Manipulating and real-time monitoring of neuronal activities with cell-type specificity and precise spatiotemporal resolution during animal behavior are fundamental technologies for exploring the functional connectivi...Manipulating and real-time monitoring of neuronal activities with cell-type specificity and precise spatiotemporal resolution during animal behavior are fundamental technologies for exploring the functional connectivity, information transmission, and physiological functions of neural circuits in vivo. However, current techniques for optogenetic stimulation and neuronal activity recording mostly operate independently. Here, we report an all-fiber-transmission photometry system for simultaneous optogenetic manipulation and multi-color recording of neuronal activities and the neurotransmitter release in a freely moving animal. We have designed and manufactured a wavelength-independent multi-branch fiber bundle to enable simultaneous optogenetic manipulation and multi-color recording at different wavelengths. Further, we combine a laser of narrow linewidth with the lock-in amplification method to suppress the optogenetic stimulation-induced artifacts and channel crosstalk. We show that the collection efficiency of our system outperforms a traditional epi-fluorescence system. Further, we demonstrate successful recording of dynamic dopamine(DA) responses to unexpected rewards in the nucleus accumbens(NAc) in a freely moving mouse. We also show simultaneous dual-color recording of neuronal Ca2+ signals and DA dynamics in the NAc upon delivering an unexpected reward and the simultaneous optogenetic activating at dopaminergic terminals in the same location. Thus, our multi-function fiber photometry system provides a compatible, efficient, and flexible solution for neuroscientists to study neural circuits and neurological diseases.展开更多
The circadian clock coordinates rhythms in numerous physiological processes to maintain organismal homeostasis. Since the suprachiasmatic nucleus(SCN) is widely accepted as the circadian pacemaker, it is critical to u...The circadian clock coordinates rhythms in numerous physiological processes to maintain organismal homeostasis. Since the suprachiasmatic nucleus(SCN) is widely accepted as the circadian pacemaker, it is critical to understand the neural mechanisms by which rhythmic information is transferred from the SCN to peripheral clocks. Here, we present the first comprehensive map of SCN efferent connections and suggest a molecular logic underlying these projections. The SCN projects broadly to most major regions of the brain, rather than solely to the hypothalamus and thalamus. The efferent projections from different subtypes of SCN neurons vary in distance and intensity, and blocking synaptic transmission of these circuits affects circadian rhythms in locomotion and feeding to different extents. We also developed a barcoding system to integrate retrograde tracing with in-situ sequencing, allowing us to link circuit anatomy and spatial patterns of gene expression. Analyses using this system revealed that brain regions functioning downstream of the SCN receive input from multiple neuropeptidergic cell types within the SCN, and that individual SCN neurons generally project to a single downstream brain region.This map of SCN efferent connections provides a critical foundation for future investigations into the neural circuits underlying SCNmediated rhythms in physiology. Further, our new barcoded tracing method provides a tool for revealing the molecular logic of neuronal circuits within heterogeneous brain regions.展开更多
G_(q)-coupled receptors regulate numerous physiological processes by activating enzymes and inducing intracellular Ca^(2+)signals.There is a strong need for an optogenetic tool that enables powerful experimental contr...G_(q)-coupled receptors regulate numerous physiological processes by activating enzymes and inducing intracellular Ca^(2+)signals.There is a strong need for an optogenetic tool that enables powerful experimental control over G_(q) signaling.Here,we present chicken opsin 5(cOpn5)as the long sought-after,single-component optogenetic tool that mediates ultra-sensitive optical control of intracellular G_(q) signaling with high temporal and spatial resolution.Expressing cOpn5 in HEK 293T cells and primary mouse astrocytes enables blue light-triggered,G_(q)-dependent Ca^(2+) release from intracellular stores and protein kinase C activation.Strong Ca^(2+) transients were evoked by brief light pulses of merely 10 ms duration and at 3 orders lower light intensity of that for common optogenetic tools.Photostimulation of cOpn5-expressing cells at the subcellular and single-cell levels generated fast intracellular Ca^(2+)transition,thus demonstrating the high spatial precision of cOpn5 optogenetics.The cOpn5-mediated optogenetics could also be applied to activate neurons and control animal behavior in a circuit-dependent manner.cOpn5 optogenetics may find broad applications in studying the mechanisms and functional relevance of G_(q) signaling in both non-excitable cells and excitable cells in all major organ systems.展开更多
基金supported by Beijing Municipal Governmentsupported by the National Natural Science Foundation of China(Grant Nos.61890952)the Director Fund of WNLO。
文摘Manipulating and real-time monitoring of neuronal activities with cell-type specificity and precise spatiotemporal resolution during animal behavior are fundamental technologies for exploring the functional connectivity, information transmission, and physiological functions of neural circuits in vivo. However, current techniques for optogenetic stimulation and neuronal activity recording mostly operate independently. Here, we report an all-fiber-transmission photometry system for simultaneous optogenetic manipulation and multi-color recording of neuronal activities and the neurotransmitter release in a freely moving animal. We have designed and manufactured a wavelength-independent multi-branch fiber bundle to enable simultaneous optogenetic manipulation and multi-color recording at different wavelengths. Further, we combine a laser of narrow linewidth with the lock-in amplification method to suppress the optogenetic stimulation-induced artifacts and channel crosstalk. We show that the collection efficiency of our system outperforms a traditional epi-fluorescence system. Further, we demonstrate successful recording of dynamic dopamine(DA) responses to unexpected rewards in the nucleus accumbens(NAc) in a freely moving mouse. We also show simultaneous dual-color recording of neuronal Ca2+ signals and DA dynamics in the NAc upon delivering an unexpected reward and the simultaneous optogenetic activating at dopaminergic terminals in the same location. Thus, our multi-function fiber photometry system provides a compatible, efficient, and flexible solution for neuroscientists to study neural circuits and neurological diseases.
基金supported by the National Natural Science Foundation of China(32171157,31971090)Ministry of Science and Technology of the People’s Republic of China(2021ZD0203400)Kuanren Talents’Project of The Second Affiliated Hospital of Chongqing Medical University。
文摘The circadian clock coordinates rhythms in numerous physiological processes to maintain organismal homeostasis. Since the suprachiasmatic nucleus(SCN) is widely accepted as the circadian pacemaker, it is critical to understand the neural mechanisms by which rhythmic information is transferred from the SCN to peripheral clocks. Here, we present the first comprehensive map of SCN efferent connections and suggest a molecular logic underlying these projections. The SCN projects broadly to most major regions of the brain, rather than solely to the hypothalamus and thalamus. The efferent projections from different subtypes of SCN neurons vary in distance and intensity, and blocking synaptic transmission of these circuits affects circadian rhythms in locomotion and feeding to different extents. We also developed a barcoding system to integrate retrograde tracing with in-situ sequencing, allowing us to link circuit anatomy and spatial patterns of gene expression. Analyses using this system revealed that brain regions functioning downstream of the SCN receive input from multiple neuropeptidergic cell types within the SCN, and that individual SCN neurons generally project to a single downstream brain region.This map of SCN efferent connections provides a critical foundation for future investigations into the neural circuits underlying SCNmediated rhythms in physiology. Further, our new barcoded tracing method provides a tool for revealing the molecular logic of neuronal circuits within heterogeneous brain regions.
基金supported by Ministry of Science and Technology China Brain Initiative Grant(2021ZD0202803)the Research Unit of Medical Neurobiology at Chinese Academy of Medical Sciences(2019RU003)Beijing Municipal Government。
文摘G_(q)-coupled receptors regulate numerous physiological processes by activating enzymes and inducing intracellular Ca^(2+)signals.There is a strong need for an optogenetic tool that enables powerful experimental control over G_(q) signaling.Here,we present chicken opsin 5(cOpn5)as the long sought-after,single-component optogenetic tool that mediates ultra-sensitive optical control of intracellular G_(q) signaling with high temporal and spatial resolution.Expressing cOpn5 in HEK 293T cells and primary mouse astrocytes enables blue light-triggered,G_(q)-dependent Ca^(2+) release from intracellular stores and protein kinase C activation.Strong Ca^(2+) transients were evoked by brief light pulses of merely 10 ms duration and at 3 orders lower light intensity of that for common optogenetic tools.Photostimulation of cOpn5-expressing cells at the subcellular and single-cell levels generated fast intracellular Ca^(2+)transition,thus demonstrating the high spatial precision of cOpn5 optogenetics.The cOpn5-mediated optogenetics could also be applied to activate neurons and control animal behavior in a circuit-dependent manner.cOpn5 optogenetics may find broad applications in studying the mechanisms and functional relevance of G_(q) signaling in both non-excitable cells and excitable cells in all major organ systems.
