Unfolding neural mechanisms by Ca2+ signaling-based research

Ca2+ signaling is one of the principal signaling mechanisms regulating myriads of cellular functions (see Figure below). Intracellular Ca2+ signals display diverse spatiotemporal dynamics, which underlie the versatility of Ca2+ in cell signaling. We showed that the intracellular Ca2+ release mechanism has an inherent regenerative property, which is essential for the generation of spatiotemporal dynamics of Ca2+ signaling.

Our research on the basic principles of Ca2+ signaling has led to the development of a new method for specific inhibition of Ca2+ signaling, which allowed us to identify hitherto unrecognized functions of Ca2+ signals in the brain such as activity-dependent synaptic maintenance mechanism and glial cell-dependent neurite growth. We are currently extending these lines of research to clarify their molecular basis and in vivo physiological roles.

We have a strength in the development of new imaging methods. We have successfully imaged neural NO dynamics and are now studying a novel (patho)physiological mechanism of NO signaling in the brain. Furthermore, we attempted to visualize glutamate dynamics in the brain and have succeeded in imaging extrasynaptic glutamate dynamics and sensory-input dependent glutamate dynamics in the cerebral cortex in vivo. Using these novel modalities of brain function imaging, we wish to clarify neural network mechanisms in the brain.

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Although Ca2+ signal can be generated by an influx of Ca2+ from the extracellular space, Ca2+ release from the intracellular stores plays very important roles. The endoplasmic reticulum functions as the intracellular Ca2+ store, and Ca2+ can be released via two types of Ca2+ release channel; ryanodine receptors and inositol 1,4,5-trisphosphate (IP3) receptors (see Figure below).

Our laboratory has clarified basic mechanisms of the Ca2+ release and Ca2+ signaling mechanisms as summarized below.

Ca2+ signals show very dynamic, temporal and spatial changes. This property allows the calcium signal to be an extremely versatile cellular switch regulating diverse cell functions. One of the most notable spatiotemporal patterns of Ca2+ signals is the oscillatory change in intracellular Ca2+ concentration ([Ca2+]i), or Ca2+ oscillation.  Many cellular functions are regulated by the Ca2+ oscillation frequency. However, fundamental questions remain. How and what for does [Ca2+]i oscillate? We have addressed these questions.

First, we studied inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release mechanism, which is one of the most important Ca2+ mobilizing mechanisms in many types of cell.  We showed that the activity of the IP3 receptor (IP3R) is dependent not only on the IP3 concentration but also on the cytoplasmic Ca2+ concentration (J. Gen. Physiol, 1990; Nature 1992). Therefore, Ca2+ release via the IP3R may be under the feedback control of mobilized Ca2+. There are three subtypes of IP3R, which have different sensitivities to IP3 and Ca2+ concentrations. W showed that these subtypes generate different patterns of Ca2+ oscillation (EMBO J. 1999). We identified the Ca2+ sensor region of the IP3R, and experimentally showed that the positive feedback regulation of IP3R via the Ca2+ sensor of IP3R indeed plays an essential role in regulating the Ca2+ signal dynamics including Ca2+ oscillation (EMBO J., 2001).  Although the positive feedback regulation underlies the rising phase of each Ca2+ oscillation, the pacemaking mechanism remained elusive. We recently showed that Ca2+ uptake and release from mitochondria plays a pivotal role in pacemaking of Ca2+ oscillations (EMBO Rep. 2006). These results provide a clue to the mechanism of Ca2+ oscillation.

Whar for then does [Ca2+]i oscillate? Transcription by the nuclear factor of activated T cells (NFAT) is one of the important cellular functions that are regulated by the Ca2+ oscillation frequency. NFAT is dephosphorylated by Ca2+-dependent phosphatase, calcineurin, and translocates from the cytoplasm to the nucleus to initiate transcription. We analyzed the kinetics of the dephosphorylation and translocation of NFAT, and found that the dephosphorylated form of NFAT functions as a working memory of transient increases in [Ca2+]i (EMBO J. 2003). With increasing frequency of Ca2+ oscillation, dephosphorylated NFAT accumulates in the cytoplasm to enhance its nuclear translocation. This is the molecular basis of the mechanism that decodes the Ca2+ oscillation frequency. We also showed that Ca2+ oscillation is more cost-effective in regulating cell functions than a continuous increase in Ca2+. These studies provide us with an insight into the secrets of Ca2+ signalling.

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Our study on Ca2+ signalling made us realize the importance of visualization of signalling molecules within living cells.  Thus, our laboratory has been involved in the generation of new indicators of signalling molecules upstream and downstream Ca2+ signals.

IP3 imaging

We generated an IP3 indicator based on the PH domain of PLC{delta}1, and have succeeded in imaging IP3 signalling in various cells including intact neurons within cerebellar slice preparations. Our study showed that IP3 signaling in cerebellar Purkinje cells are cooperatively regulated by both metabotropic and ionotropic glutamate receptors.

Nitric oxide (NO) imaging

We generated a nitric oxide (NO) indicator based on the heme-binding region of soluble guanylyl cyclase. This indicator was successfully used in cerebellar slice preparations to image NO signals in response to parallel fiber (PF) stimulation. We found that the NO signal intensity decreases steeply with distance form the activated synapse and generate synapse-specific long-term potentiation (LTP) of PF-Purkinje cell synapses. We also showed that the NO signal intensity depends biphasically on the frequency of PF stimulation. Importantly, the LTP depends similarly on the frequency of PF stimulation. Thus, our NO indicator provided us with valuable information regarding the role of NO signals in the central nervous system.

We have applied these studies to the analysis of synaptic functions of the cerebellar neurons and obtained several new findings summarized in the Figure below.

PYK2 imaging

PYK2 is a Ca2+-dependent tyrosine kinase of FAK family. We imaged GFP-tagged PYK2 at the cell-substrate interface, and found that Ca2+ dependent activation of PYK2 is important for cell retraction at the cell-cell contact interface. During the course of this study we found a new form of Ca2+ signaling "Ca2+ lightning" which seems to detect the cell-cell contact.

Glutamate imaging

Glutamate is the most important excitatory neurotransmitter in the brain. We have generated a new glutamate indicator (EOS) based on the glutamate binding site of the ionotropic glutamate receptor (GluR2). We have successfully used this indicator to image glutamate dynamics in acute brain slices and in cerebal cortex in vivo.

We use various light microsopes including two-photon microscope, confocal laser scanning microscope, evanescent microscope in addtion to conventional wide field fluorescent microscope.

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Our laboratory is searching for new cell functions that are regulated by Ca2+ signals by using a method to inhibit IP3-induced Ca2+ release. We have so far identified an activity-dependent synaptic maitenance mechanism in the crebellar cortex (see Figure above) and a neuron-astrocyte interaction in terms of the promotion of neurite growth. We have found a new NO-Ca2+ coupling mechanism that is involved in both synaptic plasticity and neuronal cell death. We hope to extend these studies to obtain further insights into the neuronal ang glial functions in the central nervous system.

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Combining new imaging tools and the specific method to inhibit Ca2+ signals, we wish to contribute to the advancement of our knowledge in cell signaling especially in conjunction with brain functions.

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