Current models emphasize that membrane voltage (Vm) depolarization-induced Ca2+ influx triggers the fusion of vesicles to the plasma membrane. In sympathetic adrenal chromaffin cells, activation of a variety of G protein coupled receptors (GPCRs) can inhibit quantal size (QS) through the direct interaction of G protein Giβγ subunits with exocytosis fusion proteins. Here we report that, independently from Ca2+, Vm (action potential) per se regulates the amount of catecholamine released from each vesicle, the QS. The Vm regulation of QS was through ATP-activated GPCR-P2Y12 receptors. D76 and D127 in P2Y12 were the voltage-sensing sites. Finally, we revealed the relevance of the Vm dependence of QS for tuning autoinhibition and target cell functions. Together, membrane voltage per se increases the quantal size of dense-core vesicle release of catecholamine via Vm → P2Y12(D76/D127) → Giβγ → QS → myocyte contractility, offering a universal Vm-GPCR signaling pathway for its functions in the nervous system and other systems containing GPCRs.

Co-release of multiple neurotransmitters from secretory vesicles is common in neurons and neuroendocrine cells. However, whether and how the transmitters co-released from a single vesicle are differentially regulated remains unknown. In matrix-containing dense-core vesicles (DCVs) in chromaffin cells, there are two modes of catecholamine (CA) release from a single DCV: quantal and sub-quantal. By combining two microelectrodes to simultaneously record co-release of the native CA and ATP from a DCV, we report that (1) CA and ATP were co-released during a DCV fusion; (2) during kiss-and-run (KAR) fusion, the co-released CA was sub-quantal, whereas the co-released ATP was quantal; and (3) knockdown and knockout of the DCV matrix led to quantal co-release of both CA and ATP even in KAR mode. These findings strongly imply that, in contrast to sub-quantal CA release in chromaffin cells, fast synaptic transmission without transmitter-matrix binding is mediated exclusively via quantal release in neurons.

Dynamin 1 (dyn1) is required for clathrin-mediated endocytosis in most secretory (neuronal and neuroendocrine) cells. There are two modes of Ca 2+ -dependent catecholamine release from single dense-core vesicles: full-quantal (quantal) and subquantal in adrenal chromaffin cells, but their relative occurrences and impacts on total secretion remain unclear. To address this fundamental question in neurotransmission area using both sexes of animals, here we report the following: (1) dyn1-KO increased quantal size (QS, but not vesicle size/content) by ≥250% in dyn1-KO mice; (2) the KO-increased QS was rescued by dyn1 (but not its deficient mutant or dyn2); (3) the ratio of quantal versus subquantal events was increased by KO; (4) following a release event, more protein contents were retained in WT versus KO vesicles; and (5) the fusion pore size ( d p ) was increased from ≤9 to ≥9 nm by KO. Therefore, Ca 2+ -induced exocytosis is generally a subquantal release in sympathetic adrenal chromaffin cells, implying that neurotransmitter release is generally regulated by dynamin in neuronal cells. SIGNIFICANCE STATEMENT Ca 2+ -dependent neurotransmitter release from a single vesicle is the primary event in all neurotransmission, including synaptic/neuroendocrine forms. To determine whether Ca 2+ -dependent vesicular neurotransmitter release is “all-or-none” (quantal), we provide compelling evidence that most Ca 2+ -induced secretory events occur via the subquantal mode in native adrenal chromaffin cells. This subquantal release mode is promoted by dynamin 1, which is universally required for most secretory cells, including neurons and neuroendocrine cells. The present work with dyn1-KO mice further confirms that Ca 2+ -dependent transmitter release is mainly via subquantal mode, suggesting that subquantal release could be also important in other types of cells.

Neuropeptides released from dorsal root ganglion (DRG) neurons play essential roles in the neurotransmission of sensory inputs, including those underlying nociception and pathological pain. Neuropeptides are released from intracellular vesicles through two modes: a partial release mode called "kiss-and-run" (KAR) and a full release mode called "full fusion-like" (FFL). Using total internal reflection fluorescence (TIRF) microscopy, we traced the release of pH-sensitive green fluorescent protein-tagged neuropeptide Y (pHluorin-NPY) from individual dense-core vesicles in the soma and axon of single DRG neurons after Ca2+ influx through either voltage-gated Ca2+ channels (VGCCs) or ligand-gated transient receptor potential vanilloid 1 (TRPV1) channels. We found that Ca2+ influx through VGCCs stimulated FFL and a greater single release of neuropeptides. In contrast, Ca2+ influx through TRPV1 channels stimulated KAR and a pulsed but prolonged release of neuropeptides that was partially mediated by Dynamin 1, which limits fusion pore expansion. Suppressing the Ca2+ gradient to an extent similar to that seen after TRPV1 activation abolished the VGCC preference for FFL. The findings suggest that by generating a steeper Ca2+ gradient, VGCCs promote a more robust fusion pore opening that facilitates FFL. Thus, KAR and FFL release modes are differentially regulated by the two principal types of Ca2+-permeable channels in DRG neurons.

Transient receptor potential A1 (TRPA1) is a nonselective cation channel implicated in thermosensation and inflammatory pain. In this study, we show that TRPA1 (activated by allyl isothiocyanate, acrolein, and 4-hydroxynonenal) elevates the intracellular Ca2+ concentration ([Ca2+]i) in dorsal root ganglion (DRG) neurons in the presence and absence of extracellular Ca2+. Pharmacological and immunocytochemical analyses revealed the presence of TRPA1 channels both on the plasma membrane and in endolysosomes. Confocal line-scan imaging demonstrated Ca2+ signals elicited from individual endolysosomes (“lysosome Ca2+ sparks”) by TRPA1 activation. In physiological solutions, the TRPA1-mediated endolysosomal Ca2+ release contributed to ∼40% of the overall [Ca2+]i rise and directly triggered vesicle exocytosis and calcitonin gene-related peptide release, which greatly enhanced the excitability of DRG neurons. Thus, in addition to working via Ca2+ influx, TRPA1 channels trigger vesicle release in sensory neurons by releasing Ca2+ from lysosome-like organelles.

Dopamine (DA) transmission plays multiple roles in movement initiation and execution, reward signaling, cognition, and motivation. It is well established that DA is released from terminals of dopaminergic neurons (DANs) in dorsal striatum, nucleus accumbens, prefrontal cortex, and many other brain regions. However, whether, how, when and what physiological relevance DA is released via vesicular exocytosis from the somata of DANs in the substantia nigra (SN) remains elusive. Here, we revealed that depolarization evoked Ca2+-dependent somatic DA release from single vesicles in SN slices with combined recordings of path-clamp and amperometry of electrochemical micro carbon fiber electrode for first time. On average, 7.8×104 DA molecules were released per vesilce. The latency of the evoked DA release was 340 ms, or 44 times slower in the somata of DANs than axonal terminal release in the striatum. D2R was expressed in SNc neural somata, and served as auto-receptor to inhibit cell activity. Thus, the longer latency of somatic DA release permits strong modulation of action potentials patterns through its autoreceptors in DA somata versus terminals. Supported by grants from NSFC, MOST and DOE 2011-program

With the upgrade of the miniaturization of the electronic equipment, heat in per area increases dramatically, which leads to the strong need for a high efficient device of heat dissipation. As the result of the nature evolution, leaf vein system is an excellent structure for heat and mass transfer but has not been widely studied. Based on the leaf vein system, a conceptual structure is designed to form the wick of a vapor chamber, in which the leaf-vein-like fractal network and the micro fin-pins are used to simulate the leaf vein network and mesophyll tissue respectively. In the experiment, the leaf-vein-like structure is manufactured by chemical etching, and two different vapor chambers (diameter is 90mm, the evaporator and condenser have the same wick structures) with and without strength boiling are compared concerning their cooling performances. The experiment result shows that when the diameter of the heating rod is 35mm, the vapor chamber can perform good temperature uniformity and small thermal resistance with the input power Q⩽90W. When the deionized water is used as the working fluid, the thermal resistance of the vapor chamber is smaller than 0.3°C/W.

Sympathetic nerve in heart is essential in cardiac physiology and diseases, because it releases catecholamines to regulate cardiac cells including ventricular myocytes, atrial myocytes and vas through GPCRs. With a modified glass-insulated micro carbon fiber electrode (pegCFE), we record stimulus (depolarization or hypoxia)-signals via either amperometric current (Iamp) or fast cyclic voltammetry from the nerve terminals in rodent hearts, by a method termed cardiac Slice of ElectroChemistry (cSEC). We found that, (1) cSEC signal Iamp is dependent on CFE-voltage and extracellular Ca2+; (2) pharmacologically, Iamp is increased 40% by Yohimbine, and decreased 60% by reserpine; (3) electronic microscope detected dese core vesicles, tyrosine hydroxylase (TH) immunostaining and TH-GFP transgenic mice showed massive TH-signal in whole heart; (4) as determined by in-situ FCV, as well as microdialysis-based HPLC, NE and/or DHPG (a NE metabolic substance) were responsible for cSEC signals. These evidences establish that the evoked cSEC signals represent catecholamine releases from sympathetic nerves in heart slices. Using cSEC we discovered that hypoxia-reperfusion triggered dual spikes of catecholamine release at pH 7.4: first peak at 10s following hypoxia perfusion, second peak at 10s following normoxia perfusion. Finally, catecholamine release were reduced by 75% in ventricle slices from syt7(Ca2+ sensor)-KO versus WT mice, indicating cSEC may serve phenotyping of any sympathetic defects in cardiac disease animal models. Supported by NSFC, MOST 973 and PKU-THU-CLS

AIMS/HYPOTHESIS:Insulin is a key metabolic regulator in health and diabetes. In pancreatic beta cells, insulin release is regulated by the major second messengers Ca(2+) and cAMP: exocytosis is triggered by Ca(2+) and mediated by the cAMP/protein kinase A (PKA) signalling pathway. However, the causal link between these two processes in primary beta cells remains undefined.METHODS:Time-resolved confocal imaging of fluorescence resonance energy transfer signals was performed to visualise PKA activity, and combined membrane capacitance recordings were used to monitor insulin secretion from patch-clamped rat beta cells.RESULTS:Membrane depolarisation-induced Ca(2+) influx caused an increase in cytosolic PKA activity via activating a Ca(2+)-sensitive adenylyl cyclase 8 (ADCY8) subpool. Glucose stimulation triggered coupled Ca(2+) oscillations and PKA activation. ADCY8 knockdown significantly reduced the level of depolarisation-evoked PKA activation and impaired replenishment of the readily releasable vesicle pool. Pharmacological inhibition of PKA by two inhibitors reduced depolarisation-induced PKA activation to a similar extent and reduced the capacity for sustained vesicle exocytosis and insulin release.CONCLUSIONS/INTERPRETATION:Our findings suggest that depolarisation-induced Ca(2+) influx plays dual roles in regulating exocytosis in rat pancreatic beta cells by triggering vesicle fusion and replenishing the vesicle pool to support sustained insulin release. Therefore, Ca(2+) influx may be important for glucose-stimulated insulin secretion.

Schizophrenia is a severely devastating mental disorder, the pathological process of which is proposed to be associated with the dysfunction of dopaminergic transmission. Our previous results have demonstrated slower kinetics of transmitter release (glutamate release in hippocampus and norepinephrine release in adrenal slice) in a schizophrenia model, dysbindin null-sandy mice. However, whether dopaminergic transmission in the nigrostriatal pathway contributes to the pathology of dysbindin-/- mice remains unknown. Here, we have provided a step-by-step protocol to be applied in the in vivo amperometric recording of dopamine (DA) release from the mouse striatum evoked by an action potential (AP) pattern. With this protocol, AP pattern-dependent DA release was recorded from dysbindin-/- mice striatum in vivo. On combining amperometric recording in slices and electrophysiology, we found that in dysbindin-/- mice, (1) presynaptically, AP-pattern dependent dopamine overflow and uptake were intact in vivo; (2) the recycling of the dopamine vesicle pool remained unchanged. (3) Postsynaptically, the excitability of medium spiny neuron (MSN) was also normal, as revealed by patch-clamp recordings in striatal slices. Taken together, in contrast to reduced norepinephrine release in adrenal chromaffin cells, the dopaminergic transmission remains unchanged in the nigrostriatal pathway in dysbindin-/- mice, providing a new insight into the functions of the schizophrenia susceptibility gene dysbindin.

Embryonic stem cell-based therapies exhibit great potential for the treatment of Parkinson's disease (PD) because they can significantly rescue PD-like behaviors. However, whether the transplanted cells themselves release dopamine in vivo remains elusive. We and others have recently induced human embryonic stem cells into primitive neural stem cells (pNSCs) that are self-renewable for massive/transplantable production and can efficiently differentiate into dopamine-like neurons (pNSC-DAn) in culture. Here, we showed that after the striatal transplantation of pNSC-DAn, (i) pNSC-DAn retained tyrosine hydroxylase expression and reduced PD-like asymmetric rotation; (ii) depolarization-evoked dopamine release and reuptake were significantly rescued in the striatum both in vitro (brain slices) and in vivo, as determined jointly by microdialysis-based HPLC and electrochemical carbon fiber electrodes; and (iii) the rescued dopamine was released directly from the grafted pNSC-DAn (and not from injured original cells). Thus, pNSC-DAn grafts release and reuptake dopamine in the striatum in vivo and alleviate PD symptoms in rats, providing proof-of-concept for human clinical translation.

Parkinson's disease (PD) is a neurodegenerative disorder due to reduced dopamine (DA) in the striatum and loss of DA neurons in the substantia nigra pars compacta. Embryonic stem cells (ESCs) are an optimal source for cell therapy for PD. We recently developed a fast (one-week) protocol using small molecules that effectively induces human ESCs to become primitive neural stem cells (pNSCs), which are then differentiated into DA-like neurons in vitro. As pNSCs are infinitely expandable, this approach offers a strategy to readily generate DA neurons on a large scale. But whether these pNSC-differentiated DA (pNSC-DA) neurons can functionally integrate into the damaged brain is unknown. Here, we transplanted pNSC-DA neurons into 6-hydroxydopamine-induced rat models of PD and monitored their behavior for 16 weeks. In striatum in vivo, we first determined that the grafted pNSC-DA neurons secreted DA(using HPLC with a 200-μm microdialysis probe at a sampling rate of 0.002 Hz); then we recorded DA release by electrochemical amperometry (using a 7-μm carbon-fiber electrode at a sampling rate of 1000 Hz), showing that the grafted pNSC-DA neurons significantly rescued DA release in slices (7%) and in vivo (16%). Furthermore, the transplanted cells survived for at least 16 weeks and dramatically rescued the apomorphine-induced asymmetric rotation behavior in PD rats. Thus, we draw the conclusion that transplanted pNSC-DA neurons can functionally integrate into the brain and partially relieve Parkinsonian symptoms in rats. Our work provides direct evidence for a therapeutic role of pNSC-DA neurons in treating Parkinsonian syndrome.

Key points The timing of synaptic transmission is critical to synaptic plasticity in the striatum. However, the timing of striatal dopamine (DA) release induced by cholinergic interneurons (ChIs) in the striatum is unclear. In this study, we focused on the temporal components of DA release and replenishment triggered by different pathways. We show that stimulation of ChIs induces DA release with a total delay of 20.6 ms, including 2.8 ms for action potential firing of ChIs, 7.0 ms for cholinergic transmission between acetylcholine terminals and DA terminals, and 10.8 ms for downstream DA release. The delay of DA release via this ChI pathway is 1.9 times that via the nigrostriatal pathways. We describe the time course of recovery of DA release via the two pathways and that of vesicle replenishment in DA terminals. Our work provides an example of unravelling the temporal building blocks during fundamental synaptic terminal–terminal transmission. Abstract Striatal dopamine (DA) is critically involved in major brain functions such as motor control and deficits such as Parkinson's disease. DA is released following stimulation by two pathways: the nigrostriatal pathway and the cholinergic interneuron (ChI) pathway. The timing of synaptic transmission is critical in striatal circuits, because millisecond latency changes can reverse synaptic plasticity from long‐term potentiation to long‐term depression in a DA‐dependent manner. Here, we determined the temporal components of ChI‐driven DA release in striatal slices from optogenetic ChAT‐ChR2‐EYFP mice. After a light stimulus at room temperature, ChIs fired an action potential with a delay of 2.8 ms. The subsequent DA release mediated by nicotinic acetylcholine (ACh) receptors had a total latency of 17.8 ms, comprising 7.0 ms for cholinergic transmission and 10.8 ms for the downstream terminal DA release. Similar latencies of DA release were also found in striatal slices from wild‐type mice. The latency of ChI‐driven DA release was regulated by inhibiting the presynaptic vesicular ACh release. Moreover, we describe the time course of recovery of DA release via the two pathways and that of vesicle replenishment in DA terminals. Our work provides an example of unravelling the temporal building blocks during fundamental synaptic terminal–terminal transmission in motor regulation.

Non‐technical summary Ca2+‐regulated exocytosis is essential for neurotransmitter and hormone release. As well as this type of exocytosis, the somata of dorsal root ganglion (DRG) neurons also show Ca2+‐independent but voltage‐dependent exocytosis. It is unclear whether these two types of exocytosis use the same or different vesicle pools in DRG neurons. Here, we found that they were separable in response to the same stimulation in low external Ca2+ solution. Depletion of the Ca2+‐dependent vesicle pool did not affect the Ca2+‐independent but voltage‐dependent exocytosis. These results show that DRG neurons exhibit two distinct types of exocytosis that use different vesicle pools.

Astrocytes release a variety of signaling molecules including glutamate, D-serine, and ATP in a regulated manner. Although the functions of these molecules, from regulating synaptic transmission to controlling specific behavior, are well documented, the identity of their cellular compartment(s) is still unclear. Here we set out to study vesicular exocytosis and glutamate release in mouse hippocampal astrocytes. We found that small vesicles and lysosomes coexisted in the same freshly isolated or cultured astrocytes. Both small vesicles and lysosome fused with the plasma membrane in the same astrocytes in a Ca2+-regulated manner, although small vesicles were exocytosed more efficiently than lysosomes. Blockade of the vesicle glutamate transporter or cleavage of synaptobrevin 2 and cellubrevin (both are vesicle-associated membrane proteins) with a clostridial toxin greatly inhibited glutamate release from astrocytes, while lysosome exocytosis remained intact. Thus, both small vesicles and lysosomes contribute to Ca2+-dependent vesicular exocytosis, and small vesicles support glutamate release from astrocytes.