Insulin secretion is a complex and highly regulated process.Although much progress has been made in understanding the cellular mechanisms of insulin secretion and regulation,it remains unclear how conclusions from the...Insulin secretion is a complex and highly regulated process.Although much progress has been made in understanding the cellular mechanisms of insulin secretion and regulation,it remains unclear how conclusions from these studies apply to living animals.That few studies have been done to address these issues is largely due to the lack of suitable tools in detecting secretory events at high spatial and temporal resolution in vivo.When combined with genetically encoded biosensor,optical imaging is a powerful tool for visualization of molecular events in vivo.In this study,we generated a DNA construct encoding a secretory granule resident protein that is linked with two spectrally separate fluorescent proteins,a highly pH-sensitive green pHluorin on the intra-granular side and a red mCherry in the cytosol.Upon exocytosis of secretory granules,the dim pHluorin inside the acidic secretory granules became highly fluorescent outside the cells at neutral pH,while mCherry fluorescence remained constant in the process,thus allowing ratiometric quantification of insulin secretory events.Furthermore,mCherry fluorescence enabled tracking the movement of secretory granules in living cells.We validated this approach in insulin-secreting cells,and generated a transgenic mouse line expressing the optical sensor specifically in pancreaticβ-cells.The transgenic mice will be a useful tool for future investigations of molecular mechanism of insulin secretion in vitro and in vivo.展开更多
Spinal cord injury （SCI） is a devastating medical condition without a cure. Reestablishment of neuronal connections after spinal cord injury is the ＂holy grail＂ of SCI research. However, grafting exogenous cells, ...Spinal cord injury （SCI） is a devastating medical condition without a cure. Reestablishment of neuronal connections after spinal cord injury is the ＂holy grail＂ of SCI research. However, grafting exogenous cells, including neural stem cells and a variety of adult somatic cells, has had very lim- ited success [1]. Two back-to-back papers published in the Proceedings of National Academy of Sciences USA by the Li and Sun groups have provided exciting information by bringing activated endogenous neurogenesis into play, using a biomaterial called chitosan that was loaded with neu- rotrophic factor 3 （NT3） to achieve slow release of the trophic factor. Moreover, these papers have provided a mechanistic insight using analyses of gene expression and neuronal function.展开更多
Currently,cellular action potentials are detected using either electrical recordings or exogenous fluorescent probes that sense the calcium concentration or transmembrane voltage.Ca imaging has a low temporal resoluti...Currently,cellular action potentials are detected using either electrical recordings or exogenous fluorescent probes that sense the calcium concentration or transmembrane voltage.Ca imaging has a low temporal resolution,while voltage indicators are vulnerable to phototoxicity,photobleaching,and heating.Here,we report full-field interferometric imaging of individual action potentials by detecting movement across the entire cell membrane.Using spike-triggered averaging of movies synchronized with electrical recordings,we demonstrate deformations up to 3 nm(0.9 mrad)during the action potential in spiking HEK-293 cells,with a rise time of 4ms.The time course of the optically recorded spikes matches the electrical waveforms.Since the shot noise limit of the camera(~2 mrad/pix)precludes detection of the action potential in a single frame,for all-optical spike detection,images are acquired at 50 kHz,and 50 frames are binned into 1 ms steps to achieve a sensitivity of 0.3 mrad in a single pixel.Using a selfreinforcing sensitivity enhancement algorithm based on iteratively expanding the region of interest for spatial averaging,individual spikes can be detected by matching the previously extracted template of the action potential with the optical recording.This allows all-optical full-field imaging of the propagating action potentials without exogeneous labels or electrodes.展开更多
脊髓损伤是尚未解决的重大医学难题.在脊髓损伤研究中,损伤后重建神经元联系被奉为研究圣杯.人们为此进行了外源细胞移植（包括神经干细胞以及各种成年体细胞）的实验研究,却鲜有成功[1].最近,李晓光和孙毅团队在Proceedings of the Nat...脊髓损伤是尚未解决的重大医学难题.在脊髓损伤研究中,损伤后重建神经元联系被奉为研究圣杯.人们为此进行了外源细胞移植（包括神经干细胞以及各种成年体细胞）的实验研究,却鲜有成功[1].最近,李晓光和孙毅团队在Proceedings of the National Academy of Sciences of the United States of America上连续发表2篇文章,成果令人兴奋.展开更多
Long-term memory(LTM)allows animals to use remote past experiences to guide current and future decisions.One of the widely-used animal models for LTM research is the fruit-fly Drosophila melanogaster,which can form di...Long-term memory(LTM)allows animals to use remote past experiences to guide current and future decisions.One of the widely-used animal models for LTM research is the fruit-fly Drosophila melanogaster,which can form different types of LTM after different learning paradigms[1-6].Spaced training-induced LTM(spLTM),perhaps the most studied Drosophila LTM,requires multiple repeated learning trials with a 15-min resting interval between each trial.For decades,spLTM has been considered the only aversive olfactory LTM that can last for>7 days[1].Recently,two other types of LTM,context-dependent LTM(cLTM)[5]and merged LTM(mLTM)[6],have been discovered with the same long duration as spLTM,and can be formed with only one aversive learning trial.展开更多
文摘Insulin secretion is a complex and highly regulated process.Although much progress has been made in understanding the cellular mechanisms of insulin secretion and regulation,it remains unclear how conclusions from these studies apply to living animals.That few studies have been done to address these issues is largely due to the lack of suitable tools in detecting secretory events at high spatial and temporal resolution in vivo.When combined with genetically encoded biosensor,optical imaging is a powerful tool for visualization of molecular events in vivo.In this study,we generated a DNA construct encoding a secretory granule resident protein that is linked with two spectrally separate fluorescent proteins,a highly pH-sensitive green pHluorin on the intra-granular side and a red mCherry in the cytosol.Upon exocytosis of secretory granules,the dim pHluorin inside the acidic secretory granules became highly fluorescent outside the cells at neutral pH,while mCherry fluorescence remained constant in the process,thus allowing ratiometric quantification of insulin secretory events.Furthermore,mCherry fluorescence enabled tracking the movement of secretory granules in living cells.We validated this approach in insulin-secreting cells,and generated a transgenic mouse line expressing the optical sensor specifically in pancreaticβ-cells.The transgenic mice will be a useful tool for future investigations of molecular mechanism of insulin secretion in vitro and in vivo.
文摘Spinal cord injury （SCI） is a devastating medical condition without a cure. Reestablishment of neuronal connections after spinal cord injury is the ＂holy grail＂ of SCI research. However, grafting exogenous cells, including neural stem cells and a variety of adult somatic cells, has had very lim- ited success [1]. Two back-to-back papers published in the Proceedings of National Academy of Sciences USA by the Li and Sun groups have provided exciting information by bringing activated endogenous neurogenesis into play, using a biomaterial called chitosan that was loaded with neu- rotrophic factor 3 （NT3） to achieve slow release of the trophic factor. Moreover, these papers have provided a mechanistic insight using analyses of gene expression and neuronal function.
基金provided by the NIH grant U01 EY025501by the Stanford Neurosciences Institute(G.G.)。
文摘Currently,cellular action potentials are detected using either electrical recordings or exogenous fluorescent probes that sense the calcium concentration or transmembrane voltage.Ca imaging has a low temporal resolution,while voltage indicators are vulnerable to phototoxicity,photobleaching,and heating.Here,we report full-field interferometric imaging of individual action potentials by detecting movement across the entire cell membrane.Using spike-triggered averaging of movies synchronized with electrical recordings,we demonstrate deformations up to 3 nm(0.9 mrad)during the action potential in spiking HEK-293 cells,with a rise time of 4ms.The time course of the optically recorded spikes matches the electrical waveforms.Since the shot noise limit of the camera(~2 mrad/pix)precludes detection of the action potential in a single frame,for all-optical spike detection,images are acquired at 50 kHz,and 50 frames are binned into 1 ms steps to achieve a sensitivity of 0.3 mrad in a single pixel.Using a selfreinforcing sensitivity enhancement algorithm based on iteratively expanding the region of interest for spatial averaging,individual spikes can be detected by matching the previously extracted template of the action potential with the optical recording.This allows all-optical full-field imaging of the propagating action potentials without exogeneous labels or electrodes.
文摘脊髓损伤是尚未解决的重大医学难题.在脊髓损伤研究中,损伤后重建神经元联系被奉为研究圣杯.人们为此进行了外源细胞移植（包括神经干细胞以及各种成年体细胞）的实验研究,却鲜有成功[1].最近,李晓光和孙毅团队在Proceedings of the National Academy of Sciences of the United States of America上连续发表2篇文章,成果令人兴奋.
基金supported by granes from the National Science Foundation of China(31970955)and the Tsinghua-Peking Center for Life Sciences.
文摘Long-term memory(LTM)allows animals to use remote past experiences to guide current and future decisions.One of the widely-used animal models for LTM research is the fruit-fly Drosophila melanogaster,which can form different types of LTM after different learning paradigms[1-6].Spaced training-induced LTM(spLTM),perhaps the most studied Drosophila LTM,requires multiple repeated learning trials with a 15-min resting interval between each trial.For decades,spLTM has been considered the only aversive olfactory LTM that can last for>7 days[1].Recently,two other types of LTM,context-dependent LTM(cLTM)[5]and merged LTM(mLTM)[6],have been discovered with the same long duration as spLTM,and can be formed with only one aversive learning trial.
