高橋 泰伽

Abstract The memory trace at the neuronal and synaptic levels remains controversial. Stable, larger spines are thought to support memory, but the high turnover of dendritic spines and the drifting of neuronal representations following memory formation suggest alternative possibilities. To elucidate a structural trace underlying memory retention, we utilize a mouse model of artificial hibernation. During hibernation, hippocampal neurons exhibited a substantial reduction in their activity and an extensive elimination of dendritic spines and synapses. Despite these changes, their memory and associated hippocampal neuronal representations are intact after arousal. We find that a subset of spines is maintained during hibernation. These spatially clustered engram-engram synapses are exclusively protected from elimination and characterized by synaptic contacts with multi-synaptic boutons. These findings suggest that synaptic engram architecture, rather than larger spines per se, is resilient to network remodeling and underlies long-term memory retention.

Abstract The mammalian brain is a thick and densely layered structure comprising a huge number of neurons that work together to process information and regulate brain functions. Although various optical methods have been developed to investigate deep brain dynamics, they are limited by technical constraints, invasiveness, suboptimal spatial resolution, and/or a restricted field of view. To overcome these limitations, we developed an implantable, optically optimized microprism interface with a refractive index matched to that of brain tissue and water, enabling minimally-invasive, wide-field two-photon imaging method with enhanced brightness and sub-micron resolution in deep prefrontal areas.

Two-photon excitation fluorescence microscopy [two-photon microscopy (2PM)] 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 the structure of the enhanced yellow fluorescent protein (EYFP)-expressing neurons at the hippocampal dentate gyrus without resecting the neocortex and neuronal activity in the hippocampal cornu ammonis (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 2PM 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 2PM and stimulated emission depletion 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 2PM.

Neural regeneration is extremely difficult to achieve. In traumatic brain injuries, the loss of brain parenchyma volume hinders neural regeneration. In this study, neuronal tissue engineering was performed by using electrically charged hydrogels composed of cationic and anionic monomers in a 1:1 ratio (C1A1 hydrogel), which served as an effective scaffold for the attachment of neural stem cells (NSCs). In the 3D environment of porous C1A1 hydrogels engineered by the cryogelation technique, NSCs differentiated into neuroglial cells. The C1A1 porous hydrogel was implanted into brain defects in a mouse traumatic damage model. The VEGF-immersed C1A1 porous hydrogel promoted host-derived vascular network formation together with the infiltration of macrophages/microglia and astrocytes into the gel. Furthermore, the stepwise transplantation of GFP-labeled NSCs supported differentiation towards glial and neuronal cells. Therefore, this two-step method for neural regeneration may become a new approach for therapeutic brain tissue reconstruction after brain damage in the future.

Multiphoton microscopy has become a powerful tool for visualizing neurobiological phenomena such as the dynamics of individual synapses and the functional activities of neurons. Owing to its near-infrared excitation laser wavelength, multiphoton microscopy achieves greater penetration depth and is less invasive than single-photon excitation. Here, we review the principles of two-photon microscopy and its technical limitations (penetration depth and spatial resolution) on brain tissue imaging. We then describe the technological improvements of two-photon microscopy that enable deeper imaging with higher spatial resolution for investigating unrevealed brain functions.