Published papers

The primary motor cortex (M1) and the dorsal striatum play a critical role in motor learning and the retention of learned behaviors. Motor representations of corticostriatal ensembles emerge during motor learning. In the coordinated reorganization of M1 and the dorsal striatum for motor learning, layer 5a (L5a), which connects M1 to the ipsi- and contra-lateral dorsal striatum, should be a key layer. Although M1 L5a neurons represent movement-related activity in the late stage of learning, it is unclear whether the activity is retained as a memory engram. Here, usingTlx3-Cremale transgenic mice, we conducted two-photon calcium imaging of striatum-projecting L5a intratelencephalic (IT) neurons in forelimb M1 during late sessions of a self-initiated lever-pull task, and in sessions after 6 days of non-training following the late sessions. We found that trained male animals exhibited stable motor performance before and after the non-training days. At the same time, we found that M1 L5a IT neurons strongly represented the well-learned forelimb movement, but not uninstructed orofacial movements. A subset of M1 L5a IT neurons consistently coded the well-learned forelimb movement before and after the non-training days. Inactivation of M1 IT neurons after learning impaired task performance when the lever was made heavier or when the target range of the pull distance was narrowed. These results suggest that a subset of M1 L5a IT neurons continuously represent skilled movement after learning and serve to fine-tune the kinematics of well-learned movement. Significance Statement Motor memory persists even when it is not used for a while. Intratelencephalic neurons in layer 5a (L5a IT neurons) of the primary motor cortex (M1) gradually come to represent skilled forelimb movements during motor learning. However, it remains to be determined whether these changes persist over a long period and how these neurons contribute to skilled movements. Here, we show that a subset of M1 L5a IT neurons retain information for skilled forelimb movements even after non-training days. Furthermore, suppressing the activity of these neurons during skilled forelimb movements impaired behavioral stability and adaptability. Our results suggest the importance of M1 L5a IT neurons for tuning skilled forelimb movements over a long period.

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.

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 alpha 1-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. (C) 2018 Elsevier Inc. All rights reserved.

Molecular diffusion in the extracellular space (ECS) plays a key role in determining tissue physiology and pharmacology. The bloodbrain barrier regulates the exchange of substances between the brain and the blood, but the diffusion properties of molecules at this bloodbrain interface, particularly around the astrocyte endfeet, are poorly characterized. In this study, we used 2-photon microscopy and acute brain slices of mouse neocortex and directly assessed the diffusion patterns of fluorescent molecules. By observing the diffusion of unconjugated and 10-kDa dextran-conjugated Alexa Fluor 488 from the ECS of the brain parenchyma to the blood vessels, we find various degrees of diffusion barriers at the endfeet: Some allow the invasion of dye inside the endfoot network while others completely block it. Detailed analyses of the time course for dye clearance support the existence of a tight endfoot network capable of acting as a diffusion barrier. Finally, we show that this diffusion pattern collapses under pathological conditions. These data demonstrate the heterogeneous nature of molecular diffusion dynamics around the endfeet and suggest that these structures can serve as the diffusion barrier. Therefore, astrocyte endfeet may add another layer of regulation to the exchange of molecules between blood vessels and brain parenchyma.

Second harmonic generation (SHG) imaging can be used to visualize unique biological phenomena, but currently available dyes limit its application owing to the strong fluorescent signals that they generate together with SHG. Here we report the first non-fluorescent and membrane potential-sensitive SHG-active organic dye Ap3. Ap3 is photostable and generates SH signals at the plasma membrane with virtually no fluorescent signals, in sharp contrast to the previously used fluorescent dye FM4-64. When tested in neurons, Ap3-SHG shows linear membrane potential sensitivity and fast responses to action potentials, and also shows significantly reduced photodamage compared with FM4-64. The SHG-specific nature of Ap3 allows simultaneous and completely independent imaging of SHG signals and fluorescent signals from various reporter molecules, including markers of cellular organelles and intracellular calcium. Therefore, this SHG-specific dye enables true multimodal two-photon imaging in biological samples.

Epilepsy is characterized by the abnormal activation of neurons in the cerebral cortex, but the molecular and cellular mechanisms contributing to the development of recurrent seizures are largely unknown. Recently, the critical involvement of astrocytes in the pathophysiology of epilepsy has been proposed. However, the nature of plastic modulations of astrocytic proteins in the epileptic cortex remains poorly understood. In this study, we utilized the zero magnesium in vitro model of epilepsy and examined the potential molecular changes of cortical astrocytes, focusing specifically on endfeet, where specialized biochemical compartments exist. We find that the continuous epileptic activation of neurons for 1 h decreases the expression level of beta-dystroglycan (beta DG) in acute cortical brain slices prepared from mice. This change is completely abolished by the pharmacological blockade of NMDA-type glutamate receptors as well as by matrix metalloproteinase inhibitors. Consistent with the highly specialized localization of beta DG at astrocytic endfeet, where it plays a pivotal role in anchoring endfeet-enriched proteins in astrocytes, the down-regulation of beta DG is accompanied by a decrease in the expression of AQP4 but not laminin. Importantly, this down-regulation of beta DG persists for at least 1 h, even after the apparent recovery of neuronal activation. Finally, we show that the down-regulation of beta DG is associated with the dysfunction of the endfeet at the blood-brain interface as a diffusion barrier. These results suggest that the sustained down-regulation of beta DG leads to dysfunctions of astrocytic endfeet in the epileptic cerebral cortex and may contribute to the pathogenesis of epilepsy.

Brain development and maintenance requires transportation of cerebral sustenance across the blood brain barrier. Disorders of this process can induce neurodegenerative diseases, which can affect numerous patients and incur significant treatment expenses. Recent scientific advances suggest that knowledge of this transportation will lead to the development of tailor-made theranostic strategies. Biotechnological tools such as biocompatible nanoparticles have promising potential in this context, however, information about their consequences in the brain is largely unknown. The present study demonstrates the application of specifically constructed cerebral sustenance nanoparticles in the form of tunable plasma lipoproteins to provide information about their pathways to the brain and delivery to glia cells. Individual cell analysis in brain sections enabled us to understand the accumulation of tunable plasma lipoproteins in the cerebral cortex, striatum, and cerebellum, which have been previously identified as the incident regions of Alzheimer's disease, Parkinson's disease, and spinocerebellar ataxia. The adjustability of the tunable plasma lipoproteins enables their use in theranostic applications against neurodegenerative diseases. (C) 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Astrocytes play pivotal roles in both the physiology and the pathophysiology of the brain. They communicate with each other via extracellular messengers as well as through gap junctions, which may exacerbate or protect against pathological processes in the brain. However, their roles during the acute phase of ischemia and the underlying cellular mechanisms remain largely unknown. To address this issue, we imaged changes in the intracellular calcium concentration ([Ca2+](i)) in astrocytes in mouse cortical slices under oxygen/glucose deprivation (OGD) condition using two-photon microscopy. Under OGD, astrocytes showed [Ca2+](i) oscillations followed by larger and sustained [Ca2+](i) increases. While the pharmacological blockades of astrocytic receptors for glutamate and ATP had no effect, the inhibitions of gap junctional intercellular coupling between astrocytes significantly advanced the onset of the sustained [Ca2+](i) increase after OGD exposure. Interestingly, the simultaneous recording of the neuronal membrane potential revealed that the onset of the sustained [Ca2+](i) increase in astrocytes was synchronized with the appearance of neuronal anoxic depolarization. Furthermore, the blockade of gap junctional coupling resulted in a concurrent faster appearance of neuronal depolarizations, which remain synchronized with the sustained [Ca2+](i) increase in astrocytes. These results indicate that astrocytes delay the appearance of the pathological responses of astrocytes and neurons through their gap junction-mediated intercellular network under OGD. Thus, astrocytic gap junctional networks provide protection against tissue damage during the acute phase of ischemia. (C) 2014 Elsevier Inc. All rights reserved.