GCaMP is one of the most widely used calcium indicators in neuronal imaging and calcium cell biology. The newly developed GCaMP6 shows superior brightness and ultrasensitivity to calcium concentration change. In this ...GCaMP is one of the most widely used calcium indicators in neuronal imaging and calcium cell biology. The newly developed GCaMP6 shows superior brightness and ultrasensitivity to calcium concentration change. In this study, we determined crystal structures of CaZ＋-bound GCaMP6 monomer and dimer and presented detailed structural analyses in comparison with its par- ent version GCaMP5G. Our analyses reveal the structural basis for the outperformance of this newly developed Ca2＋ indicator. Three substitution mutations and the resulting changes of local structure and interaction explain the ultrasensitivity and in- creased fluorescence intensity common to all three versions of GCaMP6. Each particular substitution in the three GCaMP6 is also structurally consistent with their differential sensitivity and intensity, maximizing the potential of using GCaMP6 in solving diverse problems in neuronal research and calcium signaling. Our studies shall also be beneficial to further structure-guided optimization of GCaMP and facilitate the design of novel calcium indicators.展开更多
Limb loss and spinal cord injury are two debilitating conditions that continue to grow in prevalence. Prosthetic limbs and limb reanimation present two ways of providing affected individuals with means to interact in ...Limb loss and spinal cord injury are two debilitating conditions that continue to grow in prevalence. Prosthetic limbs and limb reanimation present two ways of providing affected individuals with means to interact in the world. These techniques are both dependent on a robust interface with the peripheral nerve. Current methods for interfacing with the peripheral nerve tend to suffer from low specificity, high latency and insufficient robustness for a chronic implant. An optical peripheral nerve interface may solve some of these problems by decreasing invasiveness and providing single axon specificity. In order to implement such an interface three elements are required:(1) a transducer capable of translating light into a neural stimulus or translating neural activity into changes in fluorescence,(2) a means for delivering said transducer and(3) a microscope for providing the stimulus light and detecting the fluorescence change. There are continued improvements in both genetically encoded calcium and voltage indicators as well as new optogenetic actuators for stimulation. Similarly, improvements in specificity of viral vectors continue to improve expression in the axons of the peripheral nerve. Our work has recently shown that it is possible to virally transduce axons of the peripheral nerve for recording from small fibers. The improvements of these components make an optical peripheral nerve interface a rapidly approaching alternative to current methods.展开更多
Numerous clinical and research applications necessitate the ability to interface with peripheral nerve fibers to read and control relevant neural pathways. Visceral organ modulation and rehabilitative prosthesis are t...Numerous clinical and research applications necessitate the ability to interface with peripheral nerve fibers to read and control relevant neural pathways. Visceral organ modulation and rehabilitative prosthesis are two areas which could benefit greatly from improved neural interfacing approaches. Therapeutic neural interfacing, or ‘bioelectronic medicine’, has potential to affect a broad range of disorders given that all the major organs of the viscera are neurally innervated. However, a better understanding of the neural pathways that underlie function and a means to precisely interface with these fibers are required. Existing peripheral nerve interfaces, consisting primarily of electrode-based designs, are unsuited for highly specific （individual axon） communication and/or are invasive to the tissue. Our laboratory has explored an optogenetic approach by which optically sensitive reporters and actuators are targeted to specific cell （axon） types. The nature of such an approach is laid out in this short perspective, along with associated technologies and challenges.展开更多
Due to the complex circuitry and plethora of cell types involved in somatosensation, it is becoming increasingly important to be able to observe cellular activity at the population level. In addition, since cells rely...Due to the complex circuitry and plethora of cell types involved in somatosensation, it is becoming increasingly important to be able to observe cellular activity at the population level. In addition, since cells rely on an intricate variety of extracellular factors, it is important to strive to maintain the physiological environment. Many electrophysiological techniques require the implementation of artificially-produced physiological environments and it can be difficult to assess the activity of many cells simultane- ously. Moreover, imaging Ca^2＋ transients using Ca^2＋- sensitive dyes often requires in vitro preparations or in vivo injections, which can lead to variable expression levels. With the development of more sensitive geneticallyencoded Ca^2＋ indicators （GECIs） it is now possible to observe changes in Ca^2＋ transients in large populations of cells at the same time. Recently, groups have used a GECI called GCaMP to address fundamental questions in somatosensation. Researchers can now induce GCaMP expression in the mouse genome using viral or gene knock- in approaches and observe the activity of populations of cells in the pain pathway such as dorsal root ganglia （DRG）, spinal neurons, or glia. This approach can be used in vivo and thus maintains the organism＇s biological integrity. The implementation of GCaMP imaging has led to many advances in our understanding of somatosensation. Here, we review the current findings in pain research using GCaMP imaging as well as discussing potential method- ological considerations.展开更多
基金supported in part by an International Early Career Scientist grant from the Howard Hughes Medical Institute to Shao Fenggrant from the National Basic Research Program of China (2011CB910304 and 2011CB911103) to Wang DaChengNational Natural Science Foundation of China (31100535) to Ding JingJin
文摘GCaMP is one of the most widely used calcium indicators in neuronal imaging and calcium cell biology. The newly developed GCaMP6 shows superior brightness and ultrasensitivity to calcium concentration change. In this study, we determined crystal structures of CaZ＋-bound GCaMP6 monomer and dimer and presented detailed structural analyses in comparison with its par- ent version GCaMP5G. Our analyses reveal the structural basis for the outperformance of this newly developed Ca2＋ indicator. Three substitution mutations and the resulting changes of local structure and interaction explain the ultrasensitivity and in- creased fluorescence intensity common to all three versions of GCaMP6. Each particular substitution in the three GCaMP6 is also structurally consistent with their differential sensitivity and intensity, maximizing the potential of using GCaMP6 in solving diverse problems in neuronal research and calcium signaling. Our studies shall also be beneficial to further structure-guided optimization of GCaMP and facilitate the design of novel calcium indicators.
基金funded in part by the University of Colorado Medical Scientist Training Program and funds from the NIH SPARC initiative administered through the Office of the Director:1OT2OD023852-01
文摘Limb loss and spinal cord injury are two debilitating conditions that continue to grow in prevalence. Prosthetic limbs and limb reanimation present two ways of providing affected individuals with means to interact in the world. These techniques are both dependent on a robust interface with the peripheral nerve. Current methods for interfacing with the peripheral nerve tend to suffer from low specificity, high latency and insufficient robustness for a chronic implant. An optical peripheral nerve interface may solve some of these problems by decreasing invasiveness and providing single axon specificity. In order to implement such an interface three elements are required:(1) a transducer capable of translating light into a neural stimulus or translating neural activity into changes in fluorescence,(2) a means for delivering said transducer and(3) a microscope for providing the stimulus light and detecting the fluorescence change. There are continued improvements in both genetically encoded calcium and voltage indicators as well as new optogenetic actuators for stimulation. Similarly, improvements in specificity of viral vectors continue to improve expression in the axons of the peripheral nerve. Our work has recently shown that it is possible to virally transduce axons of the peripheral nerve for recording from small fibers. The improvements of these components make an optical peripheral nerve interface a rapidly approaching alternative to current methods.
基金financially supported in part by funds administered through VA Eastern Colorado Health Care System-Denver VA Medical Centerfunds from the NIH SPARC initiative administered through the Office of the Director:1OT2OD023852-01
文摘Numerous clinical and research applications necessitate the ability to interface with peripheral nerve fibers to read and control relevant neural pathways. Visceral organ modulation and rehabilitative prosthesis are two areas which could benefit greatly from improved neural interfacing approaches. Therapeutic neural interfacing, or ‘bioelectronic medicine’, has potential to affect a broad range of disorders given that all the major organs of the viscera are neurally innervated. However, a better understanding of the neural pathways that underlie function and a means to precisely interface with these fibers are required. Existing peripheral nerve interfaces, consisting primarily of electrode-based designs, are unsuited for highly specific （individual axon） communication and/or are invasive to the tissue. Our laboratory has explored an optogenetic approach by which optically sensitive reporters and actuators are targeted to specific cell （axon） types. The nature of such an approach is laid out in this short perspective, along with associated technologies and challenges.
基金supported by grants from the National Institutes of Health to X.D.(RO1DE022750 and ROINS054791)
文摘Due to the complex circuitry and plethora of cell types involved in somatosensation, it is becoming increasingly important to be able to observe cellular activity at the population level. In addition, since cells rely on an intricate variety of extracellular factors, it is important to strive to maintain the physiological environment. Many electrophysiological techniques require the implementation of artificially-produced physiological environments and it can be difficult to assess the activity of many cells simultane- ously. Moreover, imaging Ca^2＋ transients using Ca^2＋- sensitive dyes often requires in vitro preparations or in vivo injections, which can lead to variable expression levels. With the development of more sensitive geneticallyencoded Ca^2＋ indicators （GECIs） it is now possible to observe changes in Ca^2＋ transients in large populations of cells at the same time. Recently, groups have used a GECI called GCaMP to address fundamental questions in somatosensation. Researchers can now induce GCaMP expression in the mouse genome using viral or gene knock- in approaches and observe the activity of populations of cells in the pain pathway such as dorsal root ganglia （DRG）, spinal neurons, or glia. This approach can be used in vivo and thus maintains the organism＇s biological integrity. The implementation of GCaMP imaging has led to many advances in our understanding of somatosensation. Here, we review the current findings in pain research using GCaMP imaging as well as discussing potential method- ological considerations.
