General Design Strategy to Precisely Control the Emission of Fluorophores via a Twisted Intramolecular Charge Transfer (TICT) Process.

Fluorogenic probes for bioimaging have become essential tools for life science and medicine, and the key to their development is a precise understanding of the mechanisms available for fluorescence off/on control, such as photoinduced electron transfer (PeT) and Förster resonance energy transfer (FRET). Here we establish a new molecular design strategy to rationally develop activatable fluorescent probes, which exhibit a fluorescence off/on change in response to target biomolecules, by controlling the twisted intramolecular charge transfer (TICT) process. This approach was developed on the basis of a thorough investigation of the fluorescence quenching mechanism of -phenyl rhodamine dyes (commercially available as the QSY series) by means of time-dependent density functional theory (TD-DFT) calculations and photophysical evaluation of their derivatives.

Methodological approaches to understand the molecular mechanism of structural plasticity of dendritic spines.

Dendritic spines are tiny protrusions emanating from the neuronal dendrites, typically housing single excitatory postsynapses. Structural plasticity of dendritic spines is considered to be essential for synaptic functional plasticity and also reorganization of neural circuits during learning and memory. Structural plasticity of spines is mediated by complex biochemical signaling with various spatial and temporal scales.

Genome editing-based approaches for imaging protein localization and dynamics in the mammalian brain.

A neuron contains thousands of proteins, each of which mediates neuronal processes at distinct subcellular compartments. Thus, precise mapping of each protein with subcellular resolution in the brain is essential to understand neuronal processes at the molecular level. However, no conventional methods have provided rapid, generalizable, and high-throughput readouts for the subcellular localization of endogenous proteins in the mammalian brain.

Retrograde Signaling from Progranulin to Sort1 Counteracts Synapse Elimination in the Developing Cerebellum.

Elimination of redundant synapses formed early in development and strengthening of necessary connections are crucial for shaping functional neural circuits. Purkinje cells (PCs) in the neonatal cerebellum are innervated by multiple climbing fibers (CFs) with similar strengths. A single CF is strengthened whereas the other CFs are eliminated in each PC during postnatal development.

Virus-Mediated Genome Editing via Homology-Directed Repair in Mitotic and Postmitotic Cells in Mammalian Brain.

Precise genome editing via homology-directed repair (HDR) in targeted cells, particularly in vivo, provides an invaluable tool for biomedical research. However, HDR has been considered to be largely restricted to dividing cells, making it challenging to apply the technique in postmitotic neurons. Here we show that precise genome editing via HDR is possible in mature postmitotic neurons as well as mitotic cells in mice brain by combining CRISPR-Cas9-mediated DNA cleavage and the efficient delivery of donor template with adeno-associated virus (AAV).

Alternative splicing in the C-terminal tail of Cav2.1 is essential for preventing a neurological disease in mice.

Alternative splicing (AS) that occurs at the final coding exon (exon 47) of the Cav2.1 voltage-gated calcium channel (VGCC) gene produces two major isoforms in the brain, MPI and MPc. These isoforms differ in their splice acceptor sites; human MPI is translated into a polyglutamine tract associated with spinocerebellar ataxia type 6 (SCA6), whereas MPc splices to an immediate stop codon, resulting in a shorter cytoplasmic tail.

High-Throughput, High-Resolution Mapping of Protein Localization in Mammalian Brain by In Vivo Genome Editing.

A scalable and high-throughput method to identify precise subcellular localization of endogenous proteins is essential for integrative understanding of a cell at the molecular level. Here, we developed a simple and generalizable technique to image endogenous proteins with high specificity, resolution, and contrast in single cells in mammalian brain tissue. The technique, single-cell labeling of endogenous proteins by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated homology-directed repair (SLENDR), uses in vivo genome editing to insert a sequence encoding an epitope tag or a fluorescent protein to a gene of interest by CRISPR-Cas9-mediated homology-directed repair (HDR).

Retrograde signaling for climbing fiber synapse elimination.

Neurons form exuberant synapses with target cells early in development. Then, necessary synapses are selectively strengthened whereas unnecessary connections are weakened and eventually eliminated during postnatal development. This process is known as synapse elimination and is a crucial step for shaping immature neural circuits into functionally mature versions.

Global scaling down of excitatory postsynaptic responses in cerebellar Purkinje cells impairs developmental synapse elimination.

Synapse elimination is crucial for precise neural circuit formation during postnatal development. We examined how relative differences in synaptic strengths among competing inputs and/or absolute synaptic strengths contribute to climbing fiber (CF) to Purkinje cell (PC) synapse elimination in the cerebellum. We generated mice with PC-selective deletion of stargazin (TARP γ-2), the major AMPA receptor auxiliary subunit in PCs (γ-2 PC-KO mice).

Retrograde semaphorin signaling regulates synapse elimination in the developing mouse brain.

Neural circuits are shaped by elimination of early-formed redundant synapses during postnatal development. Retrograde signaling from postsynaptic cells regulates synapse elimination. In this work, we identified semaphorins, a family of versatile cell recognition molecules, as retrograde signals for elimination of redundant climbing fiber to Purkinje cell synapses in developing mouse cerebellum.

Arc/Arg3.1 is a postsynaptic mediator of activity-dependent synapse elimination in the developing cerebellum.

Neural circuits are shaped by activity-dependent elimination of redundant synapses during postnatal development. In many systems, postsynaptic activity is known to be crucial, but the precise mechanisms remain elusive. Here, we report that the immediate early gene Arc/Arg3.

Organotypic coculture preparation for the study of developmental synapse elimination in mammalian brain.

We developed an organotypic coculture preparation allowing fast and efficient identification of molecules that regulate developmental synapse elimination in the mammalian brain. This coculture consists of a cerebellar slice obtained from rat or mouse at postnatal day 9 (P9) or P10 and a medullary explant containing the inferior olive dissected from rat at embryonic day 15. We verified that climbing fibers (CFs), the axons of inferior olivary neurons, formed functional synapses onto Purkinje cells (PCs) in the cerebellum of cocultures.

Synapse type-independent degradation of the endocannabinoid 2-arachidonoylglycerol after retrograde synaptic suppression.

The endocannabinoid 2-arachidonoylglycerol (2-AG) mediates retrograde synaptic suppression. Although the mechanisms of 2-AG production are well characterized, how 2-AG is degraded is less clearly understood. Here we found that expression of the 2-AG hydrolyzing enzyme monoacylglycerol lipase (MGL) was highly heterogeneous in the cerebellum, being rich within parallel fiber (PF) terminals, weak in Bergman glia (BG), and absent in other synaptic terminals.

Initial and long-term effects of cloxazolam with intractable epilepsy.

Cloxazolam has been used mainly as an anxiolytic agent. The present study was designed to evaluate the effectiveness of cloxazolam as an add-on antiepileptic drug in patients with intractable epilepsy. A total of 32 patients with intractable epilepsy were treated with cloxazolam: 13 with generalized epilepsy, 15 with focal epilepsy, and 4 with undetermined type of epilepsy.

[Sulthiame treatment for patients with intractable epilepsy].

We examined the antiepileptic effect and side effects of sulthiame in 28 patients with intractable epilepsy. The patients' ages ranged from 6 months to 34 years (mean: 8 years 7 months), and 26 of them were under 18-years-old. Nineteen patients had severe physical and mental disabilities.