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Endocannabinoids (eCBs) are retrograde neuromodulators that play an important role in a wide range of physiological processes; however, the release and in vivo dynamics of eCBs remain largely unknown, due in part to a lack of suitable probes capable of detecting eCBs with sufficient spatiotemporal resolution. Here, we developed a new eCB sensor called GRABeCB2.0. This genetically encoded sensor consists of the human CB1 cannabinoid receptor fused to circular-permutated EGFP, providing cell membrane trafficking, second-resolution kinetics, high specificity for eCBs, and a robust fluorescence response at physiological eCB concentrations. Using the GRABeCB2.0 sensor, we monitored evoked changes in eCB dynamics in both cultured neurons and acute brain slices. Interestingly, in cultured neurons we also observed spontaneous compartmental eCB transients that spanned a distance of approximately 11 μm, suggesting constrained, localized eCB signaling. Moreover, by expressing GRABeCB2.0 in the mouse brain, we readily observed foot shock-elicited and running-triggered eCB transients in the basolateral amygdala and hippocampus, respectively. Lastly, we used GRABeCB2.0 in a mouse seizure model and observed a spreading wave of eCB release that followed a Ca2+ wave through the hippocampus. Thus, GRABeCB2.0 is a robust new probe for measuring the dynamics of eCB release under both physiological and pathological conditions.

The purinergic signaling molecule adenosine (Ado) modulates many physiological and pathological brain functions,but its spatiotemporal release dynamics in the brain remains largely unknown. We developed a genetically encoded GPCR-Activation–Based Ado sensor (GRABAdo) in which Ado-induced changes in the human A2A receptor are reflected by fluorescence increases. This GRABAdo revealed that neuronal activity-induced extracellular Ado elevation was due to direct Ado release from somatodendritic regions of the neuron, requiring calcium influx through L-type calcium channels, rather than the degradation of extracellular ATP. The Ado release was slow (∼30 s) and depended on equilibrative nucleoside transporters (ENTs) rather than conventional vesicular release mechanisms. Thus, GRABAdo reveals an activity-dependent slow Ado release from somatodendritic region of the neuron, potentially serving modulating functions as a retrograde signal.

Supplemental material, sj-pdf-1-jcb-10.1177_0271678X20918467 for Brain deep medullary veins on 3-T MRI in a population-based cohort by Dong-Hui Ao, Ding-Ding Zhang, Fei-Fei Zhai, Jiang-Tao Zhang, Fei Han, Ming-Li Li, Jun Ni, Ming Yao, Shu-Yang Zhang, Li-Ying Cui, Zheng-Yu Jin, Li-Xin Zhou Yi-Cheng Zhu in Journal of Cerebral Blood Flow & Metabolism

Supplemental material, sj-pdf-1-jcb-10.1177_0271678X20918467 for Brain deep medullary veins on 3-T MRI in a population-based cohort by Dong-Hui Ao, Ding-Ding Zhang, Fei-Fei Zhai, Jiang-Tao Zhang, Fei Han, Ming-Li Li, Jun Ni, Ming Yao, Shu-Yang Zhang, Li-Ying Cui, Zheng-Yu Jin, Li-Xin Zhou Yi-Cheng Zhu in Journal of Cerebral Blood Flow & Metabolism

Lung cancer is difficult to diagnose, has a high mortality rate and a high recurrence rate. By grouping and analyzing the gene expression in lung cancer samples, we selected the differentially expressed genes (DEGs) in total lung cancers or each subgroup, and then searched for the similarities and differences among these. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were performed, in addition to predictable cell proliferation or immune-related pathways, 'hemostasis', 'coagulation' and 'viral myocarditis' were also enriched in common DEGs, while specific functions or pathways were enriched in different subgroups. This may have implications for the treatment of total lung cancer or different subtypes. Through bioinformatics analysis, hub genes were obtained from total lung cancer and each subgroup respectively. Survival analysis of common hub genes led us to find that ZWINT, A2M, POLR2H and KIF11 are associated with unclassified lung cancer survival. For the construction of miRNA regulatory network, miR-16-5p was related to all of these four genes, and its expression is significantly different between lung cancers and normal samples. Combined with the hub genes of each subtype, it may have the ability of early screening and typing.

Summary Norepinephrine (NE) is a key biogenic monoamine neurotransmitter involved in a wide range of physiological processes. However, its precise dynamics and regulation remain poorly characterized, in part due to limitations of available techniques for measuring NE in vivo. Here, we developed a family of GPCR activation-based NE (GRABNE) sensors with a 230% peak ΔF/F0 response to NE, good photostability, nanomolar-to-micromolar sensitivities, sub-second kinetics, and high specificity. Viral- or transgenic-mediated expression of GRABNE sensors was able to detect electrical-stimulation-evoked NE release in the locus coeruleus (LC) of mouse brain slices, looming-evoked NE release in the midbrain of live zebrafish, as well as optogenetically and behaviorally triggered NE release in the LC and hypothalamus of freely moving mice. Thus, GRABNE sensors are robust tools for rapid and specific monitoring of in vivo NE transmission in both physiological and pathological processes. Video Abstract Download : Download video (31MB)

Cell-cell communication via gap junctions regulates a wide range of physiological processes by enabling the direct intercellular electrical and chemical coupling. However, the in vivo distribution and function of gap junctions remain poorly understood, partly due to the lack of non-invasive tools with both cell-type specificity and high spatiotemporal resolution. Here, we developed PARIS (pairing actuators and receivers to optically isolate gap junctions), a new fully genetically encoded tool for measuring the cell-specific gap junctional coupling (GJC). PARIS successfully enabled monitoring of GJC in several cultured cell lines under physiologically relevant conditions and in distinct genetically defined neurons in Drosophila brain, with ~10 s temporal resolution and sub-cellular spatial resolution. These results demonstrate that PARIS is a robust, highly sensitive tool for mapping functional gap junctions and study their regulation in both health and disease., eLife digest For the tissues and organs of our bodies to work properly, the cells within them need to communicate with each other. One important part of cellular communication is the movement of signals – usually small molecules or ions – directly from one cell to another. This happens via structures called gap junctions, a type of sealed ‘channel’ that connects two cells. Gap junctions are found throughout the body, but investigating their precise roles in health and disease has been difficult. This is due to problems with the tools available to detect and monitor gap junctions. Some are simply harmful to cells, while others cannot be restricted to specific cell populations within a tissue. This lack of specificity makes it difficult to study gap junctions in the brain, where it is important to understand the connectivity patterns between distinct types of nerve cells. Wu et al. wanted to develop a new, non-harmful method to track gap junctions in distinct groups of cells within living tissues. To do this, Wu et al. devised PARIS, a two-part, genetically encoded system. The first part comprises a light-sensitive molecular ‘pump’, which can only be turned on by shining a laser onto the cell of interest. When the pump is active, it transports hydrogen ions out of the cell. The second part of the system is a fluorescent sensor, present inside ‘receiving’ cells, which responds to the outcoming hydrogen ions (small enough to pass through gap junctions). If an illuminated ‘signaling’ cell is connected via gap junctions to cells containing the fluorescent sensor, they will light up within seconds, but other cells not connected through gap junctions will not. The researchers first tested PARIS in cultured human and rat cells that had been genetically engineered to produce both components of the system. The experiments confirmed that PARIS could both detect networks of gap junctions in healthy cells and reveal when these networks had been disrupted, for instance by drugs or genetic mutations. Experiments using fruit flies demonstrated that PARIS was stable in living tissue and could also map the gap junctions connecting specific groups of nerve cells. PARIS is a valuable addition to the toolbox available to study cell communication. In the future, it could help increase our understanding of diseases characterized by defective gap junctions, such as seizures, cardiac irregularities, and even some cancers.