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Yanwen Zhou,* Ying Liu,* Yunhua Hao, Ya Feng, Lizhen Pan, Wuchao Liu, Bing Li, Libin Xiao, Lingjing Jin, Zhiyu Nie Department of Neurology, Shanghai Tongji Hospital, Tongji University School of Medicine, Shanghai 200065, China *These authors contributed equally to this work Background: Botulinum neurotoxin type A (BoNT/A) is emerging as a treatment modality for Raynaud’s phenomenon (RP). However, the mechanism of the role of BoNT/A in antagonizing the constriction of arteriola in RP remains unclear. Materials and methods: We tested the constriction of arteriole diameter and the distribution of adrenergic receptors on the rat cremaster modle. Moreover, we measured the secretion of norepinephrine (NE), protein level changes and related receptors on cultured rat superior cervical ganglia neurons(SCGNs), a model of sympathetic neuron. Results: Based on our results, the inhibition of arteriole vasoconstriction was increased with increasing doses of BoNT/A. BoNT/Α, prazosin, and BQ123 treatment can result in significant inhibition of arteriole vasoconstriction with the same electrical stimulation. The inhibition effect of prazosin was equivalent to BoNT/A, while BQ123 has a synergistic effect with BoNT/A. After treating SCGNs using BoNT/A for 30 min, the decrease in fluorescence intensity of FM1-43 slowed down which was correlated with the doses of BoNT/A. Furthermore, release of NE in the supernatant was significantly decreased as measured by enzyme-linked immunosorbent assay, 24 h after a high dose of BoNT/A (25 µ/mL). Cleaved-SNAP-25 was detected by western blotting 24 h following BoNT/A (50 µ/mL) treatment. Moreover, receptor SV2C, GM1, and FGFR3 were detected on sympathetic neurons, similarly to cholinergic neurons. Conclusion: Our study showed that BoNT/A could significantly inhibit electrical stimulation-induced arteriole vasoconstriction through the sympathetic pathway. The mechanism was similar to the cholinergic one, in which the vesicle release of sympathetic neurons could be inhibited by cleavage of SNAP-25. The end result was blocked vesicle fusion with the presynaptic membrane after BoNT/A treatment, inhibiting the release of the NE. Keywords: botulinum neurotoxin type A, Raynaud’s phenomenon, α-adrenoceptor, arteriole diameter constrict rate, SNAP-25, SV2C, GM1, FGFR3, sympathetic neuron, vesicle cycle

We propose a generalization of the neurotransmitter release model proposed in Rodrigues et al. (2016). We increase the complexity of the underlying slow–fast system by considering a degree-four polynomial as parametrization of the critical manifold. We focus on the possible transient and asymptotic dynamics, exploiting the so-called entry–exit function to describe slow parts of the dynamics. We provide extensive numerical simulations, complemented by numerical bifurcation analysis. • We extend the neurotransmitter release model from Rodrigues et al. (2016). • We study a planar slow–fast system with a quartic critical manifold using GSPT. • We focus on the delayed response to time-dependent input and asymptotic dynamics. • We find families of canards whose branches exchange position in parameter space. • The exchange depends on the geometry of the quartic critical manifold. [ABSTRACT FROM AUTHOR]

We studied the cycling of dense core vesicles producing somatic exocytosis of serotonin. Our experiments were made using electron microscopy and vesicle staining with fluorescent dye FM1-43 in Retzius neurons of the leech, which secrete serotonin from clusters of dense core vesicles in a frequency-dependent manner. Electron micrographs of neurons at rest or after 1Hz stimulation showed two pools of dense core vesicles. A perinuclear pool near Golgi apparatuses, from which vesicles apparently form, and a peripheral pool with vesicle clusters at a distance from the plasma membrane. By contrast, after 20Hz electrical stimulation 47% of the vesicle clusters were apposed to the plasma membrane, with some omega exocytosis structures. Dense core and small clear vesicles apparently originating from endocytosis were incorporated in multivesicular bodies. In another series of experiments, neurons were stimulated at 20 Hz while bathed in a solution containing peroxidase. Electron micrographs of these neurons contained gold particles coupled to anti-peroxidase antibodies in dense core vesicles and multivesicular bodies located near the plasma membrane. Cultured neurons depolarized with high potassium in the presence of FM1-43 displayed superficial fluorescent spots, each reflecting a vesicle cluster. A partial bleaching of the spots followed by another depolarization in the presence of FM1-43 produced restaining of some spots, other spots disappeared, some remained without restaining and new spots were formed. Several hours after electrical stimulation the FM1-43 spots accumulated at the center of the somata. This correlated with electron micrographs of multivesicular bodies releasing their contents near Golgi apparatuses. Our results suggest that dense core vesicle cycling related to somatic serotonin release involves two steps: the production of clear vesicles and multivesicular bodies after exocytosis, and the formation of new dense core vesicles in the perinuclear region. [ABSTRACT FROM AUTHOR]

Presynaptic terminals are specialized compartments of neurons responsible for converting electrical signals into secreted chemicals. This self-renewing process of chemical synaptic transmission is accomplished by the calcium-triggered fusion of neurotransmitter-containing vesicles with the plasma membrane and subsequent retrieval and recycling of vesicle components. Whereas the release of neurotransmitters has been studied for over 50 years, the process of synaptic vesicle endocytosis has remained much more elusive. The advent of imaging techniques suited to monitor membrane retrieval at presynaptic terminals and the discovery of the molecules that orchestrate endocytosis have revolutionized our understanding of this critical trafficking event. [ABSTRACT FROM AUTHOR]

Retrieval of synaptic vesicles from the membrane of neurons is crucial to maintain normal rates of neurotransmitter release. Photoreceptor terminais of the fly's eye release neurotransmitter in a tonic manner. They therefore rely heavily on vesicle regeneration. Null mutations in endophilin (endo) block clathrin-mediated endocytosis at the Drosophila neuromuscular junction, where previous analysis of hypomorphic mutations has suggested a function for Endophilin (Endo) before vesicle fission, during membrane bending. Here, at fly photoreceptor synapses, we show that Endo is localized to synaptic vesicles at sites of endocytosis that are glial invaginations called capitate projections, and that when the photoreceptor synapses lack Endo they are impaired in their ability to release neurotransmitter. Detailed ultrastructural analysis of endo null mutant photoreceptor synapses fails to reveal a defect at early stages of vesicle reformation but, instead, reveals an accumulation of clusters of electron-dense, apparently nonfunctional, late endocytotic vesicles. Using dynamin; endo double-mutant photoreceptors, we provide further evidence that ultimately the function of Endophilin is required late in endocytosis, allowing vesicles to progress through the synaptic vesicle cycle. [ABSTRACT FROM AUTHOR]

Abstract In central nerve terminals transmitter release is tightly regulated and thought to occur in a number of steps. These steps include vesicle mobilization and docking prior to neurotransmitter release. Intrasynaptic changes in vesicle distribution were determined by electron microscopical analysis and neurotransmitter release was monitored by biochemical measurements. We correlated K + -induced changes in distribution of small and large vesicles with the release of their transmitters. For small synaptic vesicles, amino acid release as well as recruitment to and docking at the active zone were activated within 1 s of depolarization. In contrast, the disappearance of large dense-cored vesicles and the release of the neuropeptide cholecystokinin were much slower, and no docking was observed. Studies with diverse Ca2 + channel blockers indicated that mobilization and neurotransmitter release from both vesicle types were regulated by multiple Ca2 + channels, although in different ways. Neurotransmitter release from small synaptic vesicles was predominantly regulated by P-type Ca2 + channels, whereas primarily Q-type Ca2 + channels regulated neurotransmitter release from large dense-cored vesicles. The different Ca2 + channnel types directly regulated mobilization of and neurotransmitter release from small synaptic vesicles whereas, by their cooperativity in raising the intracellular Ca2 + concentration above release threshold, they more indirectly regulated large dense-cored vesicle exocytosis. [ABSTRACT FROM AUTHOR]

The cycle of synaptic vesicles was studied in isolated nerve terminals and in the electric tissue of Torpedo marmorata. The synaptosomes, as used in this investigation, were a pure cholinergic subcellular fraction that captured dextran particles as an extracellular marker. This endocytotic phenomenon was enhanced by potassium depolarization. Field electrical stimulation (1 Hz and 10 Hz) of the electric organ induced the appearance of membrane foldings into presynaptic terminals. Morphometric studies showed that the number of synaptic vesicles did not decline until after at least 30 min. On the other hand, at 10 Hz these changes were accompanied by an increase in length of the membrane of the terminal. At 15 min of recovery after prolonged stimulation, there was a great increase in density of synaptic vesicles with a large number of vesicles of small diameter. This increase was accompanied by a decrease of membrane length, suggesting that reformation of vesicles is related to retrieval of membrane. Pharmacological stimulation with ouabain produced changes similar to those of long-term electrical stimulation. These changes in membrane were accompanied by a decrease of the population of synaptic vesicles and a wide variation in their diameters. It is concluded that structural changes reported here could not be correlated with kinetics of the transmitter release. [ABSTRACT FROM AUTHOR]

Summary The brain is highly sensitive to proper fuel availability as evidenced by the rapid decline in neuronal function during ischemic attacks and acute severe hypoglycemia. We previously showed that sustained presynaptic function requires activity-driven glycolysis. Here, we provide strong evidence that during action potential (AP) firing, nerve terminals rely on the glucose transporter GLUT4 as a glycolytic regulatory system to meet the activity-driven increase in energy demands. Activity at synapses triggers insertion of GLUT4 into the axonal plasma membrane driven by activation of the metabolic sensor AMP kinase. Furthermore, we show that genetic ablation of GLUT4 leads to an arrest of synaptic vesicle recycling during sustained AP firing, similar to what is observed during acute glucose deprivation. The reliance on this biochemical regulatory system for “exercising” synapses is reminiscent of that occurring in exercising muscle to sustain cellular function and identifies nerve terminals as critical sites of proper metabolic control. [ABSTRACT FROM AUTHOR]