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Dual-objective two-photon microscope for volumetric imaging of dense scattering biological samples by bidirectional excitation and collection

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Photonics Research 2024年第6期12卷 1351-1361页

作者： MUyuE ZHAI jing yu YANHUI HU HANG yu BEIchen XIE YI yu DAWEI LI AIMIN WANG HEPING chenG National biomedical Imaging Center College of Future TechnologyPeking UniversityBeijing 100871China Research Unit of Mitochondria in Brain Diseases Chinese Academy of Medical SciencesPKU-Nanjing Institute of Translational MedicineNanjing 211500China beijing Transcend Vivoscope Biotech Beijing 100085China College of Engineering Peking UniversityBeijing 100871China Academy for Advanced Interdisciplinary Studies Peking UniversityBeijing 100871China Center for Quantitative Biology Peking UniversityBeijing 100871China School of Electronics Peking UniversityBeijing 100871China State Key Laboratory of Advanced Optical Communication System and Networks Peking UniversityBeijing 100871China

Full view observation throughout entire specimens over a prolonged period is crucial when exploring the physiological functions and system-level ***-photon microscopy(MPM)has been widely employed for such purposes owing to its deep penetration ***,the current MPM struggles with balancing the imaging depth and quality while avoiding photodamage for the exponential increasement of excitation power with the imaging ***,we present a dual-objective two-photon microscope(Duo-2P),characterized by bidirectional two-photon excitation and fluorescence collection,for long-duration volumetric imaging of dense scattering ***-2P effectively doubles the imaging depth,reduces the total excitation energy by an order of magnitude for samples with a thickness five times the scattering length,and enhances the signal-to-noise ratio up to 1.4 *** these advantages,we acquired volumetric images of a 380-μm suprachiasmatic nucleus slice for continuous 4-h recording at a rate of 1.67 s/volume,visualized the calcium activities over 4000 neurons,and uncovered their state-switching *** conclude that Duo-2P provides an elegant and powerful means to overcome the fundamental depth limit while mitigating photodamages for deep tissue volumetric imaging.

关键词： excitation overcome exponential

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Imbalance between excitatory and inhibitory synaptic transmission in the primary somatosensory cortex caused by persistent nociception

Imbalance between excitatory and inhibitory synaptic transmi...

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中国生理学会第23届全国会员代表大会暨生理学学术大会

作者： Fa-Le CAO~1,Rui-Rui WANG~1,jun chen~(1,2,*) 1 institute for biomedical sciences of pain and institute for Functional Brain Disorders,Tangdu Hospital,Fourth Military medical university,Xi’an 710038,china 2 institute for biomedical sciences of pain,capital medical university,beijing 100069,china

Objective:There is substantial evidence supporting the notion that the primary somatosensory cortex (S1) is an important structure involved in the perceptional component of ***,neuronal circuit and synaptic plasticity occurring within the S1 after injury has been proposed to contribute substantially to the pathophysiogenesis of chronic ***,few reports have been conducted to simultaneously investigate synaptic plasticity in the S1 in response to persistent pain at cellular and network *** present study was thus undertaken to evaluate,at the local network level,potential changes in synaptic connection and transmission in the S1 following peripheral persistent nociception in ***:Periphery subcutaneous injection of bee venom (BV) was used to mimic the state of persistent *** initial probe of underlying mechanisms was attempted at the neuronal circuit level by using multi-electrode array recording(MED-64).In electrophysiological experiments, typical neocortical neurons characterized by morphology and action potential were applied to obtain the spontaneous and miniature excitatory and inhibitory post-synaptic currents(EPSC/IPSC).Furthermore,immunohistochemistry and Western blotting were performed to investigate the changes of AMPA and GABA receptors in the state of peripheral persistent ***:By using MED-64 coupled with two-dimensional current source density imaging, we found that persistent pain produced enlargement of synaptic connection size in the S1 slice,reflecting increased spatial plasticity by ***,compared with saline control,significant increase was also found in the temporal plasticity,namely long-term *** slice patch clamp recordings on identified S1 neurons demonstrated that both of the amplitude and frequency of spontaneous and miniature EPSC were greatly increased, while the amplitude of spontaneous and miniature IPSC was markedly decreased after BV ***,the half-width

关键词： primary somatosensory cortex synaptic plasticity AMPA receptor GABA receptor bee venom test

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Activation of peripheral ERKs pathway is involved in melittin-induced pain-related behaviors and hyper-responsiveness of spinal cord dorsal horn sensory neurons in the rat

Activation of peripheral ERKs pathway is involved in melitti...

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中国神经科学学会第四次会员代表大会暨第七届全国学术会议

作者： Yao-Qing yu~1,jun chen~(1,2) 1 institute for biomedical sciences of pain,Tangdu Hospital,Fourth Military medical university,Xi''an 710038,china 2 institute for biomedical sciences of pain,capital medical university,beijing 100069,china

＜正＞Objective To investigate the roles of peripheral extracellular signaling-regulated kinases 1/2（ERKs） pathway in melittin-induced pain-related behaviors and responses of spinal wide-dynamic-range（WDR） neurons,a population of "central encoder" along the spinally-organized sensorimotor（SOSM） reflex *** In behavioral experiments, persistent spontaneous nociceptive behavioral response of rats was determined by counting the number of paw flinches during each 5 min interval for 1 h following *** *** and mechanical hypersensitivity were measured with TC-1 radiant heat stimulator and ascending graded von Frey monofilaments,*** electrophysiological experiments, typical WDR neurons characterized by increased spike firing rate to brush,pressure and noxious pinch were applied to the peripheral receptive ***（1） Pre-treatment with UOl 26（a specific MAPK kinase inhibitor,0.1,1.0,10μg） resulted in dose-related suppressions to melittin-induced persistent paw flinches（8%-,34%,42%） and heat hypersensitivity（26%. 33%and 31%）,but without any effectiveness on mechanical hypersensitivity.（2） Post-treatment of U0126（10μg） resulted in 51%suppressive effect on paw finches and 39%reversal effect on heat hypersensitivity,but without any effectiveness on mechanical hypersensitivity.（3） Treatment of U0l26（1.0μg） suppressed the tonic firing and enhanced responsiveness to heat stimulus in WDR neurons,but without any effectiveness on the increased neuronal responsiveness to either innocuous or noxious mechanical stimuli.（4） The possible systemic effect of U0126 was ruled out by intraplantar injection of U0126(10（ig） into the contralateral hind *** Peripheral ERKs pathway might be involved in pathophysiological processing associated with melittin-induced facilitation and sensitization of SOSM reflex circuitry by enhancing the ability of spinal dorsal horn WDR neurons in response to peripheral noxious thermal but not mechanical stim

关键词： ERKs melittin wide-dynamic-range neuron electrophysiology pain hypersensitivity

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Influence of expressed TRAIL on biophysical properties of the human leukemic cell line Jurkat

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Cell Research 2004年第2期14卷 161-168页

作者： Kai chen~(1,*), Dan LI~(1,*), yu Hui JIANG~1, Wei Juan YAO~1, Xin Juan WANG~2, Xiao Chao WEI~2, jing GAO~3, Li De XIE~1, Zong Yi YAN~4, Zong Yao WEN~(1**), Shu CHIEN~5 ~1Hemorheology Center, Department of Biophysics, School of Basic medical sciences, Peking university, beijing, 100083, china. ~2Department of Biochemical and Molecular Biology, School of Basic medical sciences, Peking university, beijing, 100083, china. ~3Department of Urology, medical Center, New York university, USA. ~4Department of Mechanics and Engineering Science, Peking university, beijing 100871, china. ~5Department of Bioengineering and Whitaker institute of biomedical Engineering, university of California, San Diego, La Jolla, CA 92093-0412, USA 1Hemorheology Center Department of Biophysics School of Basic Medical Sciences Peking University Beijing 100083 China. 1Hemorheology Center Department of Biophysics School of Basic Medical Sciences Peking University Beijing 100083 China. 1Hemorheology Center Department of Biophysics School of Basic Medical Sciences Peking University Beijing 100083 China. 1Hemorheology Center Department of Biophysics School of Basic Medical Sciences Peking University Beijing 100083 China. 2Department of Biochemical and Molecular Biology School of Basic Medical Sciences Peking University Beijing 100083 China. 2Department of Biochemical and Molecular Biology School of Basic Medical Sciences Peking University Beijing 100083 China. 3Department of Urology Medical Center New York University USA. 1Hemorheology Center Department of Biophysics School of Basic Medical Sciences Peking University Beijing 100083 China. 4Department of Mechanics and Engineering Science Peking University Beijing 100871 China. 1Hemorheology Center Department of Biophysics School of Basic Medical Sciences Peking University Beijing 100083 China. 5Department of Bioengineering and Whitaker institute of biomedical Engineering University of California San Diego La Jolla CA 92093-0412 USA

The cDNA fragment of human TRAIL (TNF-related apoptosis inducing ligand) was cloned into RevTet-On, a Tetregulated and high-level gene expression system. The gene expression system was constructed in a human leukemic cell line: Jurkat. By using RevTet-On TRAIL gene expression system in Jurkat as a cell model, we studied the influence of TRAIL gene on the changes of cellular apoptosis before and after the TRAIL gene expression, which was induced by adding tetracycline derivative doxycycline (Dox). The results indicated that the cellular apoptosis ratio was largely dependent on the TRAIL gene expression level. Moreover, it was found that the apoptosis-inducing TRAIL could cause significant changes in the biophysical properties of Jurkat cells. The cell surface charge density decreased, the membrane fluidity declined, the elastic coefficients K_I increased, and the proportion of α-helix in membrane protein secondary structure decreased. Thus, the apoptosis-inducing TRAIL gene caused significant changes on

关键词： TRAIL Tet gene expression system Jurkat apoptosis.

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Altered locomotor and exploratory behaviors of rats caused by subcutaneous bee venom injection:open field test

Altered locomotor and exploratory behaviors of rats caused b...

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中国生理学会第23届全国会员代表大会暨生理学学术大会

作者： Dan LU~1,jun chen~(1,2,*) 1 institute for biomedical sciences of pain,capital medical university,beijing 100069,china 2 institute for biomedical sciences of pain and institute for Functional Brain Disorders,Tangdu Hospital,Fourth Military medical university,Xi''an 710038,china

＜正＞Background:pain is usually considered to be a complex phenomenon,which requires the scoring of several behavioral *** pathological state,persistent or chronic pain can affect various higher brain functions and lead to emotional *** open field test is usually used to study the emotional state,the effect of various pharmacological agents,biological/physical environmental factors,and the impact of chronic pain on emotional ***,the spontaneous behavioral responses induced by peripheral persistent inflammatory pain in the open field were rarely ***:In this report,we investigated the altered behavioral responses induced by hind-paw bee venom（BV） injection and the relationship between pain-related behaviors and spontaneous responses in the open ***:Male Sprague-Dawley albino rats were randomly divided into four groups:naive,saline,BV-inflamed,bupivacaine-pretreated BV-inflamed *** the open field test,the spontaneous behavioral responses of the rats in these four groups were clearly and long-term ***: It was found that BV-induced persistent nociception can reduce the locomotor and exploratory behavioral responses. The BV-inflamed group also has a significantly increased traveled distance in four corners and decreased traveled distance in center and four *** addition,these altered spontaneous pain-related responses in open field can be reversed by ***:We demonstrated that bee venom induced persistent nociception can elicit alteration of spontaneous behavioral responses in open field which is relevant to pain-related behavior directly.

关键词： open field test spontaneous behavioral responses persistent nociception bee venom test

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Bee venom-induced changes in expression of NMD A receptor subunits in spinal cord,SI area and hippocampus of the rat:an immunohistochemical study

Bee venom-induced changes in expression of NMD A receptor su...

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中国神经科学学会第四次会员代表大会暨第七届全国学术会议

作者： Fu-Kang ZHANG~1,jun chen~(1,2) 1 institute for biomedical sciences of pain,capital medical university,beijing 100069,china 2 institute for biomedical sciences of pain,Tangdu Hospital,Fourth Military medical university,Xi''an 710038,china

＜正＞Using tissue immunohistochemistry technique,the present study investigated the effects of subcutaneous（s.c.） bee venom（BV） administration on the expression of three major NMDA receptor subunits（NR1,NR2A,NR2B） in rat lumbar spinal cord,S1 area and *** to *** injection（100 ug/50 ul） into the hind paw,the animal exhibited a typical behavioral expression of persistent pain and hyperalgesia,whereas *** of equal volume of normal saline produced little *** coronal sections of lumbar spinal cord and brain were sequentially prepared at 4 h after BV or saline *** was then performed and positive labeling was visualized by treatment with diaminobenzidine（DAB） and hydrogen *** of NMDA receptor subunits expression were finished by comparisons between two sides of the hindpaw or comparisons between B V-treated and saline-treated groups. As shown in our results,expression of the NR2A subunit of NMDA receptor was significantly exaggerated in ipsilateral lumbar spinal cord,S1 area and hippocampus following *** but not saline *** significant changes were detected in the expression of NR1 and NR2B subunits in each ***,the number of NR2A-positive dendric tree also markedly increased in ipsilateral hippocampus of BV-treated ***,these data suggest that the NR2A subunit of NMDA receptor might behave as a candidate molecular detector in mediating nociceptive signal processing in the lumbar spinal cord,S1 area and hippocampus following BV-induced persistent pain and *** is also speculated that BV-evoked augmentation of NR2A-positive dendric tree in the hippocampus may bear some associative relationships with the synaptic plasticity occurred in this area.

关键词： immunohistochemistry NMDA receptor subunit spinal cord S1 area hippocampus bee venom

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Distinguishable roles of tonic and phasic DRG neurons by regulating NaV1.8 and NaV1.9 and their distinct contributions to inflammatory pain

Distinguishable roles of tonic and phasic DRG neurons by reg...

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中国神经科学学会第四次会员代表大会暨第八届全国学术会议

作者： Yao-Qing yu~1 Feng ZHAO~1 jun chen~(1 2 *) 1 institute for biomedical sciences of pain and institute for Functional Brain Disorders Tangdu Hospital Fourth Military medical university Xi’an 710038 2 institute for biomedical sciences of pain capital medical university beijing 100069

＜正＞Objective According to the different firing properties to a long depolarizing current pulse,two classes of neurons can be identified in the dorsal root ganglion（DRG）.Tonic firing neuron generates multiple action potentials（Aβs）, whereas phasic firing neuron fires a few Aβ***,it is still unclear which type of the two neurons is really involved in mediation of pain *** this report,we identified the functional roles of the two types of neurons by gene regulation of TTX-resistant sodium channels,NaV1.8 and NaV1.9,which have been demonstrated to be differentially expressed in tonic and phasic *** Complete freund’s adjuvant（CFA） was used to induce peripheral inflammatory *** cell patch-clamp recording was used to identify tonic and phasic types of DRG ***-PCR, Western blot and immunocytochemistry were used to evaluate the expression levels of NaV1.8 and *** （1） Under CFA-induced inflammatory pain state（heat and mechanical hypersensitivity）,electrophysiological properties of tonic and phasic neurons were compared and it was found that tonic neurons were with decreased rheobase and increased number of spikes,while phasic type showed no changes in excitability at *** membrane potential was altered in both types of neurons by CFA.（2） RT-PCR,Western blot and immunocytochemical staining showed parallel increase in gene and protein expression level of both NaV1.8 and NaV1.9,suggesting up-regulation of TTX-resistant sodium channels in small to medium-sized DRG cells by CFA.（3） Down-regulation of NaV1.8 and NaV1.9 in DRG cells was established, respectively,by intrathecal（i.t.） administration of antisense oligonucleotides that was confirmed by both gene and protein expression *** CFA-induced decrease in rheobase and increase in spike discharges of tonic neurons were completely restored by down-regulation of NaV 1.8,while electrophysiological properties of phasic neurons were not affected by either NaV 1.8

关键词： tonic type of DRG neuron phasic type of DRG neuron inflammatory pain hypersensitivity TTX-resistant sodium channels NaV 1.8 NaV 1.9

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Metabotropic glutamate receptor subtype 1 and 5 mediate opposite effects on pain-related spatial and temporal synaptic plasticity in rat hippocampal formation:Multi-electrode array recordings

Metabotropic glutamate receptor subtype 1 and 5 mediate oppo...

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中国神经科学学会第九届全国学术会议暨第五次会员代表大会

作者： Yan WANG~1,Ming-Gang LIU~2,Dan LV~2,Rui-Rui WANG~1,Xue-Feng chen~1,Fu-Kang ZHANG~2,xiu-yu cui~2,jun chen~(1,2,*) 1 institute for biomedical sciences of pain and institute for Functional Brain Disorders,Tangdu Hospital,The Fourth Military medical university,Xi''an 710038,china 2 institute for biomedical sciences of pain,capital medical university,beijing 100069,china

＜正＞Objective It has been revealed that peripheral persistent nociception results in spatial and temporal synaptic plasticity in rat hippocampal formation（HF）,however,the underlying mechanisms are still *** the present report,we present a line of evidence implicating opposing roles of metabotropic glutamate receptor（mGluR） subtype 1 and 5 in mediation of pain-related spatial and temporal synaptic plasticity in the *** Inhibitors targeting mGluRl and mGluR5 were used,*** and temporal components of entorhinal-hippocampal synaptic connections and efficacy of rats were recorded by using a newly developed 64-channels（8×8）multi-electrode array technique（Med64 system）.Results（1）Pharmacological inhibition of mGluR5 activation,at the plateau phase of the long-term potentiation（LTP）,effectively decreased pain-enhanced hippocampal long-term potentiation（LTP） maintenance,while similar blockade of mGluRl resulted in an even higher level of potentiation.（2）The mGluR subtype-related difference was also detected when analyzing effects of these two drugs on the spatial amplification of pain,with no clear involvement of mGluR5 and a negative modulation exerted by *** These findings suggest differential and even contradictory roles of mGluRl and mGluR5 in pain-associated spatial and temporal plasticity of synaptic *** importantly,this maybe the first demonstration for a subtype-linked functional separation/opposition regarding roles of group I mGluRs in higher levels of abnormal pain processing in the HF.

关键词： persistent pain hippocampal formation synaptic plasticity multi-electrode array metabotropic glutamate receptor

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Differential roles of ERK,JNK and p38 MAPK in pain-related spatial and temporal enhancement of synaptic responses in the hippocampal formation of rats:Multi-electrode array recordings

Differential roles of ERK,JNK and p38 MAPK in pain-related s...

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中国神经科学学会第九届全国学术会议暨第五次会员代表大会

作者： Rui-Rui WANG,Ming-Gang LIU,Xue-Feng chen,Fu-Kang ZHANG,xiu-yu cui,jun chen 1 institute for biomedical sciences of pain and institute for Functional Brain Disorders,Tangdu Hospital,The Fourth Military medical university,Xi''an 710038,china 2 institute for biomedical sciences of pain,capital medical university,beijing 100069,china

＜正＞Objective It is known that chronic pain affects various higher brain functions including perception, emotion,cognition,and ***,few studies have been performed to examine pain-induced synaptic plastic changes in the hippocampal formation（HF）,an important region subserving affective-motivational component of *** previous study has revealed a strong impact of peripheral persistent nociception on synaptic connection, transmission and function in the HF of rats,"in both temporal and spatial ***,the underlying signaling mechanisms for this pain-related spatial and temporal plasticity are still less *** present study attempted to examine potential different roles of the mitogen-activated protein kinase（MAPK）members in mediating this plastic *** Inhibitors targeting extracellular signal-regulated kinase（ERK）,p38 MAPK and c-jun N-terminal kinase（JNK）were used *** and temporal components of entorhinal-hippocampal synaptic connections and efficacy of rats were recorded by using a newly developed 64-channels（8x8）multielectrode array technique（Med64 system）.Results（1）Pharmacological inhibition of the ERK-and JNK-mediated signaling pathway,at the plateau phase of the long-term potentiation（LTP）,significantly decreased pain-enhanced LTP maintenance whereas similar blockade of p38 MAPK pathway dramatically further increased the potentiation. （2）Regarding the spatial magnification of pain,ERK and p38 MAPK seemed to play opposing roles,with the former positively involved and the latter negatively involved,without any detectable effect of the JNK signaling pathway. Conclusion Together,these results suggest differential roles of the specific members of the MAPK family in mediating pain-associated spatial and temporal plasticity in the HF,which are in good agreement with previous observations.

关键词： persistent pain hippocampal formation synaptic plasticity multi-electrode-array mitogen-activated protein kinases

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Effects of peripheral nociception on neural activities of motor cortex in rats:in vivo multi-channel recordings

Effects of peripheral nociception on neural activities of mo...

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中国生理学会第23届全国会员代表大会暨生理学学术大会

作者： Chang-Ming WANG~1,Lei YANG~1,jun chen~(1,2,*) 1 institute for biomedical sciences of pain,capital medical university,beijing 100069,china 2 institute for biomedical sciences of pain and institute for Functional Brain Disorders,Tangdu Hospital,Fourth Military medical university,Xi''an 710038,china

＜正＞Objective:The present study was designed to investigate the effects of subcutaneous saline or bee venom injection on responses of the motor cortical neurons and those of behaviors in conscious,behaving ***:We examined the motor cortical neuronal activation with an in vivo multi-channel recording technique before and after saline or bee venom was delivered subcutaneously（s.c.） into the plantar surface of the hind paw contralateral to the recording ***:A total of 81 single neurons were recorded in the motor *** first analyzed the spatial sequence *** saline-induced phasic pain state,29（36%） neurons showed significant excitatory responses,whereas 19（23%） displayed significant inhibitory *** bee venom-induced tonic pain state,52（64%） indicated significant excitatory responses,however,7（9%） showed inhibitory *** then analyzed the temperoal sequence *** terms of saline-induced phasic nociception,a sharp increase of spike discharges at the start of saline injection was *** terms of bee venom-induced tonic pain state,the increase of spike discharges was during the initial half hour after *** venom ***:The present result,a microcosmic view of the brain pain-related responses,accorded well with the results of neuroimaging studies,a macroscopic view of the brain pain-related responses,indicating the role of the motor cortex in the information processing of pain.

关键词： bee venom neural ensemble coding pattern tonic nociception phasic nociception motor cortex

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