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få=îáîç images of the cerebral neocortex of an anesthetized mouse (modified from the progress report of the National Institute for Physiological Sciences, 2006). The superior tissue penetration of our newly constructed áå=îáîç two-photon microscopy can visualize neural activities in a living brain. EYFP fluorescence can be detected from layers deeper than 0.9 mm beneath the brain surface in an anaesthetized mouse. 3D-reconstruction of individual neural cells spreading in layers I-V is feasible without any degradation of the submicron spatial resolution.
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Laser light microscopy enables observation of various simultaneously occurring events in living cells. This capability is important for monitoring the spatiotemporal patterns of the molecular interactions underlying such events. Two-photon excited fluorescence microscopy (two-photon microscopy), a technology based on multiphoton excitation, is one...
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Botulinum neurotoxin type A (BoNT/A) is a major therapeutic agent that has been proven to be a successful treatment for different neurological disorders, with emerging novel therapeutic indications each year. BoNT/A exerts its action by blocking SNARE complex formation and vesicle release through the specific cleavage of SNAP-25 protein; the toxin...
Citations
... For detailed explanations, refer to other reviews (e.g. [3]). ...
Two-photon excitation fluorescence microscopy (two-photon microscopy) is a robust technique for understanding physiological phenomena from the cellular to tissue level, attributable to the nonlinear excitation process induced by near-infrared ultrashort laser light pulses. Recently, we have been promoting the use of semiconductor lasers, adaptive optics, vector beams, and nanomaterials to improve the observation depth or spatial resolution. The developed semiconductor based laser light source successfully visualized structure of EYFP-expressing neurons at the hippocampal dentate gyrus without resecting the neocortex, and neuronal activity at the hippocampal CA1 region in anesthetized mice at video rates. We also proposed using fluoropolymer nanosheets of 100-nm thickness for in vivo imaging and realized a wide field of view during anesthetized mouse brain imaging of 1-mm depth. Furthermore, the developed adaptive optical two-photon microscopy visualized single dendritic spines of EYFP-expressing neurons in cortical layer V of the secondary motor cortex, which had been difficult to observe due to the curvature of the brain surface. In addition, we combined two-photon microscopy and stimulated-emission depletion (STED) microscopy to improve spatial resolution. This combined microscopy is noninvasive and has a superior spatial resolution, exceeding the diffraction limit of the conventional light. In this review, we describe our recent results and discuss the future of two-photon microscopy.
... On the other hand, near-infrared excitations of visible fluorescent probes decrease absorption and scattering, resulting to superior penetration depth [7][8][9]. However, in conventional two-photon microscopy systems equipped with a generally used multi-alkaline photomultiplier tube (PMT) and a near-infrared Ti:Sapphire laser light source, observations of synaptic structures, somata or Ca 2+ activities were hardly performed in deeper layers than in cortical layer II/III, layer VI, or layer II/III, respectively [10][11][12]. In addition, the focal laser ablation for examining or manipulating functions of single neurons in local cortical networks was limited to cortical layer II/III at deepest [13][14][15]. ...
... In order to demonstrate such an advantage of condition E for in vivo deep imaging, we observed adult H-line mice through the cranial window (ϕ4.2 mm) implanted above the primary visual cortex. Under condition S, all cortical layers were visualized, except for hippocampal neurons, affirming consistency with previous reports (Fig 5A) [11,12]. By contrast, under condition E, not only hippocampi alveus but also hippocampal CA1 neurons were visualized with the normal multi-alkaline PMT detector (Fig 5B-5E). ...
In vivo two-photon microscopy utilizing a nonlinear optical process enables, in living mouse brains, not only the visualization of morphologies and functions of neural networks in deep regions but also their optical manipulation at targeted sites with high spatial precision. Because the two-photon excitation efficiency is proportional to the square of the photon density of the excitation laser light at the focal position, optical aberrations induced by specimens mainly limit the maximum depth of observations or that of manipulations in the microscopy. To increase the two-photon excitation efficiency, we developed a method for evaluating the focal volume in living mouse brains. With this method, we modified the beam diameter of the excitation laser light and the value of the refractive index in the immersion liquid to maximize the excitation photon density at the focal position. These two modifications allowed the successful visualization of the finer structures of hippocampal CA1 neurons, as well as the intracellular calcium dynamics in cortical layer V astrocytes, even with our conventional two-photon microscopy system. Furthermore, it enabled focal laser ablation dissection of both single apical and single basal dendrites of cortical layer V pyramidal neurons. These simple modifications would enable us to investigate the contributions of single cells or single dendrites to the functions of local cortical networks.
... La première technique de culture est la culture de tissu ex vivo. Une partie de l'embryon ou une « tranche » sont déposés dans un milieu de culture semi-solide et imagées au microscope confocal inversé (Figure 41 1 à 5) (Figure 41 6 à 12 (Fahrbach et al., 2013;Mahou et al., 2012;Nemoto, 2008;Truong et al., 2011;Zhao et al., 2014), et même récemment en neurologie (Piksarv et al., 2016) ou pour des tissus épais (Lavagnino et al., 2016). Ainsi, ma collaboration avec les membres de l'ILM a permis de mettre en place les premiers éléments physiques d'un microscope à façon afin de pouvoir visualiser des processus cellulaires au sein d'un embryon de poulet in-vivo. ...
La transition épithélio-mésenchymateuse (EMT) est un processus incontournable dans de nombreux contextes normaux et pathologiques, tels que gastrulation, organogenèse, fibroses et cancers. Cette transformation de cellule épithéliale en cellule mésenchymateuse est indissociable de l'acquisition de propriétés migratoires et est généralement associée à un changement de destin cellulaire. Différentes voies moléculaires sont impliquées selon le contexte de l'EMT concernée. Récemment, notre laboratoire a mis en évidence que la voie Notch cytoplasmique contrôle l'EMT des cellules de la lèvre dorso-médiale du somite (DML). Les crêtes neurales exprimant DLL1 activent « en passant » le récepteur NOTCH, liberant ainsi le domaine intra-cytoplasmique de NOTCH (NICD). Dans le cytoplasme, NICD inhibe la kinase GSK3ß, conduisant à la stabilisation de SNAIL, un gène maître de la transition épithélio-mésenchymateuse. Il en résulte une libération de la βcaténine des jonctions adhérentes qui, après translocation dans le noyau, active la transcription des gènes de la myogénèse (Myf5). Ainsi, l'activation de la voie Notch cytoplasmique permet une induction concomitante de l'EMT et de la myogénèse. La fonction cytoplasmique de Notch reste controversée et le mécanisme par lequel NICD inhibe GSK3ß reste obscur. Au cours de ma thèse j'ai cherché à élucider le mécanisme par lequel NICD inhibe l'activité kinase de GSK3ß. J'ai confirmé l'interaction de GSK3ß et de NICD en démontrant leur interaction via CoIP. Après avoir démontré l'implication de la sérine-thréonie kinase AKT dans la myogenèse des cellules de la DML, j'ai mis en évidence, via CoIP et électroporation, que l'inhibition GSK3ß par NICD est très certainement médiée par AKT, connue pour être impliquée dans l'EMT et inhiber GSK3ß par phosphorylation. En comparant le NICD1 de poulet et les 4 NICD de souris, j'ai montré que l'expression exogène de ces 5 molécules induit l'EMT et la différenciation myogénique de manière similaire. J'ai aussi montré que parmi des différents domaines de NICD, le domaine RAM, connu pour se lier à l'ADN (via RBPJ), est nécessaire et suffisant à l'inhibition de GSK3ß. Un second axe de ma thèse a été de tester l'implication de la voie Notch cytoplasmique dans d'autres contextes d'EMT. Pour ce faire, j'ai mis en évidence que cette voie est impliquée dans les autres lèvres du dermomyotome mais aussi dans les crêtes neurales qui délaminent du toit du tube neural. J'ai en particulier mis en évidence une co-activation des voies Wnt et Notch, une inhibition de la kinase GSK3ß par NICD cytoplasmique ainsi qu'une inhibition de la différenciation en présence d'une ß-caténine mutée, retenue à la membrane, ou en présence d'une molécule SNAIL2 dominant-négative. Le dernier axe de ma thèse a consisté à élucider le mécanisme de régulation de l'induction de l'EMT et de la myogenèse via l'activation de NICD. Il a été mis en évidence que toutes les cellules de la DML peuvent être activées via DLL1 et que la surexpression massive de NICD dans la DML provoque une différenciation massive et une déplétion du groupe de cellules progénitrices. Afin de déterminer si la régulation de cette initiation se fait avant ou après induction de NICD, j'ai créé un plasmide permettant de répondre à cette question et afin de visualiser son expression in vivo, j'ai initié une collaboration avec une équipe de l'ILM afin de créer un microscope vertical SPIM biphoton permettant l'observation d'embryon de poulets vivants [etc...]
... However, these imaging methods have limitations (i.e., limited spatial or temporal resolution), which prevent researchers from observing accurate modifications in individual neurons and synapses in the brain. To surmount these challenges, our group used state-ofthe-art imaging method; in vivo two-photon microscopy imaging with various fluorescent labeling techniques (Nemoto 2008;Holtmaat and Svoboda 2009) to observe activities of tens-hundreds neurons/glia and individual synaptic alterations present in the intact S1 cortex. ...
Tissue or nerve injury induces widespread plastic changes from the periphery and spinal cord up to the cortex, resulting in chronic pain. Although many clinicians and researchers have extensively studied altered nociceptive signaling and neural circuit plasticity at the spinal cord level, effective treatments to ameliorate chronic pain are still insufficient. For about the last two decades, the rapid development in macroscopic brain imaging studies on humans and animal models have revealed maladaptive plastic changes in the ‘pain matrix’ brain regions, which may subsequently contribute to chronic pain. Among these brain regions, our group has concentrated for many years on the primary somatosensory (S1) cortex with a help of advanced imaging techniques and has found the functional and structural changes in neurons/glia as well as individual synapses in the S1 cortex during chronic pain. Taken together, it is now believed that such S1 plasticity is one of the causes for chronic pain, not a simple and passive epiphenomenon following tissue/nerve injury as previously thought. In this small review, we discuss the relation of plasticity in the S1 cortex with chronic pain, based on clinical trials and experimental studies conducted on this field.
This article is part of the special article series “Pain” .
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... Botulinum neurotoxin C, which cleaved Syntaxin2 and (to a lesser extent) Syntaxin3, inhibited exocytotic amylase release in rat pancreatic acini (38). Syntaxin2 is the candidate factor in sequential exocytosis in the pancreatic exocrine gland (32), suggesting that Syntaxin2 is involved in the pancreatic amylase release. However, the regulation of SNAREs has not been clarified. ...
... However, the regulation of SNAREs has not been clarified. In the SNARE hypothesis, a complex formation between t-SNAREs and vesicle SNAREs is required for fusion pore opening in apical membranes (32). Considered together, these findings suggest that MARCKS displacement from the membrane lipid rafts might play a role in exocytosis via the regulation of SNAREs such as Syntaxin2. ...
Cholecystokinin (CCK) is a gastrointestinal hormone that induces exocytotic amylase release in pancreatic acinar cells. The activation of protein kinase C (PKC) is involved in the CCK-induced pancreatic amylase release. Myristoylated alanine-rich C kinase substrate (MARCKS) is a ubiquitously expressed substrate of PKC. MARCKS has been implicated in membrane trafficking in several cell types. The phosphorylation of MARCKS by PKC results in the translocation of MARCKS from the membrane to the cytosol. Here, we studied the involvement of MARCKS in the CCK-induced amylase release in rat pancreatic acini. Employing Western blotting, we detected MARCKS protein in the rat pancreatic acini. CCK induced MARCKS phosphorylation. A PKCδ inhibitor, rottlerin, inhibited the CCK-induced MARCKS phosphorylation and amylase release. In the translocation assay, we also observed CCK-induced PKCδ activation. An immunohistochemistry study showed that CCK induced MARCKS translocation from the membrane to the cytosol. When acini were lysed by a detergent, Triton X-100, CCK partially induced displacement of the MARCKS from the GM1a-rich detergent-resistant membrane fractions (DRMs) in which Syntaxin2 is distributed. A MARCKS-related peptide inhibited the CCK-induced amylase release. These findings suggest that MARCKS phosphorylation by PKCδ and then MARCKS translocation from the GM1a-rich DRMs to the cytosol are involved in the CCK-induced amylase release in pancreatic acinar cells.
... Technical advances in two-photon laser scanning microscopy and fluorescence labeling methods have enabled researchers to directly observe the structural and functional plastic changes in neuronal populations at single cell or individual synapse level [14,23,58]. The advantages of two-photon microscopy imaging are shown in Fig. 1a, compared to conventional single photon microscopy: (i) use of near-infrared laser (vs. ...
... out-focus excitation) enabling high resolution imaging with no requirement of confocal pinhole that causes some loss of emission signals. The latter two advantages that also contribute to less photodamage, together with the former advantage for deep tissue imaging, make the two-photon microscopy optimal for long-term in vivo imaging of neurons and synapses in the intact brain (for detailed explanations about the principle of two-photon microscopy in a biology field, see review articles [58][59][60]). ...
Damage in the periphery or spinal cord induces maladaptive plastic changes along the somatosensory nervous system from the periphery to the cortex, often leading to chronic pain. Although the role of neural circuit remodeling and structural synaptic plasticity in the 'pain matrix' cortices in chronic pain has been thought as a secondary epiphenomenon to altered nociceptive signaling in the spinal cord, progress in whole brain imaging studies on human patients and animal models has suggested a possibility that plastic changes in cortical neural circuits may actively contribute to chronic pain symptoms. Furthermore, recent development in two-photon microscopy and fluorescence labeling techniques have enabled us to longitudinally trace the structural and functional changes in local circuits, single neurons and even individual synapses in the brain of living animals. These technical advances has started to reveal that cortical structural remodeling following tissue or nerve damage could rapidly occur within days, which are temporally correlated with functional plasticity of cortical circuits as well as the development and maintenance of chronic pain behavior, thereby modifying the previous concept that it takes much longer periods (e.g. months or years). In this review, we discuss the relation of neural circuit plasticity in the 'pain matrix' cortices, such as the anterior cingulate cortex, prefrontal cortex and primary somatosensory cortex, with chronic pain. We also introduce how to apply long-term in vivo two-photon imaging approaches for the study of pathophysiological mechanisms of chronic pain.
... In this section, we show a newly constructed system for twophoton excitation STED (TP-STED) microscopy [9]. Twophoton excitation laser scanning microscopy (TPLSM) has several advantages [10,11]. First, excitation is spatially localized at the focus of the excitation laser light used in TPLSM, due to a non-linear dependence on photon density, which allows for the acquisition of optically sectioned fluorescence images. ...
... In general, the TPLSM system utilizes a non-descanned detector which collects the emitted light without passing a confocal pinhole [10,11]. Therefore, its spatial resolution corresponds to the square values of the intensity distribution around the focal pattern of an excitation light. ...
... We confirmed that the same tLCDs enabled both 473 and 577 nm laser beams to be converted into optical vortices [9]. One feature of TPLSM is that various fluorescent dyes can be excited simultaneously using a single wavelength NIR light [11,21]. Thus, combined with a wavelength-tunable light source for STED processes, this methodology might allow for super-resolution microscopy to visualize multiple components in living specimens using a variety of fluorophores. ...
One of the most popular super-resolution microscopies that breaks the diffraction barrier is stimulated emission depletion (STED) microscopy. As the optical set-up of STED microscopy is based on a laser scanning microscopy (LSM) system, it potentially has several merits of LSM like confocal or two-photon excitation LSM. In this article, we first describe the principles of STED microscopy and then describe the features of our newly developed two-photon excitation STED microscopy. On the basis of our recent results and those of other researchers, we conclude by discussing future research and new technologies in this field.
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... Thus, two-photon excitation laser scanning microscopy (TPLSM) has become an essential analytical method in life science and medical research. [2][3][4][5][6][7][8] Since most TPLSM systems employ a single-point laser scanning method using moving mirrors, their temporal resolution primarily depends on the speed of the physical movement of these mirrors. By utilizing resonant mirrors, video-rate scanning TPLSM has been achieved, 9 though the image quality was compromised for greater temporal resolution. ...
The temporal resolution of a two-photon excitation laser scanning microscopy (TPLSM) system is limited by the excitation laser beam's scanning speed. To improve the temporal resolution, the TPLSM system is equipped with a spinning-disk confocal scanning unit. However, the insufficient energy of a conventional Ti:sapphire laser source restricts the field of view (FOV) for TPLSM images to a narrow region. Therefore, we introduced a high-peak-power Yb-based laser in order to enlarge the FOV. This system provided three-dimensional imaging of a sufficiently deep and wide region of fixed mouse brain slices, clear four-dimensional imaging of actin dynamics in live mammalian cells and microtubule dynamics during mitosis and cytokinesis in live plant cells.
... On the other hand, a two-photon excitation laser scanning fluorescence microscope (TPLSM) is widely used because it has several advantages [8]. First, excitation is spatially localized at the focus of the excitation laser light used in TPLSM due to non-linear dependence on photon density, which allows for acquiring optically sectioned fluorescence images. ...
... This methodology achieves super-resolution microscopy more readily. Another feature of TPLSM is that various fluorescent dyes can be excited simultaneously using a single laser [8,23]. Thus, combined with a wavelength tunable light source for STED, this methodology might provide for super-resolution microscopy to visualize multiple components in living specimens by using a variety of fluorophores. ...
Transmissive liquid crystal devices (tLCDs) enable the modification of optical properties, such as phase, polarization, and laser light intensity, over a wide wavelength region at a high conversion efficiency. By utilizing tLCDs, we developed a new two-photon excitation stimulated emission depletion microscopy technique based on a conventional two-photon microscope. Spatial resolution was improved by compensating for phase shifts distributed in the optical path. Using this technique, we observed the fine structures of microtubule networks in fixed biological cells.
... Recently, the improved performance of microscopy systems enables the acquisition of large amounts of slice images from living tissues. In comparison with confocal or other optical microscopy systems, two-photon microscopy has an advantage in visualizing the morphology of neurons within deeper layers of living mouse brain [3][4][5][6]. Since the structures of tissues are stored as volume data, volume visualization techniques [7,8] are focused on the interactive exploration of the 3D images. ...
... These data capture a tomogram with a depth close to 1.4 mm from the surface layer of the cortex [4], which, to the best of our knowledge, is the deepest visualized layer that has been obtained. This is important because the visualization of these deep layers have applications in the biological and medical fields. ...
Background
Dendrites of cortical neurons are widely spread across several layers of the cortex. Recently developed two-photon microscopy systems are capable of visualizing the morphology of neurons within deeper layers of the brain and generate large amounts of volumetric imaging data from living tissue.
Method
For visual exploration of the three-dimensional (3D) structure of dendrites and the connectivity among neurons in the brain, we propose a visualization software and interface for 3D images based on a new transfer function design using volume rendered feature spaces. This software enables the visualization of multidimensional descriptors of shape and texture extracted from imaging data to characterize tissue. It also allows the efficient analysis and visualization of large data sets.
Results
We apply and demonstrate the software to two-photon microscopy images of a living mouse brain. By applying the developed visualization software and algorithms to two-photon microscope images of the mouse brain, we identified a set of feature values that distinguish characteristic structures such as soma, dendrites and apical dendrites in mouse brain. Also, the visualization interface was compared to conventional 1D/2D transfer function system.
Conclusions
We have developed a visualization tool and interface that can represent 3D feature values as textures and shapes. This visualization system allows the analysis and characterization of the higher-dimensional feature values of living tissues at the micron level and will contribute to new discoveries in basic biology and clinical medicine.
