Large-scale cranial window for in vivo mouse brain imaging utilizing fluoropolymer nanosheet and light-curable resin.

Two-photon microscopy enables in vivo imaging of neuronal activity in mammalian brains at high resolution. However, two-photon imaging tools for stable, long-term, and simultaneous study of multiple brain regions in same mice are lacking. Here, we propose a method to create large cranial windows covering such as the whole parietal cortex and cerebellum in mice using fluoropolymer nanosheets covered with light-curable resin (termed the 'Nanosheet Incorporated into light-curable REsin' or NIRE method). NIRE method can produce cranial windows conforming the curved cortical and cerebellar surfaces, without motion artifacts in awake mice, and maintain transparency for >5 months. In addition, we demonstrate that NIRE method can be used for in vivo two-photon imaging of neuronal ensembles, individual neurons and subcellular structures such as dendritic spines. The NIRE method can facilitate in vivo large-scale analysis of heretofore inaccessible neural processes, such as the neuroplastic changes associated with maturation, learning and neural pathogenesis.

Activity-dependent organization of prefrontal hub-networks for associative learning and signal transformation.

Associative learning is crucial for adapting to environmental changes. Interactions among neuronal populations involving the dorso-medial prefrontal cortex (dmPFC) are proposed to regulate associative learning, but how these neuronal populations store and process information about the association remains unclear. Here we developed a pipeline for longitudinal two-photon imaging and computational dissection of neural population activities in male mouse dmPFC during fear-conditioning procedures, enabling us to detect learning-dependent changes in the dmPFC network topology. Using regularized regression methods and graphical modeling, we found that fear conditioning drove dmPFC reorganization to generate a neuronal ensemble encoding conditioned responses (CR) characterized by enhanced internal coactivity, functional connectivity, and association with conditioned stimuli (CS). Importantly, neurons strongly responding to unconditioned stimuli during conditioning subsequently became hubs of this novel associative network for the CS-to-CR transformation. Altogether, we demonstrate learning-dependent dynamic modulation of population coding structured on the activity-dependent formation of the hub network within the dmPFC.

Erratic and blood vessel-guided migration of astrocyte progenitors in the cerebral cortex.

Astrocytes are one of the most abundant cell types in the mammalian brain. They play essential roles in synapse formation, maturation, and elimination. However, how astrocytes migrate into the gray matter to accomplish these processes is poorly understood. Here, we show that, by combinational analyses of in vitro and in vivo time-lapse observations and lineage traces, astrocyte progenitors move rapidly and irregularly within the developing cortex, which we call erratic migration. Astrocyte progenitors also adopt blood vessel-guided migration. These highly motile progenitors are generated in the restricted prenatal stages and differentiate into protoplasmic astrocytes in the gray matter, whereas postnatally generated progenitors do not move extensively and differentiate into fibrous astrocytes in the white matter. We found Cxcr4/7, and integrin ß1 regulate the blood vessel-guided migration, and their functional blocking disrupts their positioning. This study provides insight into astrocyte development and may contribute to understanding the pathogenesis caused by their defects.

Controlled activation of cortical astrocytes modulates neuropathic pain-like behaviour.

Chronic pain is a major public health problem that currently lacks effective treatment options. Here, a method that can modulate chronic pain-like behaviour induced by nerve injury in mice is described. By combining a transient nerve block to inhibit noxious afferent input from injured peripheral nerves, with concurrent activation of astrocytes in the somatosensory cortex (S1) by either low intensity transcranial direct current stimulation (tDCS) or via the chemogenetic DREADD system, we could reverse allodynia-like behaviour previously established by partial sciatic nerve ligation (PSL). Such activation of astrocytes initiated spine plasticity to reduce those synapses formed shortly after PSL. This reversal from allodynia-like behaviour persisted well beyond the active treatment period. Thus, our study demonstrates a robust and potentially translational approach for modulating pain, that capitalizes on the interplay between noxious afferents, sensitized central neuronal circuits, and astrocyte-activation induced synaptic plasticity.

Development and experience-dependent modulation of the defensive behaviors of mice to visual threats.

Rodents demonstrate defensive behaviors such as fleeing or freezing upon recognizing a looming shadow above them. Although individuals' experiences in their habitat can modulate the defensive behavior phenotype, the effects of systematically manipulating the individual's visual experience on vision-guided defensive behaviors have not been studied. We aimed to describe the developmental process of defensive behaviors in response to visual threats and the effects of visual deprivation. We found that the probability of escape response occurrence increased 3 weeks postnatally, and then stabilized. When visual experience was perturbed by dark rearing from postnatal day (P) 21 for a week, the developmental increase in escape probability was clearly suppressed, while the freezing probability increased. Intriguingly, exposure to the looming stimuli at P28 reversed the suppression of escape response development at P35. These results clearly indicate that the development of defensive behaviors in response to looming stimuli is affected by an individual's sensory experience.

PEO-CYTOP Fluoropolymer Nanosheets as a Novel Open-Skull Window for Imaging of the Living Mouse Brain.

In vivo two-photon deep imaging with a broad field of view has revealed functional connectivity among brain regions. Here, we developed a novel observation method that utilizes a polyethylene-oxide-coated CYTOP (PEO-CYTOP) nanosheet with a thickness of ∼130 nm that exhibited a water retention effect and a hydrophilized adhesive surface. PEO-CYTOP nanosheets firmly adhered to brain surfaces, which suppressed bleeding from superficial veins. By taking advantage of the excellent optical properties of PEO-CYTOP nanosheets, we performed in vivo deep imaging in mouse brains at high resolution. Moreover, PEO-CYTOP nanosheets enabled to prepare large cranial windows, achieving in vivo imaging of neural structure and Ca2+ elevation in a large field of view. Furthermore, the PEO-CYTOP nanosheets functioned as a sealing material, even after the removal of the dura. These results indicate that this method would be suitable for the investigation of neural functions that are composed of interactions among multiple regions.

CMOS-based bio-image sensor spatially resolves neural activity-dependent proton dynamics in the living brain.

Recent studies have shown that protons can function as neurotransmitters in cultured neurons. To further investigate regional and neural activity-dependent proton dynamics in the brain, the development of a device with both wide-area detectability and high spatial-ltemporal resolution is necessary. Therefore, we develop an image sensor with a high spatial-temporal resolution specifically designed for measuring protons in vivo. Here, we demonstrate that spatially deferent neural stimulation by visual stimulation induced distinct patterns of proton changes in the visual cortex. This result indicates that our biosensor can detect micrometer and millisecond scale changes of protons across a wide area. Our study demonstrates that a CMOS-based proton image sensor with high spatial and temporal precision can be used to detect pH changes associated with biological events. We believe that our sensor may have broad applicability in future biological studies.

Imaging local brain activity of multiple freely moving mice sharing the same environment.

Electrophysiological field potential dynamics have been widely used to investigate brain functions and related psychiatric disorders. Considering recent demand for its applicability to freely moving subjects, especially for animals in a group and socially interacting with each other, here we propose a new method based on a bioluminescent voltage indicator LOTUS-V. Using our fiber-free recording method based on the LOTUS-V, we succeeded in capturing dynamic change of brain activity in freely moving mice. Because LOTUS-V is the ratiometric indicator, motion and head-angle artifacts were not significantly detected. Taking advantage of our method as a fiber-free system, we further succeeded in simultaneously recording from multiple independently-locomotive mice that were freely interacting with one another. Importantly, this enabled us to find that the primary visual cortex, a center of visual processing, was activated during the interaction of mice. This methodology may further facilitate a wide range of studies in neurobiology and psychiatry.

Parvalbumin-Positive Interneurons Regulate Neuronal Ensembles in Visual Cortex.

For efficient cortical processing, neural circuit dynamics must be spatially and temporally regulated with great precision. Although parvalbumin-positive (PV) interneurons can control network synchrony, it remains unclear how they contribute to spatio-temporal patterning of activity. We investigated this by optogenetic inactivation of PV cells with simultaneous two-photon Ca2+ imaging from populations of neurons in mouse visual cortex in vivo. For both spontaneous and visually evoked activity, PV interneuron inactivation decreased network synchrony. But, interestingly, the response reliability and spatial extent of coactive neuronal ensembles during visual stimulation were also disrupted by PV-cell suppression, which reduced the functional repertoire of ensembles. Thus, PV interneurons can control the spatio-temporal dynamics of multineuronal activity by functionally sculpting neuronal ensembles and making them more different from each other. In doing so, inhibitory circuits could help to orthogonalize multicellular patterns of activity, enabling neural circuits to more efficiently occupy a higher dimensional space of potential dynamics.

Genetically encoded bioluminescent voltage indicator for multi-purpose use in wide range of bioimaging.

We report development of the first genetically encoded bioluminescent indicator for membrane voltage called LOTUS-V. Since it is bioluminescent, imaging LOTUS-V does not require external light illumination. This allows bidirectional optogenetic control of cellular activity triggered by Channelrhodopsin2 and Halorhodopsin during voltage imaging. The other advantage of LOTUS-V is the robustness of a signal-to-background ratio (SBR) wherever it expressed, even in the specimens where autofluorescence from environment severely interferes fluorescence imaging. Through imaging of moving cardiomyocyte aggregates, we demonstrated the advantages of LOTUS-V in long-term imaging are attributable to the absence of phototoxicity, and photobleaching in bioluminescent imaging, combined with the ratiometric aspect of LOTUS-V design. Collectively LOTUS-V extends the scope of excitable cell control and simultaneous voltage phenotyping, which should enable applications in bioscience, medicine and pharmacology previously not possible.

Methods for monitoring signaling molecules in cellular compartments.

Cells, irrespective of whether they are from multicellular or single-celled organisms, must communicate with the external environment through dynamic regulation of their internal metabolism, which are critical for their survival. Fluorescent and bioluminescent proteins, and related genetic engineering technologies, have provided new opportunities to investigate the molecular dynamics of cells and their internal compartments, with high spatio-temporal resolution. In this review article, since there is a sufficient number of previous reviews summarizing the history of their development and the techniques behind them, here we will focus on molecular features or technologies that have the potential to further open novel investigations of cellular and subcellular dynamics.

Social conflict resolution regulated by two dorsal habenular subregions in zebrafish.

When animals encounter conflict they initiate and escalate aggression to establish and maintain a social hierarchy. The neural mechanisms by which animals resolve fighting behaviors to determine such social hierarchies remain unknown. We identified two subregions of the dorsal habenula (dHb) in zebrafish that antagonistically regulate the outcome of conflict. The losing experience reduced neural transmission in the lateral subregion of dHb (dHbL)-dorsal/intermediate interpeduncular nucleus (d/iIPN) circuit. Silencing of the dHbL or medial subregion of dHb (dHbM) caused a stronger predisposition to lose or win a fight, respectively. These results demonstrate that the dHbL and dHbM comprise a dual control system for conflict resolution of social aggression.

A fast- and positively photoswitchable fluorescent protein for ultralow-laser-power RESOLFT nanoscopy.

Fluorescence nanoscopy has revolutionized our ability to visualize biological structures not resolvable by conventional microscopy. However, photodamage induced by intense light exposure has limited its use in live specimens. Here we describe Kohinoor, a fast-switching, positively photoswitchable fluorescent protein, and show that it has high photostability over many switching repeats. With Kohinoor, we achieved super-resolution imaging of live HeLa cells using biocompatible, ultralow laser intensity (0.004 J/cm(2)) in reversible saturable optical fluorescence transition (RESOLFT) nanoscopy.

The habenulo-raphe serotonergic circuit encodes an aversive expectation value essential for adaptive active avoidance of danger.

Anticipation of danger at first elicits panic in animals, but later it helps them to avoid the real threat adaptively. In zebrafish, as fish experience more and more danger, neurons in the ventral habenula (vHb) showed tonic increase in the activity to the presented cue and activated serotonergic neurons in the median raphe (MR). This neuronal activity could represent the expectation of a dangerous outcome and be used for comparison with a real outcome when the fish is learning how to escape from a dangerous to a safer environment. Indeed, inhibiting synaptic transmission from vHb to MR impaired adaptive avoidance learning, while panic behavior induced by classical fear conditioning remained intact. Furthermore, artificially triggering this negative outcome expectation signal by optogenetic stimulation of vHb neurons evoked place avoidance behavior. Thus, vHb-MR circuit is essential for representing the level of expected danger and behavioral programming to adaptively avoid potential hazard.

A blanket of inhibition: functional inferences from dense inhibitory connectivity.

The function of neocortical interneurons is still unclear, and, as often happens, one may be able to draw functional insights from considering the structure. In this spirit we describe recent structural results and discuss their potential functional implications. Most GABAergic interneurons innervate nearby pyramidal neurons very densely and without any apparent specificity, as if they were extending a 'blanket of inhibition', contacting pyramidal neurons often in an overlapping fashion. While subtypes of interneurons specifically target subcellular compartments of pyramidal cells, and they also target different layers selectively, they appear to treat all neighboring pyramidal cells the same and innervate them massively. We explore the functional implications and temporal properties of dense, overlapping inhibition by four interneuron populations.

Imaging of neural ensemble for the retrieval of a learned behavioral program.

The encoding of long-term associative memories for learned behaviors is a fundamental brain function. Yet, how behavior is stably consolidated and retrieved in the vertebrate cortex is poorly understood. We trained zebrafish in aversive reinforcement learning and measured calcium signals across their entire brain during retrieval of the learned response. A discrete area of dorsal telencephalon that was inactive immediately after training became active the next day. Analysis of the identified area indicated that it was specific and essential for long-term memory retrieval and contained electrophysiological responses entrained to the learning stimulus. When the behavioral rule changed, a rapid spatial shift in the functional map across the telencephalon was observed. These results demonstrate that the retrieval of long-term memories for learned behaviors can be studied at the whole-brain scale in behaving zebrafish in vivo. Moreover, the findings indicate that consolidated memory traces can be rapidly modified during reinforcement learning.

Genetic dissection of the zebrafish habenula, a possible switching board for selection of behavioral strategy to cope with fear and anxiety.

The habenula is a part of an evolutionarily highly conserved conduction pathway within the limbic system that connects telencephalic nuclei to the brain stem nuclei such as interpeduncular nucleus(IPN), the ventral tegmental area (VTA), and the raphe.In mammals, the medial habenula receives inputs from the septohippocampal system, and relaying such information to the IPN. In contrast, the lateral habenula receives inputs from the ventral pallidum, a part of the basal ganglia. The physical adjunction of these two habenular nuclei suggests that the habenula may act as an intersection of the neural circuits for controlling emotion and behavior. We have recently elucidated that zebrafish has the equivalent structure as the mammalian habenula. The transgenic zebrafish, in which the neural signal transmission from the lateral subnucleus of the dorsal habenula to the dorsal IPN was selectively impaired, showed extremely enhanced levels of freezing response to presentation of the conditioned aversive stimulus. Our observation supports that the habenula may act as the multimodal switching board for controlling emotional behaviors and/or memory inexperience dependent manners.

The habenula is crucial for experience-dependent modification of fear responses in zebrafish.

The zebrafish dorsal habenula (dHb) shows conspicuous asymmetry in its connection with the interpeduncular nucleus (IPN) and is equivalent to the mammalian medial habenula. Genetic inactivation of the lateral subnucleus of dHb (dHbL) biased fish towards freezing rather than the normal flight response to a conditioned fear stimulus, suggesting that the dHbL-IPN pathway is important for controlling experience-dependent modification of fear responses.

A metastable DWARF1 epigenetic mutant affecting plant stature in rice.

Epigenetic mutations confer heritable changes in gene expression that are not due to changes in the underlying sequence of the DNA. We identified a spontaneous rice mutant, Epi-d1, that shows a metastable dwarf phenotype. The phenotype is mitotically and meiotically inheritable and corresponds to the metastable epigenetic silencing of the DWARF1 (D1) gene. The silenced state is correlated with repressive histone and DNA methylation marks in the D1 promoter region but is not associated with DNA sequence alterations. Compared with other known epigenetic silenced loci in plants such as paramutable maize alleles and silent Arabidopsis genes, the Epi-d1 silencing phenomenon shows a high level of bidirectional metastable mutability. Epigenetic alleles such as Epi-d1 could thus provide for rapid adaptation under selective conditions.