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.

[Measuring Neural Activity Using Calcium Indicators].

Calcium imaging is a widely used imaging method to measure neuronal activity. In recent years, various highly sensitive and fast genetically encoded calcium indicators have been developed. Moreover, by utilizing these sensors with a multiphoton microscope and a microendoscope, it is possible to simultaneously measure the neural activity from multiple neurons with single-cell resolution in living animals. In this review, I will introduce features of fluorescent calcium probes and their application in vivo.

Detection of Membrane Potential-Dependent Rhodopsin Fluorescence Using Low-Intensity Light Emitting Diode for Long-Term Imaging.

Microbial rhodopsin is a family of photoreceptive membrane proteins that commonly consist of a seven-transmembrane domain and a derivative of vitamin-A, retinal, as a chromophore. In 2011, archaeorhodopsin-3 (AR3) was shown to exhibit voltage-dependent fluorescence changes in mammalian cells. Since then, AR3 and its variants have been used as genetically encoded voltage indicators, in which mostly intense laser stimulation (1-1000 W/cm2) is used for the detection of dim fluorescence of rhodopsin, leading to high spatiotemporal resolution. However, intense laser stimulation potentially causes serious cell damage, particularly during long-term imaging over minutes. In this study, we present the successful detection of voltage-sensitive fluorescence of AR3 and its high fluorescence mutant Archon1 in a variety of mammalian cell lines using low-intensity light emitting diode stimulation (0.15 W/cm2) with long exposure time (500 ms). The detection system enables real-time imaging of drug-induced slow changes in voltage within the cells for minutes harmlessly and without fluorescence bleaching. Therefore, we demonstrate a method to quantitatively understand the dynamics of slow changes in membrane voltage on long time scales.

In utero electroporation and cranial window implantation for in vivo wide-field two-photon calcium imaging using G-CaMP9a transgenic mice.

We present a protocol to prepare mouse cranial window implantation for in vivo two-photon wide-field calcium imaging. This protocol uses G-CaMP9a transgenic mice, which express a genetically encoded calcium indicator with high signal-to-noise ratio. We describe in utero electroporation, followed by headplate fixation and cranial window implantation. This protocol can be used for measuring neural activity and is suitable for long-term imaging in large populations. Moreover, this protocol does not require preparation of Flp-expressing transgenic mice. For complete details on the use and execution of this protocol, please refer to Sakamoto et al. (2022).

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.

Comparative Studies of the Fluorescence Properties of Microbial Rhodopsins: Spontaneous Emission Versus Photointermediate Fluorescence.

Rhodopsins are seven-transmembrane photoreceptor proteins that bind to the retinal chromophore and have been utilized as a genetically encoded voltage indicator (GEVI). So far, archaerhodopsin-3 (AR3) has been successfully used as a GEVI, despite its low fluorescence intensity. We performed comparative and quantitative fluorescence analyses of 15 microbial rhodopsins to explore these highly fluorescent molecules and to clarify their fluorescence mechanism. These rhodopsins showed a wide range of fluorescence intensities in mouse hippocampal neurons. Some of them, GR, HwBR, IaNaR, MR, and NpHR, showed fluorescence intensities comparable with or higher than that of AR3, suggesting their potential for GEVIs. The fluorescence intensity in neurons correlated with that of the bright fluorescent photointermediate such as a Q-intermediate (R = 0.75), suggesting that the fluorescence in neurons originates from the fluorescence of the photointermediate. Our findings provide a crucial step for producing next-generation rhodopsin-based GEVIs.

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.

Comparative Evaluation of Genetically Encoded Voltage Indicators.

Imaging voltage using fluorescent-based sensors could be an ideal technique to probe neural circuits with high spatiotemporal resolution. However, due to insufficient signal-to-noise ratio (SNR), imaging membrane potential in mammalian preparations is still challenging. In recent years, many genetically encoded voltage indicators (GEVIs) have been developed. To compare them and guide decisions on which GEVI to use, we have characterized side by side the performance of eight GEVIs that represent different families of molecular constructs. We tested GEVIs in vitro with 1-photon imaging and in vivo with 1-photon wide-field imaging and 2-photon imaging. We find that QuasAr2 exhibited the best performance in vitro, whereas only ArcLight-MT could be used to reliably detect electrical activity in vivo with 2-photon excitation. No single GEVI was ideal for every experiment. These results provide a guide for choosing optimal GEVIs for specific applications.

Attenuation of Synaptic Potentials in Dendritic Spines.

Dendritic spines receive the majority of excitatory inputs in many mammalian neurons, but their biophysical properties and exact role in dendritic integration are still unclear. Here, we study spine electrical properties in cultured hippocampal neurons using an improved genetically encoded voltage indicator (ArcLight) and two-photon glutamate uncaging. We find that back-propagating action potentials (bAPs) fully invade dendritic spines. However, uncaging excitatory post-synaptic potentials (uEPSPs) generated by glutamate photorelease, ranging from 4 to 27 mV in amplitude, are attenuated by up to 4-fold as they propagate to the parent dendrites. Finally, the simultaneous occurrence of bAPs and uEPSPs results in sublinear summation of membrane potential. Our results demonstrate that spines can behave as electric compartments, reducing the synaptic inputs injected into the cell, while receiving bAPs are unmodified. The attenuation of EPSPs by spines could have important repercussions for synaptic plasticity and dendritic integration.

Calmodulin kinases: essential regulators in health and disease.

Neuronal activity induces intracellular Ca2+ increase, which triggers activation of a series of Ca2+ -dependent signaling cascades. Among these, the multifunctional Ca2+ /calmodulin-dependent protein kinases (CaMKs, or calmodulin kinases) play key roles in neuronal transmission, synaptic plasticity, circuit development and cognition. The most investigated CaMKs for these roles in neuronal functions are CaMKI, CaMKII, CaMKIV and we will shed light on these neuronal CaMKs' functions in this review. Catalytically active members of CaMKs currently are CaMKI, CaMKII, CaMKIV and CaMKK. Although they all necessitate the binding of Ca2+ and calmodulin complex (Ca2+ /CaM) for releasing autoinhibition, each member of CaMK has distinct activation mechanisms-autophosphorylation mediated autonomy of multimeric CaMKII and CaMKK-dependent phosphoswitch-induced activation of CaMKI or CaMKIV. Furthermore, each CaMK shows distinct subcellular localization that underlies specific compartmentalized function in each activated neuron. In this review, we first summarize these molecular characteristics of each CaMK as to regulation and subcellular localization, and then describe each biological function. In the last section, we also focus on the emerging role of CaMKs in pathophysiological conditions by introducing the recent studies, especially focusing on drug addiction and depression, and discuss how dysfunctional CaMKs may contribute to the pathology of the neuropsychological disorders. This article is part of the mini review series "60th Anniversary of the Japanese Society for Neurochemistry".

The functional significance of newly born neurons integrated into olfactory bulb circuits.

The olfactory bulb (OB) is the first central processing center for olfactory information connecting with higher areas in the brain, and this neuronal circuitry mediates a variety of odor-evoked behavioral responses. In the adult mammalian brain, continuous neurogenesis occurs in two restricted regions, the subventricular zone (SVZ) of the lateral ventricle and the hippocampal dentate gyrus. New neurons born in the SVZ migrate through the rostral migratory stream and are integrated into the neuronal circuits of the OB throughout life. The significance of this continuous supply of new neurons in the OB has been implicated in plasticity and memory regulation. Two decades of huge investigation in adult neurogenesis revealed the biological importance of integration of new neurons into the olfactory circuits. In this review, we highlight the recent findings about the physiological functions of newly generated neurons in rodent OB circuits and then discuss the contribution of neurogenesis in the brain function. Finally, we introduce cutting edge technologies to monitor and manipulate the activity of new neurons.

Continuous postnatal neurogenesis contributes to formation of the olfactory bulb neural circuits and flexible olfactory associative learning.

The olfactory bulb (OB) is one of the two major loci in the mammalian brain where newborn neurons are constantly integrated into the neural circuit during postnatal life. Newborn neurons are generated from neural stem cells in the subventricular zone (SVZ) of the lateral ventricle and migrate to the OB through the rostral migratory stream. The majority of these newborn neurons differentiate into inhibitory interneurons, such as granule cells and periglomerular cells. It has been reported that prolonged supply of newborn neurons leads to continuous addition/turnover of the interneuronal populations and contributes to functional integrity of the OB circuit. However, it is not still clear how and to what extent postnatal-born neurons contribute to OB neural circuit formation, and the functional role of postnatal neurogenesis in odor-related behaviors remains elusive. To address this question, here by using genetic strategies, we first determined the unique integration mode of newly born interneurons during postnatal development of the mouse OB. We then manipulated these interneuron populations and found that continuous postnatal neurogenesis in the SVZ-OB plays pivotal roles in flexible olfactory associative learning and memory.

Genetic visualization of notch signaling in mammalian neurogenesis.

Notch signaling plays crucial roles in fate determination and the differentiation of neural stem cells in embryonic and adult brains. It is now clear that the notch pathway is under more complex and dynamic regulation than previously thought. To understand the functional details of notch signaling more precisely, it is important to reveal when, where, and how notch signaling is dynamically communicated between cells, for which the visualization of notch signaling is essential. In this review, we introduce recent technical advances in the visualization of notch signaling during neural development and in the adult brain, and we discuss the physiological significance of dynamic regulation of notch signaling.

Light-induced silencing of neural activity in Rosa26 knock-in mice conditionally expressing the microbial halorhodopsin eNpHR2.0.

Temporally precise inhibition of genetically defined cell populations in intact nervous systems has been enabled by the microbial halorhodopsin NpHR, a fast, light-activated chloride pump. Here, we report the generation of new mouse strains that express eNpHR2-EYFP fusion proteins after Cre- and/or Flp-mediated recombination to silence neural activity in vivo. In these mouse strains, Cre/Flp recombination induced a high-level of eNpHR2-EYFP expression. Slice whole-cell patch clamp experiments confirmed that eNpHR2-EYFP-expressing neurons could be optically hyperpolarized and inhibited from firing action potentials. Thus, these mouse strains offer powerful tools for light-induced silencing of neural activity in genetically defined cell populations.

A multifunctional teal-fluorescent Rosa26 reporter mouse line for Cre- and Flp-mediated recombination.

Reporters of Cre and/or Flp activity are important for defining the spatial and temporal extent of Cre/Flp-mediated recombination. Here, we describe R26-CAG-LF-mTFP1, a multifunctional fluorescent reporter mouse that strongly expresses mTFP1 (bright teal fluorescent protein) after Cre- and Flp-mediated recombination. To meet the need for single recombinase-mediated reporter expression, we generated derivatives of R26-CAG-LF-mTFP1. The germline excision of the Frt-flanked stop cassette in R26-CAG-LF-mTFP1 generated a Cre-dependent reporter (R26-CAG-LoxP-mTFP1). Similarly, R26-CAG-FRT-mTFP1, in which the loxP-flanked stop cassette was excised in the germline, requires only Flp to activate mTFP1 expression.

The role of neurogenesis in olfaction-dependent behaviors.

Newly born neurons continuously migrate into the main and accessory olfactory bulbs and modulate the output of projection neurons. Despite some contradictory results, it is becoming clear that these newly born neurons play an important role in the response to some odorant cues. In this minireview, we discuss the recent findings surrounding the functional significance of adult neurogenesis in olfaction-dependent behaviors.

Genetic methods to identify and manipulate newly born neurons in the adult brain.

Although mammalian neurogenesis is mostly completed by the perinatal period, new neurons are continuously generated in the subventricular zone of the lateral ventricle and the subgranular zone of the hippocampal dentate gyrus. Since the discovery of adult neurogenesis, many extensive studies have been performed on various aspects of adult neurogenesis, including proliferation and fate-specification of adult neural stem cells, and the migration, maturation and synaptic integration of newly born neurons. Furthermore, recent research has shed light on the intensive contribution of adult neurogenesis to olfactory-related and hippocampus-mediated brain functions. The field of adult neurogenesis progressed tremendously thanks to technical advances that facilitate the identification and selective manipulation of newly born neurons among billions of pre-existing neurons in the adult central nervous system. In this review, we introduce recent advances in the methodologies for visualizing newly generated neurons and manipulating neurogenesis in the adult brain. Particularly, the application of site-specific recombinases and Tet inducible system in combination with transgenic or gene targeting strategy is discussed in further detail.