Hiroshi Sekiya

In Vivo Visualization of Subtle, Transient, and Local Activity of Astrocytes Using an Ultrasensitive Ca2+ Indicator

Astrocytes generate local calcium (Ca(2+)) signals that are thought to regulate their functions. Visualization of these signals in the intact brain requires an imaging method with high spatiotemporal resolution. Here, we describe such a method using transgenic mice expressing the ultrasensitive ratiometric Ca(2+) indicator yellow Cameleon-Nano 50 (YC-Nano50) in astrocytes. In these mice, we detected a unique pattern of Ca(2+) signals. These occur spontaneously, predominantly in astrocytic fine processes, but not the cell body. Upon sensory stimulation, astrocytes initially responded with Ca(2+) signals at fine processes, which then propagated to the cell body. These observations suggest that astrocytic fine processes function as a high-sensitivity detector of neuronal activities. Thus, the method provides a useful tool for studying the activity of astrocytes in brain physiology and pathology.

Calcium-dependent N-cadherin up-regulation mediates reactive astrogliosis and neuroprotection after brain injury

Brain injury induces phenotypic changes in astrocytes, known as reactive astrogliosis, which may influence neuronal survival. Here we show that brain injury induces inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ signaling in astrocytes, and that the Ca2+ signaling is required for astrogliosis. We found that type 2 IP3 receptor knockout (IP3R2KO) mice deficient in astrocytic Ca2+ signaling have impaired reactive astrogliosis and increased injury-associated neuronal death. We identified N-cadherin and pumilio 2 (Pum2) as downstream signaling molecules, and found that brain injury induces up-regulation of N-cadherin around the injured site. This effect is mediated by Ca2+-dependent down-regulation of Pum2, which in turn attenuates Pum2-dependent translational repression of N-cadherin. Furthermore, we show that astrocyte-specific knockout of N-cadherin results in impairment of astrogliosis and neuroprotection. Thus, astrocytic Ca2+ signaling and the downstream function of N-cadherin play indispensable roles in the cellular responses to brain injury. These findings define a previously unreported signaling axis required for reactive astrogliosis and neuroprotection following brain injury.

Ventrolateral striatal medium spiny neurons positively regulate food-incentive, goal-directed behavior independently of D1 and D2 selectivity

The ventral striatum is involved in motivated behavior. Akin to the dorsal striatum, the ventral striatum contains two parallel pathways: the striatomesencephalic pathway consisting of dopamine receptor Type 1-expressing medium spiny neurons (D1-MSNs) and the striatopallidal pathway consisting of D2-MSNs. These two genetically identified pathways are thought to encode opposing functions in motivated behavior. It has also been reported that D1/D2 genetic selectivity is not attributed to the anatomical discrimination of two pathways. We wanted to determine whether D1- and D2-MSNs in the ventral striatum functioned in an opposing manner as previous observations claimed, and whether D1/D2 selectivity corresponded to a functional segregation in motivated behavior of mice. To address this question, we focused on the lateral portion of ventral striatum as a region implicated in food-incentive, goal-directed behavior, and recorded D1 or D2-MSN activity by using a gene-encoded ratiometric Ca2+ indicator and by constructing a fiberphotometry system, and manipulated their activities via optogenetic inhibition during ongoing behaviors. We observed concurrent event-related compound Ca2+ elevations in ventrolateral D1- and D2-MSNs, especially at trial start cue-related and first lever press-related times. D1 or D2 selective optogenetic inhibition just after the trial start cue resulted in a reduction of goal-directed behavior, indicating a shared coding of motivated behavior by both populations at this time. Only D1-selective inhibition just after the first lever press resulted in the reduction of behavior, indicating D1-MSN-specific coding at that specific time. Our data did not support opposing encoding by both populations in food-incentive, goal-directed behavior. SIGNIFICANCE STATEMENT An opposing role of dopamine receptor Type 1 or Type 2-expressing medium spiny neurons (D1-MSNs or D2-MSNs) on striatum-mediated behaviors has been widely accepted. However, this idea has been questioned by recent reports. In the present study, we measured concurrent Ca2+ activity patterns of D1- and D2-MSNs in the ventrolateral striatum during food-incentive, goal-directed behavior in mice. According to Ca2+ activity patterns, we conducted timing-specific optogenetic inhibition of each type of MSN. We demonstrated that both D1- and D2-MSNs in the ventrolateral striatum commonly and positively encoded action initiation, whereas only D1-MSNs positively encoded sustained motivated behavior. These findings led us to reconsider the prevailing notion of a functional segregation of MSN activity in the ventral striatum.

A three-dimensional single-cell-resolution whole-brain atlas using CUBIC-X expansion microscopy and tissue clearing

A three-dimensional single-cell-resolution mammalian brain atlas will accelerate systems-level identification and analysis of cellular circuits underlying various brain functions. However, its construction requires efficient subcellular-resolution imaging throughout the entire brain. To address this challenge, we developed a fluorescent-protein-compatible, whole-organ clearing and homogeneous expansion protocol based on an aqueous chemical solution (CUBIC-X). The expanded, well-cleared brain enabled us to construct a point-based mouse brain atlas with single-cell annotation (CUBIC-Atlas). CUBIC-Atlas reflects inhomogeneous whole-brain development, revealing a significant decrease in the cerebral visual and somatosensory cortical areas during postnatal development. Probabilistic activity mapping of pharmacologically stimulated Arc-dVenus reporter mouse brains onto CUBIC-Atlas revealed the existence of distinct functional structures in the hippocampal dentate gyrus. CUBIC-Atlas is shareable by an open-source web-based viewer, providing a new platform for whole-brain cell profiling.The authors developed a CUBIC tissue clearing and expansion method to generate an editable, point-based single-cell-resolution brain atlas. This atlas, termed CUBIC-Atlas, can be used for unbiased systems-level cellular analysis in whole mouse brain.

Promising techniques to illuminate neuromodulatory control of the cerebral cortex in sleeping and waking states

Sleep, a common event in daily life, has clear benefits for brain function, but what goes on in the brain when we sleep remains unclear. Sleep was long regarded as a silent state of the brain because the brain seemingly lacks interaction with the surroundings during sleep. Since the discovery of electrical activities in the brain at rest, electrophysiological methods have revealed novel concepts in sleep research. During sleep, the brain generates oscillatory activities that represent characteristic states of sleep. In addition to electrophysiology, opto/chemogenetics and two-photon Ca2+ imaging methods have clarified that the sleep/wake states organized by neuronal and glial ensembles in the cerebral cortex are transitioned by neuromodulators. Even with these methods, however, it is extremely difficult to elucidate how and when neuromodulators spread, accumulate, and disappear in the extracellular space of the cortex. Thus, real-time monitoring of neuromodulator dynamics at high spatiotemporal resolution is required for further understanding of sleep. Toward direct detection of neuromodulator behavior during sleep and wakefulness, in this review, we discuss developing imaging techniques based on the activation of G-protein-coupled receptors that allow for visualization of neuromodulator dynamics.

Whisker experience-dependent mGluR signaling maintains synaptic strength in the mouse adolescent cortex.

Sensory experience‐dependent plasticity in the somatosensory cortex is a fundamental mechanism of adaptation to the changing environment not only early in the development but also in adolescence and adulthood. Although the mechanisms underlying experience‐dependent plasticity during early development have been well documented, the corresponding understanding in the mature cortex is less complete. Here, we investigated the mechanism underlying whisker deprivation‐induced synaptic plasticity in the barrel cortex in adolescent mice. Layer 4 (L4) to L2/3 excitatory synapses play a crucial role for whisker experience‐dependent plasticity in rodent barrel cortex and whisker deprivation is known to depress synaptic strength at L4–L2/3 synapses in adolescent and adult animals. We found that whisker deprivation for 5 days or longer decreased the presynaptic glutamate release probability at L4–L2/3 synapses in the barrel cortex in adolescent mice. This whisker deprivation‐induced depression was restored by daily administration of a positive allosteric modulator of the type 5 metabotropic glutamate receptor (mGluR5). On the other hand, the administration of mGluR5 antagonists reproduced the effect of whisker deprivation in whisker‐intact mice. Furthermore, chronic and selective suppression of inositol 1,4,5‐trisphosphate (IP3) signaling in postsynaptic L2/3 neurons decreased the presynaptic release probability at L4–L2/3 synapses. These findings represent a previously unidentified mechanism of cortical plasticity, namely that whisker experience‐dependent mGluR5‐IP3 signaling in the postsynaptic neurons maintains presynaptic function in the adolescent barrel cortex.

Chemical Landscape for Tissue Clearing Based on Hydrophilic Reagents

We describe a strategy for developing hydrophilic chemical cocktails for tissue delipidation, decoloring, refractive index (RI) matching, and decalcification, based on comprehensive chemical profiling. More than 1,600 chemicals were screened by a high-throughput evaluation system for each chemical process. The chemical profiling revealed important chemical factors: salt-free amine with high octanol/water partition-coefficient (logP) for delipidation, N-alkylimidazole for decoloring, aromatic amide for RI matching, and protonation of phosphate ion for decalcification. The strategic integration of optimal chemical cocktails provided a series of CUBIC (clear,xa0unobstructed brain/body imaging cocktails and computational analysis) protocols, which efficiently clear mouse organs, mouse body including bone, and even large primate and human tissues. The updated CUBIC protocols are scalable and reproducible, and they enable three-dimensional imaging of the mammalian body and large primate and human tissues. This strategy represents a future paradigm for the rational design of hydrophilic clearing cocktails that can be used for large tissues.

Imaging glutamate spillover from synaptic clefts using the fluorescent glutamate indicator

The serine/threonine kinase SAD regulates several synaptic functions such as synapse development, axon/dendrite polarization, and neurotransmitter release. In mammalian central nervous system (CNS), SAD is localized on synaptic vesicles and at the active zone in nerve terminals where SAD appears to phosphorylate the active zone protein RIM1 implicated in neurotransmitter release. However, expression and localization of SAD in peripheral nervous system (PNS) is currently unknown. Here we have attempted to examine its expression and localization at synapses of neuromuscular junctions (NMJs). In the mouse diaphragm, quadriceps femoris muscle, and lumbrical muscle, the immunoreactivity for SAD is colocalized with that for -bungarotoxin, a major marker for NMJs, which binds to acetylcholine receptors. These results suggest that SAD is localized at NMJs and a presynaptic component at the PNS as well as the CNS.