Haiqiang Dou - Academia.edu (original) (raw)

Papers by Haiqiang Dou

International Journal of Innovative Research in Science, Engineering and Technology, 2021

Neurosecretion: Secretory Mechanisms, 2020

Insulin is a key molecule to maintain glucose hemostasis in the body. In pancreatic beta cells, i... more Insulin is a key molecule to maintain glucose hemostasis in the body. In pancreatic beta cells, insulin secretion is triggered by Ca2+ and amplified by cAMP/PKA signaling pathway. In the past few years, several studies have shown that these two signaling pathways are coupled with each other, although the causal relationship between them is still obscure. By combining FRET imaging and electrophysiological methods, a recent report confirms the role of Ca2+ to activate cAMP/PKA pathway through adenylyl cyclase 8 (AC8), a Ca2+-stimulated AC isoform. Simultaneous recordings of PKA activity and insulin granule exocytosis suggest the dual roles of Ca2+ in insulin secretion: to trigger acute exocytosis directly and to maintain sustained insulin secretion via cAMP/PKA. Here, we briefly summarize the roles of Ca2+, cAMP/PKA, and adenylyl cyclase in glucose/GLP-1-mediated insulin secretion and the unique method used in these studies.

Angewandte Chemie International Edition, 2021

Recent work has shown that chemical release during the fundamental cellular process of exocytosis... more Recent work has shown that chemical release during the fundamental cellular process of exocytosis in model cell lines is not all-or-none. We tested this theory for vesicular release from single pancreatic beta cells. The vesicles in these cells release insulin, but also serotonin, which is detectible with amperometric methods. Traditionally, it is assumed that exocytosis in beta cells is all-or-none. Here, we use a multidisciplinary approach involving nanoscale amperometric chemical methods to explore the chemical nature of insulin exocytosis. We amperometrically quantified the number of serotonin molecules stored inside of individual nanoscale vesicles (39 317 AE 1611) in the cell cytoplasm before exocytosis and the number of serotonin molecules released from single cells (13 310 AE 1127) for each stimulated exocytosis event. Thus, beta cells release only one-third of their granule content, clearly supporting partial release in this system. We discuss these observations in the context of type-2 diabetes.

Journal of Clinical Investigation, 2020

The Journal of Physiology, 2020

By secreting insulin and glucagon, the βand α-cells of the pancreatic islets play a central role ... more By secreting insulin and glucagon, the βand α-cells of the pancreatic islets play a central role in the regulation of systemic metabolism. Both cells are equipped with ATP-regulated Quan Zhang obtained his BSc at Shandong University and his PhD at the University of Lund. He currently holds a university research lectureship at the University of Oxford (funded by an RD Lawrence Fellowship from Diabetes UK). Haiqiang Dou obtained his BSc and PhD at Peking University. He currently holds a tenured research position at the University of Gothenburg in Sweden.

Nature Communications, 2020

The generation of pancreatic cell types from renewable cell sources holds promise for cell replac... more The generation of pancreatic cell types from renewable cell sources holds promise for cell replacement therapies for diabetes. Although most effort has focused on generating pancreatic beta cells, considerable evidence indicates that glucagon secreting alpha cells are critically involved in disease progression and proper glucose control. Here we report on the generation of stem cell-derived human pancreatic alpha (SC-alpha) cells from pluripotent stem cells via a transient pre-alpha cell intermediate. These pre-alpha cells exhibit a transcriptional profile similar to mature alpha cells and although they produce proinsulin protein, they do not secrete significant amounts of processed insulin. Compound screening identified a protein kinase c activator that promotes maturation of pre-alpha cells into SC-alpha cells. The resulting SC-alpha cells do not express insulin, share an ultrastructure similar to cadaveric alpha cells, express and secrete glucagon in response to glucose and some ...

Diabetes, 2019

Glucagon, the body's main hyperglycemic hormone, is produced by α-cells of the islets of Lang... more Glucagon, the body's main hyperglycemic hormone, is produced by α-cells of the islets of Langerhans. A fall in plasma glucose stimulates glucagon release and it acts by increasing hepatic glucose production. How glucose regulates glucagon secretion remains debated and both intrinsic and paracrine mechanisms have been proposed. The fact that glucagon secretion becomes dysregulated in diabetes warrants detailed studies of the cell physiology of its release. We generated mice that express the light-sensitive cation channel Channelrhodopsin-2 (ChR2) under the control of the proglucagon promoter in the α-cells. This allowed us to selectively depolarize the plasma membrane of α-cells within intact isolated islets (using 488 nm laser) and record membrane potential, cytoplasmic Ca2+ ([Ca2+]i) glucagon release in response to the optical activation. At low (1mM) glucose, optoactivation depolarized the α-cells, induced a transient increase in [Ca2+]i, decreased peak voltage of the action p...

Diabetes, 2019

Somatostatin secreted from pancreatic delta-cells is a strong paracrine inhibitor of both insulin... more Somatostatin secreted from pancreatic delta-cells is a strong paracrine inhibitor of both insulin and glucagon secretion in islets. However, little is known about the regulatory mechanisms of delta cells. This is mainly due to the scarcity of these cells (∼5-10% of islet cell population) and difficulties in maintaining their morphology and function after cell dispersion. Here, we developed an optogenetic approach, through tissue-specific expression of light-sensitive channels in delta cells, to investigate the paracrine modulation of alpha/beta cells by delta cells within intact islets. We generated a mouse model using Cre-loxp recombination to enable the specific expression of a light-sensitive cation channel Channelrhodopsin-2 (ChR2) in the pancreatic delta cells (SST-ChR2 mouse). In the presence of 1 mM glucose (when ?-cells are not electrically active), light activation of ChR2 (with 470 nm LED light) rapidly depolarised delta- cells (from -66±4 mV to -19±2 mV, n=4). Similar dep...

Physiological reports, 2018

Glucagon is the body's main hyperglycemic hormone, and its secretion is dysregulated in type ... more Glucagon is the body's main hyperglycemic hormone, and its secretion is dysregulated in type 2 diabetes mellitus (T2DM). The incretin hormone glucagon-like peptide-1 (GLP-1) is released from the gut and is used in T2DM therapy. Uniquely, it both stimulates insulin and inhibits glucagon secretion and thereby lowers plasma glucose levels. In this study, we have investigated the action of GLP-1 on glucagon release from human pancreatic islets. Immunocytochemistry revealed that only <0.5% of the α-cells possess detectable GLP-1R immunoreactivity. Despite this, GLP-1 inhibited glucagon secretion by 50-70%. This was due to a direct effect on α-cells, rather than paracrine signaling, because the inhibition was not reversed by the insulin receptor antagonist S961 or the somatostatin receptor-2 antagonist CYN154806. The inhibitory effect of GLP-1 on glucagon secretion was prevented by the PKA-inhibitor Rp-cAMPS and mimicked by the adenylate cyclase activator forskolin. Electrophysiolo...

The Analyst, 2015

This study demonstrated the unaltered dopamine release in the striatum in a schizophrenia model d... more This study demonstrated the unaltered dopamine release in the striatum in a schizophrenia model dysbindin−/− mice.

Proceedings of the National Academy of Sciences of the United States of America, Jan 4, 2014

Embryonic stem cell-based therapies exhibit great potential for the treatment of…

Nature communications, Jan 21, 2014

Striatal dopamine (DA) release can be independently triggered not only by action potentials (APs)... more 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 para...

The Journal of physiology, Jan 15, 2014

Striatal dopamine (DA) is critically involved in major brain functions such as motor control and ... more 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...

Diabetologia, 2014

Aims/hypothesis Insulin is a key metabolic regulator in health and diabetes. In pancreatic beta c... more 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 patchclamped 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 Haiqiang Dou and Changhe Wang contributed equally to this work.

Neurochemistry International, 2013

The ambient resting dopamine (DA) concentration in brain regulates cognition and motivation. Desp... more The ambient resting dopamine (DA) concentration in brain regulates cognition and motivation. Despite its importance, resting DA level in vivo remains elusive. Here, by high-frequency stimulation of the medial forebrain bundle and immediately following the stimulus-induced DA overflow, we recorded a DA ''undershoot'' which is a temporal reduction of DA concentration to a level below the baseline. Based on the DA undershoot, we predicted a resting DA concentration of $73 nM in rat striatum in vivo. Simulation studies suggested that removing basal DA by DAT during the post-stimulation inhibition of tonic DA release caused the DA undershoot, and the resting concentration of DA modulated the kinetics of the evoked DA transient. The DA undershoot was eliminated by either blocking D2 receptors with haloperidol or blocking the DA transporter (DAT) with cocaine. Therefore, the impulse-dependent resting DA concentration is in the tens of nanomolar range and is modulated by the presynaptic D2 receptors and the DAT in vivo.

Journal of Neurochemistry, 2011

The transient flow of short-term information between preand post-synaptic neurons is encoded in a... more The transient flow of short-term information between preand post-synaptic neurons is encoded in a temporal pattern of action potentials (APs) (Cazalis et al. 1985; Lisman 1997; Duan et al. 2003). In general, the patterns of presynaptic APs vary greatly from one type of neuron to another (Connors and Gutnick 1990), and they directly control transmitter release (Zhou and Misler 1995b). In addition, changes of AP pattern also occur in the same cell type (Duan et al. 2003), or in the same cell under different conditions (Huang et al. 2007). In the striatum, evoked dopamine (DA) release via APs is critical for mood, reward learning, the motor system, and drug addiction. Reports show that bursting stimulations (frequency within trains: 14 Hz) are twice as potent as regularly spaced ones having the same average frequency (5 Hz) in evoking DA overflow (Gonon 1988). However, how complex AP

Journal of Molecular Neuroscience, 2014

The genetic manipulation of the laboratory mouse has been well developed and generated more and m... more 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.

Biophysical Journal, 2013

The dorsal root ganglion (DRG) contains a subset of closely-apposed neuronal somata (NS) that are... more The dorsal root ganglion (DRG) contains a subset of closely-apposed neuronal somata (NS) that are separated solely by a thin satellite glial cell (SGC) membrane septum to form a NS-glial cell-NS (NGlN) cell trimer. We recently reported that stimulation of one NS evokes a delayed, noisy and long-duration inward current in both itself and its passive partner that was blocked by suramin, a general purinergic antagonist. Here we test the hypothesis that NGlN transmission involves purinergic activation of the SGC and its release of an excitatory transmitter. Stimulation of the NS triggered a sustained current noise in the SGC. Block of transmission through the NGlN by reactive blue 2 or thapsigargin, aCa 2þ store-depletion agent, implicated a Ca 2þ store dischargelinked P2Y receptor. P2Y2 was identified by simulation of the NGlN-like transmission by puff of UTP onto the SGC. Block of the UTP effect by BAPTA, an intracellular Ca 2þ scavenger, supported the involvement of SGC Ca 2þ stores in the signaling pathway. The response to UTP was also blocked by AP5, which, along with the N2B subunit-specific antagonist ifenprodil, inhibited NGlN transmission, implicating a glutamatergic pathway via postsynaptic NMDA receptors. Puff of glutamate could evoke transmission-like current in the NS. Immunocytochemistry localized the NMDA receptor subunit NR2B to the NS membrane, abutting staining for P2Y2 on the SGC septum. We infer that NGlN transmission involves secretion of ATP from the NS, SGC Ca 2þ store discharge via P2Y2 receptors and release of glutamate to activate NS postsynaptic NMDA receptors. Thus, the NS of the NGlN trimer communicate via a ''Sandwich Synapse'' transglial pathway, a novel signaling mechanism that may contribute to information transfer in other regions of the nervous system.

Biophysical Journal, 2010

Mutations in dysferlin cause Limb-Girdle and Miyoshi Muscular Dystrophy. Dysferlin is structurall... more Mutations in dysferlin cause Limb-Girdle and Miyoshi Muscular Dystrophy. Dysferlin is structurally related to Otoferlin, a protein involved in the calcium triggered release of neurotransmitters in cochlear hair cells. Dysferlin is a calcium-binding protein with a single membrane-spanning domain thought to be required for membrane wound repair. The specific function that dysferlin performs in wound healing is unknown. Here we report the activation of an intercellular signaling pathway in sea urchin embryos by membrane wounding that evokes calcium spikes in neighboring cells. This pathway was mimicked by ATP application, and inhibited by apyrase, cadmium, and omega-agatoxin IVA. Microinjection of dysferlin antisense morpholinos blocked this pathway; control morpholinos did not. Co-injection of mRNA encoding human dysferlin with the inhibitory morpholino rescued signaling activity. Our results indicate that membrane wounding triggers membrane depolarization, calcium influx through an agatoxin sensitive voltage-gated calcium channel, and ultimately the secretion of ATP. Moreover, dysferlin appears to mediate one of the essential steps in this signaling cascade.

Biophysical Journal, 2009

Bis(monoacylglycero)phosphate (BMP) is an unusually shaped, negatively charged phospholipid found... more Bis(monoacylglycero)phosphate (BMP) is an unusually shaped, negatively charged phospholipid found in elevated concentrations in the late endosomes. The unusual structure and stereochemistry of BMP are thought to play important roles in the endosome, including structural integrity, endosome maturation, and lipid/protein sorting and trafficking. We have utilized dynamic light scattering, fluorescence spectroscopy and transmission electron microscopy to characterize the morphology and size of BMP hydrated dispersions and extruded vesicles. We find that the morphology of hydrated BMP dispersions varies with pH, forming highly structured, clustered dispersions of 500 nm in size at neutral pH 7.4. However, at acidic pH 4.5, spontaneous hydrolysis of BMP occurs, altering the vesicle morphology to spherically shaped dispersions. BMP vesicles are also significantly smaller in diameter than palmitoyloleoylglycerophosphocholine (POPC) vesicles. In a stability assay using dynamic light scattering measurements to compare and monitor 30 nm extruded vesicles of BMP, POPC, and POPG over a 5 week period, we find that BMP vesicles do not fuse to form larger structures. BMP also forms lamellar vesicles evidenced by the fluorescence leakage assay studies. These results shed light on the possibility that the biosynthesis of BMP and the increasing acidity during the maturation process of late endosomes play an important role in the formation of intraendosomal vesicular bodies.

International Journal of Innovative Research in Science, Engineering and Technology, 2021

Neurosecretion: Secretory Mechanisms, 2020

Insulin is a key molecule to maintain glucose hemostasis in the body. In pancreatic beta cells, i... more Insulin is a key molecule to maintain glucose hemostasis in the body. In pancreatic beta cells, insulin secretion is triggered by Ca2+ and amplified by cAMP/PKA signaling pathway. In the past few years, several studies have shown that these two signaling pathways are coupled with each other, although the causal relationship between them is still obscure. By combining FRET imaging and electrophysiological methods, a recent report confirms the role of Ca2+ to activate cAMP/PKA pathway through adenylyl cyclase 8 (AC8), a Ca2+-stimulated AC isoform. Simultaneous recordings of PKA activity and insulin granule exocytosis suggest the dual roles of Ca2+ in insulin secretion: to trigger acute exocytosis directly and to maintain sustained insulin secretion via cAMP/PKA. Here, we briefly summarize the roles of Ca2+, cAMP/PKA, and adenylyl cyclase in glucose/GLP-1-mediated insulin secretion and the unique method used in these studies.

Angewandte Chemie International Edition, 2021

Recent work has shown that chemical release during the fundamental cellular process of exocytosis... more Recent work has shown that chemical release during the fundamental cellular process of exocytosis in model cell lines is not all-or-none. We tested this theory for vesicular release from single pancreatic beta cells. The vesicles in these cells release insulin, but also serotonin, which is detectible with amperometric methods. Traditionally, it is assumed that exocytosis in beta cells is all-or-none. Here, we use a multidisciplinary approach involving nanoscale amperometric chemical methods to explore the chemical nature of insulin exocytosis. We amperometrically quantified the number of serotonin molecules stored inside of individual nanoscale vesicles (39 317 AE 1611) in the cell cytoplasm before exocytosis and the number of serotonin molecules released from single cells (13 310 AE 1127) for each stimulated exocytosis event. Thus, beta cells release only one-third of their granule content, clearly supporting partial release in this system. We discuss these observations in the context of type-2 diabetes.

Journal of Clinical Investigation, 2020

The Journal of Physiology, 2020

By secreting insulin and glucagon, the βand α-cells of the pancreatic islets play a central role ... more By secreting insulin and glucagon, the βand α-cells of the pancreatic islets play a central role in the regulation of systemic metabolism. Both cells are equipped with ATP-regulated Quan Zhang obtained his BSc at Shandong University and his PhD at the University of Lund. He currently holds a university research lectureship at the University of Oxford (funded by an RD Lawrence Fellowship from Diabetes UK). Haiqiang Dou obtained his BSc and PhD at Peking University. He currently holds a tenured research position at the University of Gothenburg in Sweden.

Nature Communications, 2020

The generation of pancreatic cell types from renewable cell sources holds promise for cell replac... more The generation of pancreatic cell types from renewable cell sources holds promise for cell replacement therapies for diabetes. Although most effort has focused on generating pancreatic beta cells, considerable evidence indicates that glucagon secreting alpha cells are critically involved in disease progression and proper glucose control. Here we report on the generation of stem cell-derived human pancreatic alpha (SC-alpha) cells from pluripotent stem cells via a transient pre-alpha cell intermediate. These pre-alpha cells exhibit a transcriptional profile similar to mature alpha cells and although they produce proinsulin protein, they do not secrete significant amounts of processed insulin. Compound screening identified a protein kinase c activator that promotes maturation of pre-alpha cells into SC-alpha cells. The resulting SC-alpha cells do not express insulin, share an ultrastructure similar to cadaveric alpha cells, express and secrete glucagon in response to glucose and some ...

Diabetes, 2019

Glucagon, the body's main hyperglycemic hormone, is produced by α-cells of the islets of Lang... more Glucagon, the body's main hyperglycemic hormone, is produced by α-cells of the islets of Langerhans. A fall in plasma glucose stimulates glucagon release and it acts by increasing hepatic glucose production. How glucose regulates glucagon secretion remains debated and both intrinsic and paracrine mechanisms have been proposed. The fact that glucagon secretion becomes dysregulated in diabetes warrants detailed studies of the cell physiology of its release. We generated mice that express the light-sensitive cation channel Channelrhodopsin-2 (ChR2) under the control of the proglucagon promoter in the α-cells. This allowed us to selectively depolarize the plasma membrane of α-cells within intact isolated islets (using 488 nm laser) and record membrane potential, cytoplasmic Ca2+ ([Ca2+]i) glucagon release in response to the optical activation. At low (1mM) glucose, optoactivation depolarized the α-cells, induced a transient increase in [Ca2+]i, decreased peak voltage of the action p...

Diabetes, 2019

Somatostatin secreted from pancreatic delta-cells is a strong paracrine inhibitor of both insulin... more Somatostatin secreted from pancreatic delta-cells is a strong paracrine inhibitor of both insulin and glucagon secretion in islets. However, little is known about the regulatory mechanisms of delta cells. This is mainly due to the scarcity of these cells (∼5-10% of islet cell population) and difficulties in maintaining their morphology and function after cell dispersion. Here, we developed an optogenetic approach, through tissue-specific expression of light-sensitive channels in delta cells, to investigate the paracrine modulation of alpha/beta cells by delta cells within intact islets. We generated a mouse model using Cre-loxp recombination to enable the specific expression of a light-sensitive cation channel Channelrhodopsin-2 (ChR2) in the pancreatic delta cells (SST-ChR2 mouse). In the presence of 1 mM glucose (when ?-cells are not electrically active), light activation of ChR2 (with 470 nm LED light) rapidly depolarised delta- cells (from -66±4 mV to -19±2 mV, n=4). Similar dep...

Physiological reports, 2018

Glucagon is the body's main hyperglycemic hormone, and its secretion is dysregulated in type ... more Glucagon is the body's main hyperglycemic hormone, and its secretion is dysregulated in type 2 diabetes mellitus (T2DM). The incretin hormone glucagon-like peptide-1 (GLP-1) is released from the gut and is used in T2DM therapy. Uniquely, it both stimulates insulin and inhibits glucagon secretion and thereby lowers plasma glucose levels. In this study, we have investigated the action of GLP-1 on glucagon release from human pancreatic islets. Immunocytochemistry revealed that only <0.5% of the α-cells possess detectable GLP-1R immunoreactivity. Despite this, GLP-1 inhibited glucagon secretion by 50-70%. This was due to a direct effect on α-cells, rather than paracrine signaling, because the inhibition was not reversed by the insulin receptor antagonist S961 or the somatostatin receptor-2 antagonist CYN154806. The inhibitory effect of GLP-1 on glucagon secretion was prevented by the PKA-inhibitor Rp-cAMPS and mimicked by the adenylate cyclase activator forskolin. Electrophysiolo...

The Analyst, 2015

This study demonstrated the unaltered dopamine release in the striatum in a schizophrenia model d... more This study demonstrated the unaltered dopamine release in the striatum in a schizophrenia model dysbindin−/− mice.

Proceedings of the National Academy of Sciences of the United States of America, Jan 4, 2014

Embryonic stem cell-based therapies exhibit great potential for the treatment of…

Nature communications, Jan 21, 2014

Striatal dopamine (DA) release can be independently triggered not only by action potentials (APs)... more 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 para...

The Journal of physiology, Jan 15, 2014

Striatal dopamine (DA) is critically involved in major brain functions such as motor control and ... more 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...

Diabetologia, 2014

Aims/hypothesis Insulin is a key metabolic regulator in health and diabetes. In pancreatic beta c... more 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 patchclamped 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 Haiqiang Dou and Changhe Wang contributed equally to this work.

Neurochemistry International, 2013

The ambient resting dopamine (DA) concentration in brain regulates cognition and motivation. Desp... more The ambient resting dopamine (DA) concentration in brain regulates cognition and motivation. Despite its importance, resting DA level in vivo remains elusive. Here, by high-frequency stimulation of the medial forebrain bundle and immediately following the stimulus-induced DA overflow, we recorded a DA ''undershoot'' which is a temporal reduction of DA concentration to a level below the baseline. Based on the DA undershoot, we predicted a resting DA concentration of $73 nM in rat striatum in vivo. Simulation studies suggested that removing basal DA by DAT during the post-stimulation inhibition of tonic DA release caused the DA undershoot, and the resting concentration of DA modulated the kinetics of the evoked DA transient. The DA undershoot was eliminated by either blocking D2 receptors with haloperidol or blocking the DA transporter (DAT) with cocaine. Therefore, the impulse-dependent resting DA concentration is in the tens of nanomolar range and is modulated by the presynaptic D2 receptors and the DAT in vivo.

Journal of Neurochemistry, 2011

The transient flow of short-term information between preand post-synaptic neurons is encoded in a... more The transient flow of short-term information between preand post-synaptic neurons is encoded in a temporal pattern of action potentials (APs) (Cazalis et al. 1985; Lisman 1997; Duan et al. 2003). In general, the patterns of presynaptic APs vary greatly from one type of neuron to another (Connors and Gutnick 1990), and they directly control transmitter release (Zhou and Misler 1995b). In addition, changes of AP pattern also occur in the same cell type (Duan et al. 2003), or in the same cell under different conditions (Huang et al. 2007). In the striatum, evoked dopamine (DA) release via APs is critical for mood, reward learning, the motor system, and drug addiction. Reports show that bursting stimulations (frequency within trains: 14 Hz) are twice as potent as regularly spaced ones having the same average frequency (5 Hz) in evoking DA overflow (Gonon 1988). However, how complex AP

Journal of Molecular Neuroscience, 2014

The genetic manipulation of the laboratory mouse has been well developed and generated more and m... more 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.

Biophysical Journal, 2013

The dorsal root ganglion (DRG) contains a subset of closely-apposed neuronal somata (NS) that are... more The dorsal root ganglion (DRG) contains a subset of closely-apposed neuronal somata (NS) that are separated solely by a thin satellite glial cell (SGC) membrane septum to form a NS-glial cell-NS (NGlN) cell trimer. We recently reported that stimulation of one NS evokes a delayed, noisy and long-duration inward current in both itself and its passive partner that was blocked by suramin, a general purinergic antagonist. Here we test the hypothesis that NGlN transmission involves purinergic activation of the SGC and its release of an excitatory transmitter. Stimulation of the NS triggered a sustained current noise in the SGC. Block of transmission through the NGlN by reactive blue 2 or thapsigargin, aCa 2þ store-depletion agent, implicated a Ca 2þ store dischargelinked P2Y receptor. P2Y2 was identified by simulation of the NGlN-like transmission by puff of UTP onto the SGC. Block of the UTP effect by BAPTA, an intracellular Ca 2þ scavenger, supported the involvement of SGC Ca 2þ stores in the signaling pathway. The response to UTP was also blocked by AP5, which, along with the N2B subunit-specific antagonist ifenprodil, inhibited NGlN transmission, implicating a glutamatergic pathway via postsynaptic NMDA receptors. Puff of glutamate could evoke transmission-like current in the NS. Immunocytochemistry localized the NMDA receptor subunit NR2B to the NS membrane, abutting staining for P2Y2 on the SGC septum. We infer that NGlN transmission involves secretion of ATP from the NS, SGC Ca 2þ store discharge via P2Y2 receptors and release of glutamate to activate NS postsynaptic NMDA receptors. Thus, the NS of the NGlN trimer communicate via a ''Sandwich Synapse'' transglial pathway, a novel signaling mechanism that may contribute to information transfer in other regions of the nervous system.

Biophysical Journal, 2010

Mutations in dysferlin cause Limb-Girdle and Miyoshi Muscular Dystrophy. Dysferlin is structurall... more Mutations in dysferlin cause Limb-Girdle and Miyoshi Muscular Dystrophy. Dysferlin is structurally related to Otoferlin, a protein involved in the calcium triggered release of neurotransmitters in cochlear hair cells. Dysferlin is a calcium-binding protein with a single membrane-spanning domain thought to be required for membrane wound repair. The specific function that dysferlin performs in wound healing is unknown. Here we report the activation of an intercellular signaling pathway in sea urchin embryos by membrane wounding that evokes calcium spikes in neighboring cells. This pathway was mimicked by ATP application, and inhibited by apyrase, cadmium, and omega-agatoxin IVA. Microinjection of dysferlin antisense morpholinos blocked this pathway; control morpholinos did not. Co-injection of mRNA encoding human dysferlin with the inhibitory morpholino rescued signaling activity. Our results indicate that membrane wounding triggers membrane depolarization, calcium influx through an agatoxin sensitive voltage-gated calcium channel, and ultimately the secretion of ATP. Moreover, dysferlin appears to mediate one of the essential steps in this signaling cascade.

Biophysical Journal, 2009

Bis(monoacylglycero)phosphate (BMP) is an unusually shaped, negatively charged phospholipid found... more Bis(monoacylglycero)phosphate (BMP) is an unusually shaped, negatively charged phospholipid found in elevated concentrations in the late endosomes. The unusual structure and stereochemistry of BMP are thought to play important roles in the endosome, including structural integrity, endosome maturation, and lipid/protein sorting and trafficking. We have utilized dynamic light scattering, fluorescence spectroscopy and transmission electron microscopy to characterize the morphology and size of BMP hydrated dispersions and extruded vesicles. We find that the morphology of hydrated BMP dispersions varies with pH, forming highly structured, clustered dispersions of 500 nm in size at neutral pH 7.4. However, at acidic pH 4.5, spontaneous hydrolysis of BMP occurs, altering the vesicle morphology to spherically shaped dispersions. BMP vesicles are also significantly smaller in diameter than palmitoyloleoylglycerophosphocholine (POPC) vesicles. In a stability assay using dynamic light scattering measurements to compare and monitor 30 nm extruded vesicles of BMP, POPC, and POPG over a 5 week period, we find that BMP vesicles do not fuse to form larger structures. BMP also forms lamellar vesicles evidenced by the fluorescence leakage assay studies. These results shed light on the possibility that the biosynthesis of BMP and the increasing acidity during the maturation process of late endosomes play an important role in the formation of intraendosomal vesicular bodies.