Published papers

A variety of brain cells participates in neurovascular coupling by transmitting and modulating vasoactive signals. The present study aimed to probe cell type-dependent cerebrovascular (i.e., pial and penetrating arterial) responses with optogenetics in the cortex of anesthetized mice. Two lines of the transgenic mice expressing a step function type of light-gated cation channel (channelrhodopsine-2; ChR2) in either cortical neurons (muscarinic acetylcholine receptors) or astrocytes (Mlc1-positive) were used in the experiments. Photo-activation of ChR2-expressing astrocytes resulted in a widespread increase in cerebral blood flow (CBF), extending to the nonstimulated periphery. In contrast, photo-activation of ChR2-expressing neurons led to a relatively localized increase in CBF. The differences in the spatial extent of the CBF responses are potentially explained by differences in the involvement of the vascular compartments. In vivo imaging of the cerebrovascular responses revealed that ChR2-expressing astrocyte activation led to the dilation of both pial and penetrating arteries, whereas ChR2-expressing neuron activation predominantly caused dilation of the penetrating arterioles. Pharmacological studies showed that cell type-specific signaling mechanisms participate in the optogenetically induced cerebrovascular responses. In conclusion, pial and penetrating arterial vasodilation were differentially evoked by ChR2-expressing astrocytes and neurons.

Attention-deficit/hyperactivity disorder (ADHD) is a common neuropsychiatric disorder in children. Although animal models and human brain imaging studies indicate a significant role for glutamatergic dysfunction in ADHD, there is no direct evidence that glutamatergic dysfunction is sufficient to induce ADHD-like symptoms. The glial glutamate transporter GLT1 plays a critical role in glutamatergic neurotransmission. We report here the generation of mice expressing only 20% of normal levels of the GLT1. Unlike conventional GLT1 knockout mice, these mice survive to adulthood and exhibit ADHD-like phenotypes, including hyperactivity, impulsivity and impaired memory. These findings indicate that glutamatergic dysfunction due to GLT1 deficiency, a mechanism distinct from the dopaminergic deficit hypothesis of ADHD, underlies ADHD-like symptoms.

An artificial tool for manipulating local cerebral blood flow (CBF) is necessary for understanding how CBF controls brain function. Here, we generate vascular optogenetic tools whereby smooth muscle cells and endothelial cells express optical actuators in the brain. The illumination of channelrhodopsin-2 (ChR2)-expressing mice induces a local reduction in CBF. Photoactivated adenylyl cyclase (PAC) is an optical protein that increases intracellular cyclic adenosine monophosphate (cAMP), and the illumination of PAC-expressing mice induces a local increase in CBF. We target the ventral striatum, determine the temporal kinetics of CBF change, and optimize the illumination intensity to confine the effects to the ventral striatum. We demonstrate the utility of this vascular optogenetic manipulation in freely and adaptively behaving mice and validate the task- and actuator-dependent behavioral readouts. The development of vascular optogenetic animal models will help accelerate research linking vasculature, circuits, and behavior to health and disease.

Neural activity is diverse, and varies depending on brain regions and sleep/wakefulness states. However, whether astrocyte activity differs between sleep/wakefulness states, and whether there are differences in astrocyte activity among brain regions remain poorly understood. Therefore, in this study, we recorded astrocyte intracellular calcium (Ca21) concentrations of mice during sleep/wakefulness states in the cortex, hippocampus, hypothalamus, cerebellum, and pons using fiber photometry. For this purpose, male transgenic mice expressing the genetically encoded ratiometric Ca21 sensor YCnano50 specifically in their astrocytes were used. We demonstrated that Ca21 levels in astrocytes substantially decrease during rapid eye movement (REM) sleep, and increase after the onset of wakefulness. In contrast, differences in Ca21 levels during non-REM (NREM) sleep were observed among the different brain regions, and no significant decrease was observed in the hypothalamus and pons. Further analyses focusing on the transition between sleep/wakefulness states and correlation analysis with the duration of REM sleep showed that Ca21 dynamics differs among brain regions, suggesting the existence of several clusters, i.e., the first comprising the cortex and hippocampus, the second comprising the hypothalamus and pons, and the third comprising the cerebellum. Our study thus demonstrated that astrocyte Ca21 levels change substantially according to sleep/wakefulness states. These changes were consistent in general unlike neural activity. However, we also clarified that Ca21 dynamics varies depending on the brain region, implying that astrocytes may play various physiological roles in sleep.

The juvenile brain presents plasticity. Oligodendrocytes are the myelinating cells of the central nervous system and myelination can be adaptive. Plasticity decreases from juvenile to adulthood. The mechanisms involving oligodendrocytes underlying plasticity are unclear. Here, we show Na+-K+-Cl– co-transporter 1 (NKCC1), highly expressed in the juvenile mouse brain, regulates the oligodendrocyte activity from juvenile to adulthood in mice, as shown by optogenetic manipulation of oligodendrocytes. The reduced neuronal activity in adults was restored by Nkcc1 overexpression in oligodendrocytes. Moreover, in adult mice overexpressing Nkcc1, long-term potentiation and learning were facilitated compared to age-matched controls. These findings demonstrate that NKCC1 plays a regulatory role in the age-dependent activity of oligodendrocytes, furthermore inducing activation of NKCC1 in oligodendrocytes can restore neuronal plasticity in the adult mouse brain.

A brain atlas is necessary for analyzing structure and function in neuroimaging research. Although various annotation volumes (AVs) for the mouse brain have been proposed, it is common in magnetic resonance imaging (MRI) of the mouse brain that regions-of-interest (ROIs) for brain structures (nodes) are created arbitrarily according to each researcher’s necessity, leading to inconsistent ROIs among studies. One reason for such a situation is the fact that earlier AVs were fixed, i.e. combination and division of nodes were not implemented. This report presents a pipeline for constructing a flexible annotation atlas (FAA) of the mouse brain by leveraging public resources of the Allen Institute for Brain Science on brain structure, gene expression, and axonal projection. A mere two-step procedure with user-specified, text-based information and Python codes constructs FAA with nodes which can be combined or divided objectively while maintaining anatomical hierarchy of brain structures. Four FAAs with total node count of 4, 101, 866, and 1381 were demonstrated. Unique characteristics of FAA realized analysis of resting-state functional connectivity (FC) across the anatomical hierarchy and among cortical layers, which were thin but large brain structures. FAA can improve the consistency of whole brain ROI definition among laboratories by fulfilling various requests from researchers with its flexibility and reproducibility.

© 2018 Wiley Periodicals, Inc. Polarized light imaging (PLI) enables detecting the orientation of myelinated axon bundles in brain slices at microscopic resolution without histological staining. However, standard PLI requires labor-intensive procedures such as mounting brain cryosections on slide glasses. We developed an optical system that does not require a mounting procedure for PLI. Specifically, we developed an optical system to perform PLI in reflection mode (rPLI) instead of employing transmitted light as in standard PLI. We integrated this rPLI system with a conventional vibratome slicer whose cutting blade surface is a mirror. This combination allowed PLI measurements directly during the slicing procedure at room temperature. Thus, mounting procedure for PLI is not necessary. As a proof-of-concept experiment, a perfusion-fixed brain of a mouse was embedded in gelatin-containing agar and cut serially at 40~200 μm intervals. The slicing procedure was temporarily halted after each cut to capture the PLI images of the slice on the reflecting blade surface while the slice was still held up by the agar block. The orientation of the fiber bundle estimated with this method agreed with the results obtained from previous reports. Combination of a popular vibratome slicer and our rPLI system that uses versatile and inexpensive optical components would increase popularity of PLI and facilitates connectome studies at microscopic resolution. Research Highlights: Polarized light imaging (PLI) of brain slices was realized by using reflected light (rPLI) instead of transmitted light. The rPLI method allows detecting the myelinated fiber bundle orientation during slice preparation.

© 2020 Wiley Periodicals LLC The glymphatic system is a recently defined brain-wide network of perivascular spaces along which cerebrospinal fluid (CSF) and interstitial solutes exchange. Astrocyte endfeet encircling the perivascular space form a physical barrier in between these two compartments, and fluid and solutes that are not taken up by astrocytes move out of the perivascular space through the junctions in between astrocyte endfeet. However, little is known about the anatomical structure and the physiological roles of the astrocyte endfeet in regulating the local perivascular exchange. Here, visualizing astrocyte endfoot–endfoot junctions with immunofluorescent labeling against the protein megalencephalic leukoencephalopathy with subcortical cysts-1 (MLC1), we characterized endfoot dimensions along the mouse cerebrovascular tree. We observed marked heterogeneity in endfoot dimensions along vessels of different sizes, and of different types. Specifically, endfoot size was positively correlated with the vessel diameters, with large vessel segments surrounded by large endfeet and small vessel segments surrounded by small endfeet. This association was most pronounced along arterial, rather than venous segments. Computational modeling simulating vascular trees with uniform or varying endfeet dimensions demonstrates that varying endfoot dimensions maintain near constant perivascular-interstitial flux despite correspondingly declining perivascular pressures along the cerebrovascular tree through the cortical depth. These results describe a novel anatomical feature of perivascular astroglial endfeet and suggest that endfoot heterogeneity may be an evolutionary adaptation to maintain perivascular CSF-interstitial fluid exchange through deep brain structures.

Pharmacological intervention in the substantia nigra is known to induce repetitive behaviors in rodents, but a direct causal relationship between a specific neural circuit and repetitive behavior has not yet been established. Here we demonstrate that optogenetic activation of dopamine D1 receptor-expressing MSNs terminals in the substantia nigra pars reticulata resulted in sustained and chronic repetitive behaviors. These data show for the first time that activation of the striatonigral direct pathway is sufficient to generate motor stereotypies.

Diffusion tensor imaging (DTI) is widely used to evaluate microstructural variations in brain tissue. In particular, fractional anisotropy (FA), reflecting the magnitude and orientation of anisotropic water diffusion, allows us to detect pathological events in white matter. An ex vivo DTI study coupled with histological assessment is an efficient strategy to evaluate the myelination process, i.e. demyelination and remyelination. The relationship between DTI values and myelin content or the individual cellular components such as oligodendrocytes, microglia, and astrocytes during both processes of demyelination and remyelination are not well-understood. To address this issue, we employed a cuprizone-inducible demyelination mouse model. Demyelination can be induced in this model during cuprizone exposure and termination of cuprizone exposure induces remyelination. We fed the mice cuprizone-containing chow for 4 weeks and then normal chow for an additional 4 weeks. The ex vivo DTI was performed to evaluate the white matter profiles observed by FA, mean diffusivity (MD), and radial diffusivity (RD) at both demyelinating and remyelinating time points, and then we evaluated histological properties at the same time points. The results indicated a gradual FA decrease during the cuprizone treatment (0, 2, 3, 4 weeks). A lower peak was seen at 1 week after the normal chow was resumed, with recovery to baseline at 2 and 4 weeks. MD and RD showed an opposing pattern to that of FA. These DTI values were positively or negatively correlated with myelin content regardless of the status of the white matter. The RD value was more sensitive to myelination status than FA and MD. We have clarified the temporal changes in the DTI values coupled with histological properties over both the demyelination and remyelination processes.

© 2018 International Society for Neurochemistry Mlc1 is a causative gene for megalencephalic leukoencephalopathy with subcortical cysts, and is expressed in astrocytes. Mlc1-over-expressing mice represent an animal model of early-onset leukoencephalopathy, which manifests as astrocytic swelling followed by myelin membrane splitting in the white matter. It has been previously reported that Mlc1 is highly expressed in Bergmann glia, while the cerebellar phenotypes of Mlc1-over-expressing mouse have not been characterized. Here, we examined the cerebellum of Mlc1-over-expressing mouse and found that the distribution of Bergmann glia (BG) was normally compacted along the Purkinje cell (PC) layer until postnatal day 10 (P10), while most BG were dispersed throughout the molecular layer by P28. Ectopic BG were poorly wrapped around somatodendritic elements of PCs and exhibited reduced expression of the glutamate transporter glutamate-aspartate transporter. Extraordinarily slow and small climbing fiber (CF)-mediated excitatory post-synaptic currents, which are known to be elicited under accelerated glutamate spillover, emerged at P20-P28 when BG ectopia was severe, but not at P9-P12 when ectopia was mild. Furthermore, maturation of CF wiring, which translocates the site of innervation from somata to proximal dendrites, was also impaired. Manipulations that restricted the Mlc1-over-expressing period successfully generated mice with and without BG ectopia, depending on the over-expressing period. Together, these findings suggest that there is a critical time window for mechanisms that promote the positioning of BG in the PC layer. Once normal positioning of BG is affected, the differentiation of BG is impaired, leading to insufficient glial wrapping, exacerbated glutamate spillover, and aberrant synaptic wiring in PCs. Open Practices Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/. (Figure presented.). Cover Image for this issue: doi: 10.1111/jnc.14199.

© 2018 Elsevier Inc. Synapses are fundamental units of communication in the brain. The prototypical synapse-organizing complex neurexin-neuroligin mediates synapse development and function and is central to a shared genetic risk pathway in autism and schizophrenia. Neurexin's role in synapse development is thought to be mediated purely by its protein domains, but we reveal a requirement for a rare glycan modification. Mice lacking heparan sulfate (HS) on neurexin-1 show reduced survival, as well as structural and functional deficits at central synapses. HS directly binds postsynaptic partners neuroligins and LRRTMs, revealing a dual binding mode involving intrinsic glycan and protein domains for canonical synapse-organizing complexes. Neurexin HS chains also bind novel ligands, potentially expanding the neurexin interactome to hundreds of HS-binding proteins. Because HS structure is heterogeneous, our findings indicate an additional dimension to neurexin diversity, provide a molecular basis for fine-tuning synaptic function, and open therapeutic directions targeting glycan-binding motifs critical for brain development. Neurexins, major synaptic-organizing proteins, are heparan sulfate (HS) proteoglycans, and HS modification is required for neurexin functions in synaptic transmission, development, and behavior.

© 2018 International Society for Neurochemistry Oligodendrocytes (OLs) are myelinating cells of the central nervous system. Recent studies have shown that mechanical factors influence various cell properties. Mechanical stimulation can be transduced into intracellular biochemical signals through mechanosensors, such as integrin, p130Cas, talin and vinculin. However, the molecular mechanisms underlying the mechanical regulation of OLs by mechanosensors remain largely unknown. We found that morphology of OL was affected by knockdown of the mechanosensors p130Cas or talin1. Stretching of OL precursor cells induced the phosphorylation of p130Cas and talin-associated assembly of vinculin. Shear stress decreased the number of OL processes, whereas these effects were mechanically suppressed by dominant-negative (DN) p130Cas, but not by DN-talin1. To investigate the roles of p130Cas in post-natal OLs in vivo, we constructed a novel p130Cas knock-in mouse and found overexpression of p130Cas in vivo affected the number of mature OLs in the cortex. These results indicate that the mechanosensor p130Cas controls both OL morphogenesis and maturation. (Figure presented.).

2017 © The Authors. Optogenetics has revolutionized the experimental interrogation of neural circuits and holds promise for the treatment of neurological disorders. It is limited, however, because visible light cannot penetrate deep inside brain tissue. Upconversion nanoparticles (UCNPs) absorb tissue-penetrating near-infrared (NIR) light and emit wavelength-specific visible light. Here, we demonstrate that molecularly tailored UCNPs can serve as optogenetic actuators of transcranial NIR light to stimulate deep brain neurons. Transcranial NIR UCNP-mediated optogenetics evoked dopamine release from genetically tagged neurons in the ventral tegmental area, induced brain oscillations through activation of inhibitory neurons in the medial septum, silenced seizure by inhibition of hippocampal excitatory cells, and triggered memory recall. UCNP technology will enable less-invasive optical neuronal activity manipulation with the potential for remote therapy.

© 2018 Wiley Periodicals, Inc. Functional magnetic resonance imaging (fMRI) based on the blood oxygenation level-dependent (BOLD) signal has been used to infer sites of neuronal activation in the brain. A recent study demonstrated, however, unexpected BOLD signal generation without neuronal excitation, which led us to hypothesize the presence of another cellular source for BOLD signal generation. Collective assessment of optogenetic activation of astrocytes or neurons, fMRI in awake mice, electrophysiological measurements, and histochemical detection of neuronal activation, coherently suggested astrocytes as another cellular source. Unexpectedly, astrocyte-evoked BOLD signal accompanied oxygen consumption without modulation of neuronal activity. Imaging mass spectrometry of brain sections identified synthesis of acetyl-carnitine via oxidative glucose metabolism at the site of astrocyte-, but not neuron-evoked BOLD signal. Our data provide causal evidence that astrocytic activation alone is able to evoke BOLD signal response, which may lead to reconsideration of current interpretation of BOLD signal as a marker of neuronal activation.

© 2018 The Author(s). Recent experiments have shown that optogenetic activation of serotonin neurons in the dorsal raphe nucleus (DRN) in mice enhances patience in waiting for future rewards. Here, we show that serotonin effect in promoting waiting is maximized by both high probability and high timing uncertainty of reward. Optogenetic activation of serotonergic neurons prolongs waiting time in no-reward trials in a task with 75% food reward probability, but not with 50 or 25% reward probabilities. Serotonin effect in promoting waiting increases when the timing of reward presentation becomes unpredictable. To coherently explain the experimental data, we propose a Bayesian decision model of waiting that assumes that serotonin neuron activation increases the prior probability or subjective confidence of reward delivery. The present data and modeling point to the possibility of a generalized role of serotonin in resolving trade-offs, not only between immediate and delayed rewards, but also between sensory evidence and subjective confidence.

© 2018 the authors. Fine processes of astrocytes enwrap synapses and are well positioned to sense neuronal information via synaptic transmission. In rodents, astrocyte processes sense synaptic transmission via Gq-protein coupled receptors (GqPCR), including the P2Y1 receptor (P2Y1R), to generate Ca2+signals. Astrocytes display numerous spontaneous microdomain Ca2+signals; however, it is not clear whether such signals are due to local synaptic transmission and/or in what timeframe astrocytes sense local synaptic transmission. To ask whether GqPCRs mediate microdomain Ca2+ signals, we engineered mice (both sexes) to specifically overexpress P2Y1Rs in astrocytes, and we visualized Ca2+ signals via a genetically encoded Ca2+ indicator, GCaMP6f, in astrocytes from adult mice. Astrocytes overexpressing P2Y1Rs showed significantly larger Ca2+ signals in response to exogenously applied ligand and to repetitive electrical stimulation of axons compared with controls. However, we found no evidence of increased microdomain Ca2+signals. Instead, Ca2+waves appeared and propagated to occupy areas that were up to 80-fold larger than microdomain Ca2+ signals. These Ca2+ waves accounted for only 2% of total Ca2+ events, but they were 1.9-fold larger and 2.9-fold longer in duration than microdomain Ca2+ signals at processes. Ca2+ waves did not require action potentials for their generation and occurred in a probenecid-sensitive manner, indicating that the endogenous ligand for P2Y1R is elevated independently of synaptic transmission. Our data suggest that spontaneous microdomain Ca2+ signals occur independently of P2Y1R activation and that astrocytes may not encode neuronal information in response to synaptic transmission at a point source of neurotransmitter release.

© 2019, The Author(s). The number of patients with dry eye disease (DED) is increasing, and DED has become an urgent public health problem. A comorbidity of mental disorders has been reported in DED patients. We hypothesized that physical and psychological stressors impair tear secretion. To examine the relationship between stress loading and decreased tear secretion, we established a stress-induced DED mouse model, which permitted us to address the underlying mechanism of pathogenesis and resilience. Enriched environment (EE) was an effective intervention to prevent and alleviate stress-induced decreased tear secretion. Because stress loading resulted in decreased brain-derived neurotrophic factor (BDNF) expression while EE resulted in increased expression, we focused on the role of BDNF in tear secretion. Using two distinct Bdnf gene knockdown mice, we evaluated whether BDNF was a deterministic factor in regulating tear secretion in healthy and stressed conditions. Bdnf knockdown mice showed decreased basal tear secretion and loss of stress tolerance by EE for tear secretion. These results suggest that BDNF expression is related to tear secretion and to the pathology of DED.

© 2020, The Author(s). In rodent models of depression, (R)-ketamine has greater potency and longer-lasting antidepressant effects than (S)-ketamine; however, the precise molecular mechanisms underlying the antidepressant actions of (R)-ketamine remain unknown. Using RNA-sequencing analysis, we identified novel molecular targets that contribute to the different antidepressant effects of the two enantiomers. Either (R)-ketamine (10 mg/kg) or (S)-ketamine (10 mg/kg) was administered to susceptible mice after chronic social defeat stress (CSDS). RNA-sequencing analysis of prefrontal cortex (PFC) and subsequent GSEA (gene set enrichment analysis) revealed that transforming growth factor (TGF)-β signaling might contribute to the different antidepressant effects of the two enantiomers. (R)-ketamine, but not (S)-ketamine, ameliorated the reduced expressions of Tgfb1 and its receptors (Tgfbr1 and Tgfbr2) in the PFC and hippocampus of CSDS susceptible mice. Either pharmacological inhibitors (i.e., RepSox and SB431542) or neutralizing antibody of TGF-β1 blocked the antidepressant effects of (R)-ketamine in CSDS susceptible mice. Moreover, depletion of microglia by the colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX3397 blocked the antidepressant effects of (R)-ketamine in CSDS susceptible mice. Similar to (R)-ketamine, the recombinant TGF-β1 elicited rapid and long-lasting antidepressant effects in animal models of depression. Our data implicate a novel microglial TGF-β1-dependent mechanism underlying the antidepressant effects of (R)-ketamine in rodents with depression-like phenotype. Moreover, TGF-β1 and its receptor agonists would likely constitute a novel rapid-acting and sustained antidepressant in humans.

© 2020 American Society for Investigative Pathology Tear fluid secreted from the exocrine lacrimal gland (LG) has an essential role in maintaining a homeostatic environment for a healthy ocular surface. Tear secretion is regulated by the sympathetic and parasympathetic components of the autonomic nervous system, although the contribution of each component is not fully understood. To investigate LG innervation, we identified sympathetic and parasympathetic postganglionic nerves, specifically innervating the mouse LG, by injecting a retrograde neuronal tracer into the LG. Interruption of neural stimuli to the LG by the denervation of these postganglionic nerves immediately and chronically decreased tear secretion, leading to LG atrophy along with destruction of the lobular structure. This investigation also found that parasympathetic, but not sympathetic, innervation was involved in these alterations.