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Functional imaging of visual cortical layers and subplate in awake mice with optimized three-photon microscopy
摘要： Two-photon microscopy is used to image neuronal activity, but has severe limitations for studying deeper cortical layers. Here, we developed a custom three-photon microscope optimized to image a vertical column of the cerebral cortex > 1 mm in depth in awake mice with low (<20 mW) average laser power. Our measurements of physiological responses and tissue-damage thresholds define pulse parameters and safety limits for damage-free three-photon imaging. We image functional visual responses of neurons expressing GCaMP6s across all layers of the primary visual cortex (V1) and in the subplate. These recordings reveal diverse visual selectivity in deep layers: layer 5 neurons are more broadly tuned to visual stimuli, whereas mean orientation selectivity of layer 6 neurons is slightly sharper, compared to neurons in other layers. Subplate neurons, located in the white matter below cortical layer 6 and characterized here for the first time, show low visual responsivity and broad orientation selectivity.
关键词： subplate neurons,deep brain imaging,visual cortex,neuronal activity,three-photon microscopy,GCaMP6s
更新于2025-09-23 15:23:52
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A dimeric fluorescent protein yields a bright, red-shifted GEVI capable of population signals in brain slice
摘要： A bright, red-shifted Genetically Encoded Voltage Indicator (GEVI) was developed using a modified version of the fluorescent protein, tdTomato. Dimerization of the fluorescent domain for ArcLight-type GEVIs has been shown to affect the signal size of the voltage-dependent optical signal. For red-shifted GEVI development, tdTomato was split fusing a single dTomato chromophore to the voltage sensing domain. Optimization of the amino acid length and charge composition of the linker region between the voltage sensing domain and the fluorescent protein resulted in a probe that is an order of magnitude brighter than FlicR1 at a resting potential of ?70 mV and exhibits a ten-fold larger change in fluorescence (ΔF) upon 100 mV depolarization of the plasma membrane in HEK 293 cells. Unlike ArcLight, the introduction of charged residues to the exterior of dTomato did not substantially improve the dynamic range of the optical signal. As a result, this new GEVI, Ilmol, yields a 3-fold improvement in the signal-to-noise ratio compared to FlicR1 despite a smaller fractional change in fluorescence of 4% per 100 mV depolarization of the plasma membrane. Ilmol expresses well in neurons resolving action potentials in neuronal cultures and reporting population signals in mouse hippocampal acute brain slice recordings. Ilmol is the brightest red-shifted GEVI to date enabling imaging with 160-fold less light than Archon1 for primary neuron recordings (50 mW/cm2 versus 8 W/cm2) and 600-fold less light than QuasAr2 for mouse brain slice recordings (500 mW/cm2 versus 300 W/cm2). This new GEVI uses a distinct mechanism from other approaches, opening an alternate engineering path to improve sensitivity and speed.
关键词： fluorescence,Genetically Encoded Voltage Indicator,tdTomato,optical signal,GEVI,neuronal activity,red-shifted,voltage imaging
更新于2025-09-23 15:21:01
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High-efficiency optogenetic silencing with soma-targeted anion-conducting channelrhodopsins
摘要： Optogenetic silencing allows time-resolved functional interrogation of defined neuronal populations. However, the limitations of inhibitory optogenetic tools impose stringent constraints on experimental paradigms. The high light power requirement of light-driven ion pumps and their effects on intracellular ion homeostasis pose unique challenges, particularly in experiments that demand inhibition of a widespread neuronal population in vivo. Guillardia theta anion-conducting channelrhodopsins (GtACRs) are promising in this regard, due to their high single-channel conductance and favorable photon-ion stoichiometry. However, GtACRs show poor membrane targeting in mammalian cells, and the activity of such channels can cause transient excitation in the axon due to an excitatory chloride reversal potential in this compartment. Here, we address these problems by enhancing membrane targeting and subcellular compartmentalization of GtACRs. The resulting soma-targeted GtACRs show improved photocurrents, reduced axonal excitation and high light sensitivity, allowing highly efficient inhibition of neuronal activity in the mammalian brain.
关键词： optogenetic silencing,anion-conducting channelrhodopsins,membrane targeting,GtACRs,neuronal activity inhibition
更新于2025-09-23 15:21:01
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[IEEE 2019 IEEE 21st Electronics Packaging Technology Conference (EPTC) - Singapore, Singapore (2019.12.4-2019.12.6)] 2019 IEEE 21st Electronics Packaging Technology Conference (EPTC) - A Process Study of Laser Patterning of Different Conductive Layers for Printed Electronics
摘要： Stress-induced psychological and somatic diseases are virtually endemic nowadays. Written self-report anxiety measures are available; however, these indices tend to be time consuming to acquire. For medical patients, completing written reports can be burdensome if they are weak, in pain, or in acute anxiety states. Consequently, simple and fast non-invasive methods for assessing stress response from neurophysiological data are essential. In this paper, we report on a study that makes predictions of the state-trait anxiety inventory (STAI) index from oxyhemoglobin and deoxyhemoglobin concentration changes of the prefrontal cortex using a two-channel portable near-infrared spectroscopy device. Predictions are achieved by constructing machine learning algorithms within a Bayesian framework with nonlinear basis function together with Markov Chain Monte Carlo implementation. In this paper, prediction experiments were performed against four different data sets, i.e., two comprising young subjects, and the remaining two comprising elderly subjects. The number of subjects in each data set varied between 17 and 20 and each subject participated only once. They were not asked to perform any task; instead, they were at rest. The root mean square errors for the four groups were 6.20, 6.62, 4.50, and 6.38, respectively. There appeared to be no significant distinctions of prediction accuracies between age groups and since the STAI are defined between 20 and 80, the predictions appeared reasonably accurate. The results indicate potential applications to practical situations such as stress management and medical practice.
关键词： blood oxygenation,prevention medicine,Anxiety index,neuronal activity,regional cerebral blood flow,translational engineering,near infrared spectroscopy,health and safety,prediction methods,oxyhemoglobin
更新于2025-09-23 15:19:57