Published papers

The dopaminergic system is essential for the function of the brain in health and disease. Therefore, detailed studies focused on unraveling the mechanisms involved in dopaminergic signaling are required. However, the lack of probes that mimic dopamine in living tissues, owing to the neurotransmitter’s small size, has hampered analysis of the dopaminergic system. The current study aimed to overcome this limitation by developing alkyne-tagged dopamine compounds (ATDAs) that have a minimally invasive and uniquely identifiable alkyne group as a tag. ATDAs were established as chemically and functionally similar to dopamine and readily detectable by methods such as specific click chemistry and Raman scattering. The ATDAs developed here were verified as analogue probes that mimic dopamine in neurons and brain tissues, allowing the detailed characterization of dopamine dynamics. Therefore, ATDAs can act as safe and versatile tools with wide applicability in detailed studies of the dopaminergic system. Furthermore, our results suggest that the alkyne-tagging approach can also be applied to other small-sized neurotransmitters to facilitate characterization of their dynamics in the brain.

Visualization and measurement of drugs themselves as well as biological responses to those drugs are crucial in pharmacological research. To this end, various fluorescent dyes and proteins have been developed. Despite such progresses, there still remains technical difficulties to overcome in bioimaging that keep many pharmacological targets and phenomena invisible. Outside the fields of biology where fluorescence and luminescence prevail, variety of other optical phenomena are well known and utilized. These optical phenomena can shed unique lights on biological phenomena based on their specific physical and chemical properties. Although applications of these optical phenomena to biology are yet to be explored, they have high potentials in realizing visualization and measurement of currently invisible targets and phenomena, and thereby bringing new insights into pharmacological research. Thus, here I will introduce Raman scattering microscopy that visualize vibration of functional groups as an alternative imaging platform to fluorescence and luminescence. Special focus will be put on two recent technical advancements; namely, nonlinear Raman scattering microscopy that utilizes multi-photon effect of highly tissue penetrating near-infrared lights, and Raman-tag that realizes tagging of targets that could not have been labeled, combination of which is expected to pave a way toward imaging previously invisible targets in pharmacology.

Aquaporin-4 (AQP4) is a predominant water channel in the central nervous system. It regulates water movement in the brain and has been suggested to play critical roles in various pathological conditions. However, the molecular mechanisms underlying its regulation are not yet well understood. In this study, we biochemically characterized AQP4 in the brain using acute cortical brain slices prepared from mice. Using biochemical fractionation, we found that AQP4 is enriched in the detergent-resistant membrane (DRM) fraction that is not soluble in 1% Triton X-100. In contrast, β-dystroglycan and syntrophin, which are part of the dystrophin complex in the brain, primarily reside in the non-DRM fraction. DRM enrichment of AQP4 is insensitive to cholesterol depletion, suggesting that it is not tightly associated with lipid rafts. Furthermore, AQP4 in the DRM fraction is more enriched in the M23 isoform than in the non-DRM fraction. Finally, by employing oxygen-glucose deprivation (OGD), an in vitro model of ischemia, we examined the molecular changes of AQP4. Under OGD conditions, a reduction in AQP4 in the DRM fraction was observed before the total AQP4 protein level dropped. Our data therefore highlight the characteristics of two pools of AQP4 that are distinctly regulated under ischemic conditions.

The consensus for the precise mechanism of action of general anesthetics is through allosteric interactions with GABA receptors in neurons. However, it has been speculated that these anesthetics may also interact with the plasma membrane on some level. Owing to the small size of anesthetics, direct visualization of these interactions is difficult to achieve. We demonstrate the ability to directly visualize a deuterated analog of propofol in living cells using stimulated Raman scattering (SRS) microscopy. Our findings support the theory that propofol is highly concentrated and interacts primarily through non-specific binding to the plasma membrane of neurons. Additionally, we show that SRS microscopy can be used to monitor the dynamics of propofol binding using real-time, live-cell imaging. The strategy used to visualize propofol can be applied to other small molecule drugs that have been previously invisible to traditional imaging techniques

Neuromyelitis optica spectrum disorder (NMOSD) is characterized by the production of autoantibodies against aquaporin 4 (AQP4). Because NMOSD progressively causes irreversible and severe neurological damages, understanding the initial molecular changes induced by anti-AQP4 antibody binding is crucial for designing early interventions. However, knowledge about the effects of the antibodies before AQP4 loss in brain tissues is limited. Using acutely prepared mouse brain slices, we aimed to investigate the initial molecular impact of NMO model antibodies on AQP4 and its associated proteins. We employed two different types of NMO model antibodies, E5415A and E5415B; E5415A recognizes both M1 and M23 isoforms, whereas E5415B exclusively binds to M23. We found that E5415A but not E5415B disrupted the uniform perivascular localization of AQP4, leading to fragmentation. We further addressed the impact of these changes on AQP4-associated proteins and found that strong colocalizations between AQP4 and dystrophin-glycoprotein complex (DGC) components were preserved, even after AQP4 localization pattern became fragmented. Thus, our study reveals the initial molecular changes in the AQP4 channel at the astrocytic endfeet in response to NMO model antibodies and highlights the early pathological events occurring in NMOSD.

The parametric down-conversion process in optical parametric generators causes bunching of light due to ultrafast intensity fluctuations, which enhances the efficiency of nonlinear interactions between light and matter. However, the bunching effect in a sufficiently intense light pulse light required for biological nonlinear imaging has not yet been investigated. We demonstrate enhanced two-photon excited fluorescence by ultrafast fluctuations in intense pulse using a wavelength-tunable optical parametric generator consisting of a periodically poled lithium niobate crystal pumped by nanosecond pulses at a wavelength of 532 nm and emitting pulses with a peak power of about 1 kW. The emission wavelength is tuned to about 927 nm, which is optimal for two-photon excitation of green fluorescent protein. The effect of bunching by ultrafast intensity fluctuations in the pulse is evaluated by an autocorrelator using a green fluorescent protein solution as a two-photon absorber. We found an about 1.9-fold enhancement compared with the coherent state of light. Using this calibrated optical parametric generator, we perform two-photon imaging of green fluorescent protein in brain tissue within a timescale of seconds. These experimental results using intense pulses demonstrate that the bunching effect by ultrafast fluctuations can enhance nonlinear imaging in biology and medicine.

Cells within the brain are highly organized and coordinate complex processes with each other. The ability to simultaneously visualize the organization and interactions of cells and molecules within brain tissue remains an important issue to understand the brain comprehensively. Stimulated Raman scattering (SRS) and fluorescence, two powerful imaging modalities, can provide complementary molecular contrasts within cells and tissue samples. Here, we present a high-speed super-multiplex imaging platform that combines SRS microscopy with confocal fluorescence microscopy to perform rapid 7-color brain imaging. We show simultaneous imaging of cellular components within the brain such as astrocytes, axons, and blood vessels while also showing organelles such as the nucleus and actin cytoskeleton. Also, we demonstrate the ability to take depth-resolved images that elucidate the three-dimensional organization of diverse components within brain tissue. This platform can be adapted to explore various processes within brain tissue that can reveal critical information about the brain and how it is affected by diseases, which leads toward a deeper understanding of disease progression and potentially the development of therapeutic options for brain diseases.

Neuronal primary cilia play important roles in brain development, sensory perception, and neurogenesis. Rootletin, a fibrous protein composed of coiled-coil motifs, is a key structural component of ciliary rootlets and is essential for understanding ciliary functions. However, the precise mechanisms by which Rootletin influences ciliary dynamics and impacts neuronal function remain largely unknown, primarily due to the challenges associated with visualizing these structures. In this study, we present a label-free, rapid, and highly sensitive method for visualizing Rootletin molecules in brain tissue. This platform integrates a second harmonic generation (SHG) microscope with background reduction by a one-step chemical pretreatment. Additionally, we employed coherent anti-Stokes Raman scattering imaging to simultaneously determine the cellular regions and intracellular locations of SHG signals. Applying this multimodal multiphoton imaging approach to the mouse hippocampus revealed that neuronal ciliary rootlets exhibited highly organized and specific intracellular distributions. Moreover, measurements of cultured neurons revealed that ciliary rootlets were detected even in immature neurons. These findings highlight the utility of our label-free imaging platform in developmental and neuroscience research, providing a powerful tool for characterizing ciliary dynamics and neuronal function.

Second harmonic generation imaging is a powerful tool for visualizing molecular structures in living organisms without the need for exogenous dyes. However, SHG signal lacks molecular specificity in identifying the source. This study aimed at molecular identification of SHG sources in the mouse brain using a multimodal imaging technique combining SHG and multiplex coherent anti Stokes Raman scattering (CARS) spectroscopy. We performed multimodal imaging in two different regions, the surface and dentate gyrus of the brain tissue. For the brain surface, the SHG signal was recognized through CARS spectrum analysis, indicating its origin in collagen. In the dentate gyrus, CARS images did not unveil corresponding molecular origins; however, morphologically, the SHG signal likely originated from Rootletin within neurons. Overall, Multimodal imaging approach to molecular identification of SHG has the potential to contribute to a comprehensive understanding of the molecular and structural features of the mouse brain. These findings advance label-free imaging techniques and have implications for brain tissue analysis and functional mapping research.

The dynamics of oxytocin and its site of action in the brain are poorly understood due to the lack of appropriate tools, despite the interest in the central action of oxytocin signaling. Here, we develop and apply an oxytocin analogue probe by conjugating it with an alkyne via a widely applicable simple coupling reaction. Alkyne-tagged oxytocin behaves similarly to endogenous oxytocin while allowing specific and highly sensitive detection of extracellularly applied oxytocin. Using this probe, we find the existence of high-affinity specific binding sites of oxytocin in the hippocampus. Furthermore, characterization of oxytocin dynamics reveals the cellular basis of its volume transmission in the brain tissue. Finally, we show the wide applicability of this technique for other centrally acting peptides. Thus, the alkyne tagging strategy provides a unique opportunity to characterize the spatiotemporal dynamics of oxytocin and other small-sized peptides in the brain tissue.

Propofol is a widely used anesthetic important in clinics, but like many other bioactive molecules, it is too small to be tagged and visualized by fluorescent dyes. Here, we present a protocol to visualize deuterated propofol in living rat neurons using stimulated Raman scattering (SRS) microscopy with carbon-deuterium bonds serving as a Raman tag. We describe the preparation and culture of rat neurons, followed by optimization of the SRS system. We then detail neuron loading and real-time imaging of anesthesia dynamics. For complete details on the use and execution of this protocol, please refer to Oda et al.1

We demonstrate the sensitive detection of alkyne-terminated ethinylestradiol (EE2) in a cetyltrimethylammonium bromide (CTAB)-coated gold nanorod (AuNR) suspension by surface-enhanced stimulated Raman scattering (SRS). Since alkynes show a distinct Raman peak at the silent wavenumber region, EE2 can be detected by obtaining the alkyne-specific SRS signals. Here we find the CTAB coating uniformly adsorbs the hydrophobic EE2 and enables it to exist in the plasmonic hotspot of AuNRs without the alkyne binding to AuNR surfaces. As a result, ensemble-averaged plasmonic enhancement using a CTAB-coated AuNR suspension improved the detection limit for SRS signals from EE2 by one order of magnitude.

An understanding of the interaction between bright correlated twin beams and matter is important for expanding the applications of optical quantum technology. In particular, it is worthwhile to clarify the advantages of twin-beam correlation in spectroscopic applications, since twin-beam sources that use parametric down-conversion have a wide range of wavelength tunability. Here, we theoretically and experimentally demonstrate that the spectral-focusing technique used in coherent anti-Stokes Raman scattering (CARS) can be implemented by twin beams without the use of ultrashort-pulse lasers. In typical spectral focusing using ultrashort pulses, spectral resolution is improved by narrowing the instantaneous frequency width due to frequency chirping. Similarly, we show theoretically that the formation of a chirp structure within the temporal correlation of twin beams produces an effect similar to typical spectral focusing. To experimentally demonstrate twin-beam spectral focusing, CARS spectroscopy of polyethylene is performed when the time delay between the chirped twin beams is set within the correlation width or it is set outside the correlation width. We observe that the spectral resolution is improved when the correlation is effective. By comparing with theoretical simulations, we confirm that the improvement is caused by frequency chirping.

The biological activities of substances in the brain are shaped by their spatiotemporal dynamics in brain tissues, all of which are regulated by water dynamics. In contrast to solute dynamics, water dynamics have been poorly characterized, owing to the lack of appropriate analytical tools. To overcome this limitation, we apply stimulated Raman scattering multimodal multiphoton microscopy to live brain tissues. The microscopy system allows for the visualization of deuterated water, fluorescence-labeled solutes, and cellular structures at high spatiotemporal resolution, revealing that water moves faster than fluorescent molecules in brain tissues. Detailed analyses demonstrate that water, unlike solutes, diffuses homogeneously in brain tissues without differences between the intra- and the extracellular routes. Furthermore, we find that the water dynamics are steady during development and ischemia, when diffusions of solutes are severely affected. Thus, our approach reveals routes and uniquely robust properties of water diffusion in brain tissues.

Observing multiple molecular species simultaneously with high spatiotemporal resolution is crucial for comprehensive understanding of complex, dynamic, and heterogeneous biological systems. The recently reported super-multiplex optical imaging breaks the “color barrier” of fluorescence to achieve multiplexing number over six in living systems, while its temporal resolution is limited to several minutes mainly by slow color tuning. Herein, we report integrated stimulated Raman and fluorescence microscopy with simultaneous multimodal color tunability at high speed, enabling super-multiplex imaging covering diverse molecular contrasts with temporal resolution of seconds. We highlight this technique by demonstrating super-multiplex time-lapse imaging and image-based cytometry of live cells to investigate the dynamics and cellular heterogeneity of eight intracellular components simultaneously. Our technique provides a powerful tool to elucidate spatiotemporal organization and interactions in biological systems.

© 2018 Elsevier Inc. Norepinephrine (NE) modulates brain functions depending on both the internal and external environment. While the neuromodulatory actions of NE have been well characterized, the response and involvement of cortical astrocytes to physiological noradrenergic systems remain largely unknown, especially at the molecular level. In this study, we biochemically characterize the action of NE on astrocytes of the murine neocortex. NE stimulation of acute brain slices rapidly increase phosphorylation of connexin 43 (Cx43) at Serine (Ser) 368, in slices from both juvenile and adolescent animals. The phosphorylation is mediated by the protein kinase C (PKC) pathway under the α1-adrenergic receptor and remains elevated for tens of minutes following brief exposure to NE, well after the intracellular calcium level returns to normal level, suggesting the plastic nature of this phosphorylation event. Importantly, this phosphorylation event persists in the absence of neuronal transmissions, suggesting that the effect of NE on Cx43 phosphorylation is induced directly on astrocytes. Furthermore, these NE-induced phosphorylations are associated with biochemical dissociation of Cx43 from gap-junctional plaques to non-junctional compartments. Finally, we show that pharmacological manipulation of the noradrenergic system using psychoactive drugs modulates phosphorylation of Cx43 in the cerebral cortex in vivo. These data suggest that NE acts directly on astrocytes in parallel with neurons and modulates functionally critical connexin channel proteins in a plastic manner. Thus, plasticity of astrocytes induced by the “gliomodulatory” actions of NE may play important roles in their physiological as well as pharmacological actions in the brain.

© 2018 Elsevier Inc. Propofol is widely used as a general anesthetic and is generally considered to exert its action by regulating neuronal firing via facilitation of GABAA receptors. However, accumulating evidence suggests that propofol also acts on astrocytes, including inhibitory effects on gap junctional coupling, but the underlying molecular mechanisms remain largely unknown. Here, using acute cortical brain slices prepared from mice, we characterize propofol-induced molecular changes in astrocytic gap junction protein connexin 43 (Cx43). Propofol does not change the protein expression level of Cx43 or its incorporation into gap junctional plaques, according to biochemical and immunohistochemical analyses. However, propofol alters migration pattern of Cx43 on western blot, suggesting changes in its posttranslational modifications. Indeed, this change is accompanied by an increase in the phosphorylation of Cx43 at serine 368, which is known to reduce permeability of Cx43 gap junctions. Finally, we show that this change occurs in the absence of neuronal firing or glutamatergic transmissions. Overall, these results show that propofol induces posttranslational modification of Cx43 directly on astrocytes at the site of gap junctional plaques, exerting direct pharmacological action on astrocytes in parallel with its action on neurons.