Sasa Teng

Calcium Triggers Exocytosis from Two Types of Organelles in a Single Astrocyte

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.

Modulation of dopamine release in the striatum by physiologically relevant levels of nicotine

Striatal dopamine (DA) release can be independently triggered not only by action potentials (APs) in dopaminergic axons but also APs in cholinergic interneurons (ChIs). Nicotine causes addiction by modulating DA release, but with paradoxical findings. Here, we investigate how physiologically relevant levels of nicotine modulate striatal DA release. The optogenetic stimulation of ChIs elicits DA release, which is potently inhibited by nicotine with an IC50 of 28 nM in the dorsal striatum slice. This ChI-driven DA release is predominantly mediated by α6β2* nAChRs. Local electrical stimulus (Estim) activates both dopaminergic axons and ChIs. Nicotine does not affect the AP(DA)-dependent DA release (AP(DA), AP of dopaminergic axon). During burst Estim, nicotine permits the facilitation of DA release by prevention of DA depletion. Our work indicates that cholinergic stimulation-induced DA release is profoundly modulated by physiologically relevant levels of nicotine and resolves the paradoxical observation of nicotines effects on striatal DA release.

Calcium influx activates adenylyl cyclase 8 for sustained insulin secretion in rat pancreatic beta cells

Aims/hypothesisInsulin is a key metabolic regulator in health and diabetes. In pancreatic beta cells, insulin release is regulated by the major second messengers Ca2+ and cAMP: exocytosis is triggered by Ca2+ 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.MethodsTime-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.ResultsMembrane depolarisation-induced Ca2+ influx caused an increase in cytosolic PKA activity via activating a Ca2+-sensitive adenylyl cyclase 8 (ADCY8) subpool. Glucose stimulation triggered coupled Ca2+ 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/interpretationOur findings suggest that depolarisation-induced Ca2+ 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, Ca2+ influx may be important for glucose-stimulated insulin secretion.

Dopamine release from transplanted neural stem cells in Parkinsonian rat striatum in vivo

Significance With a combination of HPLC and carbon fiber electrodes, we demonstrate that grafted neural stem cells directly release dopamine in the damaged striatum in vivo and partially rescue a Parkinson’s disease (PD) model. (i) Primitive neural stem cell–dopamine-like neuron (pNSC–DAn) retained tyrosine hydroxylase expression and reduced the PD-like asymmetric rotation; (ii) depolarization-evoked dopamine release and reuptake were significantly rescued in striatum in vitro (brain slices) and in vivo, as determined jointly by microdialysis-based HPLC and electrochemical micro-carbon fiber electrodes; and (iii) the rescued dopamine was released directly from the grafted pNSC–DAn (not from the 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. 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.

Temporal components of cholinergic terminal to dopaminergic terminal transmission in dorsal striatum slices of mice

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.

Intracellular TRPA1 mediates Ca2+ release from lysosomes in dorsal root ganglion neurons

The temperature-sensitive TRP channel, TRPA1, is known to mediate Na+ and Ca2+ influx at the plasma membrane of sensory neurons. In this study, the authors show that TRPA1 is also present on the lysosomal membrane and mediates lysosome Ca2+ release in dorsal root ganglion neurons.

Effect of cocaine on ion channels and glutamatergic EPSCs in noradrenergic locus coeruleus neurons.

The locus coeruleus (LC) is an important brainstem area involved in cocaine addiction. However, evidence to elucidate how cocaine modulates the activity of LC neurons remains incomplete. Here, we performed whole recordings in brain slices to evaluate the effects of cocaine on the sodium (Na+), potassium (K+), calcium (Ca2+) channels, and glutamatergic synaptic transmission in the locus coeruleus neurons. Local application of cocaine significantly and reversibly reduced the spontaneous firing rate but did not affect action potential amplitude, rising time, decay time, or half width of noradrenergic locus coeruleus neurons. Moreover, cocaine attenuated the sodium current but did not affect potassium and calcium currents. The N-methyl-d-aspartate receptor mediated excitatory postsynaptic currents were reduced by neuropeptide galanin but not cocaine. All those data demonstrate that cocaine has inhibitory effect on the spontaneous activities and sodium current in locus coeruleus neurons. Therefore, neuromodulation of sodium channel in locus coeruleus neurons may play an important role in drug addiction.

Stretch‐induced Ca2+ independent ATP release in hippocampal astrocytes

Similar to neurons, astrocytes actively participate in synaptic transmission via releasing gliotransmitters. The Ca2+‐dependent release of gliotransmitters includes glutamate and ATP. Following an ‘on‐cell‐like’ mechanical stimulus to a single astrocyte, Ca2+ independent single, large, non‐quantal, ATP release occurs. Astrocytic ATP release is inhibited by either selective antagonist treatment or genetic knockdown of P2X7 receptor channels. Our work suggests that ATP can be released from astrocytes via two independent pathways in hippocampal astrocytes; in addition to the known Ca2+‐dependent vesicular release, larger non‐quantal ATP release depends on P2X7 channels following mechanical stretch.

Ca2+-Dependent and Ca2+-Independent ATP Release in Astrocytes

1 State Key Laboratory of Biomembrane and Membrane Biotechnology and Peking-Tsinghua Center for Life Sciences and PKU-IDG/McGovern Institute for Brain Research, Institute of Molecular Medicine, Peking University, Beijing, China, Department of Neurosurgery, Affiliated Hospital of The Air Force Institute of Aeromedicine, Beijing, China, Department of Anesthesiology, Beijing Friendship Hospital, Capital Medical University, Beijing, China

Importing, Caring, Breeding, Genotyping, and Phenotyping a Genetic Mouse in a Chinese University

The genetic manipulation of the laboratory mouse has been well developed and generated more and more mouse lines for biomedical research. To advance our science exploration, it is necessary to share genetically modified mouse lines with collaborators between institutions, even in different countries. The transfer process is complicated. Significant paperwork and coordination are required, concerning animal welfare, intellectual property rights, colony health status, and biohazard. Here, we provide a practical example of importing a transgenic mice line, Dynamin 1 knockout mice, from Yale University in the USA to Perking University in China for studying cell secretion. This example including the length of time that required for paper work, mice quarantine at the receiving institution, and expansion of the mouse line for experiments. The procedure described in this paper for delivery live transgenic mice from USA to China may serve a simple reference for transferring mouse lines between other countries too.