Cocaine induces locomotor sensitization through a dopamine-dependent VTA-mPFC-FrA cortico-cortical pathway in male mice.

As a central part of the mammalian brain, the prefrontal cortex (PFC) has been implicated in regulating cocaine-induced behaviors including compulsive seeking and reinstatement. Although dysfunction of the PFC has been reported in animal and human users with chronic cocaine abuse, less is known about how the PFC is involved in cocaine-induced behaviors. By using two-photon Ca2+ imaging to simultaneously record tens of intact individual networking neurons in the frontal association cortex (FrA) in awake male mice, here we report that a systematic acute cocaine exposure decreased the FrA neural activity in mice, while the chemogenetic intervention blocked the cocaine-induced locomotor sensitization. The hypoactivity of FrA neurons was critically dependent on both dopamine transporters and dopamine transmission in the ventromedial PFC (vmPFC). Both dopamine D1R and D2R neurons in the vmPFC projected to and innervated FrA neurons, the manipulation of which changed the cocaine-induced hypoactivity of the FrA and locomotor sensitization. Together, this work demonstrates acute cocaine-induced hypoactivity of FrA neurons in awake mice, which defines a cortico-cortical projection bridging dopamine transmission and cocaine sensitization.

Peptide nanotube loaded with a STING agonist, c-di-GMP, enhance cancer immunotherapy against melanoma.

The activation of the stimulating factor of the interferon gene (STING) pathway can enhance the immune response within the tumor. Cyclic diguanylate monophosphate (c-di-GMP) is a negatively charged, hydrophilic STING agonist, however, its effectiveness is limited due to the poor membrane permeability and low bioavailability. Herein, we introduced KL-7 peptide derived from Aß amyloid fibrils that can self-assemble to form nanotubes to load and deliver c-di-GMP, which significantly enhanced c-di-GMP's effectiveness and then exhibited a robust "in situ immunity" to kill melanoma cells. KL-7 peptide nanotube, also called PNT, was loaded with negatively charged c-di-GMP via electrostatic interaction, which prepared a nanocomposite named c-di-GMP-PNT. Treatment of RAW 264.7 cells (leukemia cells in mouse macrophage) with c-di-GMP-PNT markedly stimulated the secretion of IL-6 and INF-ß along with phospho-STING (Ser365) protein expression, indicating the activation of the STING pathway. In the unilateral flank B16-F10 (murine melanoma cells) tumor-bearing mouse model, compared to PNT and c-di-GMP, c-di-GMP-PNT can promote the expression of INF-ß, TNF-α, IL-6, and IL-1ß. At the same time, up-regulated CD4 and CD8 active T cells kill tumors and enhance the immune response in tumor tissues, resulting in significant inhibition of tumor growth in tumor-bearing mice. More importantly, in a bilateral flank B16-F10 tumor model, both primary and distant tumor growth can also be significantly inhibited by c-di-GMP-PNT. Moreover, c-di-GMP-PNT demonstrated no obvious biological toxicity on the main organs (heart, liver, spleen, lung, and kidney) and biochemical indexes of mice. In summary, our study provides a strategy to overcome the barriers of free c-di-GMP in the tumor microenvironment and c-di-GMP-PNT may be an attractive nanomaterial for anti-tumor immunity. Electronic Supplementary Material: Supplementary material (synthesis and characterization of KL-7 peptide; the encapsulation rate and cumulative release rate of c-di-GMP-PNT; cytotoxicity of PNT, c-di-GMP, and c-di-GMP-PNT; anti-tumor effect of c-di-GMP-PNT (equivalent to 1 and 5 µg c-di-GMP per mouse); representative immunofluorescence images; and biosafety analysis) is available in the online version of this article at 10.1007/s12274-022-5102-z.

Cu(BF4)2/AC-Catalyzed Synthesis of N-Substituted Anilines, N-Substituted 1,6-Naphthyridin-5(6H)-one, and Isoquinolin-1(2H)-one.

Herein, we report practical Cu(BF4)2/activated carbon-catalyzed amination of various anilines, isoquinolinone, and naphthyridinone with aryl boronic acids. The ultrasonic and rotary evaporation treatment of the mixture of aq. Cu(BF4)2 and activated carbon in methanol afforded a novel Cu(II)-catalyst, which is air-stable and can be effectively applied in the Chan-Lam coupling reaction. The products of N-arylation were isolated in good to excellent yields at low catalytic loading. And Cu(BF4)2/AC also showed good reusability.

Tuning the Size of Large Dense-Core Vesicles and Quantal Neurotransmitter Release via Secretogranin II Liquid-Liquid Phase Separation.

Large dense-core vesicles (LDCVs) are larger in volume than synaptic vesicles, and are filled with multiple neuropeptides, hormones, and neurotransmitters that participate in various physiological processes. However, little is known about the mechanism determining the size of LDCVs. Here, it is reported that secretogranin II (SgII), a vesicle matrix protein, contributes to LDCV size regulation through its liquid-liquid phase separation in neuroendocrine cells. First, SgII undergoes pH-dependent polymerization and the polymerized SgII forms phase droplets with Ca2+ in vitro and in vivo. Further, the Ca2+ -induced SgII droplets recruit reconstituted bio-lipids, mimicking the LDCVs biogenesis. In addition, SgII knockdown leads to significant decrease of the quantal neurotransmitter release by affecting LDCV size, which is differently rescued by SgII truncations with different degrees of phase separation. In conclusion, it is shown that SgII is a unique intravesicular matrix protein undergoing liquid-liquid phase separation, and present novel insights into how SgII determines LDCV size and the quantal neurotransmitter release.

Dynamic physiological and transcriptome changes reveal a potential relationship between the circadian clock and salt stress response in Ulmus pumila.

Despite the important role the circadian clock plays in numerous critical physiological responses in plants, such as hypocotyl elongation, leaf movement, stomatal opening, flowering, and stress responses, there have been no investigations into the effect of the circadian clock on physiological and transcriptional networks under salt stress. Ulmus pumila L. has been reported to tolerate 100-150 mM NaCl treatment. We measured the diurnal variation in photosynthesis and chlorophyll fluorescence parameters and performed a time-course transcriptome analysis of 2-years-old U. pumila seedlings under salt treatment to dissect the physiological regulation and potential relationship between the circadian network and the salt stress response. Seedlings in 150 mM NaCl treatment exhibited salt-induced physiological enhancement compared to the control group. A total of 7009 differentially expressed unigenes (DEGs) were identified under salt stress, of which 16 DEGs were identified as circadian rhythm-related DEGs (crDEGs). Further analysis of dynamic expression changes revealed that DEGs involved in four crucial pathways-photosynthesis, thiamine metabolism, abscisic acid synthesis and metabolism, and the hormone-MAPK signal crosstalk pathway-are closely related to the circadian clock. Finally, we constructed a co-expression network between the circadian clock and these four crucial pathways. Our results help shed light on the molecular link between the circadian network and salt stress tolerance in U. pumila.

Gene Coexpression Network Analysis Indicates that Hub Genes Related to Photosynthesis and Starch Synthesis Modulate Salt Stress Tolerance in Ulmus pumila.

Ulmus pumila L. is an excellent afforestation and biofuel tree that produces high-quality wood, rich in starch. In addition, U. pumila is highly adaptable to adverse environmental conditions, which is conducive to its utilization for vegetating saline soils. However, little is known about the physiological responses and transcriptional regulatory network of U. pumila under salt stress. In this study, we exposed five main cultivars in saline-alkali land (Upu2, 5, 8, 11, and 12) to NaCl stress. Of the five cultivars assessed, Upu11 exhibited the highest salt resistance. Growth and biomass accumulation in Upu11 were promoted under low salt concentrations (<150 mM). However, after 3 months of continuous treatment with 150 mM NaCl, growth was inhibited, and photosynthesis declined. A transcriptome analysis conducted after 3 months of treatment detected 7009 differentially expressed unigenes (DEGs). The gene annotation indicated that these DEGs were mainly related to photosynthesis and carbon metabolism. Furthermore, PHOTOSYNTHETIC ELECTRON TRANSFERH (UpPETH), an important electron transporter in the photosynthetic electron transport chain, and UpWAXY, a key gene controlling amylose synthesis in the starch synthesis pathway, were identified as hub genes in the gene coexpression network. We identified 25 and 62 unigenes that may interact with PETH and WAXY, respectively. Overexpression of UpPETH and UpWAXY significantly increased the survival rates, net photosynthetic rates, biomass, and starch content of transgenic Arabidopsis plants under salt stress. Our findings clarify the physiological and transcriptional regulators that promote or inhibit growth under environmental stress. The identification of salt-responsive hub genes directly responsible for photosynthesis and starch synthesis or metabolism will provide targets for future genetic improvements.

Research on the distribution of neonicotinoid and fipronil pollution in the Yangtze River by high-performance liquid chromatography.

Neonicotinoid and fipronil insecticides have been consumed worldwide, particularly in China. There is growing interest in the environmental research community about the occurrence, sources, and risks of neonicotinoids and fipronil. A new method based on high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and disc solid phase extraction (SPE) techniques were developed for the determination of neonicotinoids and fipronil in seawater in this research. The effects of the styrene divinyl benzene-reverse phase sulfonated (SDB-RPS) membrane-based solid phase extraction optimization process and the HPLC separation parameters on the detection results of the target compounds were studied. Under the optimized conditions, the detection limit of this method for 12 target compounds ranged from 0.05 ng L-1 to 0.50 ng L-1 and absolute recovery was achieved from 58.9-106.5%. The results proved that this method can effectively detect neonicotinoid and fipronil pesticides in seawater. At the same time, dinotefuran, nitenpyram, thiamethoxam, imidacloprid, thiacloprid, fipronil and its metabolites were found in seawater samples from the Yangtze River Estuary and the adjacent sea area. Among them, the total concentration of neonicotinoid insecticides ranged from ND-12.45 ng L-1 and the average total concentration was 3.5 ng L-1; the total concentration of fipronil and its metabolites ranged from ND-2.45 ng L-1, and the average total concentration was 0.79 ng L-1. The results showed that there was contamination of neonicotinoids and fipronil in the Yangtze River Estuary and its adjacent area.

Regulating quantal size of neurotransmitter release through a GPCR voltage sensor.

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 Gißγ 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) → Gißγ → QS → myocyte contractility, offering a universal Vm-GPCR signaling pathway for its functions in the nervous system and other systems containing GPCRs.

Differential Co-release of Two Neurotransmitters from a Vesicle Fusion Pore in Mammalian Adrenal Chromaffin Cells.

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 Restrains Vesicular Release to a Subquantal Mode In Mammalian Adrenal Chromaffin Cells.

Dynamin 1 (dyn1) is required for clathrin-mediated endocytosis in most secretory (neuronal and neuroendocrine) cells. There are two modes of Ca2+-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 (dp) was increased from ≤9 to ≥9 nm by KO. Therefore, Ca2+-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 Ca2+-dependent neurotransmitter release from a single vesicle is the primary event in all neurotransmission, including synaptic/neuroendocrine forms. To determine whether Ca2+-dependent vesicular neurotransmitter release is "all-or-none" (quantal), we provide compelling evidence that most Ca2+-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 Ca2+-dependent transmitter release is mainly via subquantal mode, suggesting that subquantal release could be also important in other types of cells.

Muscarinic inhibition of nicotinic transmission in rat sympathetic neurons and adrenal chromaffin cells.

Little is known about the interactions between nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs). Here we report that methacholine (MCh), a selective agonist of mAChRs, inhibited up to 80% of nicotine-induced nAChR currents in sympathetic superior cervical ganglion neurons and adrenal chromaffin cells. The muscarine-induced inhibition (MiI) substantially reduced ACh-induced membrane currents through nAChRs and quantal neurotransmitter release. The MiI was time- and temperature-dependent. The slow recovery of nAChR current after washout of MCh, as well as the high value of Q10 (3.2), suggested, instead of a direct open-channel blockade, an intracellular metabotropic process. The effects of GTP-γ-S, GDP-ß-S and pertussis toxin suggested that MiI was mediated by G-protein signalling. Inhibitors of protein kinase C (bisindolymaleimide-Bis), protein kinase A (H89) and PIP2 depletion attenuated the MiI, indicating that a second messenger pathway is involved in this process. Taken together, these data suggest that mAChRs negatively modulated nAChRs via a G-protein-mediated second messenger pathway. The time dependence suggests that MiI may provide a novel mechanism for post-synaptic adaptation in all cells/neurons and synapses expressing both types of AChRs.

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

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.

Extracellular Ca²âº per se inhibits quantal size of catecholamine release in adrenal slice chromaffin cells.

Classic calcium hypothesis states that depolarization-induced increase in intracellular Ca(2+) concentration ([Ca(2+)]i) triggers vesicle exocytosis by increasing vesicle release probability in neurons and neuroendocrine cells. The extracellular Ca(2+), in this calcium hypothesis, serves as a reservoir of Ca(2+) source. Recently we find that extracellular Ca(2+)per se inhibits the [Ca(2+)]i dependent vesicle exocytosis, but it remains unclear whether quantal size is regulated by extracellular, or intracellular Ca(2+) or both. In this work we showed that, in physiological condition, extracellular Ca(2+) per se specifically inhibited the quantal size of single vesicle release in rat adrenal slice chromaffin cells. The extracellular Ca(2+) in physiological concentration (2.5 mM) directly regulated fusion pore kinetics of spontaneous quantal release of catecholamine. In addition, removal of extracellular Ca(2+) directly triggered vesicle exocytosis without eliciting intracellular Ca(2+). We propose that intracellular Ca(2+) and extracellular Ca(2+)per se cooperately regulate single vesicle exocytosis. The vesicle release probability was jointly modulated by both intracellular and extracellular Ca(2+), while the vesicle quantal size was mainly determined by extracellular Ca(2+) in chromaffin cells physiologically.