The endoplasmic reticulum (ER) and mitochondria accumulate Ca2+ within their lumens to regulate numerous cell functions. However, determining the dynamics of intraorganellar Ca2+ has proven to be difficult. Here we describe a family of genetically encoded Ca2+ indicators, named calcium-measuring organelle-entrapped protein indicators (CEPIA), which can be utilized for intraorganellar Ca2+ imaging. CEPIA, which emit green, red or blue/green fluorescence, are engineered to bind Ca2+ at intraorganellar Ca2+ concentrations. They can be targeted to different organelles and may be used alongside other fluorescent molecular markers, expanding the range of cell functions that can be simultaneously analysed. The spatiotemporal resolution of CEPIA makes it possible to resolve Ca2+ import into individual mitochondria while simultaneously measuring ER and cytosolic Ca2+. We have used these imaging capabilities to reveal differential Ca2+ handling in individual mitochondria. CEPIA imaging is a useful new tool to further the understanding of organellar functions.

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.

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 (IP 3 )-dependent Ca 2+ signaling in astrocytes, and that the Ca 2+ signaling is required for astrogliosis. We found that type 2 IP 3 receptor knockout (IP 3 R2KO) mice deficient in astrocytic Ca 2+ signaling have impaired reactive astrogliosis and increased injuryassociated 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 Ca 2+ -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 Ca 2+ 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.calcium signal | reactive astrocyte | translational repressor | stab wound A strocytes, a major type of glial cell in the brain, play essential roles not only in physiological functions, such as synaptic plasticity and hemodynamic responses (1), but also in pathophysiological conditions, such as trauma, infection, ischemia, epileptic seizures and stroke. In response to brain injury, astrocytes undergo characteristic phenotypic changes known as reactive astrogliosis, which can exert both beneficial and detrimental effects on surrounding neurons (2-4). Despite the importance of this process, the molecular mechanisms governing astrogliosis and the role of reactive astrocytes require further clarification.Injury to the brain mobilizes astrocyte-reactivating factors, including ATP, endothelin 1 (ET1), glutamate, and inflammatory cytokines, as well as mechanical stress. These factors have been shown to evoke astrocytic Ca 2+ signals not only in culture, but also in acute brain slice preparations and in the brains of live animals (5-7). These findings raise the possibility that injury to the brain evokes astrocytic Ca 2+ signals, which in turn regulate reactive astrogliosis. However, injury-induced Ca 2+ signaling in astrocytes heretofore had not been observed in vivo, and the role of Ca 2+ signaling in pathophysiological processes after brain injury was not established.In this study, we investigated the involvement of astrocytic Ca 2+ signals and underlying molecular mechanisms in reactive astrogliosis and neuroprotection after traumatic brain injury. We found that neocortical injury evokes astrocytic Ca 2+ signals, which are required for reactive astrogliosis and neuronal protection. We identified N-cadherin and pumilio 2 (Pum2) as molecules acting downstream of astrocytic Ca 2+ signals. Around the injury site, N-cadherin is u...

IP(3) signaling in Purkinje cells is involved in the regulation of cell functions including LTD. We have used a GFP-tagged pleckstrin homology domain to visualize IP(3) dynamics in Purkinje cells. Surprisingly, IP(3) production was observed in response not only to mGluR activation, but also to AMPA receptor activation in Purkinje cells in culture. AMPA-induced IP(3) production was mediated by depolarization-induced Ca(2+) influx because it was mimicked by depolarization and was blocked by inhibition of the P-type Ca(2+) channel. Furthermore, trains of complex spikes, elicited by climbing fiber stimulation (1 Hz), induced IP(3) production in Purkinje cells in cerebellar slices. These results revealed a novel IP(3) signaling pathway in Purkinje cells that can be elicited by synaptic inputs from climbing fibers.

Glutamate is the major neurotransmitter in the brain, mediating point-to-point transmission across the synaptic cleft in excitatory synapses. Using a glutamate imaging method with fluorescent indicators, we show that synaptic activity generates extrasynaptic glutamate dynamics in the vicinity of active synapses. These glutamate dynamics had magnitudes and durations sufficient to activate extrasynaptic glutamate receptors in brain slices. We also observed crosstalk between synapses-i.e., summation of glutamate released from neighboring synapses. Furthermore, we successfully observed that sensory input from the extremities induced extrasynaptic glutamate dynamics within the appropriate sensory area of the cerebral cortex in vivo. Thus, the present study clarifies the spatiotemporal features of extrasynaptic glutamate dynamics, and opens up an avenue to directly visualizing synaptic activity in live animals.synapse | spillover | fluorescence imaging | two-photon microscopy | in vivo G lutamate is the major excitatory neurotransmitter in the mammalian brain. The conventional view is that glutamate mediates synaptically confined point-to-point transmission at excitatory synapses. However, glutamate has also been suggested to escape from the synaptic cleft, generating extrasynaptic glutamate dynamics (often referred to as glutamate spillover) (1-4). Extrasynaptic glutamate dynamics has been implicated in the activation of extrasynaptic glutamate receptors via volume transmission to regulate a variety of important neural and glial functions including synaptic transmission (5, 6), synaptic plasticity (7), synaptic crosstalk (8-11), nonsynaptic neurotransmission (12, 13), neuronal survival (14), gliotransmitter release (15-17), and hemodynamic responses (18)(19)(20).Despite the immense potential physiological importance of glutamate spillover, the spatiotemporal dynamics of extrasynaptic glutamate concentration have been only inferred indirectly, and their characteristics remain elusive because of a lack of appropriate technology. Indeed, the magnitude and spatiotemporal distribution of extrasynaptic glutamate concentrations are the key determinants of physiological functions of glutamate spillover, and they are the essential factors for understanding extrasynaptic glutamate signaling. However, we have had to indirectly estimate the spatiotemporal dynamics of the glutamate spillover from its end effects mediated by glutamate receptors using electrophysiological and other means. To overcome this problem, we set out to image extrasynaptic glutamate dynamics in the brain.We developed glutamate indicators derived from the E (glutamate) optical sensor (EOS) (21). EOS is a hybrid-type fluorescent indicator consisting of the glutamate-binding domain of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunit GluR2 and a fluorescent small molecule conjugated near the glutamate-binding pocket. EOS changes its fluorescence intensity upon binding of glutamate, for which it has both high affinity and high se...