A visualization pipeline for in vivo two-photon volumetric astrocytic calcium imaging

Astrocytes,the multi-functional glial cells with the most abundant population in the brain,integrate information across their territories to regulate neuronal synaptic and cerebrovascular activities.Astrocytic calcium(Ca^(2+))signaling is the major readout of cellular functional state of astrocytes.The conventional two-photon in vivo imaging usually focuses on a single horizontal focal plane to capture the astrocytic Ca^(2+)signals,which leaves>80%spatial information undetected.To fully probe the Ca^(2+)activity across the whole astrocytic territory,we developed a pipeline for imaging and visualizing volumetric astrocytic Ca^(2+)time-lapse images.With the pipeline,we discovered a new signal distribution pattern from three-dimensional(3D)astrocytic Ca^(2+)imaging data of mice under isoflurane anesthetic states.The tools developed in this study enable a better understanding of the spatiotemporal patterns of astrocytic activity in 3D space.

AB010. The effect of visual conditioning on cortical map plasticity:a wide-field calcium imaging study

Background:Visual conditioning can refine the response of neurons in the visual cortex and higher visual and cognitive processing of a presented stimulus.This process results in increased sensitivity for that stimulus.The development of new optical imaging technology in the field of neuroscience has led to important advances,notably to better define the functional organization and plasticity of visual areas.The objective of this project is to determine the effect of daily visual conditioning with an oblique sinusoidal grating on the distribution and amplitude of cortical responses.For this,we use wide-field calcium imaging on awake mice,allowing for the observation of responses to a stimulus throughout the entire cortex in real time.Methods:C57BL/6 mice,expressing the GCaMP6s calcium reporter gene,are used to longitudinally measure neuronal activity via wide-field calcium imaging.Spontaneous activity at rest,as well as cortical responses to visual stimuli consisting of sinusoidal networks with orientation(0,30°,60°and 90°),spatial frequency(0.03,0.12,0.24 and 0.48 cpd)and contrast(100%,75%and 50%)variables are recorded to establish cortical maps,as well as tuning curves.Subsequently,the baseline function of the cortex,as well as the cortical representation of visual stimulation(30°or 90°,0.03 cpd and a contrast of 50%,75%and 100%)are studied in the animal before,during,and after daily monocular conditioning,consisting of a specific sinusoidal network(30°,0.03 cpd and 100%)over a period of 7 days.The variations in intensity and activation specificity of various visual cortical areas are calculated according to the visual conditioning and compared to an orientation stimulus for which the animal has not been conditioned(90°).Results:The cortical activation curves show a greater sensitivity of response for stimuli having horizontal or vertical gratings(0 and 90°)than for oblique gratings(30°and 60°)at low spatial frequencies(0,0.3 and 0.12 cpd).However,this trend does not occur with high spatial frequencies(0.24 and 0.48 cpd).Finally,although the intensity of activation varies in a way that is not proportional to the contrast of the stimulation,it would have no influence on the perception of the orientation of the stimuli.Conditioning at a 30°stimulus results in greater activation of the primary visual cortex and some extra-striate visual areas,as well as greater amplification of the ipsilateral cortical responses to the presentation of the visual stimuli.Conclusions:In conclusion,the results demonstrate that visual conditioning would allow for plasticity and consolidation of higher visual pathways.

Perspective of Calcium Imaging Technology Applied to Acupuncture Research

Acupuncture, a therapeutic treatment defined as the insertion of needles into the body at specific points(ie, acupoints), has growing in popularity world-wide to treat various diseases effectively, especially acute and chronic pain. In parallel, interest in the physiological mechanisms underlying acupuncture analgesia, particularly the neural mechanisms have been increasing. Over the past decades, our understanding of how the central nervous system and peripheral nervous system process signals induced by acupuncture has developed rapidly by using electrophysiological methods. However, with the development of neuroscience, electrophysiology is being challenged by calcium imaging in view field, neuron population and visualization in vivo. Owing to the outstanding spatial resolution, the novel imaging approaches provide opportunities to enrich our knowledge about the neurophysiological mechanisms of acupuncture analgesia at subcellular, cellular, and circuit levels in combination with new labeling, genetic and circuit tracing techniques. Therefore, this review will introduce the principle and the method of calcium imaging applied to acupuncture research. We will also review the current findings in pain research using calcium imaging from in vitro to in vivo experiments and discuss the potential methodological considerations in studying acupuncture analgesia.

In vivo imaging of the neuronal response to spinal cord injury:a narrative review

Deciphering the neuronal response to injury in the spinal cord is essential for exploring treatment strategies for spinal cord injury(SCI).However,this subject has been neglected in part because appropriate tools are lacking.Emerging in vivo imaging and labeling methods offer great potential for observing dynamic neural processes in the central nervous system in conditions of health and disease.This review first discusses in vivo imaging of the mouse spinal cord with a focus on the latest imaging techniques,and then analyzes the dynamic biological response of spinal cord sensory and motor neurons to SCI.We then summarize and compare the techniques behind these studies and clarify the advantages of in vivo imaging compared with traditional neuroscience examinations.Finally,we identify the challenges and possible solutions for spinal cord neuron imaging.

Calcium imaging in Arabidopsis pollen cells using G-CaMP5

Calcium （Ca2 ＋） signaling has been implicated in poJ]en germination and pollen tube growth. To date, however, we still know very little about how exactly Ca2＋ signaling links to various physiological subcellular processes during pollen germination and pollen tube growth. Given that Ca2＋ signaling is tightly related to the cytosolic concentration and dynamics of Ca2＋, it is vital to trace the dynamic changes in Ca＋ levels in order to decode Ca2＋ signaling. Here, we demonstrate that G-CaMP5 serves well as an indicator for monitoring cytosolic Ca2＋ dynamics in pollen cells. Using this probe, we show that cytosolic Ca2＋ changes dramatically during pollen germination, and, as reported previously, Ca2＋ forms a tip-focused gradient in the pollen tube and undergoes oscillation in the tip region during pollen tube growth. In particular, using G-CaMP5 allowed us to capture the dynamic changes in the cytosolic Ca2＋ concentration （[Ca2＋ ]cyt） in pollen tubes in response to various exogenous treatments. Our data suggest that G-CaMP5 is a suitable probe for monitoring the dynamics of [Ca2＋ ]cyt in pollen cells.

Neuroprotective effects of Asiaticoside

In the central nervous system, Asiaticoside has been shown to attenuate in vitro neuronal damage caused by exposure to β-amyloid. In vivo studies demonstrated that Asiaticoside could attenuate neurobehavioral, neurochemical and histological changes in transient focal middle cerebral artery occlusion animals. In addition, Asiaticoside showed anxiolytic effects in acute and chronic stress animals. However, its potential neuroprotective properties in glutamate-induced excitotoxicity have not been fully studied. We investigated the neuroprotective effects of Asiaticoside in primary cultured mouse cortical neurons exposed to glutamate-induced excitotoxicity invoked by N-methyl-D-aspartate. Pretreatment with Asiaticoside decreased neuronal cell loss in a concentration-dependent manner and restored changes in expression of apoptotic-related proteins Bcl-2 and Bax. Asiaticoside pretreatment also attenuated the upregulation of NR2B expression, a subunit of N-methyl-D-aspartate receptors, but did not affect expression of NR2A subunits. Additionally, in cultured neurons, Asiaticoside significantly inhibited Ca^2＋ influx induced by N-methyl-D-aspartate. These experimental findings provide preliminary evidence that during excitotoxicity induced by Nmethyl-D-aspartate exposure in cultured cortical neurons, the neuroprotective effects of Asiaticoside are mediated through inhibition of calcium influx. Aside from its anti-oxidant activity, down-regulation of NR2B-contalning N-methyl-D-aspartate receptors may be one of the underlying mechanisms in Asiaticoside neuroprotection.

Live Cell Imaging with R-GEC01 Sheds Light on fig22- and Chitin-Induced Transient [Ca2＋]cyt Patterns in Arabidopsis

Intracellular Ca2＋ transients are an integral part of the signaling cascade during pathogen-associated molecular pattern （PAMP）-triggered immunity in plants. Yet, our knowledge about the spatial distribution of PAMP-induced Ca2＋ signals is limited. Investigation of cell- and tissue-specific properties of Ca2＋- dependent signaling processes requires versatile Ca2＋ reporters that are able to extract spatial information from cellular and subcellular structures, as well as from whole tissues over time periods from seconds to hours. Fluorescence-based reporters cover both a broad spatial and temporal range, which makes them ideally suited to study Ca2＋ signaling in living cells. In this study, we compared two fluorescence-based Ca2＋ sensors： the F6rster resonance energy transfer （FRET）-based reporter yellow cameleon NES-YC3.6 and the intensity-based sensor R-GECO1. We demonstrate that R-GECO1 exhibits a significantly increased signal change compared with ratiometric NES-YC3.6 in response to several stimuli. Due to its superior sensitivity, R-GECO1 is able to report fig22- and chitin-induced Ca2＋ signals on a cellular scale, which allowed identification of defined [Ca2＋]cyt oscillations in epidermal and guard cells in response to the fungal elicitor chitin. Moreover, we discovered that fig22- and chitin-induced Ca2＋ signals in the root initiate from the elongation zone.

Correction-free remotely scanned two-photon in vivo mouse retinal imaging

Non-invasive fluorescence retinal imaging in small animals is an important requirement for an array of translational vision applications.The in vivo two-photon imaging of the mouse retina may enable the long-term investigation of the structure and function of healthy and diseased retinal tissue.However,to date,this has only been possible using relatively complex adaptive-optics systems.Here,the optical modeling of the murine eye and of the imaging system is used to achieve correction-free two-photon microscopy through the pupil of a mouse eye to yield high-quality,optically sectioned fundus images.By remotely scanning the focus using an electronically tunable lens,high-resolution three-dimensional fluorescein angiograms and cellular-scale images are acquired,thus introducing a correction-free baseline performance level for two-photon in vivo retinal imaging.Moreover,the system enables functional calcium imaging of repeated retinal responses to light stimulation using the genetically encoded indicator,GCaMP6s.These results and the simplicity of the new add-on optics are an important step toward several structural,functional,and multimodal imaging applications that will benefit from the tight optical sectioning and the use of near-infrared light.

Volumetric Imaging of Neural Activity by Light Field Microscopy

Recording the highly diverse and dynamic activities in large populations of neurons in behaving animals is crucial for a better understanding of how the brain works.To meet this challenge,extensive efforts have been devoted to developing functional fluorescent indicators and optical imaging techniques to optically monitor neural activity.Indeed,optical imaging potentially has extremely high throughput due to its non-invasive access to large brain regions and capability to sample neurons at high density,but the readout speed,such as the scanning speed in two-photon scanning microscopy,is often limited by various practical considerations.Among different imaging methods,light field microscopy features a highly parallelized 3D fluorescence imaging scheme and therefore promises a novel and faster strategy for functional imaging of neural activity.Here,we briefly review the working principles of various types of light field microscopes and their recent developments and applications in neuroscience studies.We also discuss strategies and considerations of optimizing light field microscopy for different experimental purposes,with illustrative examples in imaging zebrafish and mouse brains.

CYP1B1-derived epoxides modulate the TRPA1channel in chronic pain

Pain is often debilitating,and current treatments are neither universally efficacious nor without risks.Transient receptor potential(TRP)ion channels offer alternative targets for pain relief,but little is known about the regulation or identities of endogenous TRP ligands that affect inflammation and pain.Here,transcriptomic and targeted lipidomic analysis of damaged tissue from the mouse spinal nerve ligation(SNL)-induced chronic pain model revealed a time-dependent increase in Cyp1b1 mRNA and a concurrent accumulation of 8,9-epoxyeicosatrienoic acid(EET)and 19,20-EpDPA post injury.Production of 8,9-EET and 19,20-EpDPA by human/mouse CYP1B1 was confirmed in vitro,and 8,9-EET and 19,20-EpDPA selectively and dose-dependently sensitized and activated TRPA1 in overexpressing HEK-293 cells and Trpa1-expressing/AITC-responsive cultured mouse peptidergic dorsal root ganglia(DRG)neurons.TRPA1 activation by 8,9-EET and 19,20-EpDPA was attenuated by the antagonist A967079,and mouse TRPA1 was more responsive to 8,9-EET and 19,20-EpDPA than human TRPA1.This latter effect mapped to residues Y933,G939,and S921 of TRPA1.Intra-plantar injection of 19,20-EpDPA induced acute mechanical,but not thermal hypersensitivity in mice,which was also blocked by A967079.Similarly,Cyp1b1-knockout mice displayed a reduced chronic pain phenotype following SNL injury.These data suggest that manipulation of the CYP1B1-oxylipin-TRPA1 axis might have therapeutic benefit.

Contextual Fear Learning and Extinction in the Primary Visual Cortex of Mice

Fear memory contextualization is critical for selecting adaptive behavior to survive.Contextual fear conditioning(CFC)is a classical model for elucidating related underlying neuronal circuits.The primary visual cortex(V1)is the primary cortical region for contextual visual inputs,but its role in CFC is poorly understood.Here,our experiments demonstrated that bilateral inactivation of V1 in mice impaired CFC retrieval,and both CFC learning and extinction increased the turnover rate of axonal boutons in V1.The frequency of neuronal Ca^(2+)activity decreased after CFC learning,while CFC extinction reversed the decrease and raised it to the naïve level.Contrary to control mice,the frequency of neuronal Ca^(2+)activity increased after CFC learning in microglia-depleted mice and was maintained after CFC extinction,indicating that microglial depletion alters CFC learning and the frequency response pattern of extinction-induced Ca^(2+)activity.These findings reveal a critical role of microglia in neocortical information processing in V1,and suggest potential approaches for cellular-based manipulation of acquired fear memory.

Direction Selectivityof TmY Neurites in Drosophila

The perception of motion is an important func-tion of vision.Neural wiring diagrams for extracting direc-tional information have been obtained by connectome recon-struction.Direction selectivity in Drosophila is thought to originate in T4/T5 neurons through integrating inputs with different temporal filtering properties.Through genetic screening based on synaptic distribution,we isolated a new type of TmY neuron,termed TmY-ds,that form recipro-cal synaptic connections with T4/T5 neurons.Its neurites responded to grating motion along the four cardinal direc-tions and showed a variety of direction selectivity.Intrigu-ingly,its direction selectivity originated from temporal filtering neurons rather than T4/T5.Genetic silencing and activation experiments showed that TmY-ds neurons are functionally upstream of T4/T5.Our results suggest that direction selectivity is generated in a tripartite circuit formed among these three neurons—temporal filtering,TmY-ds,and T4/T5 neurons,in which TmY-ds plays a role in the enhancement of direction selectivity in T4/T5 neurons.

Novel glycine-dependent inactivation of NMDA receptors in cultured hippocampal neurons

Objective Glycine acts as a co-agonist for the activation of N-methyl-D-aspartate receptors （NMDARs） by binding to glycine sites, thus potentiating glutamate-elicited responses and inhibiting NMDAR desensitization in a dose-dependent manner. The present study aimed to characterize the glycine-dependent inactivation of NMDARs and to explore its pathophysiological significance. Methods Primary hippocampal cell cultures from embryonic days 17-18 rats were treated with NMDA or NMDA plus glycine. Patch-clamp recording and intracellular Ca 2＋ imaging were performed to test the effects of glycine on NMDA-activated currents and increase of intracellular free Ca 2＋ respectively. Immunofluorescence staining was conducted to examine NR1 internalization. Cell damage was tested with MTT method and lactate dehydrogenase leakage. Results Glycine reduced the peak current and Ca 2＋ influx elicited by NMDA application at concentrations ≥300 μmol/L. This is a novel suppressive influence of glycine on NMDAR function, since it occurs via the NMDAR glycine-binding site, in contrast to the classic suppression, which occurs through the binding of glycine to glycine receptors. The level of membrane NMDARs was measured to evaluate whether internalization was involved. Immunohistochemical labeling showed that incubation with high concentrations of NMDA plus glycine did not change the expression of NMDARs on the cell surface when compared to the expression without glycine; hence the possibility of NMDAR internalization primed by glycine binding was excluded. Conclusion In summary, the novel suppressive effect of glycine on NMDARs was mediated via binding to the glycine site of the NMDAR and not by activation of the strychnine-sensitive glycine-receptor-gated chloride channel or by the internalization of NMDARs. The inhibitory influence of glycine on NMDARs adds a new insight to our knowledge of the complexity of synaptic transmission.

Genetically encoded neural activity indicators

Recent years have witnessed the fascinating development of imaging approaches to studying neural activities; this progress has been based on an influx of ideas and methods from molecular biology and optical engineering. Here we review the design and application of genetically encoded indicators for calcium ions, membrane potential and neurotransmitters. We also summarize common strategies for the design and optimization of genetically encoded neural activity indicators.

Restoration of FMRP expression in adult V1 neurons rescues visual deficits in a mouse model of fragile X syndrome

Many people affected by fragile X syndrome(FXS)and autism spectrum disorders have sensory processing deficits,such as hypersensitivity to auditory,tactile,and visual stimuli.Like FXS in humans,loss of Fmr1 in rodents also cause sensory,behavioral,and cognitive deficits.However,the neural mechanisms underlying sensory impairment,especially vision impairment,remain unclear.It remains elusive whether the visual processing deficits originate from corrupted inputs,impaired perception in the primary sensory cortex,or altered integration in the higher cortex,and there is no effective treatment.In this study,we used a genetic knockout mouse model(Fmr1^(KO)),in vivo imaging,and behavioral measurements to show that the loss of Fmr1 impaired signal processing in the primary visual cortex(V1).Specifically,Fmr1^(KO) mice showed enhanced responses to low-intensity stimuli but normal responses to high-intensity stimuli.This abnormality was accompanied by enhancements in local network connectivity in V1 microcircuits and increased dendritic complexity of V1 neurons.These effects were ameliorated by the acute application of GABAA receptor activators,which enhanced the activity of inhibitory neurons,or by reintroducing Fmr1 gene expression in knockout V1 neurons in both juvenile and young-adult mice.Overall,V1 plays an important role in the visual abnormalities of Fmr1^(KO) mice and it could be possible to rescue the sensory disturbances in developed FXS and autism patients.

Integrative analysis of in vivo recording with single-cell RNA-seq data reveals molecular properties of light-sensitive neurons in mouse V1

Vision formation is classically based on projections from retinal ganglion cells(RGC)to the lateral geniculate nucleus(LGN)and the primary visual cortex(V1).Neurons in the mouse V1 are tuned to light stimuli.Although the cellular information of the retina and the LGN has been widely studied,the transcriptome profiles of single light-stimulated neuron in V1 remain unknown.In our study,in vivo calcium imaging and whole-cell electrophysiological patch-clamp recording were utilized to identify 53 individual cells from layer 2/3 of V1 as lightsensitive(LS)or non-light-sensitive(NS)by single-cell light-evoked calcium evaluation and action potential spiking.The contents of each cell after functional tests were aspirated in vivo through a patch-clamp pipette for mRNA sequencing.Moreover,the three-dimensional(3-D)morphological characterizations of the neurons were reconstructed in a live mouse after the whole-cell recordings.Our sequencing results indicated that V1 neurons with a high expression of genes related to transmission regulation,such as Rtn4r and Rgs7,and genes involved in membrane transport,such as Na+/K+ATPase and NMDA-type glutamatergic receptors,preferentially responded to light stimulation.Furthermore,an antagonist that blocks Rtn4r signals could inactivate the neuronal responses to light stimulation in live mice.In conclusion,our findings of the vivo-seq analysis indicate the key role of the strength of synaptic transmission possesses neurons in V1 of light sensory.

Synthesis, Crystal Structure and Insecticidal Activities of(4aR,7aS)-6-Benzyl-1-((R)-2-(2-chlorophenyl)-4,5-dihydrothiazole-4-carbonyl)hexahydro-5H-pyrrolo[3,4-b]pyridine-5,7(6H)-dione

The title compound(4aR,7aS)-6-benzyl-1-((R)-2-(2-chlorophenyl)-4,5-dihydrothiazole-4-carbonyl)-hexahydro-5H-pyrrolo[3,4-b]pyridine-5,7(6H)-dione(C_(24)H_(22)ClN_(3)O_(3)S) was synthesized, and its chemical structure was confirmed by 1H NMR, 13C NMR and single-crystal X-ray diffraction. The crystal of the title compound belongs to monoclinic system, space group C2/c with a = 28.929(6), b = 7.858(16), c = 22.936(5) A, β = 125.20(3)°, V = 4221.4(15) ?~3, Z = 8, D_(c) = 1.473 g/cm^(3), μ(MoKa) = 0.314 mm^(–1), the final R = 0.0629 and wR = 0.1708 for 2812 observed reflections with I > 2σ(I). The preliminary insecticidal activity indicated that the title compound exhibited good and promising insecticidal activities against Mythimna separata, Plutella xylostella and Culex pipiens pallens. Moreover, the calcium imaging experiment indicated that the title compound can activate intracellular calcium channels to release the stored calcium ion from endoplasmic reticulum(ER) to cytoplasm of Mythimna separata.