
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The opener neuromuscular junction of crayfish was used to determine the affinity of the putative Ca2+ receptor(s) responsible for evoked release. Evoked, asynchronous release, and steady-state intracellular Ca2+ concentration, [Ca2+]ss, were measured concomitantly in single release boutons. It was found that, as expected, asynchronous release is highly correlated with [Ca2+]ss. Surprisingly, evoked release was also found to be highly correlated with [Ca2+]ss. The quantal content (m) and the rate of asynchronous release (S) showed sigmoidal dependence on [Ca2+]ss. The slope log m/log [Ca2+]ss varied between 1.6 and 3.3; the higher slope observed at the lower [Ca2+]o. The slope log S/log [Ca2+]ss varied between 3 and 4 and was independent of [Ca2+]o. These results are consistent with the assumption that evoked release is controlled by the sum of [Ca2+]ss and the local elevation of Ca2+ concentration near the release sites resulting from Ca2+ influx through voltage-gated Ca2+ channels (Y). On the basis of the above, we were able to estimate Y. We found Y to be significantly <10 microM even for [Ca2+]o = 13.5 mM. The dissociation constant (Kd) of the Ca2+ receptor(s) associated with evoked release was calculated to be in the range of 4-5 microM. This value of Kd is similar to that found previously for asynchronous release. (+info)
Characterization of K+ currents underlying pacemaker potentials of fish gonadotropin-releasing hormone cells.
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Endogenous pacemaker activities are important for the putative neuromodulator functions of the gonadotropin-releasing hormone (GnRH)-immunoreactive terminal nerve (TN) cells. We analyzed several types of voltage-dependent K+ currents to investigate the ionic mechanisms underlying the repolarizing phase of pacemaker potentials of TN-GnRH cells by using the whole brain in vitro preparation of fish (dwarf gourami, Colisa lalia). TN-GnRH cells have at least four types of voltage-dependent K+ currents: 1) 4-aminopyridine (4AP)-sensitive K+ current, 2) tetraethylammonium (TEA)-sensitive K+ current, and 3) and 4) two types of TEA- and 4AP-resistant K+ currents. A transient, low-threshold K+ current, which was 4AP sensitive and showed significant steady-state inactivation in the physiological membrane potential range (-40 to -60 mV), was evoked from a holding potential of -100 mV. This current thus cannot contribute to the repolarizing phase of pacemaker potentials. TEA-sensitive K+ current evoked from a holding potential of -100 mV was slowly activating, long lasting, and showed comparatively low threshold of activation. This current was only partially inactivated at steady state of -60 to -40 mV, which is equivalent to the resting membrane potential. TEA- and 4AP-resistant sustained K+ currents were evoked from a holding potential of -100 mV and were suggested to consist of two types, based on the analysis of activation curves. From the inactivation and activation curves, it was suggested that one of them with low threshold of activation may be partly involved in the repolarizing phase of pacemaker potentials. Bath application of TEA together with tetrodotoxin reversibly blocked the pacemaker potentials in current-clamp recordings. We conclude that the TEA-sensitive K+ current is the most likely candidate that contributes to the repolarizing phase of the pacemaker potentials of TN-GnRH cells. (+info)