Probing neuronal activity with genetically encoded calcium and voltage fluorescent indicators.

Monitoring neural activity in individual neurons is crucial for understanding neural circuits and brain functions. The emergence of optical imaging technologies has dramatically transformed the field of neuroscience, enabling detailed observation of large-scale neuronal populations with both cellular and subcellular resolution. This transformation will be further accelerated by the integration of these imaging technologies and advanced big data analysis. Genetically encoded fluorescent indicators to detect neural activity with high signal-to-noise ratios are pivotal in this advancement. In recent years, these indicators have undergone significant developments, greatly enhancing the understanding of neural dynamics and networks. This review highlights the recent progress in genetically encoded calcium and voltage indicators and discusses the future direction of imaging techniques with big data analysis that deepens our understanding of the complexities of the brain.

The A-kinase anchoring protein Yotiao decrease the ER calcium content by inhibiting the store operated calcium entry.

The meticulous regulation of ER calcium (Ca2+) homeostasis is indispensable for the proper functioning of numerous cellular processes. Disrupted ER Ca2+ balance is implicated in diverse diseases, underscoring the need for a systematic exploration of its regulatory factors in cells. Our recent genomic-scale screen identified a scaffolding protein A-kinase anchoring protein 9 (AKAP9) as a regulator of ER Ca2+ levels, but the underlying molecular mechanisms remain elusive. Here, we reveal that Yotiao, the smallest splicing variant of AKAP9 decreased ER Ca2+ content in animal cells. Additional testing using a combination of Yotiao truncations, knock-out cells and pharmacological tools revealed that, Yotiao does not require most of its interactors, including type 1 inositol 1,4,5-trisphosphate receptors (IP3R1), protein kinase A (PKA), protein phosphatase 1 (PP1), adenylyl cyclase type 2 (AC2) and so on, to reduce ER Ca2+ levels. However, adenylyl cyclase type 9 (AC9), which is known to increases its cAMP generation upon interaction with Yotiao for the modulation of potassium channels, plays an essential role for Yotiao's ER-Ca2+-lowering effect. Mechanistically, Yotiao may work through AC9 to act on Orai1-C terminus and suppress store operated Ca2+ entry, resulting in reduced ER Ca2+ levels. These findings not only enhance our comprehension of the interplay between Yotiao and AC9 but also contribute to a more intricate understanding of the finely tuned mechanisms governing ER Ca2+ homeostasis.

A multicolor suite for deciphering population coding of calcium and cAMP in vivo.

cAMP is a universal second messenger regulated by various upstream pathways including Ca2+ and G-protein-coupled receptors (GPCRs). To decipher in vivo cAMP dynamics, we rationally designed cAMPinG1, a sensitive genetically encoded green cAMP indicator that outperformed its predecessors in both dynamic range and cAMP affinity. Two-photon cAMPinG1 imaging detected cAMP transients in the somata and dendritic spines of neurons in the mouse visual cortex on the order of tens of seconds. In addition, multicolor imaging with a sensitive red Ca2+ indicator RCaMP3 allowed simultaneous measurement of population patterns in Ca2+ and cAMP in hundreds of neurons. We found Ca2+-related cAMP responses that represented specific information, such as direction selectivity in vision and locomotion, as well as GPCR-related cAMP responses. Overall, our multicolor suite will facilitate analysis of the interaction between the Ca2+, GPCR and cAMP signaling at single-cell resolution both in vitro and in vivo.

A Flp-dependent G-CaMP9a transgenic mouse for neuronal imaging in vivo.

Genetically encoded calcium indicators (GECIs) are widely used to measure calcium transients in neuronal somata and processes, and their use enables the determination of action potential temporal series in a large population of neurons. Here, we generate a transgenic mouse line expressing a highly sensitive green GECI, G-CaMP9a, in a Flp-dependent manner in excitatory and inhibitory neuronal subpopulations downstream of a strong CAG promoter. Combining this reporter mouse with viral or mouse genetic Flp delivery methods produces a robust and stable G-CaMP9a expression in defined neuronal populations without detectable detrimental effects. In vivo two-photon imaging reveals spontaneous and sensory-evoked calcium transients in excitatory and inhibitory ensembles with cellular resolution. Our results show that this reporter line allows long-term, cell-type-specific investigation of neuronal activity with enhanced resolution in defined populations and facilitates dissecting complex dynamics of neural networks in vivo.

Imaging Voltage with Microbial Rhodopsins.

Membrane potential is the critical parameter that reflects the excitability of a neuron, and it is usually measured by electrophysiological recordings with electrodes. However, this is an invasive approach that is constrained by the problems of lacking spatial resolution and genetic specificity. Recently, the development of a variety of fluorescent probes has made it possible to measure the activity of individual cells with high spatiotemporal resolution. The adaptation of this technique to image electrical activity in neurons has become an informative method to study neural circuits. Genetically encoded voltage indicators (GEVIs) can be used with superior performance to accurately target specific genetic populations and reveal neuronal dynamics on a millisecond scale. Microbial rhodopsins are commonly used as optogenetic actuators to manipulate neuronal activities and to explore the circuit mechanisms of brain function, but they also can be used as fluorescent voltage indicators. In this review, we summarize recent advances in the design and the application of rhodopsin-based GEVIs.

A Photodeactivatable Antagonist for Controlling CREB-Dependent Gene Expression.

A novel photodeactivation strategy for controlling gene expression has been developed based on light-induced activation of cAMP response element binding protein (CREB). Light-induced cleavage of the photoresponsive protecting group of an antagonist of CREB binding protein (CBP) results in photocleaved products with weak binding affinity for CBP. This photodissociation reaction enables protein-protein interactions between CBP and CREB that trigger the formation of a multiprotein transcription complex to turn gene expression "on". This enables irradiation of antagonist-treated HEK293T cells to be used to trigger temporal recovery of CREB-dependent transcriptional activity and endogenous gene expression under photolytic control.

Photolytic Release of a Caged Inhibitor of an Endogenous Transcription Factor Enables Optochemical Control of CREB-Mediated Gene Expression.

A direct optochemical method for regulating gene function has been developed based on uncaging of an inactive caged precursor that fragments to produce a CREB (cAMP-response element binding protein) inhibitor that binds to an endogenous transcription factor responsible for regulating CREB-mediated gene expression levels.

Rational Engineering of XCaMPs, a Multicolor GECI Suite for In Vivo Imaging of Complex Brain Circuit Dynamics.

To decipher dynamic brain information processing, current genetically encoded calcium indicators (GECIs) are limited in single action potential (AP) detection speed, combinatorial spectral compatibility, and two-photon imaging depth. To address this, here, we rationally engineered a next-generation quadricolor GECI suite, XCaMPs. Single AP detection was achieved within 3-10 ms of spike onset, enabling measurements of fast-spike trains in parvalbumin (PV)-positive interneurons in the barrel cortex in vivo and recording three distinct (two inhibitory and one excitatory) ensembles during pre-motion activity in freely moving mice. In vivo paired recording of pre- and postsynaptic firing revealed spatiotemporal constraints of dendritic inhibition in layer 1 in vivo, between axons of somatostatin (SST)-positive interneurons and apical tufts dendrites of excitatory pyramidal neurons. Finally, non-invasive, subcortical imaging using red XCaMP-R uncovered somatosensation-evoked persistent activity in hippocampal CA1 neurons. Thus, the XCaMPs offer a critical enhancement of solution space in studies of complex neuronal circuit dynamics. VIDEO ABSTRACT.

A case of recurrent depressive disorder presenting with Alice in Wonderland syndrome: psychopathology and pre- and post-treatment FDG-PET findings.

BACKGROUND: Alice in Wonderland syndrome (AIWS) is a rare neuropsychiatric syndrome that typically manifests in distortion of extrapersonal visual image, altered perception of one's body image, and a disturbed sense of the passage of distance and time. Several conditions have been reported to contribute to AIWS, although its biological basis is still unknown. Here, we present the first case demonstrating a clear concurrence of recurrent depressive disorder and AIWS. The clinical manifestations and pre- and post-treatment fluorodeoxyglucose positron-emission tomographic (FDG-PET) images provide insights into the psychopathological and biological basis of AIWS. CASE PRESENTATION: We describe a 63-year-old Japanese male who developed two distinct episodes of major depression concurrent with AIWS. In addition to typical AIWS perceptual symptoms, he complained of losing the ability to intuitively grasp the seriousness of news and the value of money, which implies disturbance of high-order cognition related to estimating magnitude and worth. Both depression and AIWS remitted after treatment in each episode. Pre-treatment FDG-PET images showed significant hypometabolism in the frontal cortex and hypermetabolism in the occipital and parietal cortex. Post-treatment images showed improvement of these abnormalities. CONCLUSIONS: The clinical co-occurrence of depressive episodes and presentation of AIWS can be interpreted to mean that they have certain functional disturbances in common. In view of incapacity, indifference, devitalization, altered perception of one's body image, and disturbed sense of time and space, the features of AIWS analogous to those of psychotic depression imply a common psychopathological basis. These high-order brain dysfunctions are possibly associated with the metabolic abnormalities in visual and parietotemporal association cortices that we observed on the pre- and post-treatment FDG-PET images in this case, while the hypometabolism in the frontal cortex is probably associated with depressive symptoms.